US20140121263A1 - Lipid formulated dsrna targeting the pcsk9 gene - Google Patents

Lipid formulated dsrna targeting the pcsk9 gene Download PDF

Info

Publication number
US20140121263A1
US20140121263A1 US14/058,052 US201314058052A US2014121263A1 US 20140121263 A1 US20140121263 A1 US 20140121263A1 US 201314058052 A US201314058052 A US 201314058052A US 2014121263 A1 US2014121263 A1 US 2014121263A1
Authority
US
United States
Prior art keywords
lipid
dsrna
peg
composition
cholesterol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US14/058,052
Inventor
Kevin Fitzgerald
Gregory Hinkle
Akin Akinc
Stuart Milstein
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Alnylam Pharmaceuticals Inc
Original Assignee
Alnylam Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Alnylam Pharmaceuticals Inc filed Critical Alnylam Pharmaceuticals Inc
Priority to US14/058,052 priority Critical patent/US20140121263A1/en
Assigned to ALNYLAM PHARMACEUTICALS, INC. reassignment ALNYLAM PHARMACEUTICALS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: AKINC, AKIN, FITZGERALD, KEVIN, HINKLE, GREGORY, MILSTEIN, STUART
Assigned to ALNYLAM PHARMACEUTICALS, INC. reassignment ALNYLAM PHARMACEUTICALS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: AKINC, AKIN, FITZGERALD, KEVIN, HINKLE, GREGORY, MILSTEIN, STUART
Publication of US20140121263A1 publication Critical patent/US20140121263A1/en
Priority to US15/060,492 priority patent/US20170000815A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1273—Polymersomes; Liposomes with polymerisable or polymerised bilayer-forming substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol

Definitions

  • compositions comprising lipid formulated dsRNA targeting a PCSK9 gene and methods for treating diseases caused by PCSK9 gene expression.
  • This application includes a Sequence Listing submitted electronically as a text file named 24757US_sequencelisting.txt, created on Oct. 18, 2013, with a size of 569,344 bytes. The sequence listing is incorporated by reference.
  • PCSK9 Proprotein convertase subtilisin kexin 9
  • PCSK1-PCSK8 also called PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, and S1P/SKI-1
  • PCSK1-PCSK8 also called PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, and S1P/SKI-1
  • PCSK1-PCSK8 also called PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, and S1P/SKI-1
  • PCSK1-PCSK8 also called PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, and S1P/SKI-1
  • PCSK9 has been proposed to play a role in cholesterol metabolism.
  • PCSK9 mRNA expression is down-regulated by dietary cholesterol feeding in mice (Maxwell, K. N., (2003) J. Lipid Res. 44, 2109-2119), up-regulated by statins in HepG2 cells (Dubuc, G., (2004) Arterioscler. Thromb. Vasc. Biol. 24, 1454-1459), and up-regulated in sterol regulatory element binding protein (SREBP) transgenic mice (Horton, J. D., (2003) Proc. Natl. Acad. Sci.
  • SREBP sterol regulatory element binding protein
  • Hchola3 autosomal dominant hypercholesterolemia
  • 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).
  • SNPs single-nucleotide polymorphisms
  • Autosomal dominant hypercholesterolemias are monogenic diseases in which patients exhibit elevated total and LDL cholesterol levels, tendon xanthomas, and premature atherosclerosis (Rader, D. J., (2003) J. Clin. Invest. 111, 1795-1803).
  • the pathogenesis of ADHs and a recessive form, autosomal recessive hypercholesterolemia (ARH) 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.
  • 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.
  • PCSK9 Loss of function mutations in PCSK9 have been designed in mouse models (Rashid et al., (2005) PNAS, 102, 5374-5379), and identified in human individuals (Cohen et al. (2005) Nature Genetics 37:161-165). In both cases loss of PCSK9 function lead to lowering of total and LDLc cholesterol. In a retrospective outcome study over 15 years, loss of one copy of PCSK9 was shown to shift LDLc levels lower and to lead to an increased risk-benefit protection from developing cardiovascular heart disease (Cohen et al., (2006) N. Engl. J. Med., 354:1264-1272).
  • dsRNA double-stranded RNA molecules
  • RNAi RNA interference
  • WO 99/32619 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 .
  • siRNA targeting PCSK9 can be found in U.S. Pat. No. 7,605,251 and WO 2007/134161. Additional disclosure can be found in U.S. Patent Publication No. 2010/0010066 and WO 2009/134487
  • compositions comprising lipid formulated siRNA targeting PCSK9, e.g., MC3 formulated siRNA targeting PCSK9. Also disclosed are methods of using the compositions for inhibition of PCSK9 expression and for treatment of pathologies related to PCSK9 expression, e.g., hyperlipidemia
  • one aspect of the invention is a compositing comprising a nucleic acid lipid particle comprising a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human PCSK9 gene in a cell
  • the nucleic acid lipid particle comprises a lipid formulation comprising 45-65 mol % of a cationic lipid, 5 mol % to about 10 mol %, of a non-cationic lipid, 25-40 mol % of a sterol, and 0.5-5 mol % of a PEG or PEG-modified lipid
  • the dsRNA consists of a sense strand and an antisense strand
  • the sense strand comprises a first sequence
  • the antisense strand comprises a second sequence complementary to at least 15 contiguous nucleotides of SEQ ID NO:1523 (5′-UUCUAGACCUGUUUUGCUU-3′), wherein the first sequence is complementary to the second sequence and wherein the d
  • the composition includes a cationic lipid.
  • the cationic lipid comprises MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate.
  • the lipid formulation can be selected from the following:
  • the cationic lipid comprises formula A wherein formula A is
  • the cationic lipid comprises formula A and is XTC (2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane).
  • the lipid formulation can include the cationic lipid XTC, the non-cationic lipid DSPC, the sterol cholesterol and the PEG lipid PEG-DMG.
  • the cationic lipid comprises XTC and the formulation is selected from the group consisting of:
  • the cationic lipid comprises ALNY-100 ((3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)).
  • the cationic lipid comprises ALNY-100 and the formulation consists of ALNY-100/DSPC/Cholesterol/PEG-DMG in a ratio of 50/10/38.5/1.5
  • the composition includes a dsRNA targeting PCSK9.
  • the sense strand comprises SEQ ID NO:1227 and the antisense strand comprises SEQ ID NO:1228.
  • the sense strand consists of SEQ ID NO:1227 and the antisense strand consists of SEQ ID NO:1228.
  • One or both strands can be modified, e.g., each strand is modified as follows to include a 2′-O-methyl ribonucleotide as indicated by a lower case letter “c” or “u” and a phosphorothioate as indicated by a lower case letter “s”: the dsRNA consists of a sense strand consisting of
  • SEQ ID NO: 1230 (5′- AAGcAAAAcAGGUCuAGAATsT-3′).
  • the dsRNA comprises at least one modified nucleotide, e.g., a modified nucleotide chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, and/or, e.g., the modified nucleotide is chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
  • compositions include a dsRNA between 15 and 30 base pairs in length.
  • each strand of the dsRNA is 19-23 bases in length, or, e.g., 21-23 bases in length, or, e.g. 21 bases in length.
  • the compositions include a lipoprotein, e.g., apolipoprotein E (ApoE).
  • the compositions include a lipoprotein and the dsRNA is conjugated to a lipophile, e.g., a cholesterol.
  • the ApoE can be reconstituted with at least one amphiphilic agent, e.g., a phospholipid, e.g., a phospholipid selected from the group consisting of dimyristoyl phosphatidyl choline (DMPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), -phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carbox
  • compositions e.g., lipid formulated dsRNA targeting PCSK9
  • a cell or subject e.g., a primate, e.g., a human.
  • administration of the compositions inhibits expression of PCSK9 by at least 40% compared to administration of a control and/or reduces PCSK9 protein levels in the mammal compared to administration of a control, and/or reduces LDLc levels in a mammal compared to administration of a control and/or reduces both PCSK9 hepatic mRNA and total serum cholesterol at a dosage of less than 0.1 mg/kg compared to administration of a control and/or reduces PCSK9 hepatic mRNA at an ED50 of about 0.2 mg/kg and reduces total serum cholesterol with an ED50 of about 0.08 mg/kg compared to administration of a control and/or reduces serum LDL particle numbers by more than 90% or reduces serum HDL particle numbers by more than 75% compared to administration of a cell or subject, a
  • the invention also provides methods comprising administering the lipid formulated PCSK9 targeted dsRNA compositions described herein.
  • the invention includes a method for inhibiting the expression of PCSK9 in a cell comprising administering the compositions to the cell.
  • the invention includes a method for reducing LDLc levels in a mammal in need of treatment comprising administering the compositions to the mammal.
  • the methods can include any appropriate dosage, e.g., between 0.25 mg/kg and 4 mg/kg dsRNA, or e.g., at about 0.01, 0.1, 0.5, 1.0, 2.5, or 5.0 mg/kg dsRNA.
  • Also described herein are methods for inhibiting expression of a PCSK9 gene in a subject comprising administering to the subject the lipid formulated PCSK9 targeted dsRNA compositions described herein 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.
  • the subject can be, .e.g., a rat or a non-human primate or a human.
  • the subsequent dose can be about 1.0 mg/kg or about 0.3 mg/kg.
  • the subsequent dose is administered once a week for four weeks.
  • administration of the first dose decreases total cholesterol levels by about 15-60%.
  • AD- “DP-” and “AL-DP-” are used interchangeably e.g., AL-DP-9327 and AD-9237.
  • FIG. 1 shows the structure of the ND-98 lipid.
  • FIG. 2 shows the results of the in vivo screen of 16 mouse specific (AL-DP-9327 through AL-DP-9342) PCSK9 siRNAs directed against different ORF regions of PCSK9 mRNA (having the first nucleotide corresponding to the ORF position indicated on the graph) in C57/BL6 mice (5 animals/group).
  • the ratio of PCSK9 mRNA to GAPDH mRNA in liver lysates was averaged over each treatment group and compared to a control group treated with PBS or a control group treated with an unrelated siRNA (blood coagulation factor VII).
  • FIG. 3 shows the results of the in vivo screen of 16 human/mouse/rat 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).
  • FIG. 4 shows the results of the in vivo screen of 16 mouse specific (AL-DP-9327 through AL-DP-9342) PCSK9 siRNAs in C57/BL6 mice (5 animals/group). Total serum cholesterol levels were averaged over each treatment group and compared to a control group treated with PBS or a control group treated with an unrelated siRNA (blood coagulation factor VII).
  • FIG. 5 shows the results of the in vivo screen of 16 human/mouse/rat 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 siRNA (blood coagulation factor VII).
  • FIGS. 6A and 6B compare in vitro and in vivo results, respectively, for silencing PCSK9.
  • FIG. 7A and FIG. 7B are an example of in vitro results for silencing PCSK9 using monkey primary hepatocytes.
  • FIG. 7C show results for silencing of PCSK9 in monkey primary hepatocytes using AL-DP-9680 and chemically modified version of AL-DP-9680.
  • FIG. 8 shows in vivo activity of LNP-01 formulated siRNAs to PCSK-9.
  • 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.
  • 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 10 A.
  • 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. 10C shows the effect of PCSK9 siRNAs on hepatic cholesterol and triglyceride levels in the same experiment as 10 A.
  • FIG. 11 is a Western blot showing that liver LDL receptor levels were upregulated following administration of PCSK9 siRNAs in rat.
  • 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.
  • FIG. 13A is a graph showing that unmodified siRNA-AD-A1A (AD-9314), but not 2′OMe modified siRNA-AD-1A2 (AD-10792), induced IFN-alpha in human primary blood monocytes.
  • FIG. 13B is a graph showing that unmodified siRNA-AD-A1A (AD-9314), but not 2′OMe modified siRNA-AD-1A2 (AD-10792), also induced TNF-alpha in human primary blood monocytes.
  • FIG. 14A is a graph showing that the PCSK9 siRNA siRNA-AD-1A2 (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-1A2 decreased serum total cholesterol levels in mice within 48 hours.
  • FIG. 15A is a graph showing that PCSK9 siRNAs 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.
  • FIG. 16 shows the structure of an siRNA conjugated to Chol-p-(GalNAc) 3 via phosphate linkage at the 3′ end.
  • FIG. 17 shows the structure of an siRNA conjugated to LCO(GalNAc) 3 (a (GalNAc)3-3′-Lithocholic-oleoyl siRNA Conjugate).
  • FIG. 18 is a graph showing the results of conjugated siRNAs on PCSK9 transcript levels and total serum cholesterol in mice.
  • FIG. 19 is a graph showing the results of lipid formulated siRNAs on PCSK9 transcript levels and total serum cholesterol in rats.
  • 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.
  • 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.
  • FIG. 22 is a graph with the results of PCSK9 targeted siRNA transfection of Hep3B cells and the effects on PCSK9 and off-target gene levels.
  • FIG. 23 shows the results of treatment in rats with a maintenance dose of PCSK9 targeted siRNA.
  • FIG. 24 is a dose response curve of treatment of HeLa cells with modified siRNAs.
  • FIG. 25 is a graph of average IC50 of siRNA vs. target position in human PCSK9 transcript. The large blue dot indicates the IC50 and location of AD-9680.
  • FIG. 26 is a graph with the results of administration of rEHDL formulated cholesterol conjugated siRNA.
  • FIG. 27A is a graph with results of administration of second generation LNP formulated PCSK9 targeted siRNA (AD-9680 in LNP11) to non-human primates, demonstrating a reduction in both PCSK9 protein and LDLc levels.
  • LDLc low density lipoprotein cholesterol
  • mpk mg per kg.
  • FIG. 27B is a bar graph showing dose dependent PCSK mRNA silencing in non-human primates after treatment with LNP formulated siRNA targeting PCSK9.
  • FIG. 27C is a graph with the results of administration of second generation LNP formulated PCSK9 targeted siRNA (AD-9680) to non-human primates, demonstrating a no change in HDLc levels.
  • FIG. 28 illustrates the chemical structures of the cationic lipids MC3 and ALNY-100.
  • FIG. 29 is a graph of effects on PCSK9 mRNA and serum cholesterol levels in rats after administration of LNP-09 formulated AD-10792, an siRNA targeting rodent PCSK9.
  • FIG. 30 are graphs of the effects on PCSK9 mRNA and LDL/HDL particle numbers in CETP/ApoB tg mice after administration of LNP-09 formulated AD-10792, an siRNA targeting rodent PCSK9.
  • the invention provides a solution to the problem of treating diseases that can be modulated by the down regulation of the PCSK9 gene, such as hyperlipidemia, by using double-stranded ribonucleic acid (dsRNA) to silence the PCSK9 gene.
  • diseases that can be modulated by the down regulation of the PCSK9 gene, such as hyperlipidemia, by using double-stranded ribonucleic acid (dsRNA) to silence the PCSK9 gene.
  • dsRNA double-stranded ribonucleic acid
  • the invention provides compositions and methods for inhibiting the expression of the PCSK9 gene in a subject using a dsRNA.
  • the invention also provides compositions and methods for treating pathological conditions and diseases, such as hyperlipidemia, that can be modulated by down regulating the expression of the PCSK9 gene.
  • dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).
  • the dsRNA useful for the compositions and methods of an invention include an RNA strand (the antisense strand) having a region that is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an mRNA transcript of the PCSK9 gene.
  • the use of these dsRNAs enables the targeted degradation of an mRNA that is involved in the regulation of the LDL Receptor and circulating cholesterol levels.
  • the present inventors Using cell-based and animal assays, the present inventors have demonstrated that very low dosages of these dsRNAs can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of the PCSK9 gene.
  • methods and compositions including these dsRNAs are useful for treating pathological processes that can be mediated by down regulating PCSK9, such as in the treatment of hyperlipidemia.
  • compositions of the invention include a dsRNA having an antisense strand having a region of complementarity that is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and that is substantially complementary to at least part of an RNA transcript of the PCSK9 gene, together with a pharmaceutically acceptable carrier.
  • compositions including the dsRNA that targets PCSK9 together with a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of the PCSK9 gene, and methods of using the pharmaceutical compositions to treat diseases by down regulating the expression of PCSK9.
  • 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.
  • ribonucleotide or “nucleotide” or “deoxyribonucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety.
  • 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.
  • a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil.
  • 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.
  • PCSK9 refers to the proprotein convertase subtilisin kexin 9 gene or protein (also known as FH3, HCHOLA3, NARC-1, NARC1).
  • Examples of mRNA sequences to PCSK9 include but are not limited to the following: human: NM — 174936; mouse: NM — 153565, and rat: NM — 199253. Additional examples of PCSK9 mRNA sequences are readily available using, e.g., GenBank.
  • target sequence refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the PCSK9 gene, including mRNA that is a product of RNA processing of a primary transcription product.
  • 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.
  • 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.
  • sequences can be referred to as “fully complementary” with respect to each other.
  • 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.
  • 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.”
  • “Complementary” sequences 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.
  • a polynucleotide which is “substantially complementary to at least part of” a messenger RNA 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.
  • 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.
  • double-stranded RNA refers a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands.
  • 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.
  • 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.
  • 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”.
  • the connecting structure is referred to as a “linker”.
  • the RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex.
  • a dsRNA may comprise one or more nucleotide overhangs.
  • each or both strands can also include at least one non-ribonucleotide, e.g., a deoxyribonucleotide and/or a modified nucleotide.
  • 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.
  • 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.
  • 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.
  • antisense strand refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence.
  • 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.
  • sense strand refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.
  • Introducing into a cell means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be “introduced into a cell”, wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.
  • the degree of inhibition is usually expressed in terms of
  • 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.
  • target gene silencing can be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay.
  • the assays provided in the Examples below shall serve as such reference.
  • 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.
  • 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.
  • treatment will involve a decrease in serum lipid levels.
  • therapeutically effective amount refers 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.
  • a “pharmaceutical composition” includes a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier.
  • pharmaceutically effective amount refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.
  • 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.
  • a “transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.
  • Double-Stranded Ribonucleic Acid dsRNA
  • the invention provides methods and composition having double-stranded ribonucleic acid (dsRNA) molecules 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.
  • 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.
  • expression of a PCSK9 gene in cell culture can be assayed by measuring PCSK9 mRNA levels, such as by bDNA or TaqMan assay, or by measuring protein levels, such as by ELISA assay.
  • the dsRNA of the invention can further include one or more single-stranded nucleotide overhangs.
  • 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.
  • 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 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.
  • 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 is 19 base pairs in length.
  • the duplex is 21 base pairs in length.
  • the duplex lengths can be identical or can differ.
  • 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. In other embodiments, each is strand is 25-30 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.
  • the dsRNA of the invention can include one or more single-stranded overhang(s) of one or more nucleotides.
  • at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides.
  • the antisense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ end and the 5′ end over the sense strand.
  • the sense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ end and the 5′ end over the antisense strand.
  • a dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties than the blunt-ended counterpart.
  • 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.
  • 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.
  • 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.
  • the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt.
  • one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
  • 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.
  • the PCSK9 gene is a human PCSK9 gene.
  • the antisense strand of the dsRNA includes a first strand selected from the sense sequences of Table 1a, Table 2a, and Table 5a, and a second strand selected from the antisense sequences of Table 1a, Table 2a, and Table 5a.
  • Alternative antisense agents that target elsewhere in the target sequence provided in Table 1a, Table 2a, and Table 5a can readily be determined using the target sequence and the flanking PCSK9 sequence.
  • the dsRNA AD-9680 targets the PCSK 9 gene at 3530-3548; therefore the target sequence is as follows: 5′ UUCUAGACCUGUUUUGCUU 3′ (SEQ ID NO:1523).
  • the dsRNA AD-10792 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 that have regions of complementarity to SEQ ID NO:1523 and SEQ ID NO:1524.
  • the dsRNA includes at least one nucleotide sequence selected from the groups of sequences provided in Table 1a, Table 2a, and Table 5a. In other embodiments, the dsRNA includes at least two sequences selected from this group, where one of the at least two sequences is complementary to another of the at least two sequences, and one of the at least two sequences is substantially complementary to a sequence of an mRNA generated in the expression of the PCSK9 gene.
  • the dsRNA includes two oligonucleotides, where one oligonucleotide is described as the sense strand in Table 1a, Table 2a, and Table 5a and the second oligonucleotide is described as the antisense strand in Table 1a, Table 2a, and Table 5a
  • the dsRNAs of the invention can include at least one strand of a length of minimally 21 nt.
  • dsRNAs having one of the sequences of Table 1a, Table 2a, and Table 5a minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above.
  • dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Table 1a, Table 2a, and Table 5a, and differing in their ability to inhibit the expression of the PCSK9 gene in a FACS assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence are contemplated by the invention.
  • Further dsRNAs that cleave within the target sequence provided in Table 1a, Table 2a, and Table 5a can readily be made using the PCSK9 sequence and the target sequence provided.
  • RNAi agents provided in Table 1a, Table 2a, and Table 5a identify a site in the PCSK9 mRNA that is susceptible to RNAi based cleavage.
  • the present invention further includes RNAi agents that target within the sequence targeted by one of the agents of the present invention.
  • a second RNAi agent is said to target within the sequence of a first RNAi agent if the second RNAi agent cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first RNAi agent.
  • Such a second agent will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Table 1a, Table 2a, and Table 5a coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in the PCSK9 gene.
  • 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 1a, Table 2a, and Table 5a.
  • the dsRNA of the invention can contain one or more mismatches to the target sequence.
  • the dsRNA of the invention contains no more than 1, no more than 2, or no more than 3 mismatches.
  • 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.
  • 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.
  • 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.
  • At least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides.
  • dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties than their blunt-ended counterparts.
  • the present inventors have discovered that the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability.
  • dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum.
  • the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand.
  • the dsRNA may also have a blunt end, generally located at the 5′-end of the antisense strand.
  • Such dsRNAs have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day.
  • the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt.
  • one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
  • the dsRNA is chemically modified to enhance stability.
  • the nucleic acids of the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference.
  • Chemical modifications may include, but are not limited to 2′ modifications, modifications at other sites of the sugar or base of an oligonucleotide, introduction of non-natural bases into the oligonucleotide chain, covalent attachment to a ligand or chemical moiety, and replacement of internucleotide phosphate linkages with alternate linkages such as thiophosphates. More than one such modification may be employed.
  • Chemical linking of the two separate dsRNA strands may be achieved by any of a variety of well-known techniques, for example by introducing covalent, ionic or hydrogen bonds; hydrophobic interactions, van der Waals or stacking interactions; by means of metal-ion coordination, or through use of purine analogues.
  • the chemical groups that can be used to modify the dsRNA include, without limitation, methylene blue; bifunctional groups, generally bis-(2-chloroethyl)amine; N-acetyl-N′-(p-glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen.
  • the linker is a hexa-ethylene glycol linker.
  • the dsRNA are produced by solid phase synthesis and the hexa-ethylene glycol linker is incorporated according to standard methods (e.g., Williams, D. J., and K. B. Hall, Biochem . (1996) 35:14665-14670).
  • the 5′-end of the antisense strand and the 3′-end of the sense strand are chemically linked via a hexaethylene glycol linker.
  • at least one nucleotide of the dsRNA comprises a phosphorothioate or phosphorodithioate groups.
  • the chemical bond at the ends of the dsRNA is generally formed by triple-helix bonds.
  • Table 1a, Table 2a, and Table 5a provides examples of modified RNAi agents of the invention.
  • the nucleotides at one or both of the two single strands may be modified to prevent or inhibit the degradation activities of cellular enzymes, such as, for example, without limitation, certain nucleases.
  • cellular enzymes such as, for example, without limitation, certain nucleases.
  • Techniques for inhibiting the degradation activity of cellular enzymes against nucleic acids are known in the art including, but not limited to, 2′-amino modifications, 2′-amino sugar modifications, 2′-F sugar modifications, 2′-F modifications, 2′-alkyl sugar modifications, uncharged backbone modifications, morpholino modifications, 2′-O-methyl modifications, and phosphoramidate (see, e.g., Wagner, Nat. Med . (1995) 1:1116-8).
  • At least one 2′-hydroxyl group of the nucleotides on a dsRNA is replaced by a chemical group, generally by a 2′-amino or a 2′-methyl group.
  • at least one nucleotide may be modified to form a locked nucleotide.
  • Such locked nucleotide contains a methylene bridge that connects the 2′-oxygen of ribose with the 4′-carbon of ribose.
  • Oligonucleotides containing the locked nucleotide are described in Koshkin, A. A., et al., Tetrahedron (1998), 54: 3607-3630) and Obika, S. et al., Tetrahedron Lett .
  • Conjugating a ligand to a dsRNA can enhance its cellular absorption as well as targeting to a particular tissue or uptake by specific types of cells such as liver cells.
  • a hydrophobic ligand is conjugated to the dsRNA to facilitate direct permeation of the cellular membrane and or uptake across the liver cells.
  • the ligand conjugated to the dsRNA is a substrate for receptor-mediated endocytosis.
  • oligonucleotides include 1-pyrene butyric acid, 1,3-bis-O-(hexadecyl)glycerol, and menthol.
  • a ligand for receptor-mediated endocytosis is folic acid. Folic acid enters the cell by folate-receptor-mediated endocytosis. dsRNA compounds bearing folic acid would be efficiently transported into the cell via the folate-receptor-mediated endocytosis.
  • Li and coworkers report that attachment of folic acid to the 3′-terminus of an oligonucleotide resulted in an 8-fold increase in cellular uptake of the oligonucleotide.
  • Other ligands that have been conjugated to oligonucleotides include polyethylene glycols, carbohydrate clusters, cross-linking agents, porphyrin conjugates, delivery peptides and lipids such as cholesterol and cholesterylamine.
  • carbohydrate clusters examples include Chol-p-(GalNAc) 3 (N-acetyl galactosamine cholesterol) and LCO(GalNAc) 3 (N-acetyl galactosamine-3′-Lithocholic-oleoyl.
  • conjugation of a cationic ligand to oligonucleotides results in improved resistance to nucleases.
  • Representative examples of cationic ligands are propylammonium and dimethylpropylammonium.
  • antisense oligonucleotides were reported to retain their high binding affinity to mRNA when the cationic ligand was dispersed throughout the oligonucleotide. See M. Manoharan Antisense & Nucleic Acid Drug Development 2002, 12, 103 and references therein.
  • a ligand can be multifunctional and/or a dsRNA can be conjugated to more than one ligand.
  • the dsRNA can be conjugated to one ligand for improved uptake and to a second ligand for improved release.
  • the ligand-conjugated dsRNA of the invention may be synthesized by the use of a dsRNA that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the dsRNA.
  • This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.
  • the methods of the invention facilitate the synthesis of ligand-conjugated dsRNA by the use of, in some embodiments, nucleoside monomers that have been appropriately conjugated with ligands and that may further be attached to a solid-support material.
  • Such ligand-nucleoside conjugates are prepared according to certain embodiments of the methods described herein via reaction of a selected serum-binding ligand with a linking moiety located on the 5′ position of a nucleoside or oligonucleotide.
  • a dsRNA bearing an aralkyl ligand attached to the 3′-terminus of the dsRNA is prepared by first covalently attaching a monomer building block to a controlled-pore-glass support via a long-chain aminoalkyl group. Then, nucleotides are bonded via standard solid-phase synthesis techniques to the monomer building-block bound to the solid support.
  • the monomer building block may be a nucleoside or other organic compound that is compatible with solid-phase synthesis.
  • the dsRNA used in the conjugates of the invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis.
  • Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.
  • 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. 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.
  • 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.
  • nucleotide-conjugate precursors that already bear a linking moiety
  • the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide.
  • Oligonucleotide conjugates bearing a variety of molecules such as steroids, vitamins, lipids and reporter molecules, has previously been described (see Manoharan et al., PCT Application WO 93/07883).
  • 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.
  • oligonucleotide confers enhanced hybridization properties to the oligonucleotide. Further, oligonucleotides containing phosphorothioate backbones have enhanced nuclease stability.
  • functionalized, linked nucleosides of the invention can be augmented to include either or both a phosphorothioate backbone or a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′- ⁇ -aminoalkyl, 2′-O-allyl or 2′-deoxy-2′-fluoro group.
  • a phosphorothioate backbone or a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′- ⁇ -aminoalkyl, 2′-O-allyl or 2′-deoxy-2′-fluoro group.
  • 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.
  • 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.
  • 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.
  • 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.
  • modified internucleoside linkages or backbones that do not include a phosphorus atom therein i.e., oligonucleosides
  • 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.
  • 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
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • Representative United States patents relating to the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.
  • the oligonucleotide may be modified by a non-ligand group.
  • 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.
  • a thioether e.g., hexyl-5-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., 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).
  • oligonucleotide conjugates 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.
  • 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).
  • a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell.
  • each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid.
  • a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
  • the recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors.
  • dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol . (1992) 158:97-129)); adenovirus (see, for example, Berkner, et al., BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or alphavirus as well as others known in the art.
  • 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 (
  • 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 et al., 1991, Proc. Natl. Acad. Sci.
  • 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.
  • susceptible hosts e.g., rat, hamster, dog, and chimpanzee
  • 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.
  • AV adenovirus
  • AAV adeno-associated virus
  • 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.
  • 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.
  • 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.
  • 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.
  • Preferred viral vectors are those derived from AV and AAV.
  • 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 H1 RNA promoters, or the cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • a suitable AV vector for expressing the dsRNA of the invention a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
  • Suitable AAV vectors for expressing the dsRNA of the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
  • the promoter driving dsRNA expression in either a DNA plasmid or viral vector of the invention may be a eukaryotic RNA polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter or actin promoter or U1 snRNA promoter) or generally RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter.
  • RNA polymerase I e.g. ribosomal RNA promoter
  • RNA polymerase II e.g. CMV early promoter or actin promoter or U1 snRNA promoter
  • RNA polymerase III promoter e.g. U6 snRNA or 7SK RNA promoter
  • a prokaryotic promoter for example
  • 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)).
  • 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).
  • inducible expression systems suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (EPTG).
  • ETG isopropyl-beta-D1-thiogalactopyranoside
  • recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells.
  • viral vectors can be used that provide for transient expression of dsRNA molecules.
  • Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
  • dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g. Oligofectamine) or non-cationic lipid-based carriers (e.g. Transit-TKOTM).
  • cationic lipid carriers e.g. Oligofectamine
  • non-cationic lipid-based carriers e.g. Transit-TKOTM
  • Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single PCSK9 gene or multiple PCSK9 genes over a period of a week or more are also contemplated by the invention.
  • Successful introduction of the vectors of the invention into host cells can be monitored using various known methods. For example, transient transfection. can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of ex vivo cells can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as
  • the PCSK9 specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • the invention provides pharmaceutical compositions containing a dsRNA, as described herein, and a pharmaceutically acceptable carrier and methods of administering the same.
  • the pharmaceutical composition containing the dsRNA 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.
  • compositions featured herein are administered in dosages sufficient to inhibit expression of PCSK9 genes.
  • a suitable dose of dsRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day.
  • 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.
  • the dosage is between 0.01 and 0.2 mg/kg.
  • 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.
  • the dosage is between 0.2 mg/kg and 1.5 mg/kg.
  • the dsRNA 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, or 1.5 mg/kg.
  • the dsRNA can be administered at a dose of 0.03, 0.1, 0.3, or 1.3, or 3.0 mg/kg.
  • the pharmaceutical composition can be administered once daily, or the dsRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day.
  • the effect of a single dose on PCSK9 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.
  • the lipid formulated PCSK9 targeted dsRNA 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.
  • the subsequent dose can be administered, e.g., once a week for four weeks.
  • the dsRNA is administered using continuous infusion or delivery through a controlled release formulation.
  • the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage.
  • the dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention.
  • the dosage unit contains a corresponding multiple of the daily dose.
  • treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments.
  • Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
  • a suitable mouse model is, for example, a mouse containing a plasmid expressing human PCSK9.
  • Another suitable mouse model is a transgenic mouse carrying a transgene that expresses human PCSK9.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds that exhibit high therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • 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.
  • a target sequence e.g., achieving a decreased concentration of the polypeptide
  • the IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • the dsRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by target gene expression.
  • 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.
  • compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, 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.
  • the dsRNA molecules 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.
  • dsRNAs conjugated or unconjugate or formulated with or without liposomes
  • a dsRNA molecule can be formulated into compositions such as sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions in liquid or solid oil bases.
  • Such solutions also can contain buffers, diluents, and other suitable additives.
  • a dsRNA molecule can be formulated into compositions such as sterile aqueous solutions, which also can contain buffers, diluents, and other suitable additives (e.g., penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers). Formulations are described in more detail herein.
  • the dsRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).
  • a particular tissue such as the liver (e.g., the hepatocytes of the liver).
  • the pharmaceutical formulations of the present invention 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.
  • 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.
  • 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.
  • dsRNA that target the PCSK9 gene can be formulated into compositions containing the dsRNA admixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of nucleic acids.
  • a composition containing one or more dsRNA agents that target the PCSK9 gene can contain other therapeutic agents, such as other cancer therapeutics or one or more dsRNA compounds that target non-PCSK9 genes.
  • compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators.
  • Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof.
  • Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate.
  • DCA chenodeoxycholic acid
  • UDCA ursodeoxychenodeoxycholic acid
  • cholic acid dehydrocholic acid
  • deoxycholic 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).
  • arachidonic acid 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, gly
  • combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts.
  • One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA.
  • Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
  • dsRNAs featured in the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles.
  • dsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches.
  • Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG).
  • TDAE polythiodiethylamino
  • 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.
  • 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 dsRNAs 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).
  • neutral e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline
  • negative e.g., dimyristoylphosphatidyl glycerol DMPG
  • cationic e.g., dioleoyltetramethylaminopropyl DOTAP and
  • dsRNAs may be complexed to lipids, in particular to cationic lipids.
  • Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C 1-10 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.
  • oleic acid eicosanoic acid
  • lauric acid caprylic acid
  • capric acid myristic acid, palmitic acid
  • dsRNA molecules can be administered to a mammal as biologic or abiologic means as described in, for example, U.S. Pat. No. 6,271,359.
  • Abiologic delivery can be accomplished by a variety of methods including, without limitation, (1) loading liposomes with a dsRNA acid molecule provided herein and (2) complexing a dsRNA molecule with lipids or liposomes to form nucleic acid-lipid or nucleic acid-liposome complexes.
  • the liposome can be composed of cationic and neutral lipids commonly used to transfect cells in vitro.
  • Cationic lipids can complex (e.g., charge-associate) with negatively charged nucleic acids to form liposomes.
  • 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.
  • lipophilic agents are commercially available, including LipofectinTM (Invitrogen/Life Technologies, Carlsbad, Calif.) and EffecteneTM (Qiagen, Valencia, Calif.).
  • 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.
  • liposomes such as those described by Templeton et al. (Nature Biotechnology, 15: 647-652 (1997) can be used.
  • polycations such as polyethyleneimine can be used to achieve delivery in vivo and ex vivo (Boletta et al., J. Am Soc. Nephrol.
  • Biologic delivery can be accomplished by a variety of methods including, without limitation, the use of viral vectors.
  • viral vectors e.g., adenovirus and herpes virus vectors
  • 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.
  • liposome means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
  • Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.
  • lipid vesicles 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.
  • 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.
  • 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.
  • 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.
  • liposomes to deliver agents including high-molecular weight DNA into the skin.
  • Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis
  • Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).
  • Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).
  • liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine.
  • Neutral liposome compositions 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).
  • DOPE dioleoyl phosphatidylethanolamine
  • Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC.
  • PC phosphatidylcholine
  • Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
  • 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 NovasomeTM I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and NovasomeTM II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al., S.T.P. Pharma. Sci., 1994, 4, 6, 466).
  • Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids.
  • sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G M1 , or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
  • PEG polyethylene glycol
  • Liposomes comprising (1) sphingomyelin and (2) the ganglioside G M1 or a galactocerebroside sulfate ester.
  • U.S. Pat. No. 5,543,152 discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Lim et al.).
  • liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art.
  • Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C 1215G , that contains a PEG moiety.
  • Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives.
  • Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S.
  • Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher.
  • Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1).
  • Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No.
  • a number of liposomes comprising nucleic acids are known in the art.
  • WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes.
  • U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA.
  • U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes.
  • WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.
  • Transfersomes are yet another type of liposomes and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes, it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
  • HLB hydrophile/lipophile balance
  • Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure.
  • Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters.
  • Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class.
  • the polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
  • 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.
  • Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
  • amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
  • a dsRNA featured in the invention is fully encapsulated in the lipid formulation, e.g., to form a nucleic acid-lipid particle, e.g., 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).
  • i.v. intravenous
  • nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease.
  • Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.
  • 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.
  • 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. Pat. No. 6,320,017), peptides, proteins, detergents, lipid-derivatives, such as PEG coupled to phosphatidylethanolamine and PEG conjugated to ceramides (see, U.S. Pat. No. 5,885,613).
  • bilayer stabilizing components such as polyamide oligomers (see, e.g., U.S. Pat. No. 6,320,017), peptides, proteins, detergents, lipid-derivatives, such as PEG coupled to phosphatidylethanolamine and PEG conjugated to ceramides (see, U.S. Pat. No. 5,885,613).
  • 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.
  • Nucleic acid-lipid particles include, e.g., a SPLP, pSPLP, and SNALP.
  • SNALP refers to a stable nucleic acid-lipid particle, including SPLP.
  • 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.
  • 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 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, or 33:1.
  • the nucleic acid-lipid particles of the invention typically include a cationic lipid.
  • the cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),
  • 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.
  • 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”); 1,2-Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.C1”); 3 ⁇ -(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”)
  • cationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECTAMINE (comprising DOSPA and DOPE, available from GIBCO/BRL).
  • LIPOFECTIN including DOTMA and DOPE, available from GIBCO/BRL
  • LIPOFECTAMINE comprising DOSPA and DOPE, available from GIBCO/BRL
  • a cationic lipid is an amino lipid.
  • 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.
  • 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).
  • R 11 and R 12 are both long chain alkyl or acyl groups, they can be the same or different.
  • 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 C 14 to C 22 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.
  • 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.
  • physiological pH e.g. pH 7.4
  • second pH preferably at or above physiological pH.
  • 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.
  • 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.
  • pKa is that at least some nucleic acid associated with the outside surface of the particle will lose its electrostatic interaction at physiological pH and be removed by simple dialysis; thus greatly reducing the particle's susceptibility to clearance.
  • a cationic lipid is 1,2-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 Apr. 15, 2009.
  • the cationic lipid XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane) is used to prepare nucleic acid-lipid particles. Synthesis of XTC is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.
  • the cationic lipids have the structure
  • R 1 and R 2 are each independently for each occurrence optionally substituted C 10 -C 30 alkyl, optionally substituted C 10 -C 30 alkenyl, optionally substituted C 10 -C 30 alkynyl, optionally substituted C 10 -C 30 acyl, or -linker-ligand;
  • R 3 is H, optionally substituted C 1 -C 10 alkyl, optionally substituted C 2 -C 10 alkenyl, optionally substituted C 2 -C 10 alkynyl, alkylhetrocycle, alkylphosphate, alkylphosphorothioate, alkylphosphorodithioate, alkylphosphonates, alkylamines, hydroxyalkyls, ⁇ -aminoalkyls, ⁇ -(substituted)aminoalkyls, ⁇ -phosphoalkyls, ⁇ -thiophosphoalkyls, optionally substituted polyethylene glycol (PEG,
  • the cationic lipid MC3 ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-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. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, and U.S. Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, and U.S. patent application Ser. No. 12/813/448 filed on Jun. 10, 2010, which are hereby incorporated by reference.
  • the cationic lipid ALNY-100 ((3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]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 Nov. 10, 2009, which is herein incorporated by reference.
  • FIG. 28 illustrates the structures of ALNY-100 and MC3.
  • the cationic lipid may comprise from about 20 mol % to about 70 mol % or about 45-65 mol % or about 10, 20, 30, 40, 50, 60, or 70 mol % of the total lipid present in the particle.
  • 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-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidy
  • 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.
  • 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 diacylphosphatidylcholine, diacylphosphatidylethanolamine, 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.
  • the neutral lipid component is a lipid having two acyl groups, (i.e., diacylphosphatidylcholine and diacylphosphatidylethanolamine).
  • 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.
  • lipids containing saturated fatty acids with carbon chain lengths in the range of C 14 to C 22 are preferred.
  • lipids with mono- or di-unsaturated fatty acids with carbon chain lengths in the range of C 14 to C 22 are used.
  • lipids having mixtures of saturated and unsaturated fatty acid chains can be used.
  • 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.
  • non-cationic lipid is distearoylphosphatidylcholine (DSPC). In another embodiment the non-cationic lipid is dipalmitoylphosphatidylcholine (DPPC).
  • DSPC distearoylphosphatidylcholine
  • DPPC dipalmitoylphosphatidylcholine
  • 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.
  • Conjugated lipids can be used in nucleic acid-lipid particle to prevent aggregation, including polyethylene glycol (PEG)-modified lipids, monosialoganglioside Gm1, and polyamide oligomers (“PAO”) such as (described in U.S. Pat. No. 6,320,017).
  • PEG polyethylene glycol
  • PAO polyamide oligomers
  • Other compounds with uncharged, hydrophilic, steric-barrier moieties, which prevent aggregation during formulation, like PEG, Gm1 or ATTA, 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. Pat. No.
  • the concentration of the lipid component selected to reduce aggregation is about 1 to 15% (by mole percent of lipids).
  • PEG-modified lipids or lipid-polyoxyethylene conjugates
  • 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 U.S. Ser. No. 08/486,214, incorporated herein by reference, PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. Particularly preferred are PEG-modified diacylglycerols and dialkylglycerols.
  • lipid anchor 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 1/2 less than 60 minutes in some assays. As illustrated in U.S. patent application Ser. No.
  • Compounds having suitable variations of these features may be useful for the invention.
  • Exemplary lipid anchors include those having lengths of from about C 14 to about C 22 , preferably from about C 14 to about C 16 .
  • a PEG moiety for example an mPEG-NH 2 , has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 Daltons.
  • 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.
  • 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.
  • PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci 2 ), a PEG-dimyristyloxypropyl (Ci 4 ), a PEG-dipalmityloxypropyl (Ci 6 ), or a PEG-distearyloxypropyl (C] 8 ).
  • 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)propyl1-(methoxy poly(ethylene 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)propyl1-(methoxy poly(ethylene glycol)2000)propylcarbamate)) (GalNAc-PEG-DSG); and polyethylene glycol-dipalmitoylglycerol (PEG-DPG).
  • PEG-DMG poly
  • 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.
  • 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 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.
  • 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.
  • 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 formulations of the invention further comprise an apolipoprotein.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. Vasc. 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.
  • the apolipoprotein can be a fragment, variant or isoform of the apolipoprotein.
  • 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.
  • 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.
  • 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.
  • conservative substitutions include the substitution of at least one hydrophobic residue such as isoleucine, valine, leucine or methionine for another.
  • 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).
  • 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.
  • the methods and compositions of the present invention include the use of a chimeric construction of an apolipoprotein.
  • 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.
  • a specific character e.g., lipid binding, receptor binding, enzymatic, enzyme activating, antioxidant or reduction-oxidation property
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the apolipoprotein can be a mixture of apolipoproteins.
  • the apolipoprotein can be a homogeneous mixture, that is, a single type of apolipoprotein.
  • the apolipoprotein can be a heterogeneous mixture of apolipoproteins, that is, a mixture of two or more different apolipoproteins.
  • Embodiments of heterogeneous mixtures of apolipoproteins can comprise, for example, a mixture of an apolipoprotein from an animal source and an apolipoprotein from a semi-synthetic source.
  • a heterogeneous 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.
  • 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 apolipoprotein 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.
  • 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 sphingolipids.
  • Representative phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatdylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleylphosphatidylcholine.
  • phosphorus-lacking compounds such as sphingolipids, glycosphingolipid families, diacylglycerols, and ⁇ -acyloxyacids, can also be used. Additionally, such amphipathic lipids can be readily mixed with other lipids, such as triglycerides and sterols.
  • 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.
  • 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 14 to about C 22 , preferably from about C 14 to about C 16 .
  • a PEG moiety for example an mPEG-NH 2 , has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 Daltons.
  • 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.
  • a targeting moiety e.g., a targeting moiety that is specific to a cell type or tissue.
  • 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. Pat. 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, T M, Prog. Lipid Res. 42(5):439-62 (2003); and Abra, R M et al., J. Liposome Res. 12:1-3, (2002).
  • lipid particles i.e., liposomes
  • hydrophilic polymer chains such as polyethylene glycol (PEG) chains
  • a ligand such as an antibody, for targeting the lipid particle is linked to the polar head group of lipids forming the lipid particle.
  • 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)).
  • Standard methods for coupling the target agents can be used.
  • phosphatidylethanolamine which can be activated for attachment of target agents
  • derivatized lipophilic compounds such as lipid-derivatized bleomycin
  • 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. Pat. No.
  • 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.
  • the nucleic acid-lipid particle formulations of the invention are produced via an extrusion method or an in-line mixing method.
  • 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.
  • 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.
  • the lipid-nucleic acid compositions which are formed can be used without any sizing.
  • 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 U.S. Pat. No. 6,534,018 and U.S. Pat. No. 6,855,277; US publication 2007/0042031 and Pharmaceuticals Research , Vol. 22, No. 3, March 2005, p. 362-372, which are hereby incorporated by reference in their entirety.
  • formulations of the invention can be prepared by any methods known to one of ordinary skill in the art.
  • Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners.
  • 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-X100.
  • 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%.
  • the formulations of the invention are entrapped by at least 75%, at least 80% or at least 90%.
  • 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.
  • nucleic acid-lipid particles are synthesized using the lipidoid ND98.4HCl (MW 1487) (Formula 1), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids). This nucleic acid-lipid particle is sometimes referred to as a LNP01 particle.
  • Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml.
  • the ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio.
  • the combined lipid solution can be mixed with aqueous 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.
  • aqueous siRNA e.g., in sodium acetate pH 5
  • Lipid-siRNA nanoparticles typically form spontaneously upon mixing.
  • 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).
  • a thermobarrel extruder such as Lipex Extruder (Northern Lipids, Inc).
  • 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.
  • PBS phosphate buffered saline
  • LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.
  • nucleic acid-lipid particle formulations are described in the following table. It is to be understood that the name of the nucleic acid-lipid particle in the table is not meant to be limiting.
  • SNALP refers to formulations that include the cationic lipid DLinDMA.
  • lipid:siRNA ratio SNALP DLinDMA/DPPC/Cholesterol/PEG-cDMA 57.1/7.1/34.4/1.4
  • XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, which is hereby incorporated by reference.
  • MC3 comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, and U.S. Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, which are hereby incorporated by reference.
  • ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.
  • Lipid refers to a cationic lipid.
  • any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles of the invention may be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. All substituents are as defined below unless indicated otherwise.
  • Alkyl means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.
  • Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.
  • saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.
  • Alkenyl means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.
  • Alkynyl means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons.
  • Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.
  • Acyl means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below.
  • —C( ⁇ O)alkyl, —C( ⁇ O)alkenyl, and —C( ⁇ O)alkynyl are acyl groups.
  • Heterocycle means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring.
  • the heterocycle may be attached via any heteroatom or carbon atom.
  • Heterocycles include heteroaryls as defined below.
  • Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
  • optionally substituted alkyl means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent ( ⁇ O) two hydrogen atoms are replaced.
  • substituents include oxo, halogen, heterocycle, —CN, —OR x , —NR x R y , —NR x C( ⁇ O)R y , —NR x SO 2 R y , —C( ⁇ O)R x , —C( ⁇ O)OR x , —C( ⁇ O)NR x R y , —SO n R x and —SO n NR x R y , wherein n is 0, 1 or 2, R x and R y are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl, —OR x , heterocycle, —NR x R y , —NR x C( ⁇ O)R y , —NR x SO 2 R y , —
  • Halogen means fluoro, chloro, bromo and iodo.
  • the methods of the invention may require the use of protecting groups.
  • protecting group methodology is well known to those skilled in the art (see, for example, P ROTECTIVE G ROUPS IN O RGANIC S YNTHESIS , Green, T. W. et al., Wiley-Interscience, New York City, 1999).
  • protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group.
  • a protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group.
  • an “alcohol protecting group” is used.
  • An “alcohol protecting group” is any group which decreases or eliminates unwanted reactivity of an alcohol functional group.
  • Protecting groups can be added and removed using techniques well known in the art.
  • nucleic acid-lipid particles of the invention are formulated using a cationic lipid of formula A:
  • the cationic lipid is XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane).
  • the lipid of formula A above may be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.
  • Lipid A where R 1 and R 2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R 3 and R 4 are independently lower alkyl or R 3 and R 4 can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1.
  • Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A.
  • the lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.
  • the ketone 1 starting material can be prepared according to Scheme 2.
  • Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.
  • the cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction ( ⁇ 3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature.
  • compositions comprising a lipid particle of the invention and an active agent, wherein the active agent is associated with the lipid particle.
  • the active agent is a therapeutic agent.
  • the active agent is encapsulated within an aqueous interior of the lipid particle.
  • the active agent is present within one or more lipid layers of the lipid particle.
  • the active agent is bound to the exterior or interior lipid surface of a lipid particle.
  • “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, Calif.). 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.
  • Active agents 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 PrimatizedTM 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.
  • nucleic acids e.g., nucleic acids, peptides and polypeptides
  • antibodies such as, e.g., polyclonal antibodies, monoclonal antibodies, antibody fragments
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, de
  • 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 ⁇ m 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.
  • Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other.
  • emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety.
  • 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.
  • compositions 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.
  • a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
  • Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion.
  • Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
  • Synthetic surfactants also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (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.
  • HLB hydrophile/lipophile balance
  • 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).
  • 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.
  • 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.
  • non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
  • Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
  • polysaccharides for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth
  • cellulose derivatives for example, carboxymethylcellulose and carboxypropylcellulose
  • synthetic polymers for example, carbomers, cellulose ethers, and
  • 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.
  • 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.
  • free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite
  • antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
  • Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants.
  • ionic surfactants non-ionic surfactants
  • Brij 96 polyoxyethylene oleyl ethers
  • polyglycerol fatty acid esters tetraglycerol monolaurate (ML310),
  • the cosurfactant usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules.
  • Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art.
  • the aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol.
  • the oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
  • materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
  • Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs.
  • Lipid based microemulsions both o/w and w/o have been proposed to enhance the oral bioavailability of drugs, including peptides (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.
  • 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.
  • 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 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.
  • surfactants 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.
  • 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).
  • Fatty acids Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C 1-10 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 e
  • 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).
  • 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
  • Chelating agents 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.
  • 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).
  • EDTA disodium ethylenediaminetetraacetate
  • citric acid e.g., citric acid
  • salicylates e.g., sodium salicylate, 5-methoxysalicylate and homovanilate
  • N-acyl derivatives of collagen e.g., laureth-9 and N-amino acyl derivatives of
  • 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 includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).
  • Agents that enhance uptake of dsRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention.
  • 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.
  • 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.
  • glycols such as ethylene glycol and propylene glycol
  • pyrrols such as 2-pyrrol
  • azones such as 2-pyrrol
  • terpenes such as limonene and menthone.
  • dsRNAs of the present invention can be formulated in a pharmaceutically acceptable carrier or diluent.
  • a “pharmaceutically acceptable carrier” (also referred to herein as an “excipient”) is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle.
  • Pharmaceutically acceptable carriers can be liquid or solid, and can be selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other pertinent transport and chemical properties.
  • Typical pharmaceutically acceptable carriers include, by way of example and not limitation: water; saline solution; binding agents (e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, gelatin, or calcium sulfate); lubricants (e.g., starch, polyethylene glycol, or sodium acetate); disintegrates (e.g., starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).
  • 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
  • wetting agents e.g., sodium lau
  • compositions of the present invention also incorporate carrier compounds in the formulation.
  • 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.
  • a nucleic acid and a carrier compound 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.
  • the recovery of a partially phosphorothioate dsRNA 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.
  • a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal.
  • the excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition.
  • Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).
  • binding agents e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropy
  • compositions of the present invention can also be used to formulate the compositions of the present invention.
  • suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases.
  • the solutions may also contain buffers, diluents and other suitable additives.
  • Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
  • Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels.
  • 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.
  • 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.
  • 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.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • a composition of the invention can be used in combination therapy.
  • the term “combination therapy” includes the administration of the subject compounds in further combination with other biologically active ingredients (such as, but not limited to, a second and different antineoplastic agent) and non-drug therapies (such as, but not limited to, surgery or radiation treatment).
  • the compounds of the invention can be used in combination with other pharmaceutically active compounds, preferably compounds that are able to enhance the effect of the compounds of the invention.
  • the compounds of the invention can be administered simultaneously (as a single preparation or separate preparation) or sequentially to the other drug therapy.
  • a combination therapy envisions administration of two or more drugs during a single cycle or course of therapy.
  • the subject compounds may be administered in combination with one or more separate agents that modulate protein kinases involved in various disease states.
  • kinases may include, but are not limited to: serine/threonine specific kinases, receptor tyrosine specific kinases and non-receptor tyrosine specific kinases.
  • Serine/threonine kinases include mitogen activated protein kinases (MAPK), meiosis specific kinase (MEK), RAF and aurora kinase.
  • receptor kinase families include epidermal growth factor receptor (EGFR) (e.g., HER2/neu, HER3, HER4, ErbB, ErbB2, ErbB3, ErbB4, Xmrk, DER, Let23); fibroblast growth factor (FGF) receptor (e.g. FGF-R1, GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF, KGF-R); hepatocyte growth/scatter factor receptor (HGFR) (e.g., MET, RON, SEA, SEX); insulin receptor (e.g. IGFI-R); Eph (e.g.
  • Non-receptor tyrosine kinase families include, but are not limited to, BCR-ABL (e.g. p43 abl , ARG); BTK (e.g. ITK/EMT, TEC); CSK, FAK, FPS, JAK, SRC, BMX, FER, CDK and SYK.
  • the subject compounds may be administered in combination with one or more agents that modulate non-kinase biological targets or processes.
  • targets include histone deacetylases (HDAC), DNA methyltransferase (DNMT), heat shock proteins (e.g., HSP90), and proteosomes.
  • subject compounds may be combined with antineoplastic agents (e.g. small molecules, monoclonal antibodies, antisense RNA, and fusion proteins) that inhibit one or more biological targets such as Zolinza, Tarceva, Iressa, Tykerb, Gleevec, Sutent, Sprycel, Nexavar, Sorafenib, CNF2024, RG108, BMS387032, Affmitak, Avastin, Herceptin, Erbitux, AG24322, PD325901, ZD6474, PD 184322, Obatodax, ABT737 and AEE788.
  • antineoplastic agents e.g. small molecules, monoclonal antibodies, antisense RNA, and fusion proteins
  • antineoplastic agents e.g. small molecules, monoclonal antibodies, antisense RNA, and fusion proteins
  • the compounds of the invention are administered in combination with a chemotherapeutic agent.
  • chemotherapeutic agents encompass a wide range of therapeutic treatments in the field of oncology. These agents are administered at various stages of the disease for the purposes of shrinking tumors, destroying remaining cancer cells left over after surgery, inducing remission, maintaining remission and/or alleviating symptoms relating to the cancer or its treatment.
  • alkylating agents such as mustard gas derivatives (Mechlorethamine, cylophosphamide, chlorambucil, melphalan, ifosfamide), ethylenimines (thiotepa, hexamethylmelanine), Alkylsulfonates (Busulfan), Hydrazines and Triazines (Altretamine, Procarbazine, dacarbazine and Temozolomide), Nitrosoureas (Carmustine, Lomustine and Streptozocin), Ifosfamide and metal salts (Carboplatin, Cisplatin, and Oxaliplatin); plant alkaloids such as Podophyllotoxins (Etoposide and Tenisopide), Taxanes (Paclitaxel and Docetaxel), Vinca alkaloids (Vincristine, Vinblastine, Vindesine and Vinorelbine), and Camptothecan analogs (Iri)
  • the compounds of the invention are administered in combination with a chemoprotective agent.
  • chemoprotective agents act to protect the body or minimize the side effects of chemotherapy. Examples of such agents include, but are not limited to, amfostine, mesna, and dexrazoxane.
  • the subject compounds are administered in combination with radiation therapy.
  • Radiation is commonly delivered internally (implantation of radioactive material near cancer site) or externally from a machine that employs photon (x-ray or gamma-ray) or particle radiation.
  • the combination therapy further comprises radiation treatment
  • the radiation treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and radiation treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the radiation treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.
  • compounds of the invention can be used in combination with an immunotherapeutic agent.
  • immunotherapy is the generation of an active systemic tumor-specific immune response of host origin by administering a vaccine composition at a site distant from the tumor.
  • Various types of vaccines have been proposed, including isolated tumor-antigen vaccines and anti-idiotype vaccines.
  • Another approach is to use tumor cells from the subject to be treated, or a derivative of such cells (reviewed by Schirrmacher et al. (1995) J. Cancer Res. Clin. Oncol. 121:487).
  • Schirrmacher et al. (1995) J. Cancer Res. Clin. Oncol. 121:487) In U.S. Pat. No. 5,484,596, Hanna Jr. et al.
  • a method for treating a resectable carcinoma to prevent recurrence or metastases comprising surgically removing the tumor, dispersing the cells with collagenase, irradiating the cells, and vaccinating the patient with at least three consecutive doses of about 10 7 cells.
  • the compounds of the invention may advantageously be used in conjunction with one or more adjunctive therapeutic agents.
  • suitable agents for adjunctive therapy include steroids, such as corticosteroids (amcinonide, betamethasone, betamethasone dipropionate, betamethasone valerate, budesonide, clobetasol, clobetasol acetate, clobetasol butyrate, clobetasol 17-propionate, cortisone, deflazacort, desoximetasone, diflucortolone valerate, dexamethasone, dexamethasone sodium phosphate, desonide, furoate, fluocinonide, fluocinolone acetonide, halcinonide, hydrocortisone, hydrocortisone butyrate, hydrocortisone sodium succinate, hydrocortisone valerate, methyl prednisolone, mometasone, prednicarbate, predni
  • steroids such
  • adenosine A1 agonist such as an EP ligand; an NMDA modulator, such as a glycine antagonist; a sodium channel blocker (e.g. lamotrigine); a substance P antagonist (e.g. an NKi antagonist); a cannabinoid; acetaminophen or phenacetin; a 5-lipoxygenase inhibitor; a leukotriene receptor antagonist; a DMARD (e.g. methotrexate); gabapentin and related compounds; a tricyclic antidepressant (e.g.
  • amitryptilline a neurone stabilizing antiepileptic drug
  • a mono-aminergic uptake inhibitor e.g. venlafaxine
  • a matrix metalloproteinase inhibitor e.g. a nitric oxide synthase (NOS) inhibitor, such as an iNOS or an nNOS inhibitor
  • NOS nitric oxide synthase
  • an antibody therapy such as a monoclonal antibody therapy
  • an antiviral agent such as a nucleoside inhibitor (e.g. lamivudine) or an immune system modulator (e.g.
  • an opioid analgesic e.g. a local anaesthetic; a stimulant, including caffeine; an H2-antagonist (e.g. ranitidine); a proton pump inhibitor (e.g. omeprazole); an antacid (e.g. aluminium or magnesium hydroxide; an antiflatulent (e.g. simethicone); a decongestant (e.g. phenylephrine, phenylpropanolamine, pseudoephedrine, oxymetazoline, epinephrine, naphazoline, xylometazoline, propylhexedrine, or levo-desoxyephedrine); an antitussive (e.g. codeine, hydrocodone, carmiphen, carbetapentane, or dextramethorphan); a diuretic; or a sedating or non-sedating antihistamine.
  • an antitussive e.g. codeine, hydroco
  • the compounds of the invention can be co-administered with siRNA that target other genes.
  • a compound of the invention can be co-administered with an siRNA targeted to a c-Myc gene.
  • AD-12115 can be co-administered with a c-Myc siRNA. Examples of c-Myc targeted siRNAs are disclosed in U.S. patent application Ser. No. 12/373,039 which is herein incorporated by reference.
  • 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.
  • the present invention provides methods of preparing lipid particles, including those associated with a therapeutic agent, e.g., a nucleic acid.
  • a mixture of lipids is combined with a buffered aqueous solution of nucleic acid to produce an intermediate mixture containing nucleic acid encapsulated in lipid particles wherein the encapsulated nucleic acids are present in a nucleic acid/lipid 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-nucleic acid particles, thus providing an at least partially surface-neutralized lipid-encapsulated nucleic acid composition.
  • lipid vesicles can be formed at the lower pH with titratable cationic lipids and other vesicle components in the presence of nucleic acids. In this manner, the vesicles will encapsulate and entrap the nucleic acids.
  • 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 a resultant nucleic acid 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 nucleic acid-lipid particles are provided in U.S. Pat. No. 6,287,591 and U.S. Pat. No. 6,858,225, incorporated herein by reference.
  • the vesicles formed in this manner provide formulations of uniform vesicle size with high content of nucleic acids. Additionally, the vesicles have a size range of from about 30 to about 150 nm, more preferably about 30 to about 90 nm.
  • nucleic acid encapsulation is a result of electrostatic interaction at low pH.
  • acidic pH e.g. pH 4.0
  • the vesicle surface is charged and binds a portion of the nucleic acids through electrostatic interactions.
  • 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.
  • the present invention provides methods of preparing lipid/nucleic acid formulations.
  • a mixture of lipids is combined with a buffered aqueous solution of nucleic acid to produce an intermediate mixture containing nucleic acid encapsulated in lipid particles, e.g., wherein the encapsulated nucleic acids are present in a nucleic acid/lipid ratio of about 10 wt % to about 20 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-nucleic acid particles, thus providing an at least partially surface-neutralized lipid-encapsulated nucleic acid composition.
  • 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-nucleic acid particle formation.
  • the amino lipid is a novel cationic lipid of the present 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.
  • 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.
  • the alcohol is used in the form in which it is commercially available.
  • ethanol can be used as absolute ethanol (100%), or as 95% ethanol, the remainder being water. This method is described in more detail in U.S. Pat. No. 5,976,567).
  • the lipid mixture is combined with a buffered aqueous solution that may contain the nucleic acids.
  • 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.
  • 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., U.S. Pat. No. 6,287,591 and U.S. Pat. No.
  • nucleic acid 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.
  • the mixture of lipids and the buffered aqueous solution of therapeutic nucleic acids is combined to provide an intermediate mixture.
  • the intermediate mixture is typically a mixture of lipid particles having encapsulated nucleic acids. Additionally, the intermediate mixture may also contain some portion of nucleic acids 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 pK a of the protonatable group on the lipid).
  • the mixture of lipids is an alcohol solution of lipids and the volumes of each of the solutions are 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.
  • the lipid-encapsulated therapeutic agent e.g., nucleic acid
  • 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.
  • multilamellar vesicles are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed.
  • the particle size distribution can be monitored by conventional laser-beam particle size determination.
  • extrusion is used to obtain a uniform vesicle size.
  • Extrusion of liposome compositions through a small-pore polycarbonate membrane or an asymmetric ceramic membrane results in a relatively well-defined size distribution.
  • 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.
  • the lipid-nucleic acid compositions which are formed can be used without any sizing.
  • 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-nucleic acid compositions.
  • unencapsulated nucleic acid is freed from the lipid particle surface and can be removed from the composition using conventional techniques.
  • unencapsulated and surface adsorbed nucleic acids are removed from the resulting compositions through exchange of buffer solutions.
  • 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 release from the surface.
  • the released nucleic acid 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.
  • the lipid vesicles 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.
  • the aqueous buffer should be of a pH below the pKa of the amino lipid.
  • a solution of the nucleic acids can then be added to these sized, preformed 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).
  • nucleic acid encapsulation process 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. 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 in the lipid vesicles will require manipulation of variable such as ethanol concentration and temperature. Examples of suitable conditions for nucleic acid encapsulation are provided in the Examples.
  • the invention provides a method for inhibiting the expression of the PCSK9 gene in a mammal.
  • the method includes administering a composition of the invention to the mammal such that expression of the target PCSK9 gene is decreased for an extended duration, e.g., at least one week, two weeks, three weeks, or four weeks or longer.
  • 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.
  • the PCSK9 gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide.
  • the PCSK9 gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide.
  • Table 1b, Table 2b, and Table 5b provide a wide range of values for inhibition of expression obtained in an in vitro assay using various PCSK9 dsRNA molecules at various concentrations.
  • the effect of the decreased target PCSK9 gene preferably results in a decrease in LDLc (low density lipoprotein cholesterol) levels in the blood, and more particularly in the serum, of the mammal.
  • LDLc levels are decreased by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as compared to pretreatment levels.
  • the method includes administering a composition containing a dsRNA, where the dsRNA has a nucleotide sequence that is complementary to at least a part of an RNA transcript of the PCSK9 gene of the mammal to be treated.
  • 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.
  • the compositions are administered by intravenous infusion or injection.
  • compositions described herein can be used to treat diseases and conditions that can be modulated by down regulating PCSK9 gene expression.
  • 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.
  • a patient treated with a PCSK9 dsRNA is also administered a non-dsRNA therapeutic agent, such as an agent known to treat lipid disorders.
  • the invention provides a method of inhibiting the expression of the PCSK9 gene in a subject, e.g., a human.
  • the method includes administering a first single 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 optionally, administering a second single dose of dsRNA, wherein the second single dose is administered at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days after the first single dose is administered, thereby inhibiting the expression of the PCSK9 gene in a subject.
  • 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
  • a second single dose of dsRNA wherein the second single dose is administered at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days after the first single dose is administered, thereby inhibiting the expression of the PCSK9 gene in a
  • doses of dsRNA 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.
  • administration can be provided when Low Density Lipoprotein cholesterol (LDLc) levels reach or surpass a predetermined minimal level, such as greater than 70 mg/dL, 130 mg/dL, 150 mg/dL, 200 mg/dL, 300 mg/dL, or 400 mg/dL.
  • LDLc Low Density Lipoprotein cholesterol
  • 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.
  • the dsRNA does not activate the immune system, e.g., it does not increase cytokine levels, such as TNF-alpha or IFN-alpha levels.
  • cytokine levels such as TNF-alpha or IFN-alpha levels.
  • 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.
  • the invention provides a method for treating, preventing or managing a disorder, pathological process or symptom, which, for example, can be mediated by down regulating PCSK9 gene expression in a subject, such as a human subject.
  • the disorder is hyperlipidemia.
  • the method includes administering a first single 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 optionally, administering a second single dose of dsRNA, wherein the second single dose is administered at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days after the first single dose is administered, thereby inhibiting the expression of the PCSK9 gene in a subject.
  • 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
  • a second single dose of dsRNA wherein the second single dose is administered at least 5, more preferably 7,
  • composition containing a dsRNA featured in the invention is administered with a non-dsRNA therapeutic agent, such as an agent known to treat a lipid disorders, such as hypercholesterolemia, atherosclerosis or dyslipidemia.
  • a non-dsRNA therapeutic agent such as an agent known to treat a lipid disorders, such as hypercholesterolemia, atherosclerosis or dyslipidemia.
  • a dsRNA featured in the invention can be administered with, e.g., an HMG-CoA reductase inhibitor (e.g., a statin), a fibrate, a bile acid sequestrant, niacin, an antiplatelet agent, an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist (e.g., losartan potassium, such as Merck & Co.'s Cozaar®), an acylCoA cholesterol acetyltransferase (ACAT) inhibitor, a cholesterol absorption inhibitor, a cholesterol ester transfer protein (CETP) inhibitor, a microsomal triglyceride transfer protein (MTTP) inhibitor, a cholesterol modulator, a bile acid modulator, a peroxisome proliferation activated receptor (PPAR) agonist, a gene-based therapy, a composite vascular protectant (e.g., AGI-1067, from Atherogenics), a glycoprotein IIb/IIIa inhibitor,
  • 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 Crest
  • 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®).
  • bezafibrate e.g., Roche's Befizal®/Cedur®/Bezalip®, Kissei's Bezatol
  • clofibrate e.g., Wyeth's Atromid-S®
  • fenofibrate e.g.,
  • Exemplary bile acid sequestrants include, e.g., cholestyramine (Bristol-Myers Squibb's Questran® and Questran LightTM), colestipol (e.g., Pharmacia's Colestid), and colesevelam (Genzyme/Sankyo's WelCholTM).
  • 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.
  • 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).
  • aspirin e.g., Bayer's aspirin
  • clopidogrel Sanofi-Synthelabo/Bristol-Myers Squibb's Plavix
  • 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/Ange
  • Exemplary angiotensin-converting enzyme inhibitors include, e.g., ramipril (e.g., Aventis' Altace) and enalapril (e.g., Merck & Co.'s Vasotec).
  • Exemplary acyl CoA cholesterol acetyltransferase (ACAT) inhibitors include, e.g., avasimibe (Pfizer), eflucimibe (BioM ⁇ acute over ( ⁇ ) ⁇ rieux Pierre Fabre/Eli Lilly), CS-505 (Sankyo and Kyoto), and SMP-797 (Sumito).
  • Exemplary cholesterol absorption inhibitors include, e.g., ezetimibe (Merck/Schering-Plough Pharmaceuticals Zetia®) and Pamaqueside (Pfizer).
  • Exemplary CETP inhibitors include, e.g., Torcetrapib (also called CP-529414, Pfizer), JTT-705 (Japan Tobacco), and CETi-I (Avant Immunotherapeutics).
  • Exemplary microsomal triglyceride transfer protein (MTTP) inhibitors include, e.g., implitapide (Bayer), R-103757 (Janssen), and CP-346086 (Pfizer).
  • 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 Pharmaceuticals/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), ApoA1 (UCB Pharma/Groupe Fournier), EG-004 (Trinam) (Ark Therapeutics), and ATP-binding cassette transporter-A1 (ABCA1) (CV Therapeutics/Incyte, Aventis, Xenon).
  • Exemplary Glycoprotein Ilb/IIIa inhibitors include, e.g., roxifiban (also called DMP754, Bristol-Myers Squibb), Gantofiban (Merck KGaA/Yamanouchi), and Cromafiban (Millennium Pharmaceuticals).
  • Exemplary squalene synthase inhibitors include, e.g., BMS-1884941 (Bristol-Myers Squibb), CP-210172 (Pfizer), CP-295697 (Pfizer), CP-294838 (Pfizer), and TAK-475 (Takeda).
  • An exemplary MCP-I inhibitor is, e.g., RS-504393 (Roche Bioscience).
  • the anti-atherosclerotic agent BO-653 (Chugai Pharmaceuticals), and the nicotinic acid derivative Nyclin (Yamanouchi Pharmaceuticals) 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).
  • advicor Niacin/lovastatin from Kos Pharmaceuticals
  • Amlodipine/atorvastatin Pfizer
  • 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
  • a dsRNA targeting PCSK9 is administered in combination with an ezetimibe/simvastatin combination (e.g., Vytorin® (Merck/Schering-Plough Pharmaceuticals)).
  • an ezetimibe/simvastatin combination e.g., Vytorin® (Merck/Schering-Plough Pharmaceuticals)
  • 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.
  • the invention features, a method of instructing an end user, e.g., a caregiver or a subject, on how to administer a dsRNA described herein.
  • the method includes, optionally, providing the end user with one or more doses of the dsRNA, and instructing the end user to administer the dsRNA on a regimen described herein, thereby instructing the end user.
  • 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 dsRNA targeting PCSK9 in an amount sufficient to lower the patient's LDL levels or ApoB levels, e.g., without substantially lowering HDL levels.
  • 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 dsRNA targeting PCSK9 in an amount sufficient to lower the patient's ApoB levels.
  • the amount of PCSK9 is sufficient to lower LDL levels as well as ApoB levels.
  • administration of the PCSK9 dsRNA does not affect the level of HDL cholesterol in the patient.
  • siRNA design was carried out to identify in two separate selections
  • siRNAs targeting PCSK9 human and either mouse or rat mRNA a) siRNAs targeting PCSK9 human and either mouse or rat mRNA and
  • 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.
  • SiRNAs specifically targeting human PCSK9 were identified in a second selection. All potential 19mer sequences of human PCSK9 were extracted and defined as candidate target sequences. Sequences cross-reactive to human, monkey, and those cross-reactive to mouse, rat, human and monkey are all listed in Tables 1a and 2a. Chemically modified versions of those sequences and their activity in both in vitro and in vivo assays are also listed in Tables 1a and 2a. The data is described in the examples and in FIGS. 2-8 .
  • siRNAs with low off-target potential were defined as preferable and assumed to be more specific in vivo.
  • 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)
  • positions 10 and 11 may contribute more to off-target potential than non-seed region
  • positions 1 and 19 of each strand are not relevant for off-target interactions
  • 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
  • off-target scores are to be considered more relevant for off-target potential than numbers of off-targets
  • the off-target score was calculated for considering assumption 1 to 3 as follows:
  • Off-target score number of seed mismatches*10+number of cleavage site mismatches*1.2+number of non-seed mismatches*1
  • the most relevant off-target gene for each siRNA corresponding to the input 19mer sequence was defined as the gene with the lowest off-target score. Accordingly, the lowest off-target score was defined as the relevant off-target score for each siRNA.
  • such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.
  • RNAs Single-stranded RNAs were produced by solid phase synthesis on a scale of 1 ⁇ mole using an Expedite 8909 synthesizer (Applied Biosystems, Appleratechnik GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 ⁇ , 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).
  • RNA synthesis For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referred to as -Chol-3), an appropriately modified solid support was used for RNA synthesis.
  • the modified solid support was prepared as follows:
  • Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved in dichloromethane (50 ml) and cooled with ice.
  • Diisopropylcarbodiimde (3.25 g, 3.99 ml, 25.83 mmol) was added to the solution at 0° C. It was then followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). The solution was brought to room temperature and stirred further for 6 h. Completion of the reaction was ascertained by TLC.
  • Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 ml of dry toluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) of diester AD was added slowly with stirring within 20 mins. The temperature was kept below 5° C. during the addition. The stirring was continued for 30 mins at 0° C.
  • Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2 ⁇ 5 ml) in vacuo.
  • the reaction was carried out at room temperature overnight.
  • the reaction was quenched by the addition of methanol.
  • the reaction mixture was concentrated under vacuum and to the residue dichloromethane (50 ml) was added.
  • the organic layer was washed with 1M aqueous sodium bicarbonate.
  • the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated.
  • HuH-7 cells were obtained from JCRB Cell Bank (Japanese Collection of Research Bioresources) (Shinjuku, Japan, cat. No.: JCRB0403) Cells were cultured in Dulbecco's MEM (Biochrom AG, Berlin, Germany, cat. No. F0435) supplemented to contain 10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany, cat. No. S0115), Penicillin 100 U/ml, Streptomycin 100 ⁇ g/ml (Biochrom AG, Berlin, Germany, cat. No. A2213) and 2 mM L-Glutamin (Biochrom AG, Berlin, Germany, cat. No K0282) at 37° C.
  • FCS fetal calf serum
  • HepG2 and HeLa cells were obtained from American Type Culture Collection (Rockville, Md., cat. No. HB-8065) and cultured in MEM (Gibco Invitrogen, Düsseldorf, Germany, cat. No. 21090-022) supplemented to contain 10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany, cat. No. S0115), Penicillin 100 U/ml, Streptomycin 100 ⁇ g/ml (Biochrom AG, Berlin, Germany, cat. No. A2213), 1 ⁇ Non Essential Amino Acids (Biochrom AG, Berlin, Germany, cat.
  • FCS fetal calf serum
  • HuH7, HepG2, or HeLa cells were seeded at a density of 2.0 ⁇ 10 4 cells/well in 96-well plates and transfected directly.
  • Transfection of siRNA (30 nM for single dose screen) was carried out with lipofectamine 2000 (Invitrogen GmbH, Düsseldorf, Germany, cat. No. 11668-019) as described by the manufacturer.
  • PCSK9 mRNA levels were quantified with the Quantigene Explore Kit (Genosprectra, Dumbarton Circle Fremont, USA, cat. No. QG-000-02) according to the protocol.
  • PCSK9 mRNA levels were normalized to GAP-DH mRNA.
  • siRNA duplexes unrelated to PCSK9 gene were used as control.
  • the activity of a given PCSK9 specific siRNA duplex was expressed as percent PCSK9 mRNA concentration in treated cells relative to PCSK9 mRNA concentration in cells treated with the control siRNA duplex.
  • cryopreserved Primary cynomolgus monkey hepatocytes (cryopreserved) were obtained from In vitro Technologies, Inc. (Baltimore, Md., USA, cat No M00305) and cultured in InVitroGRO CP Medium (cat No Z99029) at 37° C. in an atmosphere with 5% CO 2 in a humidified incubator.
  • siRNA For transfection with siRNA, primary cynomolgus monkey cells were seeded on Collagen coated plates (Fisher Scientific, cat. No. 08-774-5) at a density of 3.5 ⁇ 10 4 cells/well in 96-well plates and transfected directly. Transfection of siRNA (eight 2-fold dilution series starting from 30 nM) in duplicates was carried out with lipofectamine 2000 (Invitrogen GmbH, Düsseldorf, Germany, cat. No. 11668-019) as described by the manufacturer.
  • PCSK9 mRNA levels were quantified with the Quantigene Explore Kit (Genosprectra, Dumbarton Circle Fremont, USA, cat. No. QG-000-02) according to the protocol.
  • PCSK9 mRNA levels were normalized to GAPDH mRNA. Normalized PCSK9/GAPDH ratios were then compared to PCSK9/GAPDH ratio of lipofectamine 2000 only control.
  • Tables 1b and 2b summarize the results and provide examples of in vitro screens in different cell lines at different doses. Silencing of PCSK9 transcript was expressed as percentage of remaining transcript at a given dose.
  • Highly active sequences are those with less than 70% transcript remaining post treatment with a given siRNA at a dose less than or equal to 100 nM.
  • Very active sequences are those that have less than 60% of transcript remaining after treatment with a dose less than or equal to 100 nM.
  • Active sequences are those that have less than 90% transcript remaining after treatment with a high dose (100 nM).
  • active siRNAs were also screened in vivo in mouse in lipidoid formulations as described below. Active sequences in vitro were also generally active in vivo (See FIGS. 6A and 6B and example 4).
  • LNP01 is a lipidoid formulation formed from cholesterol, mPEG2000-C14 Glyceride, and dsRNA.
  • the LNP01 formulation is useful for delivering dsRNAs to the liver.
  • the lipidoid LNP-01.4HCl (MW 1487) ( FIG. 1 ), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) were used to prepare lipid-siRNA nanoparticles.
  • Stock solutions of each in ethanol were prepared: LNP-01, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml.
  • LNP-01, Cholesterol, and PEG-Ceramide C16 stock solutions were then combined in a 42:48:10 molar ratio.
  • Combined lipid solution was mixed rapidly with aqueous siRNA (in sodium acetate pH 5) such that the final ethanol concentration was 35-45% and the final sodium acetate concentration was 100-300 mM.
  • Lipid-siRNA nanoparticles formed spontaneously upon mixing.
  • the resultant nanoparticle mixture was in some cases extruded through a polycarbonate membrane (100 nm cut-off) using a thermobarrel extruder (Lipex Extruder, Northern Lipids, Inc). In other cases, the extrusion step was omitted. Ethanol removal and simultaneous buffer exchange was accomplished by either dialysis or tangential flow filtration. Buffer was exchanged to phosphate buffered saline (PBS) pH 7.2.
  • PBS phosphate buffered saline
  • Formulations prepared by either the standard or extrusion-free method are characterized in a similar manner.
  • Formulations are first characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles are measured by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be 20-300 nm, and ideally, 40-100 nm in size. The particle size distribution should be unimodal.
  • the total siRNA concentration in the formulation, as well as the entrapped fraction is estimated using a dye exclusion assay.
  • a sample of the formulated siRNA is incubated with the RNA-binding dye Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, 0.5% Triton-X100.
  • the total siRNA in the formulation is determined by the signal from the sample containing the surfactant, relative to a standard curve.
  • the entrapped fraction is determined by subtracting the “free” siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically >85%.
  • siRNAs were formulated in LNP-01 (and then dialyzed against PBS) at 0.5 mg/ml concentration allowing the delivery of the 5 mg/kg dose in 10 ⁇ l/g body weight. Mice were kept under an infrared lamp for approximately 3 min prior to dosing to ease injection.
  • mice 48 hour post dosing mice were sacrificed by CO 2 -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. ⁇ l
  • Frozen livers were grinded using 6850 Freezer/Mill Cryogenic Grinder (SPEX CentriPrep, Inc) and powders stored at ⁇ 80° C. until analysis.
  • PCSK9 mRNA levels were detected using the branched-DNA technology based kit from QuantiGene Reagent System (Genospectra) according to the protocol. 10-20 mg of frozen liver powders was lysed in 600 ⁇ l of 0.16 ⁇ g/ml Proteinase K (Epicentre, #MPRK092) in Tissue and Cell Lysis Solution (Epicentre, #MTC096H) at 65° C. for 3 hours. Then 10 ⁇ l of the lysates were added to 90 ⁇ l of Lysis Working Reagent (1 volume of stock Lysis Mixture in two volumes of water) and incubated at 52° C. overnight on Genospectra capture plates with probe sets specific to mouse PCSK9 and mouse GAPDH or cyclophilin B.
  • Lysis Working Reagent 1 volume of stock Lysis Mixture in two volumes of water
  • Nucleic acid sequences for Capture Extender (CE), Label Extender (LE) and blocking (BL) probes were selected from the nucleic acid sequences of PCSK9, GAPDH and cyclophilin B with the help of the QuantiGene ProbeDesigner Software 2.0 (Genospectra, Fremont, Calif., USA, cat. No. QG-002-02). Chemo luminescence was read on a Victor2-Light (Perkin Elmer) as Relative light units. The ratio of PCSK9 mRNA to GAPDH or cyclophilin B mRNA in liver lysates was averaged over each treatment group and compared to a control group treated with PBS or a control group treated with an unrelated siRNA (blood coagulation factor VII).
  • CE Capture Extender
  • LE Label Extender
  • BL blocking
  • Total serum cholesterol in mouse serum was measured using the StanBio Cholesterol LiquiColor kit (StanBio Laboratory, Boerne, Tex., USA) according to manufacturer's instructions. Measurements were taken on a Victor2 1420 Multilabel Counter (Perkin Elmer) at 495 nm.
  • PCSK9 siRNAs showed more than 40% PCSK9 mRNA knock down compared to a control group treated with PBS, while control group treated with an unrelated siRNA (blood coagulation factor VII) had no effect ( FIGS. 2-3 ).
  • Silencing of PCSK9 transcript also correlated with a lowering of total serum cholesterol in these animals ( FIGS. 4-5 ).
  • 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 ).
  • FIGS. 6A and 6B shows a strong correlation between those molecules that were active in vitro and those active in vivo.
  • FIG. 8 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-0792.
  • 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 SEQ ID NO: 1521 5′- GccuGGAGuuuAuucGGAAdTsdT
  • Antisense strand SEQ ID NO: 1522 5′- puUCCGAAuAAACUCcAGGCdTsdT
  • Rats were treated via tail vein injection with 5 mg/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. 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.
  • Cynomolgus monkeys were treated with LNP01 formulated dsRNA 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.
  • 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.
  • 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 ⁇ L. If the total sample volume collected was under 150 ⁇ L, 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.
  • 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.
  • PCSK9 siRNAs cause Decreased PCSK mRNA in Liver Extracts, and Lower Serum Cholesterol Levels in Mice and Rats
  • siRNA molecule AD-1a2 (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-1a2 (LNP01-1a2) 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.
  • mice livers were harvested 48 hours post-injection, and levels of PCSK9 transcript were determined.
  • blood was harvested and subjected to a total cholesterol analysis.
  • LNP01-1a2 displayed a clear dose response with maximal PCSK9 message suppression ( ⁇ 60-70%) as compared to a control siRNA targeting luciferase (LNP01-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.
  • 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.
  • 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).
  • 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 al., Curr. Opin. Lipidol. 18:397-400, 2007).
  • Increased clearance of plasma cholesterol induced by PCSK9 silencing in the liver was not predicted to result in accumulation of liver triglycerides.
  • liver cholesterol and triglyceride concentrations in livers of the treated or control animals were quantified. As shown in FIG. 10C , 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.
  • AD-1a2, AD-1a3, AD-1a5, AD-1a4, 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 LNP01-1a2 ( 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.
  • LNP01-1a2 and LNP01-3a1 Silence Human PCSK9 and Circulating Human PCSK9 Protein in Transgenic Mice
  • LNP01-1a2 i.e., PCS-A2 or AD-10792
  • AD-3a1 i.e., PCS-C2 or AD-9736
  • PCSK9 a line of transgenic mice expressing human PCSK9 under the ApoE promoter was used (Lagace et al., 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 LNP01-1a2 (a.k.a.
  • LNP-PCS-A2 or LNP01-3a1 were able to decrease the human PCSK9 transcript levels by >70% ( FIG. 15A ), and this transcript down-regulation resulted in significantly lower levels of circulating human PCSK9 protein as measured by ELISA ( FIG. 15B ).
  • siRNAs were capable of silencing the human transcript and subsequently reducing the amount of circulating plasma human PCSK9 protein.
  • PCSK9 mRNA levels are regulated by the transcription factor sterol regulatory element binding protein-2 and are reduced by fasting.
  • 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.
  • Cyno monkeys were acclimated to a twice daily feeding schedule during which food was removed after a one hour period. Animals were fed from 9-10 am in the morning, after which food was removed. The animals were next fed once again for an hour between 5 pm-6 pm with subsequent food removal. Blood was drawn after an overnight fast (6 pm until 9 am the next morning), and again, 2 and 4 hours following the 9 am 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-1a2 and another PCSK9 siRNA (AD-2a1) that was highly active in primary Cyno hepatocytes.
  • PCSK9 siRNAs Reduce Circulating LDLc, ApoB, and PCSK9, but not HDLc in Non-Human Primates (NHPs)
  • siRNA 1 LNP01-10792
  • siRNA 2 LNP-01-9680
  • 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
  • siRNA 1 caused ⁇ 60% LDLc lowering for at least 7 days.
  • LNP01-1a2 or LNP01-2a1 resulted in a statistically significant reduction of LDLc beginning at day 3 post-dose that returned to baseline over ⁇ 14 days (for LNP01-1a2) and ⁇ 21 days (LNP01-2a1). This effect was not seen in either the PBS, the control siRNA groups, or the 1 mg/kg treatment groups.
  • LNP01-2a1 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 .
  • PCSK9 protein levels were also measured in treated and control animals. As shown in FIG. 11 , LNP01-1a2 and LNP01-2a1 treatment each resulted in trends toward decreased circulating PCSK9 protein levels versus pre-dose. Specifically, the more active siRNA LNP01-2a1 demonstrated significant reduction of circulating PCSK9 protein versus both PBS (day 3-21) and LNP01-ctrl siRNA control (day 4, day 7).
  • 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-1a1 was found to induce both IFN-alpha and TNF-alpha. However, chemically modified versions of this sequence (AD-1a2, AD-1a3, AD-1a5, and AD-1a4) as well as AD-2a1 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-1a2, nor AD-2a1 activated IFN-alpha when formulated into liposomes and tested in mice.
  • 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.
  • 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.
  • 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 ⁇ l/g body weight. Mice were kept under an infrared lamp for approximately 3 min prior to dosing to ease injection.
  • mice 24 hour post last dose mice were sacrificed by CO2-asphyxiation.
  • 0.2 ml blood was collected by retro-orbital bleeding and the liver was harvested and frozen in liquid nitrogen. Serum and livers were stored at ⁇ 80° C. Frozen livers were grinded using 6850 Freezer/Mill Cryogenic Grinder (SPEX CentriPrep, Inc) and powders stored at ⁇ 80° C. until analysis.
  • PCSK9 mRNA levels were detected using the branched-DNA technology based kit from QuantiGene Reagent System (Panomics, USA) according to the protocol. 10-20 mg of frozen liver powders was lysed in 600 ⁇ l of 0.16 ⁇ g/ml Proteinase K (Epicentre, #MPRK092) in Tissue and Cell Lysis Solution (Epicentre, #MTC096H) at 65° C. for 3 hours. Then 10 ⁇ l of the lysates were added to 90 ⁇ l of Lysis Working Reagent (1 volume of stock Lysis Mixture in two volumes of water) and incubated at 52° C. overnight on Genospectra capture plates with probe sets specific to mouse PCSK9 and mouse GAPDH.
  • Lysis Working Reagent 1 volume of stock Lysis Mixture in two volumes of water
  • 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).
  • 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.
  • mice 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.
  • 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 ⁇ l/g body weight. Rats were kept under an infrared lamp for approximately 3 min prior to dosing to ease injection.
  • PCSK9 mRNA levels were detected using the branched-DNA technology based kit from QuantiGene Reagent System (Panomics, USA) according to the protocol. 10-20 mg of frozen liver powders was lysed in 600 ⁇ l of 0.16 ⁇ g/ml Proteinase K (Epicentre, #MPRK092) in Tissue and Cell Lysis Solution (Epicentre, #MTC096H) at 65° C. for 3 hours. Then 10 ⁇ l of the lysates were added to 90 ⁇ l 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.
  • Lysis Working Reagent 1 volume of stock Lysis Mixture in two volumes of water
  • 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).
  • 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.
  • AD-9680, AD-14676, and AD-10792 were assayed in HeLa cells.
  • a number of variants were synthesized as shown in Table 6 and include adding DFT (2,4-Difluorotoluoyl, a thymidine triphosphate shape analog lacking Watson-Crick pairing); adding single or combination mismatches; and testing two different backbone chemistries: leading with a 2′-O methyl, or alternating with 2′F.
  • HeLa were plated in 96-well plates (8,000-10,000 cells/well) in 100 ⁇ l 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 ⁇ l of transfection reagent LipofectamineTM 2000 (Invitrogen Corporation, Carlsbad, Calif.) was used per well and transfections were performed according to the manufacturer's protocol. Namely, the siRNA: LipofectamineTM 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.
  • DMEM Dulbecco's Modified Eagle Medium
  • the LipofectamineTM 2000 was mixed gently before use, then for each well of a 96 well plate 0.4 ⁇ l was diluted in 25 ⁇ l 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 ⁇ l of the diluted siRNA was combined with the diluted LipofectamineTM 2000 (total volume is 26.4 ⁇ l). The complex was mixed gently and incubated for 20 minutes at room temperature to allow the siRNA: LipofectamineTM 2000 complexes to form. Then 100 ⁇ l of 10% fetal bovine serum in DMEM was added to each of the siRNA:LipofectamineTM 2000 complexes and mixed gently by rocking the plate back and forth.
  • 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 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.
  • FIG. 24 is a dose response curve comparing the efficiency of parent duplexes AD-9680 and AD-10792 with modified duplexes wherein a DFT is inserted at position 10 of the sense strand. This modification improves the efficiency by about 2 fold in HeLa cells.
  • a lipid formulated PCSK9 targeted siRNA was transfected into Hep3B cells at concentrations of 250 nM, 1 uM and 5 uM in triplicates using the reagent RNAiMAX (Invitrogen) according to the manufacture's instruction: 1 ul of transfection reagent; reverse transfection protocol. Samples were collected 48 hrs post transfection.
  • Transcript levels were measured for the following genes having the closest homology to the target sequence: ORMDL2, BMP6, TAPT1, MYEF2, LOC442252, RFT1, and PCSK9.
  • AD-9680 S 1531 uucuAGAccuGuuuuGcuudTsdT AS 1532 AAGcAAAAcAGGUCuAGAAdTsdT
  • Rats were treated with 3 mg/kg bolus dose of SNALP-Dlin DMA 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 repeat dosing and total serum cholesterol levels were determined. The negative control was PBS.
  • Rats were treated with four different lipid formulations of AD-10792 including SNALP and LNP08, described herein. At day 3, total serum cholesterol levels were determined. The experiment was performed using the methods described herein. Administration of LNP-08 formulated AD-10792 results in the lowest EC50 of 0.08 mg/kg compared to LNP01 formulated (EC50 of 2.0 mg/kg) and SNALP formulated (EC50 of 1.0 mg/kg). (data not shown).
  • Sense and antisense oligomers were designed to target the human PCSK9 transcript in the flanking regions immediately upstream and downstream of the 19 base target region of ALN-PCSK9 (AD-9680).
  • AD-9680 We used the NCBI Refseq NM — 174936.2 as the reference human transcript for the PCSK9 gene.
  • the antisense oligo of AD-9680 contains 19 contiguous bases complementary to the bases in the region of NM — 174936 from positions 3530 through 3548 relative to the start of the mRNA.
  • a set of siRNA molecules was designed to each unique 19mer of the subset of the transcript sequence defined by 10 bases upstream from the 5′ end to 10 bases downstream from the 3′ end of the target region of AD-9680.
  • the first base at the 5′ position of the sense oligo 19mer extends from positions 3520 to positions 3558 (Tables 7 and 8).
  • PCSK9 sequences were synthesized on MerMade 192 synthesizer. Two sets of sequences were made. The first set contained no chemical modifications (unmodified) and a second set was made with endolight chemical modifications. In sequences containing endolight chemical modification, all pyrimidines (cytosine and uridine) in the sense strand were replaced with corresponding 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U). In the antisense strand, pyrimidines adjacent to (towards 5′ position) ribo A nucleoside were replaced with their corresponding 2-O-Methyl nucleosides. A two base dTsdT extension at the 3′ end of both sense and anti sense sequences was introduced. The sequence file was converted to a text file to make it compatible for loading in the MerMade 192 synthesis software.
  • PCSK9 sequences used solid supported oligonucleotide synthesis using phosphoramidite chemistry.
  • the synthesis of the above sequences was performed at 1 ⁇ m scale in 96 well plates.
  • the amidite solutions were prepared at 0.1 M concentration and ethyl thio tetrazole (0.6M in Acetonitrile) was used as activator.
  • the synthesized sequences were cleaved and deprotected in 96 well plates, using methylamine in the first step and Fluoride ion in the second step.
  • the crude sequences thus obtained were precipitated using acetone: ethanol mix and the pellet were re-suspended in 0.2M sodium acetate buffer.
  • Hela cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO 2 in Eagle's Minimum Essential Medium (EMEM, ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization.
  • EMEM Eagle's Minimum Essential Medium
  • FBS FBS
  • streptomycin Septomycin
  • glutamine ATCC
  • Reverse transfection was carried out by adding 5 ⁇ l of Opti-MEM to 5 ⁇ l of siRNA duplexes per well into a 96-well plate along with 10 ⁇ l of Opti-MEM plus 0.2 ⁇ l of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) and incubated at room temperature for 15 minutes.
  • HeLa cells were transfected with siRNAs over seven, ten-fold serial dilutions from 1 nM to 1 fM.
  • RNA Rebinding Solution 100 ⁇ l was added and mixed for 3 minutes.
  • Supernatant was removed and magnetic beads were washed again with 150 ⁇ l Wash Solution 2 and mixed for 1 minute and supernatant was removed completely. The magnetic beads were mixed for 2 minutes to dry before RNA was eluted with 50 ⁇ l of water.
  • cDNA was synthesized using ABI High capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, Calif., Cat #4368813). A master mix of 2 ⁇ l 10 ⁇ Buffer, 0.8 ⁇ l 25 ⁇ dNTPs, 2 ⁇ l Random primers, 1 ⁇ l Reverse Transcriptase, 1 ⁇ l RNase inhibitor and 3.2 ⁇ l of H2O per reaction were added into 10 ⁇ l total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.
  • Real time PCR was performed as follows. 2 ⁇ l of cDNA were added to a master mix containing 1 ⁇ l GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 1 ⁇ l PCSK9 TaqMan probe (Applied Biosystems cat #HS03037355_M1) and 10 ⁇ l Roche Probes Master Mix (Roche Cat #04887301001) per well in a LightCycler 480 384 well plate (Roche cat #0472974001). Real time PCR was done in a LightCycler 480 Real Time PCR machine (Roche). Each duplex was tested in two independent transfections and each transfections was assayed in duplicate.
  • the data for the dose response screen is shown in Table 10. Data are expressed as dose in pM that results in 50% inhibition relative to AD-1955. Each dose response was repeated twice (Rep1 and Rep2). Average of the IC50s generated in the two dose response screens is shown.
  • the average IC50 for siRNA flanking AD-9680 was plotted vs. the starting position of the target region in the human PCSK9 transcript FIG. 25 .
  • RNAi agent targeting nucleotide region 3520-3555 of PCSK9 with an RNAi agent is highly effective at inhibiting PCSK9.
  • C57BL6 mice were administered 30 mg/kg rEHDL/chol-siPCSK9 by intravenous administration (tail vein injection) in a single bolus dose.
  • Chol-siPCSK9 (dsRNA Duplex AD-20583) has the following sequence:
  • L10 The structure of L10 is:
  • mice were fasted overnight ( ⁇ 14 hours), and then sacrificed at 48 h post-injection.
  • mRNA levels from liver were determined by bDNA assay, and normalized to GAPDH mRNA levels.
  • siRNA targeting PCSK9 was formulated in a LNP-11 formulation (described herein) and administered to cynomologous monkeys. Control was AD-1955. The lipid formulated siRNAs were administered via a 30 minute infusion on day 1 at dosages of 0.03, 0.1, 0.3, and 1.0 mg/kg. Control was administered at 1.0 mg/kg. On day 3, liver biopsies were performed for measurement of PCSK9 transcript. Blood samples were collected on days ⁇ 3, ⁇ 1, 3, 4, 5, 7, 9, 11, 12, 15, 22, 30, and 37 and PCSK9 protein levels and LDLc numbers and HDLc numbers were determined.
  • FIG. 27A , FIG. 27B , and FIG. 27C The results are shown in FIG. 27A , FIG. 27B , and FIG. 27C .
  • the dsRNA AD-10792 (targeting rate PCSK9) was encapsulated in a XTC containing formulation, e.g., a LNP09 formulation.
  • LNP09 formulation was XTC/DSPC/Cholesterol/PEG-DMG at a % mol ratio of 50/10/38.5/1.5 and a lipid:siRNA ratio of 10:1.
  • Formulations were injected via tail vein, single dose (DRC) into rats. Livers and plasma were harvested 72 hours post-injection (5 animals per group). PCSK9 transcript levels were measured via bDNA in livers prepared as manufacturer's protocol. GAPDH transcript levels were also measured and the PCSK9 to GAPDH ratios were normalized to those of PBS control and graphed. Total cholesterol was measured in serum using cholesterol kit from WAKO TX.
  • CETP/ApoB double humanized transgenic mice were purchased from Taconic labs. Animals were injected through tail vein (single injection) of 5 mg/kg of LNP09 formulated AD-10792 (standard formulation procedure), or AD-1955 Luciferase control (4 animals per group). Livers and plasma were harvested 72 hours post-injection (5 animals per group) and liver PCSK9 mRNA, LDL particle, and HDL particle number were determined.
  • PCSK9 transcript levels were measured via bDNA in livers prepared according to manufacturer's protocol. GAPDH transcript levels were also measured and the PCSK9 to GAPDH ratios were graphed, normalized to those of PBS control. LDL and HDL particle numbers/concentration were measured by NMR (Liposciences Inc.) based on their SOP.
  • a human subject is treated with a lipid formulated dsRNA targeted to a PCSK9 gene, described herein, to inhibit expression of the PCSK9 gene and lower cholesterol levels for an extended period of time following a single dose.
  • the lipid formulated dsRNA includes the lipid MC3.
  • 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.
  • a suitable first dose of an anti-PCSK9 siRNA is subcutaneously administered to the subject.
  • the dsRNA is formulated as described herein.
  • 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 in said subject, and/or the products of the successful siRNA-targeting of PCSK9 mRNA. Other relevant criteria can also be measured.
  • the number and strength of doses are adjusted according to the subject's needs.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Dispersion Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Dermatology (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Cardiology (AREA)
  • Vascular Medicine (AREA)
  • Urology & Nephrology (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)

Abstract

This invention relates to composition and methods using lipid formulated siRNA targeted to a PCSK9 gene.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This application a continuation of pending U.S. patent application Ser. No. 13/568,898, filed Aug. 7, 2012 (allowed), which is a continuation of U.S. patent application Ser. No. 12/816,207, filed Jun. 15, 2010, now U.S. Pat. No. 8,273,869, issued Sep. 25, 2012, which claims the benefit of U.S. Provisional Application Ser. No. 61/187,169, filed Jun. 15, 2009; and U.S. Provisional Application Ser. No. 61/218,350, filed Jun. 18, 2009; and U.S. Provisional Application Ser. No. 61/244,790, filed Sep. 22, 2009; and U.S. Provisional Application Ser. No. 61/285,598, filed Dec. 11, 2009; and U.S. Provisional Application Ser. No. 61/293,474, filed Jan. 8, 2010; and U.S. Provisional Application Ser. No. 61/313,578, filed Mar. 12, 2010, all of which are incorporated herein by reference, in their entirety, for all purposes.
    • FIELD OF THE INVENTION
  • This invention relates to compositions comprising lipid formulated dsRNA targeting a PCSK9 gene and methods for treating diseases caused by PCSK9 gene expression.
    • REFERENCE TO A SEQUENCE LISTING
  • This application includes a Sequence Listing submitted electronically as a text file named 24757US_sequencelisting.txt, created on Oct. 18, 2013, with a size of 569,344 bytes. The sequence listing is incorporated by reference.
    • BACKGROUND OF THE INVENTION
  • Proprotein convertase subtilisin kexin 9 (PCSK9) is a member of the subtilisin serine protease family. The other eight mammalian subtilisin proteases, PCSK1-PCSK8 (also called PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, and S1P/SKI-1) are proprotein convertases that process a wide variety of proteins in the secretory pathway and play roles in diverse biological processes (Bergeron, F. (2000) J. Mol. Endocrinol. 24, 1-22, Gensberg, K., (1998) Semin. Cell Dev. Biol. 9, 11-17, Seidah, N. G. (1999) Brain Res. 848, 45-62, Taylor, N. A., (2003) FASEB J. 17, 1215-1227, and Zhou, A., (1999) J. Biol. Chem. 274, 20745-20748). PCSK9 has been proposed to play a role in cholesterol metabolism. PCSK9 mRNA expression is down-regulated by dietary cholesterol feeding in mice (Maxwell, K. N., (2003) J. Lipid Res. 44, 2109-2119), up-regulated by statins in HepG2 cells (Dubuc, G., (2004) Arterioscler. Thromb. Vasc. Biol. 24, 1454-1459), and up-regulated in sterol regulatory element binding protein (SREBP) transgenic mice (Horton, J. D., (2003) Proc. Natl. Acad. Sci. USA 100, 12027-12032), similar to the cholesterol biosynthetic enzymes and the low-density lipoprotein receptor (LDLR). Furthermore, PCSK9 missense mutations have been found to be associated with a form of autosomal dominant hypercholesterolemia (Hchola3) (Abifadel, M., et al. (2003) Nat. Genet. 34, 154-156, Timms, K. M., (2004) Hum. Genet. 114, 349-353, Leren, T. P. (2004) Clin. Genet. 65, 419-422). PCSK9 may also play a role in determining LDL cholesterol levels in the general population, because single-nucleotide polymorphisms (SNPs) have been associated with cholesterol levels in a Japanese population (Shioji, K., (2004) J. Hum. Genet. 49, 109-114).
  • Autosomal dominant hypercholesterolemias (ADHs) are monogenic diseases in which patients exhibit elevated total and LDL cholesterol levels, tendon xanthomas, and premature atherosclerosis (Rader, D. J., (2003) J. Clin. Invest. 111, 1795-1803). The pathogenesis of ADHs and a recessive form, autosomal recessive hypercholesterolemia (ARH) (Cohen, J. C., (2003) Curr. Opin. Lipidol. 14, 121-127), is due to defects in LDL uptake by the liver. ADH may be caused by LDLR mutations, which prevent LDL uptake, or by mutations in the protein on LDL, apolipoprotein B, which binds to the LDLR. ARH is caused by mutations in the ARH protein that are necessary for endocytosis of the LDLR-LDL complex via its interaction with clathrin. Therefore, if PCSK9 mutations are causative in Hchola3 families, it seems likely that PCSK9 plays a role in receptor-mediated LDL uptake.
  • Overexpression studies point to a role for PCSK9 in controlling LDLR levels and, hence, LDL uptake by the liver (Maxwell, K. N. (2004) Proc. Natl. Acad. Sci. USA 101, 7100-7105, Benjannet, S., et al. (2004) J. Biol. Chem. 279, 48865-48875, Park, S. W., (2004) J. Biol. Chem. 279, 50630-50638). Adenoviral-mediated overexpression of mouse or human PCSK9 for 3 or 4 days in mice results in elevated total and LDL cholesterol levels; this effect is not seen in LDLR knockout animals (Maxwell, K. N. (2004) Proc. Natl. Acad. Sci. USA 101, 7100-7105, Benjannet, S., et al. (2004) J. Biol. Chem. 279, 48865-48875, Park, S. W., (2004) J. Biol. Chem. 279, 50630-50638). In addition, PCSK9 overexpression results in a severe reduction in hepatic LDLR protein, without affecting LDLR mRNA levels, SREBP protein levels, or SREBP protein nuclear to cytoplasmic ratio.
  • Loss of function mutations in PCSK9 have been designed in mouse models (Rashid et al., (2005) PNAS, 102, 5374-5379), and identified in human individuals (Cohen et al. (2005) Nature Genetics 37:161-165). In both cases loss of PCSK9 function lead to lowering of total and LDLc cholesterol. In a retrospective outcome study over 15 years, loss of one copy of PCSK9 was shown to shift LDLc levels lower and to lead to an increased risk-benefit protection from developing cardiovascular heart disease (Cohen et al., (2006) N. Engl. J. Med., 354:1264-1272).
  • 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.
  • A description of siRNA targeting PCSK9 can be found in U.S. Pat. No. 7,605,251 and WO 2007/134161. Additional disclosure can be found in U.S. Patent Publication No. 2010/0010066 and WO 2009/134487
    • SUMMARY OF THE INVENTION
  • As described in more detail below, disclosed herein are compositions comprising lipid formulated siRNA targeting PCSK9, e.g., MC3 formulated siRNA targeting PCSK9. Also disclosed are methods of using the compositions for inhibition of PCSK9 expression and for treatment of pathologies related to PCSK9 expression, e.g., hyperlipidemia
  • Accordingly, one aspect of the invention is a compositing comprising a nucleic acid lipid particle comprising a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human PCSK9 gene in a cell, wherein the nucleic acid lipid particle comprises a lipid formulation comprising 45-65 mol % of a cationic lipid, 5 mol % to about 10 mol %, of a non-cationic lipid, 25-40 mol % of a sterol, and 0.5-5 mol % of a PEG or PEG-modified lipid, the dsRNA consists of a sense strand and an antisense strand, and the sense strand comprises a first sequence and the antisense strand comprises a second sequence complementary to at least 15 contiguous nucleotides of SEQ ID NO:1523 (5′-UUCUAGACCUGUUUUGCUU-3′), wherein the first sequence is complementary to the second sequence and wherein the dsRNA is between 15 and 30 base pairs in length.
  • As described herein the composition includes a cationic lipid. In one embodiment, the cationic lipid comprises MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate. For example, the lipid formulation can be selected from the following:
  •
    LNP11 MC3/DSPC/Cholesterol/PEG-DMG
    50/10/38.5/1.5
    LNP14 MC3/DSPC/Cholesterol/PEG-DMG
    40/15/40/5
    LNP15 MC3/DSPC/Cholesterol/PEG-DSG/GalNAc-PEG-DSG
    50/10/35/4.5/0.5
    LNP16 MC3/DSPC/Cholesterol/PEG-DMG
    50/10/38.5/1.5
    LNP17 MC3/DSPC/Cholesterol/PEG-DSG
    50/10/38.5/1.5
    LNP18 MC3/DSPC/Cholesterol/PEG-DMG
    50/10/38.5/1.5
    LNP19 MC3/DSPC/Cholesterol/PEG-DMG
    50/10/35/5
    LNP20 MC3/DSPC/Cholesterol/PEG-DPG
    50/10/38.5/1.5
  • In other embodiments, the cationic lipid comprises formula A wherein formula A is
  • Figure US20140121263A1-20140501-C00001
  • where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments the cationic lipid comprises formula A and is XTC (2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane). The lipid formulation can include the cationic lipid XTC, the non-cationic lipid DSPC, the sterol cholesterol and the PEG lipid PEG-DMG. In other embodiments the cationic lipid comprises XTC and the formulation is selected from the group consisting of:
  •
    LNP05 XTC/DSPC/Cholesterol/PEG-DMG
    57.5/7.5/31.5/3.5
    LNP06 XTC/DSPC/Cholesterol/PEG-DMG
    57.5/7.5/31.5/3.5
    LNP07 XTC/DSPC/Cholesterol/PEG-DMG
    60/7.5/31/1.5,
    LNP08 XTC/DSPC/Cholesterol/PEG-DMG
    60/7.5/31/1.5
    LNP09 XTC/DSPC/Cholesterol/PEG-DMG
    50/10/38.5/1.5
    LNP13 XTC/DSPC/Cholesterol/PEG-DMG
    50/10/38.5/1.5
    LNP22 XTC/DSPC/Cholesterol/PEG-DSG
    50/10/38.5/1.5
  • In still further embodiments, the cationic lipid comprises ALNY-100 ((3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)). For example, the cationic lipid comprises ALNY-100 and the formulation consists of ALNY-100/DSPC/Cholesterol/PEG-DMG in a ratio of 50/10/38.5/1.5
  • The composition includes a dsRNA targeting PCSK9. In some embodiments, the sense strand comprises SEQ ID NO:1227 and the antisense strand comprises SEQ ID NO:1228. In other embodiments, the sense strand consists of SEQ ID NO:1227 and the antisense strand consists of SEQ ID NO:1228. One or both strands can be modified, e.g., each strand is modified as follows to include a 2′-O-methyl ribonucleotide as indicated by a lower case letter “c” or “u” and a phosphorothioate as indicated by a lower case letter “s”: the dsRNA consists of a sense strand consisting of
  • SEQ ID NO: 1229
    (5′- uucuAGAccuGuuuuGcuuTsT -3′)
  • and an antisense strand consisting of
  • SEQ ID NO: 1230
    (5′- AAGcAAAAcAGGUCuAGAATsT-3′).
  • In other embodiments, the dsRNA comprises at least one modified nucleotide, e.g., a modified nucleotide chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, and/or, e.g., the modified nucleotide is chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. In one embodiment, dsRNA comprises at least one 2′-O-methyl modified ribonucleotide and at least one nucleotide comprising a 5′-phosphorothioate group.
  • The compositions include a dsRNA between 15 and 30 base pairs in length. In one embodiment, each strand of the dsRNA is 19-23 bases in length, or, e.g., 21-23 bases in length, or, e.g. 21 bases in length.
  • In one aspect, the compositions include a lipoprotein, e.g., apolipoprotein E (ApoE). In some embodiments, the compositions include a lipoprotein and the dsRNA is conjugated to a lipophile, e.g., a cholesterol. The ApoE can be reconstituted with at least one amphiphilic agent, e.g., a phospholipid, e.g., a phospholipid selected from the group consisting of dimyristoyl phosphatidyl choline (DMPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), -phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), and combinations thereof. In some embodiments, the ApoE is reconstituted high density lipoprotein (rHDL).
  • The compositions, e.g., lipid formulated dsRNA targeting PCSK9, can be administered to a cell or subject, e.g., a primate, e.g., a human. In one aspect, administration of the compositions inhibits expression of PCSK9 by at least 40% compared to administration of a control and/or reduces PCSK9 protein levels in the mammal compared to administration of a control, and/or reduces LDLc levels in a mammal compared to administration of a control and/or reduces both PCSK9 hepatic mRNA and total serum cholesterol at a dosage of less than 0.1 mg/kg compared to administration of a control and/or reduces PCSK9 hepatic mRNA at an ED50 of about 0.2 mg/kg and reduces total serum cholesterol with an ED50 of about 0.08 mg/kg compared to administration of a control and/or reduces serum LDL particle numbers by more than 90% or reduces serum HDL particle numbers by more than 75% compared to administration of a control.
  • The invention also provides methods comprising administering the lipid formulated PCSK9 targeted dsRNA compositions described herein. In one embodiment, the invention includes a method for inhibiting the expression of PCSK9 in a cell comprising administering the compositions to the cell. In another embodiment, the invention includes a method for reducing LDLc levels in a mammal in need of treatment comprising administering the compositions to the mammal.
  • As described in more detail below, the methods can include any appropriate dosage, e.g., between 0.25 mg/kg and 4 mg/kg dsRNA, or e.g., at about 0.01, 0.1, 0.5, 1.0, 2.5, or 5.0 mg/kg dsRNA.
  • Also described herein are methods for inhibiting expression of a PCSK9 gene in a subject, comprising administering to the subject the lipid formulated PCSK9 targeted dsRNA compositions described herein 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. The subject can be, .e.g., a rat or a non-human primate or a human. The subsequent dose can be about 1.0 mg/kg or about 0.3 mg/kg. In some embodiments, the subsequent dose is administered once a week for four weeks. In some embodiments, administration of the first dose decreases total cholesterol levels by about 15-60%.
  • The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • The prefixes “AD-” “DP-” and “AL-DP-” are used interchangeably e.g., AL-DP-9327 and AD-9237.
  • FIG. 1 shows the structure of the ND-98 lipid.
  • FIG. 2 shows the results of the in vivo screen of 16 mouse specific (AL-DP-9327 through AL-DP-9342) PCSK9 siRNAs directed against different ORF regions of PCSK9 mRNA (having the first nucleotide corresponding to the ORF position indicated on the graph) in C57/BL6 mice (5 animals/group). The ratio of PCSK9 mRNA to GAPDH mRNA in liver lysates was averaged over each treatment group and compared to a control group treated with PBS or a control group treated with an unrelated siRNA (blood coagulation factor VII).
  • FIG. 3 shows the results of the in vivo screen of 16 human/mouse/rat 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).
  • FIG. 4 shows the results of the in vivo screen of 16 mouse specific (AL-DP-9327 through AL-DP-9342) PCSK9 siRNAs in C57/BL6 mice (5 animals/group). Total serum cholesterol levels were averaged over each treatment group and compared to a control group treated with PBS or a control group treated with an unrelated siRNA (blood coagulation factor VII).
  • FIG. 5 shows the results of the in vivo screen of 16 human/mouse/rat 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 siRNA (blood coagulation factor VII).
  • FIGS. 6A and 6B compare in vitro and in vivo results, respectively, for silencing PCSK9.
  • FIG. 7A and FIG. 7B are an example of in vitro results for silencing PCSK9 using monkey primary hepatocytes.
  • FIG. 7C show results for silencing of PCSK9 in monkey primary hepatocytes using AL-DP-9680 and chemically modified version of AL-DP-9680.
  • FIG. 8 shows in vivo activity of LNP-01 formulated siRNAs to PCSK-9.
  • 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.
  • 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. 10C shows the effect of PCSK9 siRNAs on hepatic cholesterol and triglyceride levels in the same experiment as 10A.
  • FIG. 11 is a Western blot showing that liver LDL receptor levels were upregulated following administration of PCSK9 siRNAs in rat.
  • 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.
  • FIG. 13A is a graph showing that unmodified siRNA-AD-A1A (AD-9314), but not 2′OMe modified siRNA-AD-1A2 (AD-10792), induced IFN-alpha in human primary blood monocytes. FIG. 13B is a graph showing that unmodified siRNA-AD-A1A (AD-9314), but not 2′OMe modified siRNA-AD-1A2 (AD-10792), also induced TNF-alpha in human primary blood monocytes.
  • FIG. 14A is a graph showing that the PCSK9 siRNA siRNA-AD-1A2 (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-1A2 decreased serum total cholesterol levels in mice within 48 hours.
  • FIG. 15A is a graph showing that PCSK9 siRNAs 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.
  • FIG. 16 shows the structure of an siRNA conjugated to Chol-p-(GalNAc)3 via phosphate linkage at the 3′ end.
  • FIG. 17 shows the structure of an siRNA conjugated to LCO(GalNAc)3 (a (GalNAc)3-3′-Lithocholic-oleoyl siRNA Conjugate).
  • FIG. 18 is a graph showing the results of conjugated siRNAs on PCSK9 transcript levels and total serum cholesterol in mice.
  • FIG. 19 is a graph showing the results of lipid formulated siRNAs on PCSK9 transcript levels and total serum cholesterol in rats.
  • 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.
  • 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.
  • FIG. 22 is a graph with the results of PCSK9 targeted siRNA transfection of Hep3B cells and the effects on PCSK9 and off-target gene levels.
  • FIG. 23 shows the results of treatment in rats with a maintenance dose of PCSK9 targeted siRNA.
  • FIG. 24 is a dose response curve of treatment of HeLa cells with modified siRNAs.
  • FIG. 25 is a graph of average IC50 of siRNA vs. target position in human PCSK9 transcript. The large blue dot indicates the IC50 and location of AD-9680.
  • FIG. 26 is a graph with the results of administration of rEHDL formulated cholesterol conjugated siRNA.
  • FIG. 27A is a graph with results of administration of second generation LNP formulated PCSK9 targeted siRNA (AD-9680 in LNP11) to non-human primates, demonstrating a reduction in both PCSK9 protein and LDLc levels. LDLc: low density lipoprotein cholesterol; mpk: mg per kg.
  • FIG. 27B is a bar graph showing dose dependent PCSK mRNA silencing in non-human primates after treatment with LNP formulated siRNA targeting PCSK9.
  • FIG. 27C is a graph with the results of administration of second generation LNP formulated PCSK9 targeted siRNA (AD-9680) to non-human primates, demonstrating a no change in HDLc levels.
  • FIG. 28 illustrates the chemical structures of the cationic lipids MC3 and ALNY-100.
  • FIG. 29 is a graph of effects on PCSK9 mRNA and serum cholesterol levels in rats after administration of LNP-09 formulated AD-10792, an siRNA targeting rodent PCSK9.
  • FIG. 30 are graphs of the effects on PCSK9 mRNA and LDL/HDL particle numbers in CETP/ApoB tg mice after administration of LNP-09 formulated AD-10792, an siRNA targeting rodent PCSK9.
    •  DETAILED DESCRIPTION OF THE INVENTION
  • The invention provides a solution to the problem of treating diseases that can be modulated by the down regulation of the PCSK9 gene, such as hyperlipidemia, by using double-stranded ribonucleic acid (dsRNA) to silence the PCSK9 gene.
  • The invention provides compositions and methods for inhibiting the expression of the PCSK9 gene in a subject using a dsRNA. The invention also provides compositions and methods for treating pathological conditions and diseases, such as hyperlipidemia, that can be modulated by down regulating the expression of the PCSK9 gene. dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).
  • The dsRNA useful for the compositions and methods of an invention include an RNA strand (the antisense strand) having a region that is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an mRNA transcript of the PCSK9 gene. The use of these dsRNAs enables the targeted degradation of an mRNA that is involved in the regulation of the LDL Receptor and circulating cholesterol levels. Using cell-based and animal assays, the present inventors have demonstrated that very low dosages of these dsRNAs can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of the PCSK9 gene. Thus, methods and compositions including these dsRNAs are useful for treating pathological processes that can be mediated by down regulating PCSK9, such as in the treatment of hyperlipidemia.
  • The following detailed description discloses how to make and use the dsRNA and compositions containing dsRNA to inhibit the expression of the target PCSK9 gene, as well as compositions and methods for treating diseases that can be modulated by down regulating the expression of PCSK9, such as hyperlipidemia. The pharmaceutical compositions of the invention include a dsRNA having an antisense strand having a region of complementarity that is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and that is substantially complementary to at least part of an RNA transcript of the PCSK9 gene, together with a pharmaceutically acceptable carrier.
  • Accordingly, certain aspects of the invention provide pharmaceutical compositions including the dsRNA that targets PCSK9 together with a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of the PCSK9 gene, and methods of using the pharmaceutical compositions to treat diseases by down regulating the expression of PCSK9.
    • DEFINITIONS
  • For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.
  • “G,” “C,” “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. “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.
  • 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—174936; mouse: NM—153565, and rat: NM—199253. Additional examples of PCSK9 mRNA sequences are readily available using, e.g., GenBank.
  • As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the PCSK9 gene, including mRNA that is a product of RNA processing of a primary transcription product.
  • As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
  • As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
  • This includes base-pairing of the oligonucleotide or polynucleotide 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.”
  • “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.
  • The terms “complementary”, “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.
  • As used herein, a polynucleotide which is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide 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.
  • 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.
  • The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where separate RNA molecules, such dsRNA are often referred to in the literature as siRNA (“short interfering RNA”). Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop”, “short hairpin RNA” or “shRNA”. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker”. The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs. In 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.
  • As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule. For clarity, chemical caps or non-nucleotide chemical moieties conjugated to the 3′ end or 5′ end of an siRNA are not considered in determining whether an siRNA has an overhang or is blunt ended.
  • The term “antisense strand” refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches 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.
  • The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.
  • “Introducing into a cell”, when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be “introduced into a cell”, wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.
  • The terms “silence,” “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 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 ) ( mRNA in control cells ) · 100 %
  • Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to 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.
  • 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.
  • 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.
  • As used herein, a “pharmaceutical composition” includes a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.
  • The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof and are described in more detail below. The term specifically excludes cell culture medium.
  • As used herein, a “transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.
  • Double-Stranded Ribonucleic Acid (dsRNA)
  • As described in more detail below, the invention provides methods and composition having double-stranded ribonucleic acid (dsRNA) molecules 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. For example, expression of a PCSK9 gene in cell culture, such as in HepB3 cells, can be assayed by measuring PCSK9 mRNA levels, such as by bDNA or TaqMan assay, or by measuring protein levels, such as by ELISA assay. The dsRNA of the invention can further include one or more single-stranded nucleotide overhangs.
  • 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. 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. 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. 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.
  • 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. In other embodiments, each is strand is 25-30 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.
  • 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, generally 1 or 2 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.
  • 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.
  • The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. In one embodiment, the PCSK9 gene is a human PCSK9 gene. In other embodiments, the antisense strand of the dsRNA includes a first strand selected from the sense sequences of Table 1a, Table 2a, and Table 5a, and a second strand selected from the antisense sequences of Table 1a, Table 2a, and Table 5a. Alternative antisense agents that target elsewhere in the target sequence provided in Table 1a, Table 2a, and Table 5a, can readily be determined using the target sequence and the flanking PCSK9 sequence.
  • For example, the dsRNA AD-9680 (from Table 1a) targets the PCSK 9 gene at 3530-3548; therefore the target sequence is as follows: 5′ UUCUAGACCUGUUUUGCUU 3′ (SEQ ID NO:1523). The dsRNA AD-10792 (from Table 1a) 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 that have regions of complementarity to SEQ ID NO:1523 and SEQ ID NO:1524.
  • In further embodiments, the dsRNA includes at least one nucleotide sequence selected from the groups of sequences provided in Table 1a, Table 2a, and Table 5a. In other embodiments, the dsRNA includes at least two sequences selected from this group, where one of the at least two sequences is complementary to another of the at least two sequences, and one of the at least two sequences is substantially complementary to a sequence of an mRNA generated in the expression of the PCSK9 gene. Generally, the dsRNA includes two oligonucleotides, where one oligonucleotide is described as the sense strand in Table 1a, Table 2a, and Table 5a and the second oligonucleotide is described as the antisense strand in Table 1a, Table 2a, and Table 5a
  • 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 1a, Table 2a, and Table 5a, the dsRNAs of the invention can include at least one strand of a length of minimally 21 nt. It can be reasonably expected that shorter dsRNAs having one of the sequences of Table 1a, Table 2a, 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, or more contiguous nucleotides from one of the sequences of Table 1a, Table 2a, and Table 5a, and differing in their ability to inhibit the expression of the PCSK9 gene in a FACS assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further dsRNAs that cleave within the target sequence provided in Table 1a, Table 2a, and Table 5a can readily be made using the PCSK9 sequence and the target sequence provided.
  • In addition, the RNAi agents provided in Table 1a, Table 2a, and Table 5a identify a site in the PCSK9 mRNA that is susceptible to RNAi based cleavage. As such the present invention further includes RNAi agents that target within the sequence targeted by one of the agents of the present invention. As used herein a second RNAi agent is said to target within the sequence of a first RNAi agent if the second RNAi agent cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first RNAi agent. Such a second agent will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Table 1a, Table 2a, 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 1a, Table 2a, and Table 5a.
  • 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.
  • In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties than their blunt-ended counterparts. Moreover, the present inventors have discovered that the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Generally, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA may also have a blunt end, generally located at the 5′-end of the antisense strand. Such dsRNAs have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
  • Chemical Modifications and Conjugates
  • In yet another embodiment, the dsRNA is chemically modified to enhance stability. The nucleic acids of the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Chemical modifications may include, but are not limited to 2′ modifications, modifications at other sites of the sugar or base of an oligonucleotide, introduction of non-natural bases into the oligonucleotide chain, covalent attachment to a ligand or chemical moiety, and replacement of internucleotide phosphate linkages with alternate linkages such as thiophosphates. More than one such modification may be employed.
  • Chemical linking of the two separate dsRNA strands may be achieved by any of a variety of well-known techniques, for example by introducing covalent, ionic or hydrogen bonds; hydrophobic interactions, van der Waals or stacking interactions; by means of metal-ion coordination, or through use of purine analogues. Generally, the chemical groups that can be used to modify the dsRNA include, without limitation, methylene blue; bifunctional groups, generally bis-(2-chloroethyl)amine; N-acetyl-N′-(p-glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen. In one embodiment, the linker is a hexa-ethylene glycol linker. In this case, the dsRNA are produced by solid phase synthesis and the hexa-ethylene glycol linker is incorporated according to standard methods (e.g., Williams, D. J., and K. B. Hall, Biochem. (1996) 35:14665-14670). In a particular embodiment, the 5′-end of the antisense strand and the 3′-end of the sense strand are chemically linked via a hexaethylene glycol linker. In another embodiment, at least one nucleotide of the dsRNA comprises a phosphorothioate or phosphorodithioate groups. The chemical bond at the ends of the dsRNA is generally formed by triple-helix bonds. Table 1a, Table 2a, and Table 5a provides examples of modified RNAi agents of the invention.
  • In yet another embodiment, the nucleotides at one or both of the two single strands may be modified to prevent or inhibit the degradation activities of cellular enzymes, such as, for example, without limitation, certain nucleases. Techniques for inhibiting the degradation activity of cellular enzymes against nucleic acids are known in the art including, but not limited to, 2′-amino modifications, 2′-amino sugar modifications, 2′-F sugar modifications, 2′-F modifications, 2′-alkyl sugar modifications, uncharged backbone modifications, morpholino modifications, 2′-O-methyl modifications, and phosphoramidate (see, e.g., Wagner, Nat. Med. (1995) 1:1116-8). Thus, at least one 2′-hydroxyl group of the nucleotides on a dsRNA is replaced by a chemical group, generally by a 2′-amino or a 2′-methyl group. Also, at least one nucleotide may be modified to form a locked nucleotide. Such locked nucleotide contains a methylene bridge that connects the 2′-oxygen of ribose with the 4′-carbon of ribose. Oligonucleotides containing the locked nucleotide are described in Koshkin, A. A., et al., Tetrahedron (1998), 54: 3607-3630) and Obika, S. et al., Tetrahedron Lett. (1998), 39: 5401-5404). Introduction of a locked nucleotide into an oligonucleotide improves the affinity for complementary sequences and increases the melting temperature by several degrees (Braasch, D. A. and D. R. Corey, Chem. Biol. (2001), 8:1-7).
  • Conjugating a ligand to a dsRNA can enhance its cellular absorption as well as targeting to a particular tissue or uptake by specific types of cells such as liver cells. In certain instances, a hydrophobic ligand is conjugated to the dsRNA to facilitate direct permeation of the cellular membrane and or uptake across the liver cells. Alternatively, the ligand conjugated to the dsRNA is a substrate for receptor-mediated endocytosis. These approaches have been used to facilitate cell permeation of antisense oligonucleotides as well as dsRNA agents. For example, cholesterol has been conjugated to various antisense oligonucleotides resulting in compounds that are substantially more active compared to their non-conjugated analogs. See M. Manoharan Antisense & Nucleic Acid Drug Development 2002, 12, 103. Other lipophilic compounds that have been conjugated to oligonucleotides include 1-pyrene butyric acid, 1,3-bis-O-(hexadecyl)glycerol, and menthol. One example of a ligand for receptor-mediated endocytosis is folic acid. Folic acid enters the cell by folate-receptor-mediated endocytosis. dsRNA compounds bearing folic acid would be efficiently transported into the cell via the folate-receptor-mediated endocytosis. Li and coworkers report that attachment of folic acid to the 3′-terminus of an oligonucleotide resulted in an 8-fold increase in cellular uptake of the oligonucleotide. Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res. 1998, 15, 1540. Other ligands that have been conjugated to oligonucleotides include polyethylene glycols, carbohydrate clusters, cross-linking agents, porphyrin conjugates, delivery peptides and lipids such as cholesterol and cholesterylamine. Examples of carbohydrate clusters include Chol-p-(GalNAc)3 (N-acetyl galactosamine cholesterol) and LCO(GalNAc)3 (N-acetyl galactosamine-3′-Lithocholic-oleoyl.
  • In certain instances, conjugation of a cationic ligand to oligonucleotides results in improved resistance to nucleases. Representative examples of cationic ligands are propylammonium and dimethylpropylammonium. Interestingly, antisense oligonucleotides were reported to retain their high binding affinity to mRNA when the cationic ligand was dispersed throughout the oligonucleotide. See M. Manoharan Antisense & Nucleic Acid Drug Development 2002, 12, 103 and references therein.
  • In some cases, a ligand can be multifunctional and/or a dsRNA can be conjugated to more than one ligand. For example, the dsRNA can be conjugated to one ligand for improved uptake and to a second ligand for improved release.
  • The ligand-conjugated dsRNA of the invention may be synthesized by the use of a dsRNA that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the dsRNA. This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto. The methods of the invention facilitate the synthesis of ligand-conjugated dsRNA by the use of, in some embodiments, nucleoside monomers that have been appropriately conjugated with ligands and that may further be attached to a solid-support material. Such ligand-nucleoside conjugates, optionally attached to a solid-support material, are prepared according to certain embodiments of the methods described herein via reaction of a selected serum-binding ligand with a linking moiety located on the 5′ position of a nucleoside or oligonucleotide. In certain instances, a dsRNA bearing an aralkyl ligand attached to the 3′-terminus of the dsRNA is prepared by first covalently attaching a monomer building block to a controlled-pore-glass support via a long-chain aminoalkyl group. Then, nucleotides are bonded via standard solid-phase synthesis techniques to the monomer building-block bound to the solid support. The monomer building block may be a nucleoside or other organic compound that is compatible with solid-phase synthesis.
  • The dsRNA used in the conjugates of the invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.
  • Synthesis
  • Teachings regarding the synthesis of particular modified oligonucleotides may be found in the following U.S. patents: U.S. Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugated oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for the preparation of oligonucleotides having chiral phosphorus linkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn to oligonucleotides having modified backbones; U.S. Pat. No. 5,386,023, drawn to backbone-modified oligonucleotides and the preparation thereof through reductive coupling; U.S. Pat. No. 5,457,191, drawn to modified nucleobases based on the 3-deazapurine ring system and methods of synthesis thereof; U.S. Pat. No. 5,459,255, drawn to modified nucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302, drawn to processes for preparing oligonucleotides having chiral phosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides having β-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods and materials for the synthesis of oligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides having alkylthio groups, wherein such groups may be used as linkers to other moieties attached at any of a variety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides having phosphorothioate linkages of high chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for the preparation of 2′-O-alkyl guanosine and related compounds, including 2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2 substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat. No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to conjugated 4′-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn to backbone-modified oligonucleotide analogs; U.S. Pat. Nos. 6,262,241, and 5,459,255, drawn to, inter alia, methods of synthesizing 2′-fluoro-oligonucleotides.
  • In the ligand-conjugated dsRNA and ligand-molecule bearing sequence-specific linked nucleosides of the invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.
  • When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. Oligonucleotide conjugates bearing a variety of molecules such as steroids, vitamins, lipids and reporter molecules, has previously been described (see Manoharan et al., PCT Application WO 93/07883). In 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.
  • The incorporation of a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-allyl, 2′-β-aminoalkyl or 2′-deoxy-2′-fluoro group in nucleosides of an oligonucleotide confers enhanced hybridization properties to the oligonucleotide. Further, oligonucleotides containing phosphorothioate backbones have enhanced nuclease stability. Thus, functionalized, linked nucleosides of the invention can be augmented to include either or both a phosphorothioate backbone or a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-β-aminoalkyl, 2′-O-allyl or 2′-deoxy-2′-fluoro group. A summary listing of some of the oligonucleotide modifications known in the art is found at, for example, PCT Publication WO 200370918.
  • In some embodiments, functionalized nucleoside sequences of the invention possessing an amino group at the 5′-terminus are prepared using a DNA synthesizer, and then reacted with an active ester derivative of a selected ligand. Active ester derivatives are well known to those skilled in the art. Representative active esters include N-hydrosuccinimide esters, tetrafluorophenolic esters, pentafluorophenolic esters and pentachlorophenolic esters. The reaction of the amino group and the active ester produces an oligonucleotide in which the selected ligand is attached to the 5′-position through a linking group. The amino group at the 5′-terminus can be prepared utilizing a 5′-Amino-Modifier C6 reagent. In one embodiment, ligand molecules may be conjugated to oligonucleotides at the 5′-position by the use of a ligand-nucleoside phosphoramidite wherein the ligand is linked to the 5′-hydroxy group directly or indirectly via a linker. Such ligand-nucleoside phosphoramidites are typically used at the end of an automated synthesis procedure to provide a ligand-conjugated oligonucleotide bearing the ligand at the 5′-terminus.
  • Examples of modified internucleoside linkages or backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free-acid forms are also included.
  • Representative United States patents relating to the preparation of the above phosphorus-atom-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; and 5,697,248, each of which is herein incorporated by reference.
  • Examples of modified internucleoside linkages or backbones that do not include a phosphorus atom therein (i.e., oligonucleosides) have backbones that are formed by short chain alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short chain heteroatomic or heterocyclic intersugar linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
  • Representative United States patents relating to the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.
  • In certain instances, the oligonucleotide may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to oligonucleotides in order to enhance the activity, cellular distribution or cellular uptake of the oligonucleotide, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such oligonucleotide conjugates have been listed above. Typical conjugation protocols involve the synthesis of oligonucleotides bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the oligonucleotide still bound to the solid support or following cleavage of the oligonucleotide in solution phase. Purification of the oligonucleotide conjugate by HPLC typically affords the pure conjugate. The use of a cholesterol conjugate is particularly preferred since such a moiety can increase targeting liver cells, a site of PCSK9 expression.
  • Vector Encoded RNAi Agents
  • In another aspect of the invention, PCSK9 specific dsRNA molecules that modulate PCSK9 gene expression activity are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).
  • The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In 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.
  • 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 et 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; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.
  • Any viral vector capable of accepting the coding sequences for the dsRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.
  • For example, lentiviral vectors of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors of the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
  • Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the dsRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat. Genet. 33: 401-406, the entire disclosures of which are herein incorporated by reference.
  • Preferred viral vectors are those derived from AV and AAV. In a particularly preferred embodiment, the dsRNA of the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter.
  • A suitable AV vector for expressing the dsRNA of the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
  • Suitable AAV vectors for expressing the dsRNA of the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
  • The promoter driving dsRNA expression in either a DNA plasmid or viral vector of the invention may be a eukaryotic RNA polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter or actin promoter or U1 snRNA promoter) or generally RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the pancreas (see, e.g., the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).
  • In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.
  • Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
  • dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g. Oligofectamine) or non-cationic lipid-based carriers (e.g. Transit-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.
  • The PCSK9 specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • Pharmaceutical Compositions Containing dsRNA
  • In one embodiment, the invention provides pharmaceutical compositions containing a dsRNA, as described herein, and a pharmaceutically acceptable carrier and methods of administering the same. The pharmaceutical composition containing the dsRNA 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
  • The pharmaceutical compositions featured herein are administered in dosages sufficient to inhibit expression of PCSK9 genes. In general, a suitable dose of dsRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.01 mg/kg, 0.05 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.
  • 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.
  • In one embodiment, the dosage is between 0.2 mg/kg and 1.5 mg/kg. For example, the dsRNA 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, or 1.5 mg/kg.
  • The dsRNA can be administered at a dose of 0.03, 0.1, 0.3, or 1.3, or 3.0 mg/kg.
  • The pharmaceutical composition can be administered once daily, or the dsRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day. The effect of a single dose on PCSK9 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.
  • In one embodiment the lipid formulated PCSK9 targeted dsRNA 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. The subsequent dose can be administered, e.g., once a week for four weeks.
  • In some embodiments the dsRNA is administered using continuous infusion or delivery through a controlled release formulation. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
  • The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
  • Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by PCSK9 expression. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose. A suitable mouse model is, for example, a mouse containing a plasmid expressing human PCSK9. Another suitable mouse model is a transgenic mouse carrying a transgene that expresses human PCSK9.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.
  • The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • In addition to their administration, as discussed above, the dsRNAs 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
  • The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, 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.
  • Typically, when treating a mammal with hyperlipidemia, the dsRNA molecules 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, dsRNAs, conjugated or unconjugate or formulated with or without liposomes, can be administered intravenously to a patient. For such, a dsRNA molecule can be formulated into compositions such as sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions in liquid or solid oil bases. Such solutions also can contain buffers, diluents, and other suitable additives. For parenteral, intrathecal, or intraventricular administration, a dsRNA molecule can be formulated into compositions such as sterile aqueous solutions, which also can contain buffers, diluents, and other suitable additives (e.g., penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers). Formulations are described in more detail herein.
  • The dsRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).
  • Formulations
  • The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
  • 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.
  • In addition, dsRNA that target the PCSK9 gene can be formulated into compositions containing the dsRNA admixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of nucleic acids. For example, a composition containing one or more dsRNA agents that target the PCSK9 gene can contain other therapeutic agents, such as other cancer therapeutics or one or more dsRNA compounds that target non-PCSK9 genes.
  • Oral, Parenteral, Topical, and Biologic Formulations
  • Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. dsRNAs featured in the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. dsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, U.S. Patent Publication. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.
  • Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
  • Pharmaceutical compositions 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 dsRNAs 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). dsRNAs featured in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, dsRNAs may be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1-10 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference. In addition, dsRNA molecules can be administered to a mammal as biologic or abiologic means as described in, for example, U.S. Pat. No. 6,271,359. Abiologic delivery can be accomplished by a variety of methods including, without limitation, (1) loading liposomes with a dsRNA acid molecule provided herein and (2) complexing a dsRNA molecule with lipids or liposomes to form nucleic acid-lipid or nucleic acid-liposome complexes. The liposome can be composed of cationic and neutral lipids commonly used to transfect cells in vitro. Cationic lipids can complex (e.g., charge-associate) with negatively charged nucleic acids to form liposomes. Examples of cationic liposomes include, without limitation, lipofectin, lipofectamine, lipofectace, and DOTAP. Procedures for forming liposomes are well known in the art. Liposome compositions can be formed, for example, from phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, or dioleoyl phosphatidylethanolamine. Numerous lipophilic agents are commercially available, including Lipofectin™ (Invitrogen/Life Technologies, Carlsbad, Calif.) and 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 al., J. Am Soc. Nephrol. 7: 1728 (1996)). Additional information regarding the use of liposomes to deliver nucleic acids can be found in U.S. Pat. No. 6,271,359, PCT Publication WO 96/40964 and Morrissey, D. et al. 2005. Nat Biotechnol. 23(8):1002-7.
  • Biologic delivery can be accomplished by a variety of methods including, without limitation, the use of viral vectors. For example, viral vectors (e.g., adenovirus and 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
  • There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
  • Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.
  • In order to 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.
  • 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.
  • Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
  • Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.
  • Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis
  • Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).
  • Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).
  • One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
  • Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).
  • Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al., S.T.P. Pharma. Sci., 1994, 4, 6, 466).
  • Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).
  • Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Lim et al.).
  • Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C1215G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.
  • A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.
  • Transfersomes are yet another type of liposomes and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes, it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
  • Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
  • If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
  • If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
  • If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
  • If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
  • The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
  • Nucleic Acid Lipid Particles
  • In one embodiment, a dsRNA featured in the invention is fully encapsulated in the lipid formulation, e.g., to form a nucleic acid-lipid particle, e.g., 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. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.
  • 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.
  • 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. Pat. No. 6,320,017), peptides, proteins, detergents, lipid-derivatives, such as PEG coupled to phosphatidylethanolamine and PEG conjugated to ceramides (see, U.S. Pat. No. 5,885,613).
  • 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.
  • 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.
  • 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
  • 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, or 33:1.
  • Cationic Lipids
  • The nucleic acid-lipid particles of the invention typically include a cationic lipid. The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.C1), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.C1), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALNY-100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), or a mixture thereof.
  • 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”); 1,2-Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.C1”); 3β-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine (“DOGS”), 1,2-dileoyl-sn-3-phosphoethanolamine (“DOPE”), 1,2-dioleoyl-3-dimethylammonium propane (“DODAP”), N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), and N-(1,2-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 LIPOFECTAMINE (comprising DOSPA and DOPE, available from GIBCO/BRL). In particular embodiments, a cationic lipid is an amino lipid.
  • 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.
  • 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.
  • 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.
  • 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 pH and be removed by simple dialysis; thus greatly reducing the particle's susceptibility to clearance.
  • One example of a cationic lipid is 1,2-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 Apr. 15, 2009.
  • In one embodiment, the cationic lipid XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane) is used to prepare nucleic acid-lipid particles. Synthesis of XTC is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.
  • In one aspect, the cationic lipids have the structure
  • Figure US20140121263A1-20140501-C00002
  • and salts or isomers thereof, wherein R1 and R2 are each independently for each occurrence optionally substituted C10-C30 alkyl, optionally substituted C10-C30 alkenyl, optionally substituted C10-C30 alkynyl, optionally substituted C10-C30 acyl, or -linker-ligand; R3 is H, optionally substituted C1-C10 alkyl, optionally substituted C2-C10 alkenyl, optionally substituted C2-C10 alkynyl, alkylhetrocycle, alkylphosphate, alkylphosphorothioate, alkylphosphorodithioate, alkylphosphonates, alkylamines, hydroxyalkyls, ω-aminoalkyls, ω-(substituted)aminoalkyls, ω-phosphoalkyls, ω-thiophosphoalkyls, optionally substituted polyethylene glycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K), heteroaryl, heterocycle, or linker-ligand; and E is C(O)O or OC(O). Synthesis and use of this lipid family is described in WO 2010/054401 (PCTUS2009/063927 filed on Nov. 10, 2009. The cationic lipid MC3 is one embodiment of this family of cationic lipids.
  • In another embodiment, the cationic lipid MC3 ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-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. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, and U.S. Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, and U.S. patent application Ser. No. 12/813/448 filed on Jun. 10, 2010, which are hereby incorporated by reference.
  • 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][1,3]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 Nov. 10, 2009, which is herein incorporated by reference.
  • FIG. 28 illustrates the structures of ALNY-100 and MC3.
  • The cationic lipid may comprise from about 20 mol % to about 70 mol % or about 45-65 mol % or about 10, 20, 30, 40, 50, 60, or 70 mol % of the total lipid present in the particle.
  • Non-Cationic Lipids
  • 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-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof.
  • 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.
  • 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 diacylphosphatidylcholine, diacylphosphatidylethanolamine, 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., diacylphosphatidylcholine and diacylphosphatidylethanolamine). 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.
  • In one embodiment the non-cationic lipid is distearoylphosphatidylcholine (DSPC). In another embodiment the non-cationic lipid is dipalmitoylphosphatidylcholine (DPPC).
  • 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.
  • Conjugated Lipids
  • Conjugated lipids can be used in nucleic acid-lipid particle to prevent aggregation, including polyethylene glycol (PEG)-modified lipids, monosialoganglioside Gm1, and polyamide oligomers (“PAO”) such as (described in U.S. Pat. No. 6,320,017). Other compounds with uncharged, hydrophilic, steric-barrier moieties, which prevent aggregation during formulation, like PEG, Gm1 or ATTA, 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. Pat. No. 6,320,017, and PEG-lipid conjugates are described, e.g., in U.S. Pat. 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).
  • 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 U.S. Ser. No. 08/486,214, incorporated herein by reference, PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. Particularly preferred are PEG-modified diacylglycerols and dialkylglycerols.
  • 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 T1/2 less than 60 minutes in some assays. As illustrated in U.S. patent application Ser. No. 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.
  • 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.
  • The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci2), a PEG-dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Ci6), or a PEG-distearyloxypropyl (C]8). 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)propyl1-(methoxy poly(ethylene 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)propyl1-(methoxy poly(ethylene glycol)2000)propylcarbamate)) (GalNAc-PEG-DSG); and polyethylene glycol-dipalmitoylglycerol (PEG-DPG).
  • 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.
  • 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 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.
  • 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.
  • Lipoproteins
  • 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.
  • 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-IM) and ApoA-I Paris (ApoA-IP) 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 Ral1, 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.)
  • 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. Vasc. 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.
  • 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(1):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(1):80-8; Sacre et al., 2003, FEBS Lett. 540(1-3):181-7; Weers, et al., 2003, Biophys. Chem. 100(1-3):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).
  • 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(1):468-74; Powell et al., 1987, Cell 50(6):831-40; Aviram et al., 1998, Arterioscler. Thromb. Vasc. 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(1):80-8; Sorenson et al., 1999, Arterioscler. Thromb. Vasc. Biol. 19(9):2214-25; Palgunachari 1996, Arterioscler. Throb. Vasc. Biol. 16(2):328-38: Thurberg et al., J. Biol. Chem. 271(11):6062-70; Dyer 1991, J. Biol. Chem. 266(23):150009-15; Hill 1998, J. Biol. Chem. 273(47):30979-84).
  • 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).
  • 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-IM), ApoA-I Paris (ApoA-IP), 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.
  • 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, Ill., (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.
  • 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 heterogeneous mixtures of apolipoproteins can comprise, for example, a mixture of an apolipoprotein from an animal source and an apolipoprotein from a semi-synthetic source. In certain embodiments, a heterogeneous 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.
  • 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 apolipoprotein 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
  • 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 sphingolipids. 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.
  • 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 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.
  • 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. Pat. 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, T M, Prog. Lipid Res. 42(5):439-62 (2003); and Abra, R M et al., J. Liposome Res. 12:1-3, (2002).
  • 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. Pat. No. 5,013,556; 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 Fla. (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)).
  • 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. Pat. 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
  • In one embodiment, the nucleic acid-lipid particle formulations of the invention are produced via an extrusion method or an in-line mixing method.
  • 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 U.S. Pat. No. 5,008,050; U.S. Pat. No. 4,927,637; U.S. Pat. No. 4,737,323; Biochim Biophys Acta. 1979 Oct. 19; 557(1):9-23; Biochim Biophys Acta. 1980 Oct. 2; 601(3):559-7; Biochim Biophys Acta. 1986 Jun. 13; 858(1):161-8; and Biochim. Biophys. Acta 1985 812, 55-65, which are hereby incorporated by reference in their entirety.
  • 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 U.S. Pat. No. 6,534,018 and U.S. Pat. No. 6,855,277; US publication 2007/0042031 and Pharmaceuticals Research, Vol. 22, No. 3, March 2005, p. 362-372, which are hereby incorporated by reference in their entirety.
  • 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
  • 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-X100. 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%.
  • 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
  • One example of synthesis of a nucleic acid-lipid particle is as follows. Nucleic acid-lipid particles are synthesized using the lipidoid ND98.4HCl (MW 1487) (Formula 1), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids). This nucleic acid-lipid particle is sometimes referred to as a LNP01 particle. Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous 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.
  • Figure US20140121263A1-20140501-C00003
  • LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.
  • Additional exemplary nucleic acid-lipid particle formulations are described in the following table. 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.
  • cationic lipid/non-cationic lipid/cholesterol/
    PEG-lipid conjugate mol % ratio
    Name Lipid:siRNA ratio
    SNALP DLinDMA/DPPC/Cholesterol/PEG-cDMA
    (57.1/7.1/34.4/1.4)
    lipid:siRNA ~7:1
    LNP-S-X XTC/DPPC/Cholesterol/PEG-cDMA
    57.1/7.1/34.4/1.4
    lipid:siRNA ~7:1
    LNP05 XTC/DSPC/Cholesterol/PEG-DMG
    57.5/7.5/31.5/3.5
    lipid:siRNA ~6:1
    LNP06 XTC/DSPC/Cholesterol/PEG-DMG
    57.5/7.5/31.5/3.5
    lipid:siRNA ~11:1
    LNP07 XTC/DSPC/Cholesterol/PEG-DMG
    60/7.5/31/1.5,
    lipid:siRNA ~6:1
    LNP08 XTC/DSPC/Cholesterol/PEG-DMG
    60/7.5/31/1.5,
    lipid:siRNA ~11:1
    LNP09 XTC/DSPC/Cholesterol/PEG-DMG
    50/10/38.5/1.5
    lipid:siRNA ~10:1
    LNP10 ALNY-100/DSPC/Cholesterol/PEG-DMG
    50/10/38.5/1.5
    lipid:siRNA ~10:1
    LNP11 MC3/DSPC/Cholesterol/PEG-DMG
    50/10/38.5/1.5
    lipid:siRNA ~10:1
    LNP13 XTC/DSPC/Cholesterol/PEG-DMG
    50/10/38.5/1.5
    lipid:siRNA ~33:1
    LNP14 MC3/DSPC/Cholesterol/PEG-DMG
    40/15/40/5
    lipid:siRNA ~11:1
    LNP15 MC3/DSPC/Cholesterol/PEG-DSG/GalNAc-PEG-DSG
    50/10/35/4.5/0.5
    lipid:siRNA ~11:1
    LNP16 MC3/DSPC/Cholesterol/PEG-DMG
    50/10/38.5/1.5
    lipid:siRNA ~7:1
    LNP17 MC3/DSPC/Cholesterol/PEG-DSG
    50/10/38.5/1.5
    lipid:siRNA ~10:1
    LNP18 MC3/DSPC/Cholesterol/PEG-DMG
    50/10/38.5/1.5
    lipid:siRNA ~12:1
    LNP19 MC3/DSPC/Cholesterol/PEG-DMG
    50/10/35/5
    lipid:siRNA ~8:1
    LNP20 MC3/DSPC/Cholesterol/PEG-DPG
    50/10/38.5/1.5
    lipid:siRNA ~10:1
    LNP22 XTC/DSPC/Cholesterol/PEG-DSG
    50/10/38.5/1.5
    lipid:siRNA ~10:1
  • XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, which is hereby incorporated by reference.
  • MC3 comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, and U.S. Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, which are hereby incorporated by reference.
  • ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.
  • Additional representative formulations delineated in Tables 11 and 12. Lipid refers to a cationic lipid.
  • TABLE 11
    Composition of exemplary nucleic acid-lipid particle (mole %)
    prepared via extrusion methods.
    Lipid
    (mol %) DSPC (mol %) Chol (mol %) PEG (mol %) Lipid/siRNA
    20 30 40 10 2.13
    20 30 40 10 2.35
    20 30 40 10 2.37
    20 30 40 10 3.23
    20 30 40 10 3.91
    30 20 40 10 2.89
    30 20 40 10 3.34
    30 20 40 10 3.34
    30 20 40 10 4.10
    30 20 40 10 5.64
    40 10 40 10 3.02
    40 10 40 10 3.35
    40 10 40 10 3.74
    40 10 40 10 5.80
    40 10 40 10 8.00
    45 5 40 10 3.27
    45 5 40 10 3.30
    45 5 40 10 4.45
    45 5 40 10 7.00
    45 5 40 10 9.80
    50 0 40 10 27.03
    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
    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 (DOPC) 35 5 4.52
    45 15 (POPC) 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
    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
    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 12
    Composition of exemplary nucleic acid-lipid particles prepared via in-
    line mixing
    DSPC Chol
    Lipid (mol %) (mol %) (mol %) PEG (mol %) Lipid A/siRNA
    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 25 10 6.79
    60 5 32.5 2.5 3.96
    60 5 34 1 3.75
    60 5 34.5 0.5 3.28
    50 5 40 5 3.96
    60 5 30 5 4.75
    70 5 20 5 5.00
    80 5 10 5 5.18
    60 5 30 5 13.60
    60 5 30 5 14.51
    60 5 30 5 6.20
    60 5 30 5 4.60
    60 5 30 5 6.20
    60 5 30 5 5.82
    40 5 54 1 3.39
    40 7.5 51.5 1 3.39
    40 10 49 1 3.39
    50 5 44 1 3.39
    50 7.5 41.5 1 3.43
    50 10 39 1 3.35
    60 5 34 1 3.52
    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
  • Synthesis of Cationic Lipids.
  • Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles of the invention may be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. All substituents are as defined below unless indicated otherwise.
  • “Alkyl” means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.
  • “Alkenyl” means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.
  • “Alkynyl” means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.
  • “Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acyl groups.
  • “Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
  • The terms “optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted acyl”, and “optionally substituted heterocycle” means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (═O) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, —CN, —ORx, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy.
  • “Halogen” means fluoro, chloro, bromo and iodo.
  • In some embodiments, the methods of the invention may require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, T. W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an “alcohol protecting group” is used. An “alcohol protecting group” is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.
  • Synthesis of MC3
  • Preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions were dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue was passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed, yielding a colorless oil (0.54 g). Further description is provided in WO 2010/054401 (PCTUS2009/063927 filed on Nov. 10, 2009 and U.S. patent application Ser. No. 12/813/448 filed on Jun. 10, 2010.
  • Synthesis of Formula A
  • In one embodiment, nucleic acid-lipid particles of the invention are formulated using a cationic lipid of formula A:
  • Figure US20140121263A1-20140501-C00004
  • where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, the lipid of formula A above may be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.
  • Figure US20140121263A1-20140501-C00005
  • Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.
  • Figure US20140121263A1-20140501-C00006
  • Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.
  • Synthesis of ALNY-100
  • Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3:
  • Figure US20140121263A1-20140501-C00007
  • Synthesis of 515:
  • To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 0° C. and quenched with careful addition of saturated Na2SO4 solution. Reaction mixture was stirred for 4 h at room temperature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).
  • Synthesis of 516:
  • To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with 1N HCl solution (1×100 mL) and saturated NaHCO3 solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m, 2H). LC-MS [M+H] −232.3 (96.94%).
  • Synthesis of 517A and 517B:
  • The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (˜3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2×100 mL) followed by saturated NaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (1×50 mL). Organic phase was dried over an.Na2SO4 and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield: −6 g crude
  • 517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS−[M+H]−266.3, [M+NH4+]−283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.
  • Synthesis of 518:
  • Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.
  • General Procedure for the Synthesis of Compound 519:
  • A solution of compound 518 (1 eq) in hexane (15 mL) was added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40 C over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR=130.2, 130.1 (×2), 127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc. 654.6, Found 654.6.
  • Therapeutic Agent-Lipid Particle Compositions and Formulations
  • 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.
  • “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, Calif.). 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.
  • 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.
  • 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.
  • 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.
  • 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, VP16, 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
  • The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
  • Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
  • Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (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).
  • 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.
  • Large varieties of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
  • Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
  • Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture has 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.
  • 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).
  • 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.
  • Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
  • Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (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.
  • 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
  • In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly dsRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
  • Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.
  • Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of 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).
  • Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C1-10 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).
  • 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).
  • 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).
  • 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 includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).
  • Agents that enhance uptake of dsRNAs 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.
  • 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
  • dsRNAs of the present invention can be formulated in a pharmaceutically acceptable carrier or diluent. A “pharmaceutically acceptable carrier” (also referred to herein as an “excipient”) is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle. Pharmaceutically acceptable carriers can be liquid or solid, and can be selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other pertinent transport and chemical properties. Typical pharmaceutically acceptable carriers include, by way of example and not limitation: water; saline solution; binding agents (e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, gelatin, or calcium sulfate); lubricants (e.g., starch, polyethylene glycol, or sodium acetate); disintegrates (e.g., starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).
  • 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 dsRNA 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
  • In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).
  • Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
  • Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • Other Components
  • The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
  • Combination Therapy
  • In one aspect, a composition of the invention can be used in combination therapy. The term “combination therapy” includes the administration of the subject compounds in further combination with other biologically active ingredients (such as, but not limited to, a second and different antineoplastic agent) and non-drug therapies (such as, but not limited to, surgery or radiation treatment). For instance, the compounds of the invention can be used in combination with other pharmaceutically active compounds, preferably compounds that are able to enhance the effect of the compounds of the invention. The compounds of the invention can be administered simultaneously (as a single preparation or separate preparation) or sequentially to the other drug therapy. In general, a combination therapy envisions administration of two or more drugs during a single cycle or course of therapy.
  • In one aspect of the invention, the subject compounds may be administered in combination with one or more separate agents that modulate protein kinases involved in various disease states. Examples of such kinases may include, but are not limited to: serine/threonine specific kinases, receptor tyrosine specific kinases and non-receptor tyrosine specific kinases. Serine/threonine kinases include mitogen activated protein kinases (MAPK), meiosis specific kinase (MEK), RAF and aurora kinase. Examples of receptor kinase families include epidermal growth factor receptor (EGFR) (e.g., HER2/neu, HER3, HER4, ErbB, ErbB2, ErbB3, ErbB4, Xmrk, DER, Let23); fibroblast growth factor (FGF) receptor (e.g. FGF-R1, GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF, KGF-R); hepatocyte growth/scatter factor receptor (HGFR) (e.g., MET, RON, SEA, SEX); insulin receptor (e.g. IGFI-R); Eph (e.g. CEK5, CEK8, EBK, ECK, EEK, EHK-I, EHK-2, ELK, EPH, ERK, HEK, MDK2, MDK5, SEK); AxI (e.g. Mer/Nyk, Rse); RET; and platelet-derived growth factor receptor (PDGFR) (e.g. PDGFα-R, PDGβ-R, CSF1-R/FMS, SCF-R/C-KIT, VEGF-R/FLT, NEK/FLK1, FLT3/FLK2/STK-1). Non-receptor tyrosine kinase families include, but are not limited to, BCR-ABL (e.g. p43abl, ARG); BTK (e.g. ITK/EMT, TEC); CSK, FAK, FPS, JAK, SRC, BMX, FER, CDK and SYK.
  • In another aspect of the invention, the subject compounds may be administered in combination with one or more agents that modulate non-kinase biological targets or processes. Such targets include histone deacetylases (HDAC), DNA methyltransferase (DNMT), heat shock proteins (e.g., HSP90), and proteosomes.
  • In one embodiment, subject compounds may be combined with antineoplastic agents (e.g. small molecules, monoclonal antibodies, antisense RNA, and fusion proteins) that inhibit one or more biological targets such as Zolinza, Tarceva, Iressa, Tykerb, Gleevec, Sutent, Sprycel, Nexavar, Sorafenib, CNF2024, RG108, BMS387032, Affmitak, Avastin, Herceptin, Erbitux, AG24322, PD325901, ZD6474, PD 184322, Obatodax, ABT737 and AEE788. Such combinations may enhance therapeutic efficacy over efficacy achieved by any of the agents alone and may prevent or delay the appearance of resistant mutational variants.
  • In certain preferred embodiments, the compounds of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents encompass a wide range of therapeutic treatments in the field of oncology. These agents are administered at various stages of the disease for the purposes of shrinking tumors, destroying remaining cancer cells left over after surgery, inducing remission, maintaining remission and/or alleviating symptoms relating to the cancer or its treatment. Examples of such agents include, but are not limited to, alkylating agents such as mustard gas derivatives (Mechlorethamine, cylophosphamide, chlorambucil, melphalan, ifosfamide), ethylenimines (thiotepa, hexamethylmelanine), Alkylsulfonates (Busulfan), Hydrazines and Triazines (Altretamine, Procarbazine, Dacarbazine and Temozolomide), Nitrosoureas (Carmustine, Lomustine and Streptozocin), Ifosfamide and metal salts (Carboplatin, Cisplatin, and Oxaliplatin); plant alkaloids such as Podophyllotoxins (Etoposide and Tenisopide), Taxanes (Paclitaxel and Docetaxel), Vinca alkaloids (Vincristine, Vinblastine, Vindesine and Vinorelbine), and Camptothecan analogs (Irinotecan and Topotecan); anti-tumor antibiotics such as Chromomycins (Dactinomycin and Plicamycin), Anthracyclines (Doxorubicin, Daunorubicin, Epirubicin, Mitoxantrone, Valrubicin and Idarubicin), and miscellaneous antibiotics such as Mitomycin, Actinomycin and Bleomycin; anti-metabolites such as folic acid antagonists (Methotrexate, Pemetrexed, Raltitrexed, Aminopterin), pyrimidine antagonists (5-Fluorouracil, Floxuridine, Cytarabine, Capecitabine, and Gemcitabine), purine antagonists (6-Mercaptopurine and 6-Thioguanine) and adenosine deaminase inhibitors (Cladribine, Fludarabine, Mercaptopurine, Clofarabine, Thioguanine, Nelarabine and Pentostatin); topoisomerase inhibitors such as topoisomerase I inhibitors (Ironotecan, topotecan) and topoisomerase II inhibitors (Amsacrine, etoposide, etoposide phosphate, teniposide); monoclonal antibodies (Alemtuzumab, Gemtuzumab ozogamicin, Rituximab, Trastuzumab, Ibritumomab Tioxetan, Cetuximab, Panitumumab, Tositumomab, Bevacizumab); and miscellaneous anti-neoplasties such as ribonucleotide reductase inhibitors (Hydroxyurea); adrenocortical steroid inhibitor (Mitotane); enzymes (Asparaginase and Pegaspargase); anti-microtubule agents (Estramustine); and retinoids (Bexarotene, Isotretinoin, Tretinoin (ATRA). In certain preferred embodiments, the compounds of the invention are administered in combination with a chemoprotective agent. Chemoprotective agents act to protect the body or minimize the side effects of chemotherapy. Examples of such agents include, but are not limited to, amfostine, mesna, and dexrazoxane.
  • In one aspect of the invention, the subject compounds are administered in combination with radiation therapy. Radiation is commonly delivered internally (implantation of radioactive material near cancer site) or externally from a machine that employs photon (x-ray or gamma-ray) or particle radiation. Where the combination therapy further comprises radiation treatment, the radiation treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and radiation treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the radiation treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.
  • It will be appreciated that compounds of the invention can be used in combination with an immunotherapeutic agent. One form of immunotherapy is the generation of an active systemic tumor-specific immune response of host origin by administering a vaccine composition at a site distant from the tumor. Various types of vaccines have been proposed, including isolated tumor-antigen vaccines and anti-idiotype vaccines. Another approach is to use tumor cells from the subject to be treated, or a derivative of such cells (reviewed by Schirrmacher et al. (1995) J. Cancer Res. Clin. Oncol. 121:487). In U.S. Pat. No. 5,484,596, Hanna Jr. et al. claim a method for treating a resectable carcinoma to prevent recurrence or metastases, comprising surgically removing the tumor, dispersing the cells with collagenase, irradiating the cells, and vaccinating the patient with at least three consecutive doses of about 107 cells.
  • It will be appreciated that the compounds of the invention may advantageously be used in conjunction with one or more adjunctive therapeutic agents. Examples of suitable agents for adjunctive therapy include steroids, such as corticosteroids (amcinonide, betamethasone, betamethasone dipropionate, betamethasone valerate, budesonide, clobetasol, clobetasol acetate, clobetasol butyrate, clobetasol 17-propionate, cortisone, deflazacort, desoximetasone, diflucortolone valerate, dexamethasone, dexamethasone sodium phosphate, desonide, furoate, fluocinonide, fluocinolone acetonide, halcinonide, hydrocortisone, hydrocortisone butyrate, hydrocortisone sodium succinate, hydrocortisone valerate, methyl prednisolone, mometasone, prednicarbate, prednisolone, triamcinolone, triamcinolone acetonide, and halobetasol proprionate); a 5HTi agonist, such as a triptan (e.g. sumatriptan or naratriptan); an adenosine A1 agonist; an EP ligand; an NMDA modulator, such as a glycine antagonist; a sodium channel blocker (e.g. lamotrigine); a substance P antagonist (e.g. an NKi antagonist); a cannabinoid; acetaminophen or phenacetin; a 5-lipoxygenase inhibitor; a leukotriene receptor antagonist; a DMARD (e.g. methotrexate); gabapentin and related compounds; a tricyclic antidepressant (e.g. amitryptilline); a neurone stabilizing antiepileptic drug; a mono-aminergic uptake inhibitor (e.g. venlafaxine); a matrix metalloproteinase inhibitor; a nitric oxide synthase (NOS) inhibitor, such as an iNOS or an nNOS inhibitor; an inhibitor of the release, or action, of tumour necrosis factor α; an antibody therapy, such as a monoclonal antibody therapy; an antiviral agent, such as a nucleoside inhibitor (e.g. lamivudine) or an immune system modulator (e.g. interferon); an opioid analgesic; a local anaesthetic; a stimulant, including caffeine; an H2-antagonist (e.g. ranitidine); a proton pump inhibitor (e.g. omeprazole); an antacid (e.g. aluminium or magnesium hydroxide; an antiflatulent (e.g. simethicone); a decongestant (e.g. phenylephrine, phenylpropanolamine, pseudoephedrine, oxymetazoline, epinephrine, naphazoline, xylometazoline, propylhexedrine, or levo-desoxyephedrine); an antitussive (e.g. codeine, hydrocodone, carmiphen, carbetapentane, or dextramethorphan); a diuretic; or a sedating or non-sedating antihistamine.
  • The compounds of the invention can be co-administered with siRNA that target other genes. For example, a compound of the invention can be co-administered with an siRNA targeted to a c-Myc gene. In one example, AD-12115 can be co-administered with a c-Myc siRNA. Examples of c-Myc targeted siRNAs are disclosed in U.S. patent application Ser. No. 12/373,039 which is herein incorporated by reference.
  • Methods of Preparing Lipid Particles
  • 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 to produce an intermediate mixture containing nucleic acid encapsulated in lipid particles wherein the encapsulated nucleic acids are present in a nucleic acid/lipid 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-nucleic acid particles, thus providing an at least partially surface-neutralized lipid-encapsulated nucleic acid composition.
  • As described above, several of these cationic lipids are amino lipids that are charged at a pH below the pKa of the amino group and substantially neutral at a pH above the pKa. 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. In this manner, the vesicles will encapsulate and entrap the nucleic acids. Second, the surface charge of the newly formed vesicles can be neutralized by increasing the pH of the medium to a level above the pKa 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 a resultant nucleic acid 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 nucleic acid-lipid particles are provided in U.S. Pat. No. 6,287,591 and U.S. Pat. No. 6,858,225, incorporated herein by reference.
  • It is further noted that the vesicles formed in this manner provide formulations of uniform vesicle size with high content of nucleic acids. Additionally, the vesicles have a size range of from about 30 to about 150 nm, more preferably about 30 to about 90 nm.
  • 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., U.S. Pat. No. 6,287,591 and U.S. Pat. No. 6,858,225).
  • In view of the above, the present invention provides methods of preparing lipid/nucleic acid formulations. In the methods described herein, a mixture of lipids is combined with a buffered aqueous solution of nucleic acid to produce an intermediate mixture containing nucleic acid encapsulated in lipid particles, e.g., wherein the encapsulated nucleic acids are present in a nucleic acid/lipid ratio of about 10 wt % to about 20 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-nucleic acid particles, thus providing an at least partially surface-neutralized lipid-encapsulated nucleic acid composition.
  • 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-nucleic acid particle formation. In particular embodiments, the amino lipid is a novel cationic lipid of the present invention.
  • In preparing the nucleic acid-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 U.S. Pat. No. 5,976,567).
  • In accordance with the invention, the lipid mixture is combined with a buffered aqueous solution that may contain the nucleic acids. The buffered aqueous solution of is typically a solution in which the buffer has a pH of less than the pKa 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., U.S. Pat. No. 6,287,591 and U.S. Pat. No. 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 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.
  • The mixture of lipids and the buffered aqueous solution of therapeutic nucleic acids is combined to provide an intermediate mixture. The intermediate mixture is typically a mixture of lipid particles having encapsulated nucleic acids. Additionally, the intermediate mixture may also contain some portion of nucleic acids 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 are 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.
  • 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) 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.
  • 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.
  • 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-nucleic acid compositions. By at least partially neutralizing the surface charges, unencapsulated nucleic acid is freed from the lipid particle surface and can be removed from the composition using conventional techniques. Preferably, unencapsulated and surface adsorbed nucleic acids 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 release from the surface. The released nucleic acid 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.
  • 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. As described above, the aqueous buffer should be of a pH below the pKa of the amino lipid. A solution of the nucleic acids can then be added to these sized, preformed vesicles. To allow encapsulation of nucleic acids 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. 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 in the lipid vesicles will require manipulation of variable such as ethanol concentration and temperature. Examples of suitable conditions for nucleic acid encapsulation are provided in the Examples. Once the nucleic acids 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 can then be removed as described above.
  • Methods for Inhibiting Expression of the PCSK9 Gene
  • In yet another aspect, the invention provides a method for inhibiting the expression of the PCSK9 gene in a mammal. The method includes 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.
  • 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 1b, Table 2b, and Table 5b provide a wide range of values for inhibition of expression obtained in an in vitro assay using various PCSK9 dsRNA molecules at various concentrations.
  • The effect of the decreased target PCSK9 gene preferably results in a decrease in LDLc (low density lipoprotein cholesterol) levels in the blood, and more particularly in the serum, of the mammal. In some embodiments, LDLc levels are decreased by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as compared to pretreatment levels.
  • The method includes administering a composition containing a dsRNA, where the dsRNA has a nucleotide sequence that is complementary to at least a part of an RNA transcript of the PCSK9 gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, and airway (aerosol) administration. In some embodiments, the compositions are administered by intravenous infusion or injection.
  • The methods and compositions described herein can be used to treat diseases and conditions that can be modulated by down regulating PCSK9 gene expression. For example, the compositions described herein can be used to treat hyperlipidemia and other forms of lipid imbalance such as hypercholesterolemia, hypertriglyceridemia and the pathological conditions associated with these disorders such as heart and circulatory diseases. 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.
  • In one aspect, the invention provides a method of inhibiting the expression of the PCSK9 gene in a subject, e.g., a human. The method includes administering a first single 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 optionally, administering a second single dose of dsRNA, wherein the second single dose is administered at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days after the first single dose is administered, thereby inhibiting the expression of the PCSK9 gene in a subject.
  • In one embodiment, doses of dsRNA are administered not more than once every four weeks, not more than once every three weeks, not more than once every two weeks, or not more than once every week. In another embodiment, the administrations can be maintained for one, two, three, or six months, or one year or longer.
  • In another embodiment, administration can be provided when Low Density Lipoprotein cholesterol (LDLc) levels reach or surpass a predetermined minimal level, such as greater than 70 mg/dL, 130 mg/dL, 150 mg/dL, 200 mg/dL, 300 mg/dL, or 400 mg/dL.
  • In 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.
  • 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.
  • In one aspect, the invention provides a method for treating, preventing or managing a disorder, pathological process or symptom, which, for example, can be mediated by down regulating PCSK9 gene expression in a subject, such as a human subject. In one embodiment, the disorder is hyperlipidemia. The method includes administering a first single 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 optionally, administering a second single dose of dsRNA, wherein the second single dose is administered at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days after the first single dose is administered, thereby inhibiting the expression of the PCSK9 gene in a subject.
  • In 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 HMG-CoA reductase inhibitor (e.g., a statin), a fibrate, a bile acid sequestrant, niacin, an antiplatelet agent, an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist (e.g., losartan potassium, such as Merck & Co.'s Cozaar®), an acylCoA cholesterol acetyltransferase (ACAT) inhibitor, a cholesterol absorption inhibitor, a cholesterol ester transfer protein (CETP) inhibitor, a microsomal triglyceride transfer protein (MTTP) inhibitor, a cholesterol modulator, a bile acid modulator, a peroxisome proliferation activated receptor (PPAR) agonist, a gene-based therapy, a composite vascular protectant (e.g., AGI-1067, from Atherogenics), a glycoprotein IIb/IIIa inhibitor, aspirin or an aspirin-like compound, an IBAT inhibitor (e.g., S-8921, from Shionogi), a squalene synthase inhibitor, or a monocyte chemoattractant protein (MCP)-I inhibitor. Exemplary HMG-CoA reductase inhibitors include atorvastatin (Pfizer's Lipitor®/Tahor/Sortis/Torvast/Cardyl), pravastatin (Bristol-Myers Squibb's Pravachol, Sankyo's Mevalotin/Sanaprav), simvastatin (Merck's Zocor®/Sinvacor, Boehringer Ingelheim's Denan, Banyu's Lipovas), lovastatin (Merck's Mevacor/Mevinacor, Bexal's Lovastatina, Cepa; Schwarz Pharma's Liposcler), fluvastatin (Novartis' Lescol®/Locol/Lochol, Fujisawa's Cranoc, Solvay's Digaril), cerivastatin (Bayer's Lipobay/GlaxoSmithKline's Baycol), rosuvastatin (AstraZeneca's Crestor®), and pitivastatin (itavastatin/risivastatin) (Nissan Chemical, Kowa Kogyo, Sankyo, and Novartis). Exemplary fibrates include, e.g., bezafibrate (e.g., Roche's Befizal®/Cedur®/Bezalip®, Kissei's Bezatol), clofibrate (e.g., Wyeth's Atromid-S®), fenofibrate (e.g., Fournier's Lipidil/Lipantil, Abbott's Tricor®, Takeda's Lipantil, generics), gemfibrozil (e.g., Pfizer's Lopid/Lipur) and ciprofibrate (Sanofi-Synthelabo's Modalim®). Exemplary bile acid sequestrants include, e.g., cholestyramine (Bristol-Myers Squibb's Questran® and Questran Light™), colestipol (e.g., Pharmacia's Colestid), and colesevelam (Genzyme/Sankyo's WelChol™). Exemplary niacin therapies include, e.g., immediate release formulations, such as Aventis' Nicobid, Upsher-Smith's Niacor, Aventis' Nicolar, and Sanwakagaku's Perycit. Niacin extended release formulations include, e.g., Kos Pharmaceuticals' Niaspan and Upsher-Smith's SIo-Niacin. Exemplary antiplatelet agents include, e.g., aspirin (e.g., Bayer's aspirin), clopidogrel (Sanofi-Synthelabo/Bristol-Myers Squibb's Plavix), and ticlopidine (e.g., Sanofi-Synthelabo's Ticlid and Daiichi's Panaldine). Other aspirin-like compounds useful in combination with a dsRNA targeting PCSK9 include, e.g., Asacard (slow-release aspirin, by Pharmacia) and Pamicogrel (Kanebo/Angelini Ricerche/CEPA). Exemplary angiotensin-converting enzyme inhibitors include, e.g., ramipril (e.g., Aventis' Altace) and enalapril (e.g., Merck & Co.'s Vasotec). Exemplary acyl CoA cholesterol acetyltransferase (ACAT) inhibitors include, e.g., avasimibe (Pfizer), eflucimibe (BioM{acute over (ε)}rieux Pierre Fabre/Eli Lilly), CS-505 (Sankyo and Kyoto), and SMP-797 (Sumito). Exemplary cholesterol absorption inhibitors include, e.g., ezetimibe (Merck/Schering-Plough Pharmaceuticals Zetia®) and Pamaqueside (Pfizer). Exemplary CETP inhibitors include, e.g., Torcetrapib (also called CP-529414, Pfizer), JTT-705 (Japan Tobacco), and CETi-I (Avant Immunotherapeutics). Exemplary microsomal triglyceride transfer protein (MTTP) inhibitors include, e.g., implitapide (Bayer), R-103757 (Janssen), and CP-346086 (Pfizer). Other exemplary cholesterol modulators include, e.g., NO-1886 (Otsuka/TAP Pharmaceutical), CI-1027 (Pfizer), and WAY-135433 (Wyeth-Ayerst). Exemplary bile acid modulators include, e.g., HBS-107 (Hisamitsu/Banyu), Btg-511 (British Technology Group), BARI-1453 (Aventis), S-8921 (Shionogi), SD-5613 (Pfizer), and AZD-7806 (AstraZeneca). Exemplary peroxisome proliferation activated receptor (PPAR) agonists include, e.g., tesaglitazar (AZ-242) (AstraZeneca), Netoglitazone (MCC-555) (Mitsubishi/Johnson & Johnson), GW-409544 (Ligand Pharmaceuticals/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), ApoA1 (UCB Pharma/Groupe Fournier), EG-004 (Trinam) (Ark Therapeutics), and ATP-binding cassette transporter-A1 (ABCA1) (CV Therapeutics/Incyte, Aventis, Xenon). Exemplary Glycoprotein Ilb/IIIa inhibitors include, e.g., roxifiban (also called DMP754, Bristol-Myers Squibb), Gantofiban (Merck KGaA/Yamanouchi), and Cromafiban (Millennium Pharmaceuticals). Exemplary squalene synthase inhibitors include, e.g., BMS-1884941 (Bristol-Myers Squibb), CP-210172 (Pfizer), CP-295697 (Pfizer), CP-294838 (Pfizer), and TAK-475 (Takeda). An exemplary MCP-I inhibitor is, e.g., RS-504393 (Roche Bioscience). The anti-atherosclerotic agent BO-653 (Chugai Pharmaceuticals), and the nicotinic acid derivative Nyclin (Yamanouchi Pharmaceuticals) are also appropriate for administering in combination with a dsRNA featured in the invention. Exemplary combination therapies suitable for administration with a dsRNA targeting PCSK9 include, e.g., advicor (Niacin/lovastatin from Kos Pharmaceuticals), amlodipine/atorvastatin (Pfizer), and ezetimibe/simvastatin (e.g., Vytorin® 10/10, 10/20, 10/40, and 10/80 tablets by Merck/Schering-Plough Pharmaceuticals). Agents for treating hypercholesterolemia, and suitable for administration in combination with a dsRNA targeting PCSK9 include, e.g., lovastatin, niacin Altoprev® Extended-Release Tablets (Andrx Labs), lovastatin Caduet® Tablets (Pfizer), amlodipine besylate, atorvastatin calcium Crestor® Tablets (AstraZeneca), rosuvastatin calcium Lescol® Capsules (Novartis), fluvastatin sodium Lescol® (Reliant, Novartis), fluvastatin sodium Lipitor® Tablets (Parke-Davis), atorvastatin calcium Lofibra® Capsules (Gate), Niaspan Extended-Release Tablets (Kos), niacin Pravachol Tablets (Bristol-Myers Squibb), pravastatin sodium TriCor® Tablets (Abbott), fenofibrate Vytorin® 10/10 Tablets (Merck/Schering-Plough Pharmaceuticals), ezetimibe, simvastatin WelChol™ Tablets (Sankyo), colesevelam hydrochloride Zetia® Tablets (Schering), ezetimibe Zetia® Tablets (Merck/Schering-Plough Pharmaceuticals), and ezetimibe Zocor® Tablets (Merck).
  • In one embodiment, a dsRNA targeting PCSK9 is administered in combination with an ezetimibe/simvastatin combination (e.g., Vytorin® (Merck/Schering-Plough Pharmaceuticals)).
  • 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.
  • 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 described herein. The method includes, optionally, providing the end user with one or more doses of the dsRNA, and instructing the end user to administer the dsRNA on a regimen described herein, thereby instructing the end user.
  • 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 dsRNA targeting PCSK9 in an amount sufficient to lower the patient's LDL levels or ApoB levels, e.g., without substantially lowering HDL levels.
  • 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 dsRNA 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 dsRNA does not affect the level of HDL cholesterol in the patient.
  • Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
    • EXAMPLES Example 1 Gene Walking of the PCSK9 Gene
  • siRNA design was carried out to identify in two separate selections
  • a) siRNAs targeting PCSK9 human and either mouse or rat mRNA and
  • b) all human reactive siRNAs with predicted specificity to the target gene PCSK9.
  • mRNA sequences to human, mouse and rat PCSK9 were used: Human sequence NM—174936.2 was used as reference sequence during the complete siRNA selection procedure.
  • 19 mer stretches conserved in human and mouse, and human and rat PCSK9 mRNA sequences were identified in the first step, resulting in the selection of siRNAs cross-reactive to human and mouse, and siRNAs cross-reactive to human and rat targets
  • SiRNAs specifically targeting human PCSK9 were identified in a second selection. All potential 19mer sequences of human PCSK9 were extracted and defined as candidate target sequences. Sequences cross-reactive to human, monkey, and those cross-reactive to mouse, rat, human and monkey are all listed in Tables 1a and 2a. Chemically modified versions of those sequences and their activity in both in vitro and in vivo assays are also listed in Tables 1a and 2a. The data is described in the examples and in FIGS. 2-8.
  • In order to rank candidate target sequences and their corresponding siRNAs and select appropriate ones, their predicted potential for interacting with irrelevant targets (off-target potential) was taken as a ranking parameter. siRNAs with low off-target potential were defined as preferable and assumed to be more specific in vivo.
  • For predicting siRNA-specific off-target potential, the following assumptions were made:
  • 1) positions 2 to 9 (counting 5′ to 3′) of a strand (seed region) may contribute more to off-target potential than rest of sequence (non-seed and cleavage site region)
  • 2) positions 10 and 11 (counting 5′ to 3′) of a strand (cleavage site region) may contribute more to off-target potential than non-seed region
  • 3) positions 1 and 19 of each strand are not relevant for off-target interactions
  • 4) an off-target score can be calculated for each gene and each strand, based on complementarity of siRNA strand sequence to the gene's sequence and position of mismatches
  • 5) number of predicted off-targets as well as highest off-target score must be considered for off-target potential
  • 6) off-target scores are to be considered more relevant for off-target potential than numbers of off-targets
  • 7) assuming potential abortion of sense strand activity by internal modifications introduced, only off-target potential of antisense strand will be relevant To identify potential off-target genes, 19mer candidate sequences were subjected to a homology search against publically available human mRNA sequences.
  • The following off-target properties for each 19mer input sequence were extracted for each off-target gene to calculate the off-target score:
  • Number of mismatches in non-seed region
  • Number of mismatches in seed region
  • Number of mismatches in cleavage site region
  • The off-target score was calculated for considering assumption 1 to 3 as follows:

  • Off-target score=number of seed mismatches*10+number of cleavage site mismatches*1.2+number of non-seed mismatches*1
  • The most relevant off-target gene for each siRNA corresponding to the input 19mer sequence was defined as the gene with the lowest off-target score. Accordingly, the lowest off-target score was defined as the relevant off-target score for each siRNA.
    • Example 2 dsRNA Synthesis
  • Source of Reagents
  • Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.
  • siRNA Synthesis
  • Single-stranded RNAs were produced by solid phase synthesis on a scale of 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support. RNA and RNA containing 2′-O-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and 2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).
  • Deprotection and purification of the crude oligoribonucleotides by anion exchange HPLC were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, Unterschlei
    Figure US20140121263A1-20140501-P00001
    heim, 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.
  • Conjugated siRNAs
  • For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referred to as -Chol-3), an appropriately modified solid support was used for RNA synthesis. The modified solid support was prepared as follows:
    • Diethyl-2-azabutane-1,4-dicarboxylate AA
  • Figure US20140121263A1-20140501-C00008
  • A 4.7 M aqueous solution of sodium hydroxide (50 ml) was added into a stirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g, 0.23 mole) in water (50 ml). Then, ethyl acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred at room temperature until completion of the reaction was ascertained by TLC. After 19 h the solution was partitioned with dichloromethane (3×100 ml). The organic layer was dried with anhydrous sodium sulfate, filtered and evaporated. The residue was distilled to afford AA (28.8 g, 61%).
    • 3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionic acid ethyl ester AB
  • Figure US20140121263A1-20140501-C00009
  • Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved in dichloromethane (50 ml) and cooled with ice. Diisopropylcarbodiimde (3.25 g, 3.99 ml, 25.83 mmol) was added to the solution at 0° C. It was then followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). The solution was brought to room temperature and stirred further for 6 h. Completion of the reaction was ascertained by TLC. The reaction mixture was concentrated under vacuum and ethyl acetate was added to precipitate diisopropyl urea. The suspension was filtered. The filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. The combined organic layer was dried over sodium sulfate and concentrated to give the crude product which was purified by column chromatography (50% EtOAC/Hexanes) to yield 11.87 g (88%) of AB.
    • 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC
  • Figure US20140121263A1-20140501-C00010
  • 3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidine in dimethylformamide at 0° C. The solution was continued stirring for 1 h. The reaction mixture was concentrated under vacuum, water was added to the residue, and the product was extracted with ethyl acetate. The crude product was purified by conversion into its hydrochloride salt.
    • 3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD
  • Figure US20140121263A1-20140501-C00011
  • The hydrochloride salt of 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. The suspension was cooled to 0° C. on ice. To the suspension diisopropylethylamine (3.87 g, 5.2 ml, 30 mmol) was added. To the resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) was added. The reaction mixture was stirred overnight. The reaction mixture was diluted with dichloromethane and washed with 10% hydrochloric acid. The product was purified by flash chromatography (10.3 g, 92%).
    • 1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester AE
  • Figure US20140121263A1-20140501-C00012
  • Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 ml of dry toluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) of diester AD was added slowly with stirring within 20 mins. The temperature was kept below 5° C. during the addition. The stirring was continued for 30 mins at 0° C. and 1 ml of glacial acetic acid was added, immediately followed by 4 g of NaH2PO4.H2O in 40 ml of water The resultant mixture was extracted twice with 100 ml of dichloromethane each and the combined organic extracts were washed twice with 10 ml of phosphate buffer each, dried, and evaporated to dryness. The residue was dissolved in 60 ml of toluene, cooled to 0° C. and extracted with three 50 ml portions of cold pH 9.5 carbonate buffer. The aqueous extracts were adjusted to pH 3 with phosphoric acid, and extracted with five 40 ml portions of chloroform which were combined, dried and evaporated to dryness. The residue was purified by column chromatography using 25% ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).
    • [6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AF
  • Figure US20140121263A1-20140501-C00013
  • Methanol (2 ml) was added dropwise over a period of 1 h to a refluxing mixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 ml). Stirring was continued at reflux temperature for 1 h. After cooling to room temperature, 1 N HCl (12.5 ml) was added, the mixture was extracted with ethylacetate (3×40 ml). The combined ethylacetate layer was dried over anhydrous sodium sulfate and concentrated under vacuum to yield the product which was purified by column chromatography (10% MeOH/CHCl3) (89%).
    • (6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AG
  • Figure US20140121263A1-20140501-C00014
  • Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5 ml) in vacuo. Anhydrous pyridine (10 ml) and 4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added with stirring. The reaction was carried out at room temperature overnight. The reaction was quenched by the addition of methanol. The reaction mixture was concentrated under vacuum and to the residue dichloromethane (50 ml) was added. The organic layer was washed with 1M aqueous sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residual pyridine was removed by evaporating with toluene. The crude product was purified by column chromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl3) (1.75 g, 95%).
    • Succinic acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13- dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) ester AH
  • Figure US20140121263A1-20140501-C00015
  • Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloroethane (3 ml), triethylamine (0.318 g, 0.440 ml, 3.15 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (40 ml) and washed with ice cold aqueous citric acid (5 wt %, 30 ml) and water (2×20 ml). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was used as such for the next step.
  • Cholesterol Derivatised CPG AI
  • Figure US20140121263A1-20140501-C00016
  • Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture of dichloromethane/acetonitrile (3:2, 3 ml). To that solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 ml), 2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in acetonitrile/dichloroethane (3:1, 1.25 ml) were added successively. To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using a wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) was added. The suspension was agitated for 2 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The achieved loading of the CPG was measured by taking UV measurement (37 mM/g).
  • The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamide group (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivative group (herein referred to as “5′-Chol-”) was performed as described in WO 2004/065601, except that, for the cholesteryl derivative, the oxidation step was performed using the Beaucage reagent in order to introduce a phosphorothioate linkage at the 5′-end of the nucleic acid oligomer.
  • 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 U.S. patent application Ser. No. 12/328,528, filed on Dec. 4, 2008, which is hereby incorporated by reference.
    • Example 3 PCSK9 siRNA Screening in HuH7, HepG2, HeLa and Primary Monkey Hepatocytes Discovers Highly Active Sequences
  • HuH-7 cells were obtained from JCRB Cell Bank (Japanese Collection of Research Bioresources) (Shinjuku, Japan, cat. No.: JCRB0403) Cells were cultured in Dulbecco's MEM (Biochrom AG, Berlin, Germany, cat. No. F0435) supplemented to contain 10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany, cat. No. S0115), Penicillin 100 U/ml, Streptomycin 100 μg/ml (Biochrom AG, Berlin, Germany, cat. No. A2213) and 2 mM L-Glutamin (Biochrom AG, Berlin, Germany, cat. No K0282) at 37° C. in an atmosphere with 5% CO2 in a humidified incubator (Heraeus HERAcell, Kendro Laboratory Products, Langenselbold, Germany). HepG2 and HeLa cells were obtained from American Type Culture Collection (Rockville, Md., cat. No. HB-8065) and cultured in MEM (Gibco Invitrogen, Karlsruhe, Germany, cat. No. 21090-022) supplemented to contain 10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany, cat. No. S0115), Penicillin 100 U/ml, Streptomycin 100 μg/ml (Biochrom AG, Berlin, Germany, cat. No. A2213), 1× Non Essential Amino Acids (Biochrom AG, Berlin, Germany, cat. No. K-0293), and 1 mM Sodium Pyruvate (Biochrom AG, Berlin, Germany, cat. No. L-0473) at 37° C. in an atmosphere with 5% CO2 in a humidified incubator (Heraeus HERAcell, Kendro Laboratory Products, Langenselbold, Germany).
  • For transfection with siRNA, HuH7, HepG2, or HeLa cells were seeded at a density of 2.0×104 cells/well in 96-well plates and transfected directly. Transfection of siRNA (30 nM for single dose screen) was carried out with lipofectamine 2000 (Invitrogen GmbH, Karlsruhe, Germany, cat. No. 11668-019) as described by the manufacturer.
  • 24 hours after transfection HuH7 and HepG2 cells were lysed and PCSK9 mRNA levels were quantified with the Quantigene Explore Kit (Genosprectra, Dumbarton Circle Fremont, USA, cat. No. QG-000-02) according to the protocol. PCSK9 mRNA levels were normalized to GAP-DH mRNA. For each siRNA eight individual datapoints were collected. siRNA duplexes unrelated to PCSK9 gene were used as control. The activity of a given PCSK9 specific siRNA duplex was expressed as percent PCSK9 mRNA concentration in treated cells relative to PCSK9 mRNA concentration in cells treated with the control siRNA duplex.
  • Primary cynomolgus monkey hepatocytes (cryopreserved) were obtained from In vitro Technologies, Inc. (Baltimore, Md., USA, cat No M00305) and cultured in InVitroGRO CP Medium (cat No Z99029) at 37° C. in an atmosphere with 5% CO2 in a humidified incubator.
  • For transfection with siRNA, primary cynomolgus monkey cells were seeded on Collagen coated plates (Fisher Scientific, cat. No. 08-774-5) at a density of 3.5×104 cells/well in 96-well plates and transfected directly. Transfection of siRNA (eight 2-fold dilution series starting from 30 nM) in duplicates was carried out with lipofectamine 2000 (Invitrogen GmbH, Karlsruhe, Germany, cat. No. 11668-019) as described by the manufacturer.
  • 16 hours after transfection medium was changed to fresh InVitroGRO CP Medium with Torpedo Antibiotic Mix (In vitro Technologies, Inc, cat. No Z99000) added.
  • 24 hours after medium change primary cynomolgus monkey cells were lysed and PCSK9 mRNA levels were quantified with the Quantigene Explore Kit (Genosprectra, Dumbarton Circle Fremont, USA, cat. No. QG-000-02) according to the protocol. PCSK9 mRNA levels were normalized to GAPDH mRNA. Normalized PCSK9/GAPDH ratios were then compared to PCSK9/GAPDH ratio of lipofectamine 2000 only control.
  • Tables 1b and 2b (and FIG. 6A) summarize the results and provide examples of in vitro screens in different cell lines at different doses. Silencing of PCSK9 transcript was expressed as percentage of remaining transcript at a given dose.
  • Highly active sequences are those with less than 70% transcript remaining post treatment with a given siRNA at a dose less than or equal to 100 nM. Very active sequences are those that have less than 60% of transcript remaining after treatment with a dose less than or equal to 100 nM. Active sequences are those that have less than 90% transcript remaining after treatment with a high dose (100 nM).
  • Examples of active siRNAs were also screened in vivo in mouse in lipidoid formulations as described below. Active sequences in vitro were also generally active in vivo (See FIGS. 6A and 6B and example 4).
    • Example 4 In Vivo Efficacy Screen of PCSK9 siRNAs in Mice
  • 32 PCSK9 siRNAs formulated in LNP-01 liposomes were tested in vivo in a mouse model. LNP01 is a lipidoid formulation formed from cholesterol, mPEG2000-C14 Glyceride, and dsRNA. The LNP01 formulation is useful for delivering dsRNAs to the liver.
  • Formulation Procedure
  • The lipidoid LNP-01.4HCl (MW 1487) (FIG. 1), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) were used to prepare lipid-siRNA nanoparticles. Stock solutions of each in ethanol were prepared: LNP-01, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. LNP-01, Cholesterol, and PEG-Ceramide C16 stock solutions were then combined in a 42:48:10 molar ratio. Combined lipid solution was mixed rapidly with aqueous siRNA (in sodium acetate pH 5) such that the final ethanol concentration was 35-45% and the final sodium acetate concentration was 100-300 mM. Lipid-siRNA nanoparticles formed spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture was in some cases extruded through a polycarbonate membrane (100 nm cut-off) using a thermobarrel extruder (Lipex Extruder, Northern Lipids, Inc). In other cases, the extrusion step was omitted. Ethanol removal and simultaneous buffer exchange was accomplished by either dialysis or tangential flow filtration. Buffer was exchanged to phosphate buffered saline (PBS) pH 7.2.
  • Characterization of Formulations
  • Formulations prepared by either the standard or extrusion-free method are characterized in a similar manner. Formulations are first characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles are measured by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be 20-300 nm, and ideally, 40-100 nm in size. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA is incubated with the RNA-binding dye Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, 0.5% Triton-X100. The total siRNA in the formulation is determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically >85%.
  • Bolus Dosing
  • Bolus dosing of formulated siRNAs in C57/BL6 mice (5/group, 8-10 weeks old, Charles River Laboratories, MA) was performed by tail vein injection using a 27 G needle. SiRNAs were formulated in LNP-01 (and then dialyzed against PBS) at 0.5 mg/ml concentration allowing the delivery of the 5 mg/kg dose in 10 μl/g body weight. Mice were kept under an infrared lamp for approximately 3 min prior to dosing to ease injection.
  • 48 hour post dosing mice were sacrificed by CO2-asphyxiation. 0.2 ml blood was collected by retro-orbital bleeding and the liver was harvested and frozen in liquid nitrogen. Serum and livers were stored at −80° C. μl
  • Frozen livers were grinded using 6850 Freezer/Mill Cryogenic Grinder (SPEX CentriPrep, Inc) and powders stored at −80° C. until analysis.
  • PCSK9 mRNA levels were detected using the branched-DNA technology based kit from QuantiGene Reagent System (Genospectra) according to the protocol. 10-20 mg of frozen liver powders was lysed in 600 μl of 0.16 μg/ml Proteinase K (Epicentre, #MPRK092) in Tissue and Cell Lysis Solution (Epicentre, #MTC096H) at 65° C. for 3 hours. Then 10 μl of the lysates were added to 90 μl of Lysis Working Reagent (1 volume of stock Lysis Mixture in two volumes of water) and incubated at 52° C. overnight on Genospectra capture plates with probe sets specific to mouse PCSK9 and mouse GAPDH or cyclophilin B. Nucleic acid sequences for Capture Extender (CE), Label Extender (LE) and blocking (BL) probes were selected from the nucleic acid sequences of PCSK9, GAPDH and cyclophilin B with the help of the QuantiGene ProbeDesigner Software 2.0 (Genospectra, Fremont, Calif., USA, cat. No. QG-002-02). Chemo luminescence was read on a Victor2-Light (Perkin Elmer) as Relative light units. The ratio of PCSK9 mRNA to GAPDH or cyclophilin B mRNA in liver lysates was averaged over each treatment group and compared to a control group treated with PBS or a control group treated with an unrelated siRNA (blood coagulation factor VII).
  • Total serum cholesterol in mouse serum was measured using the StanBio Cholesterol LiquiColor kit (StanBio Laboratory, Boerne, Tex., USA) according to manufacturer's instructions. Measurements were taken on a Victor2 1420 Multilabel Counter (Perkin Elmer) at 495 nm.
  • Results
  • At least 10 PCSK9 siRNAs showed more than 40% PCSK9 mRNA knock down compared to a control group treated with PBS, while control group treated with an unrelated siRNA (blood coagulation factor VII) had no effect (FIGS. 2-3). Silencing of PCSK9 transcript also correlated with a lowering of total serum cholesterol in these animals (FIGS. 4-5). 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 FIGS. 6A and 6B).
  • Sequences containing different chemical modifications were also screened in vitro (Tables 1 and 2) 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); 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 (also see Tables 1 and 2) 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-0792.
  • 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:
    SEQ ID NO: 1521
    5′- GccuGGAGuuuAuucGGAAdTsdT
    Antisense strand:
    SEQ ID NO: 1522
    5′- puUCCGAAuAAACUCcAGGCdTsdT
    • Example 5 PCSK9 Duration of Action Experiments in Rats and NHP
  • Rats
  • Rats were treated via tail vein injection with 5 mg/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. 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
  • Cynomolgus monkeys were treated with LNP01 formulated dsRNA 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.
  • 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.
  • 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.
  • 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 μL. If the total sample volume collected was under 150 μL, 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.
  • 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 in Mice and Rats
  • 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-1a2 (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-1a2 (LNP01-1a2) 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.
  • 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. LNP01-1a2 displayed a clear dose response with maximal PCSK9 message suppression (˜60-70%) as compared to a control siRNA targeting luciferase (LNP01-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 Apr. 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.
  • 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. LNP01-1a2 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 LNP01-1a2, but not PBS or LNP01-ctrl control animals. (Frank-Kamanetsky et al. (2008) PNAS 105:119715-11920) This result demonstrated that the effects of LNP01-1a2 observed were due to cleavage of the PCSK9 transcript via an siRNA specific mechanism.
  • 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 5 mg/kg LNP01-1a2. As shown in FIG. 11, LNP01-1a2 treated animals had a significant (˜3-5 fold average) induction of LDLR levels 48 hours post a single dose of LNP01-1a2 compared to PBS or LNP01-ctrl control siRNA treated animals.
  • 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 al., 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. 10C, 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
  • 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-1a2 (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-1a2 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-1a2 had a T1/2 of ˜7 hours in pooled human serum. Sequences AD-1a3, AD-1a5, and AD-1a4, which were more heavily modified (see chemical modifications in Table 5) all had T 1/2's greater than 24 hours. To test whether the differences in chemical modification or stability resulted in changes in efficacy, AD-1a2, AD-1a3, AD-1a5, AD-1a4, 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 LNP01-1a2 (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 LNP01-1a2 and LNP01-3a1 Silence Human PCSK9 and Circulating Human PCSK9 Protein in Transgenic Mice
  • The efficacy of LNP01-1a2 (i.e., PCS-A2 or AD-10792) and another molecule, AD-3a1 (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 al., 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 LNP01-1a2 (a.k.a. LNP-PCS-A2) or LNP01-3a1 (a.k.a. LNP-PCS-C2), and 48 hours later both livers and blood were collected. A single dose of LNP01-1a2 or LNP01-3a1 was able to decrease the human PCSK9 transcript levels by >70% (FIG. 15A), 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
  • 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.
  • Cyno monkeys were acclimated to a twice daily feeding schedule during which food was removed after a one hour period. Animals were fed from 9-10 am in the morning, after which food was removed. The animals were next fed once again for an hour between 5 pm-6 pm with subsequent food removal. Blood was drawn after an overnight fast (6 pm until 9 am the next morning), and again, 2 and 4 hours following the 9 am 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-1a2 and another PCSK9 siRNA (AD-2a1) 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)
  • 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.
  • 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.
  • Male Cynos were first pre-screened for those that had LDLc of 40 mg/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 LNP01-1a2 and 5 mg/kg LNP01-2a1 were injected, along with PBS and 1 mg/kg LNP01-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 5 mg/kg LNP01-ctrl control siRNA, which could then serve as an additional control for the high dose groups of 5 mg/kg LNP01-1a2 and 5 mg/kg LNP01-2a1. 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 LNP01-1a2, LNP01-2a1 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).
  • As shown in FIG. 12A, a single dose of LNP01-1a2 or LNP01-2a1 resulted in a statistically significant reduction of LDLc beginning at day 3 post-dose that returned to baseline over ˜14 days (for LNP01-1a2) and ˜21 days (LNP01-2a1). This effect was not seen in either the PBS, the control siRNA groups, or the 1 mg/kg treatment groups. LNP01-2a1 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.
  • Neither LNP01-1a2 nor LNP01-2a1 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.
  • PCSK9 protein levels were also measured in treated and control animals. As shown in FIG. 11, LNP01-1a2 and LNP01-2a1 treatment each resulted in trends toward decreased circulating PCSK9 protein levels versus pre-dose. Specifically, the more active siRNA LNP01-2a1 demonstrated significant reduction of circulating PCSK9 protein versus both PBS (day 3-21) and LNP01-ctrl siRNA control (day 4, day 7).
    • Example 11 Modified siRNA and Activation of Immune Responses in hPBMCs
  • 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-1a1 was found to induce both IFN-alpha and TNF-alpha. However, chemically modified versions of this sequence (AD-1a2, AD-1a3, AD-1a5, and AD-1a4) as well as AD-2a1 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-1a2, nor AD-2a1 activated IFN-alpha when formulated into liposomes and tested in mice.
    • Example 12 Evaluation of siRNA Conjugates in Mice
  • 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.
  • 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.
  • The results are shown in FIG. 18. Compared to control PBS, both siRNA conjugates demonstrated activity with an ED50 of 3×50 mg/kg for GalNAc)3/Cholesterol conjugated AD-10792 and 3×100 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
  • 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 27 G 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 μl/g body weight. Mice were kept under an infrared lamp for approximately 3 min prior to dosing to ease injection.
  • 24 hour post last dose mice were sacrificed by CO2-asphyxiation. 0.2 ml blood was collected by retro-orbital bleeding and the liver was harvested and frozen in liquid nitrogen. Serum and livers were stored at −80° C. Frozen livers were grinded using 6850 Freezer/Mill Cryogenic Grinder (SPEX CentriPrep, Inc) and powders stored at −80° C. until analysis.
  • PCSK9 mRNA levels were detected using the branched-DNA technology based kit from QuantiGene Reagent System (Panomics, USA) according to the protocol. 10-20 mg of frozen liver powders was lysed in 600 μl of 0.16 μg/ml Proteinase K (Epicentre, #MPRK092) in Tissue and Cell Lysis Solution (Epicentre, #MTC096H) at 65° C. for 3 hours. Then 10 μl of the lysates were added to 90 μl of Lysis Working Reagent (1 volume of stock Lysis Mixture in two volumes of water) and incubated at 52° C. overnight on Genospectra capture plates with probe sets specific to mouse PCSK9 and mouse GAPDH. 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).
  • 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 Lipid Formulated siRNAs in Rats
  • 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
  • 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 27 G 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 μl/g body weight. Rats were kept under an infrared lamp for approximately 3 min prior to dosing to ease injection.
  • 72 hour post last dose rats were sacrificed by CO2-asphyxiation. 0.2 ml blood was collected by retro-orbital bleeding and the liver was harvested and frozen in liquid nitrogen. Serum and livers were stored at −80° C. Frozen livers were grinded using 6850 Freezer/Mill Cryogenic Grinder (SPEX CentriPrep, Inc) and powders stored at −80° C. until analysis.
  • PCSK9 mRNA levels were detected using the branched-DNA technology based kit from QuantiGene Reagent System (Panomics, USA) according to the protocol. 10-20 mg of frozen liver powders was lysed in 600 μl of 0.16 μg/ml Proteinase K (Epicentre, #MPRK092) in Tissue and Cell Lysis Solution (Epicentre, #MTC096H) at 65° C. for 3 hours. Then 10 μl of the lysates were added to 90 μl 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).
  • 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 in HeLa Cells of Mismatch Walk of AD-9680 and AD-14676
  • The effects of variations in sequence or modification on the effectiveness of AD-9680, AD-14676, and AD-10792 were assayed in HeLa cells. A number of variants were synthesized as shown in Table 6 and include adding DFT (2,4-Difluorotoluoyl, a thymidine triphosphate shape analog lacking Watson-Crick pairing); adding single or combination mismatches; and testing two different backbone chemistries: leading with a 2′-O methyl, or alternating with 2′F.
  • Sequences of the 3 parent duplexes can be found in Table 1a and are duplicated as follows:
  • SEQ SEQ
    target ID ID
    region Sense strand (5′ to 3′) NO: Antisense strand (5′ to 3′) NO: Duplex
    3530- uucuAGAccuGuuuuGcuuTsT 1229 AAGcAAAAcAGGUCuAGAATsT 1230 AD-
    3548 9680
    3530- UfuCfuAfgAfcCfuGfuUfuUfg 1231 p- 1232 AD-
    3548 CfuUfTsT aAfgCfaAfaAfcAfgGfuCfuAfgA 14676
    faTsT
    1091- GccuGGAGuuuAuucGGAATsT 459 UUCCGAAuAAACUCcAGGCTsT 460 AD-
    1109 10792
  • HeLa were plated in 96-well plates (8,000-10,000 cells/well) in 100 μl 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 μl of transfection reagent Lipofectamine™ 2000 (Invitrogen Corporation, Carlsbad, Calif.) 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 μl was diluted in 25 μl 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 μl of the diluted siRNA was combined with the diluted Lipofectamine™ 2000 (total volume is 26.4 μl). The complex was mixed gently and incubated for 20 minutes at room temperature to allow the siRNA: Lipofectamine™ 2000 complexes to form. Then 100 μl 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. 100 μl of the above mixture was added to each well containing the cells and the plates were incubated at 37° C. in a CO2 incubator for 24 hours, then the culture medium was removed and 100 μA 10% fetal bovine serum in DMEM was added.
  • 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.
  • 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 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. FIG. 24 is a dose response curve comparing the efficiency of parent duplexes AD-9680 and AD-10792 with modified duplexes wherein a DFT is inserted at position 10 of the sense strand. This modification improves the efficiency by about 2 fold in HeLa cells.
  • 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.
    • Example 15 Lack of Off Target Effects in Hep3B Cells at High Concentrations
  • A lipid formulated PCSK9 targeted siRNA (AD-9680) was transfected into Hep3B cells at concentrations of 250 nM, 1 uM and 5 uM in triplicates using the reagent RNAiMAX (Invitrogen) according to the manufacture's instruction: 1 ul of transfection reagent; reverse transfection protocol. Samples were collected 48 hrs post transfection. Total RNA was purified using MagMAX™-96 Total RNA Isolation Kit (Ambion); cDNA was synthesized with High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (ABI) from 13.5 μl of RNA prep; ABI Gene Expression Taqman assays were used; q-PCR reactions were set up according to manufacturer's instruction using TaqMan® Gene Expression Master Mix (ABI) and run on ABI 7900 machine. Delta delta Ct method was used to calculate values. Samples were normalized to hGAPDH and calibrated to mock transfection.
  • Transcript levels were measured for the following genes having the closest homology to the target sequence: ORMDL2, BMP6, TAPT1, MYEF2, LOC442252, RFT1, and PCSK9.
  • The results are shown in FIG. 22. No off target effects were observed at high concentrations of dsRNA (PCS-B2=AD-9680).
  • AD-9680
    S 1531 uucuAGAccuGuuuuGcuudTsdT
    AS 1532 AAGcAAAAcAGGUCuAGAAdTsdT
    • Example 16 Maintenance of Decrease in Total Cholesterol Levels in Rats by Lower Dosage of AD-10792
  • Rats were treated with 3 mg/kg bolus dose of SNALP-Dlin DMA 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 repeat dosing and total serum cholesterol levels were determined. The negative control was PBS.
  • 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.
  • 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. 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 subsequent administration AD-10792.
    • Example 17 Assay of Effects on Cholesterol Levels in Rats after Administration of Various Lipid Formulations of AD-10792
  • Rats were treated with four different lipid formulations of AD-10792 including SNALP and LNP08, described herein. At day 3, total serum cholesterol levels were determined. The experiment was performed using the methods described herein. Administration of LNP-08 formulated AD-10792 results in the lowest EC50 of 0.08 mg/kg compared to LNP01 formulated (EC50 of 2.0 mg/kg) and SNALP formulated (EC50 of 1.0 mg/kg). (data not shown).
    • Example 18 PCSK9 siRNA Tiling Experiment
  • Bioinformatic Selection of PCSK9 Tiling Set
  • Sense and antisense oligomers were designed to target the human PCSK9 transcript in the flanking regions immediately upstream and downstream of the 19 base target region of ALN-PCSK9 (AD-9680). We used the NCBI Refseq NM—174936.2 as the reference human transcript for the PCSK9 gene. The antisense oligo of AD-9680 contains 19 contiguous bases complementary to the bases in the region of NM—174936 from positions 3530 through 3548 relative to the start of the mRNA. A set of siRNA molecules was designed to each unique 19mer of the subset of the transcript sequence defined by 10 bases upstream from the 5′ end to 10 bases downstream from the 3′ end of the target region of AD-9680. With respect to the NM—174936.2 transcript, the first base at the 5′ position of the sense oligo 19mer extends from positions 3520 to positions 3558 (Tables 7 and 8).
  • Synthesis of PCSK9 Tiling Sequences:
  • PCSK9 sequences were synthesized on MerMade 192 synthesizer. Two sets of sequences were made. The first set contained no chemical modifications (unmodified) and a second set was made with endolight chemical modifications. In sequences containing endolight chemical modification, all pyrimidines (cytosine and uridine) in the sense strand were replaced with corresponding 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U). In the antisense strand, pyrimidines adjacent to (towards 5′ position) ribo A nucleoside were replaced with their corresponding 2-O-Methyl nucleosides. A two base dTsdT extension at the 3′ end of both sense and anti sense sequences was introduced. The sequence file was converted to a text file to make it compatible for loading in the MerMade 192 synthesis software.
  • The synthesis of PCSK9 sequences used solid supported oligonucleotide synthesis using phosphoramidite chemistry. The synthesis of the above sequences was performed at 1 μm scale in 96 well plates. The amidite solutions were prepared at 0.1 M concentration and ethyl thio tetrazole (0.6M in Acetonitrile) was used as activator. The synthesized sequences were cleaved and deprotected in 96 well plates, using methylamine in the first step and Fluoride ion in the second step. The crude sequences thus obtained were precipitated using acetone: ethanol mix and the pellet were re-suspended in 0.2M sodium acetate buffer. Samples from each sequence were analyzed by LC-MS and the resulting mass data confirmed the identity of the sequences. A selected set of samples were also analyzed by IEX chromatography. All sequences were purified on AKTA explorer purification system using Source 15Q column. A single peak corresponding to the full length sequence was collected in the eluent and was subsequently analyzed for purity by ion exchange chromatography. The purified sequences were desalted on a Sephadex G25 column using AKTA purifier. The desalted PCSK9 sequences were analyzed for concentration and purity. The single strands were then submitted for annealing.
  • In Vitro Screening of PCSK9 Tiling siRNAs:
  • Cell Culture and Transfection:
  • Hela cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO2 in Eagle's Minimum Essential Medium (EMEM, ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Reverse transfection was carried out by adding 5 μl of Opti-MEM to 5 μl of siRNA duplexes per well into a 96-well plate along with 10 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotic containing 2.0×104 Hela cells were then added. Cells were incubated for 24 hours prior to RNA purification. Experiments were performed at 0.1 or 10 nM final duplex concentration. For dose response screens, HeLa cells were transfected with siRNAs over seven, ten-fold serial dilutions from 1 nM to 1 fM.
  • Total RNA was isolated using MagMAX-96 Total RNA Isolation Kit (Applied Biosystem, Forer City Calif., part #: AM1830). Cells were harvested and lysed in 140 μl of Lysis/Binding Solution then mixed for 1 minute at 850 rpm using and Eppendorf Thermomixer (the mixing speed was the same throughout the process). Twenty micro liters of magnetic beads and Lysis/Binding Enhancer mixture were added into cell-lysate and mixed for 5 minutes. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, magnetic beads were washed with Wash Solution 1 (isopropanol added) and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 μl Wash Solution 2 (Ethanol added), captured and supernatant was removed. 50 μl of DNase mixture (MagMax turbo DNase Buffer and Turbo DNase) was then added to the beads and they were mixed for 10 to 15 minutes. After mixing, 100 μl of RNA Rebinding Solution was added and mixed for 3 minutes. Supernatant was removed and magnetic beads were washed again with 150 μl Wash Solution 2 and mixed for 1 minute and supernatant was removed completely. The magnetic beads were mixed for 2 minutes to dry before RNA was eluted with 50 μl of water.
  • cDNA was synthesized using ABI High capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, Calif., Cat #4368813). A master mix of 2 μl 10× Buffer, 0.8 μl 25× dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.
  • Real time PCR was performed as follows. 2 μl of cDNA were added to a master mix containing 1 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 1 μl PCSK9 TaqMan probe (Applied Biosystems cat #HS03037355_M1) and 10 μl Roche Probes Master Mix (Roche Cat #04887301001) per well in a LightCycler 480 384 well plate (Roche cat #0472974001). Real time PCR was done in a LightCycler 480 Real Time PCR machine (Roche). Each duplex was tested in two independent transfections and each transfections was assayed in duplicate.
  • Real time data were analyzed using the ΔΔ Ct method. Each sample was normalized to GAPDH expression and knockdown was assessed relative to cells transfected with the non-targeting duplex AD-1955. IC50s were defined using a 4 parameter fit model in XLfit.
  • The data for the single dose experiments are shown in Table 9. Data are expressed as the percent of message remaining relative to cells targeted with control AD-1955.
  • The data for the dose response screen is shown in Table 10. Data are expressed as dose in pM that results in 50% inhibition relative to AD-1955. Each dose response was repeated twice (Rep1 and Rep2). Average of the IC50s generated in the two dose response screens is shown.
  • The average IC50 for siRNA flanking AD-9680 was plotted vs. the starting position of the target region in the human PCSK9 transcript FIG. 25.
  • Thus, targeting nucleotide region 3520-3555 of PCSK9 with an RNAi agent is highly effective at inhibiting PCSK9.
    • Example 19 ApoE3-Based Reconstituted HDL Complexed with dsRNAs Targeting PCSK9
  • C57BL6 mice were administered 30 mg/kg rEHDL/chol-siPCSK9 by intravenous administration (tail vein injection) in a single bolus dose.
  • Chol-siPCSK9 (dsRNA Duplex AD-20583) has the following sequence:
  • Sense:
    (SEQ ID NO: 1729)
    GccuGGAGuuuAuucGGAAdTsdTL10
    Antisense:
    (SEQ ID NO: 1730)
    PuUfcCfgAfaUfaAfaCfuCfcAfgGfcdTsdT
  • The structure of L10 is:
  • Figure US20140121263A1-20140501-C00017
  • After injection, mice were fasted overnight (˜14 hours), and then sacrificed at 48 h post-injection. mRNA levels from liver were determined by bDNA assay, and normalized to GAPDH mRNA levels.
  • Results
  • The results of the bDNA assays are shown in FIG. 26, which indicate that there was a significant reduction in PCSK9 following administration of rEHDL/chol-siPCSK9, but not following administration of uncomplexed siRNAs (chol-siPCSK9). rEHDL/chol-siPCSK9 decreased PCSK9 mRNA levels by about 80%.
    • Example 20 LNP-11 Formulated siRNA in Non-Human Primates (NHPs)
  • An siRNA targeting PCSK9 (AD-9680) was formulated in a LNP-11 formulation (described herein) and administered to cynomologous monkeys. Control was AD-1955. The lipid formulated siRNAs were administered via a 30 minute infusion on day 1 at dosages of 0.03, 0.1, 0.3, and 1.0 mg/kg. Control was administered at 1.0 mg/kg. On day 3, liver biopsies were performed for measurement of PCSK9 transcript. Blood samples were collected on days −3, −1, 3, 4, 5, 7, 9, 11, 12, 15, 22, 30, and 37 and PCSK9 protein levels and LDLc numbers and HDLc numbers were determined.
  • The results are shown in FIG. 27A, FIG. 27B, and FIG. 27C.
  • As shown in FIG. 27A and FIG. 27B, administration resulted in a rapid and durable dose dependent reduction in PCSK9 protein levels and resulted in >50% reduction in LDLc (LDL cholesterol) levels. These effects were very potent with ED50 dose levels between 30 and 100 micrograms per kilogram. As shown in FIG. 27C, administration resulted in no change in HDLc levels.
    • Example 21 Dose Response in Rats with LNP-09 Formulated PCSK9 dsRNA
  • The dsRNA AD-10792 (targeting rate PCSK9) was encapsulated in a XTC containing formulation, e.g., a LNP09 formulation. LNP09 formulation was XTC/DSPC/Cholesterol/PEG-DMG at a % mol ratio of 50/10/38.5/1.5 and a lipid:siRNA ratio of 10:1.
  • Formulations were injected via tail vein, single dose (DRC) into rats. Livers and plasma were harvested 72 hours post-injection (5 animals per group). PCSK9 transcript levels were measured via bDNA in livers prepared as manufacturer's protocol. GAPDH transcript levels were also measured and the PCSK9 to GAPDH ratios were normalized to those of PBS control and graphed. Total cholesterol was measured in serum using cholesterol kit from WAKO TX.
  • The results are shown in FIG. 29. With this formulation PCSK9 silencing and total cholesterol lowering in rats was achieved at doses <0.1 mg/kg. The ED50 for was 0.2 mg/kg for lowering PCSK9 mRNA and 0.2 mg/kg and 0.08 for lowering serum cholesterol.
    • Example 22 Treatment of Transgenic Mice with LNP-09 Formulated PCSK9 dsRNA
  • Transgenic mice that overexpress human CETP and ApoB 100 (CETP/ApoB double humanized transgenic mice, Taconic Labs) more closely mimic the LDL/HDL ratios found in man.
  • CETP/ApoB double humanized transgenic mice were purchased from Taconic labs. Animals were injected through tail vein (single injection) of 5 mg/kg of LNP09 formulated AD-10792 (standard formulation procedure), or AD-1955 Luciferase control (4 animals per group). Livers and plasma were harvested 72 hours post-injection (5 animals per group) and liver PCSK9 mRNA, LDL particle, and HDL particle number were determined.
  • PCSK9 transcript levels were measured via bDNA in livers prepared according to manufacturer's protocol. GAPDH transcript levels were also measured and the PCSK9 to GAPDH ratios were graphed, normalized to those of PBS control. LDL and HDL particle numbers/concentration were measured by NMR (Liposciences Inc.) based on their SOP.
  • The results are shown in FIG. 30. Silencing of PCSK9 lowered LDL particle concentrations ˜90%, while HDL levels were modestly lower (as compared to those treated animals treated with PBS controls). This demonstrates significant lowering of PCSK9 levels with subsequent LDLc lowering in these animals.
    • Example 23 Inhibition of PCSK9 Expression in Humans
  • A human subject is treated with a lipid formulated dsRNA targeted to a PCSK9 gene, described herein, to inhibit expression of the PCSK9 gene and lower cholesterol levels for an extended period of time following a single dose. In one embodiment, the lipid formulated dsRNA includes the lipid MC3.
  • 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.
  • At time zero, a suitable first dose of an anti-PCSK9 siRNA is subcutaneously administered to the subject. The dsRNA is 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 in said subject, and/or the products of the successful siRNA-targeting of PCSK9 mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.
  • After treatment, the subject's 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.
  • Those skilled in the art are familiar with methods and compositions in addition to those specifically set out in the present disclosure which will allow them to practice this invention to the full scope of the claims hereinafter appended.
  • TABLE 1a
    dsRNA sequences targeted to PCSK9
    position
    in
    human
    access. SEQ SEQ
    # NM_ ID ID Duplex
    174936 Sense strand sequence (5'-3')1 NO: Antisense-strand sequence (5'-3')1 NO: name
       2-20 AGCGACGUCGAGGCGCUCATT 1 UGAGCGCCUCGACGUCGCUTT 2 AD-
    15220
      15-33 CGCUCAUGGUUGCAGGCGGTT 3 CCGCCUGCAACCAUGAGCGTT 4 AD-
    15275
      16-34 GCUCAUGGUUGCAGGCGGGTT 5 CCCGCCUGCAACCAUGAGCTT 6 AD-
    15301
      30-48 GCGGGCGCCGCCGUUCAGUTT 7 ACUGAACGGCGGCGCCCGCTT 8 AD-
    15276
      31-49 CGGGCGCCGCCGUUCAGUUTT 9 AACUGAACGGCGGCGCCCGTT 10 AD-
    15302
      32-50 GGGCGCCGCCGUUCAGUUCTT 11 GAACUGAACGGCGGCGCCCTT 12 AD-
    15303
      40-58 CCGUUCAGUUCAGGGUCUGTT 13 CAGACCCUGAACUGAACGGTT 14 AD-
    15221
      43-61 UUCAGUUCAGGGUCUGAGCTT 15 GCUCAGACCCUGAACUGAATT 16 AD-
    15413
      82-100 GUGAGACUGGCUCGGGCGGTT 17 CCGCCCGAGCCAGUCUCACTT 18 AD-
    15304
     100-118 GGCCGGGACGCGUCGUUGCTT 19 GCAACGACGCGUCCCGGCCTT 20 AD-
    15305
     101-119 GCCGGGACGCGUCGUUGCATT 21 UGCAACGACGCGUCCCGGCTT 22 AD-
    15306
     102-120 CCGGGACGCGUCGUUGCAGTT 23 CUGCAACGACGCGUCCCGGTT 24 AD-
    15307
     105-123 GGACGCGUCGUUGCAGCAGTT 25 CUGCUGCAACGACGCGUCCTT 26 AD-
    15277
     135-153 UCCCAGCCAGGAUUCCGCGTsT 27 CGCGGAAUCCUGGCUGGGATsT 28 AD-
    9526
     135-153 ucccAGccAGGAuuccGcGTsT 29 CGCGGAAUCCUGGCUGGGATsT 30 AD-
    9652
     136-154 CCCAGCCAGGAUUCCGCGCTsT 31 GCGCGGAAUCCUGGCUGGGTsT 32 AD-
    9519
     136-154 cccAGccAGGAuuccGcGcTsT 33 GCGCGGAAUCCUGGCUGGGTsT 34 AD-
    9645
     138-156 CAGCCAGGAUUCCGCGCGCTsT 35 GCGCGCGGAAUCCUGGCUGTsT 36 AD-
    9523
     138-156 cAGccAGGAuuccGcGcGcTsT 37 GCGCGCGGAAUCCUGGCUGTsT 38 AD-
    9649
     185-203 AGCUCCUGCACAGUCCUCCTsT 39 GGAGGACUGUGCAGGAGCUTsT 40 AD-
    9569
     185-203 AGcuccuGcAcAGuccuccTsT 41 GGAGGACUGUGcAGGAGCUTsT 42 AD-
    9695
     205-223 CACCGCAAGGCUCAAGGCGTT 43 CGCCUUGAGCCUUGCGGUGTT 44 AD-
    15222
     208-226 CGCAAGGCUCAAGGCGCCGTT 45 CGGCGCCUUGAGCCUUGCGTT 46 AD-
    15278
     210-228 CAAGGCUCAAGGCGCCGCCTT 47 GGCGGCGCCUUGAGCCUUGTT 48 AD-
    15178
     232-250 GUGGACCGCGCACGGCCUCTT 49 GAGGCCGUGCGCGGUCCACTT 50 AD-
    15308
     233-251 UGGACCGCGCACGGCCUCUTT 51 AGAGGCCGUGCGCGGUCCATT 52 AD-
    15223
     234-252 GGACCGCGCACGGCCUCUATT 53 UAGAGGCCGUGCGCGGUCCTT 54 AD-
    15309
     235-253 GACCGCGCACGGCCUCUAGTT 55 CUAGAGGCCGUGCGCGGUCTT 56 AD-
    15279
     236-254 ACCGCGCACGGCCUCUAGGTT 57 CCUAGAGGCCGUGCGCGGUTT 58 AD-
    15194
     237-255 CCGCGCACGGCCUCUAGGUTT 59 ACCUAGAGGCCGUGCGCGGTT 60 AD-
    15310
     238-256 CGCGCACGGCCUCUAGGUCTT 61 GACCUAGAGGCCGUGCGCGTT 62 AD-
    15311
     239-257 GCGCACGGCCUCUAGGUCUTT 63 AGACCUAGAGGCCGUGCGCTT 64 AD-
    15392
     240-258 CGCACGGCCUCUAGGUCUCTT 65 GAGACCUAGAGGCCGUGCGTT 66 AD-
    15312
     248-266 CUCUAGGUCUCCUCGCCAGTT 67 CUGGCGAGGAGACCUAGAGTT 68 AD-
    15313
     249-267 UCUAGGUCUCCUCGCCAGGTT 69 CCUGGCGAGGAGACCUAGATT 70 AD-
    15280
     250-268 CUAGGUCUCCUCGCCAGGATT 71 UCCUGGCGAGGAGACCUAGTT 72 AD-
    15267
     252-270 AGGUCUCCUCGCCAGGACATT 73 UGUCCUGGCGAGGAGACCUTT 74 AD-
    15314
     258-276 CCUCGCCAGGACAGCAACCTT 75 GGUUGCUGUCCUGGCGAGGTT 76 AD-
    15315
     300-318 CGUCAGCUCCAGGCGGUCCTsT 77 GGACCGCCUGGAGCUGACGTsT 78 AD-
    9624
     300-318 cGucAGcuccAGGcGGuccTsT 79 GGACCGCCUGGAGCUGACGTsT 80 AD-
    9750
     301-319 GUCAGCUCCAGGCGGUCCUTsT 81 AGGACCGCCUGGAGCUGACTsT 82 AD-
    9623
     301-319 GucAGcuccAGGcGGuccuTsT 83 AGGACCGCCUGGAGCUGACTsT 84 AD-
    9749
     370-388 GGCGCCCGUGCGCAGGAGGTT 85 CCUCCUGCGCACGGGCGCCTT 86 AD-
    15384
     408-426 GGAGCUGGUGCUAGCCUUGTsT 87 CAAGGCUAGCACCAGCUCCTsT 88 AD-
    9607
     408-426 GGAGcuGGuGcuAGccuuGTsT 89 cAAGGCuAGcACcAGCUCCTsT 90 AD-
    9733
     411-429 GCUGGUGCUAGCCUUGCGUTsT 91 ACGCAAGGCUAGCACCAGCTsT 92 AD-
    9524
     411-429 GcuGGuGcuAGccuuGcGuTsT 93 ACGcAAGGCuAGcACcAGCTsT 94 AD-
    9650
     412-430 CUGGUGCUAGCCUUGCGUUTsT 95 AACGCAAGGCUAGCACCAGTsT 96 AD-
    9520
     412-430 CUGGUGCUAGCCUUGCGUUTsT 97 AACGCAAGGCUAGCACCAGTsT 98 AD-
    9520
     412-430 cuGGuGcuAGccuuGcGuuTsT 99 AACGcAAGGCuAGcACcAGTsT 100 AD-
    9646
     416-434 UGCUAGCCUUGCGUUCCGATsT 101 UCGGAACGCAAGGCUAGCATsT 102 AD-
    9608
     416-434 uGcuAGccuuGcGuuccGATsT 103 UCGGAACGcAAGGCuAGcATsT 104 AD-
    9734
     419-437 UAGCCUUGCGUUCCGAGGATsT 105 UCCUCGGAACGCAAGGCUATsT 106 AD-
    9546
     419-437 uAGccuuGcGuuccGAGGATsT 107 UCCUCGGAACGcAAGGCuATsT 108 AD-
    9672
     439-457 GACGGCCUGGCCGAAGCACTT 109 GUGCUUCGGCCAGGCCGUCTT 110 AD-
    15385
     447-465 GGCCGAAGCACCCGAGCACTT 111 GUGCUCGGGUGCUUCGGCCTT 112 AD-
    15393
     448-466 GCCGAAGCACCCGAGCACGTT 113 CGUGCUCGGGUGCUUCGGCTT 114 AD-
    15316
     449-467 CCGAAGCACCCGAGCACGGTT 115 CCGUGCUCGGGUGCUUCGGTT 116 AD-
    15317
     458-476 CCGAGCACGGAACCACAGCTT 117 GCUGUGGUUCCGUGCUCGGTT 118 AD-
    15318
     484-502 CACCGCUGCGCCAAGGAUCTT 119 GAUCCUUGGCGCAGCGGUGTT 120 AD-
    15195
     486-504 CCGCUGCGCCAAGGAUCCGTT 121 CGGAUCCUUGGCGCAGCGGTT 122 AD-
    15224
     487-505 CGCUGCGCCAAGGAUCCGUTT 123 ACGGAUCCUUGGCGCAGCGTT 124 AD-
    15188
     489-507 CUGCGCCAAGGAUCCGUGGTT 125 CCACGGAUCCUUGGCGCAGTT 126 AD-
    15225
     500-518 AUCCGUGGAGGUUGCCUGGTT 127 CCAGGCAACCUCCACGGAUTT 128 AD-
    15281
     509-527 GGUUGCCUGGCACCUACGUTT 129 ACGUAGGUGCCAGGCAACCTT 130 AD-
    15282
     542-560 AGGAGACCCACCUCUCGCATT 131 UGCGAGAGGUGGGUCUCCUTT 132 AD-
    15319
     543-561 GGAGACCCACCUCUCGCAGTT 133 CUGCGAGAGGUGGGUCUCCTT 134 AD-
    15226
     544-562 GAGACCCACCUCUCGCAGUTT 135 ACUGCGAGAGGUGGGUCUCTT 136 AD-
    15271
     549-567 CCACCUCUCGCAGUCAGAGTT 137 CUCUGACUGCGAGAGGUGGTT 138 AD-
    15283
     552-570 CCUCUCGCAGUCAGAGCGCTT 139 GCGCUCUGACUGCGAGAGGTT 140 AD-
    15284
     553-571 CUCUCGCAGUCAGAGCGCATT 141 UGCGCUCUGACUGCGAGAGTT 142 AD-
    15189
     554-572 UCUCGCAGUCAGAGCGCACTT 143 GUGCGCUCUGACUGCGAGATT 144 AD-
    15227
     555-573 CUCGCAGUCAGAGCGCACUTsT 145 AGUGCGCUCUGACUGCGAGTsT 146 AD-
    9547
     555-573 cucGcAGucAGAGcGcAcuTsT 147 AGUGCGCUCUGACUGCGAGTsT 148 AD-
    9673
     558-576 GCAGUCAGAGCGCACUGCCTsT 149 GGCAGUGCGCUCUGACUGCTsT 150 AD-
    9548
     558-576 GcAGucAGAGcGcAcuGccTsT 151 GGcAGUGCGCUCUGACUGCTsT 152 AD-
    9674
     606-624 GGGAUACCUCACCAAGAUCTsT 153 GAUCUUGGUGAGGUAUCCCTsT 154 AD-
    9529
     606-624 GGGAuAccucAccAAGAucTsT 155 GAUCUUGGUGAGGuAUCCCTsT 156 AD-
    9655
     659-677 UGGUGAAGAUGAGUGGCGATsT 157 UCGCCACUCAUCUUCACCATsT 158 AD-
    9605
     659-677 uGGuGAAGAuGAGuGGcGATsT 159 UCGCcACUcAUCUUcACcATsT 160 AD-
    9731
     663-681 GAAGAUGAGUGGCGACCUGTsT 161 CAGGUCGCCACUCAUCUUCTsT 162 AD-
    9596
     663-681 GAAGAuGAGuGGcGAccuGTsT 163 cAGGUCGCcACUcAUCUUCTsT 164 AD-
    9722
     704-722 CCCAUGUCGACUACAUCGATsT 165 UCGAUGUAGUCGACAUGGGTsT 166 AD-
    9583
     704-722 cccAuGucGAcuAcAucGATsT 167 UCGAUGuAGUCGAcAUGGGTsT 168 AD-
    9709
     718-736 AUCGAGGAGGACUCCUCUGTsT 169 CAGAGGAGUCCUCCUCGAUTsT 170 AD-
    9579
     718-736 AucGAGGAGGAcuccucuGTsT 171 cAGAGGAGUCCUCCUCGAUTsT 172 AD-
    9705
     758-776 GGAACCUGGAGCGGAUUACTT 173 GUAAUCCGCUCCAGGUUCCTT 174 AD-
    15394
     759-777 GAACCUGGAGCGGAUUACCTT 175 GGUAAUCCGCUCCAGGUUCTT 176 AD-
    15196
     760-778 AACCUGGAGCGGAUUACCCTT 177 GGGUAAUCCGCUCCAGGUUTT 178 AD-
    15197
     777-795 CCCUCCACGGUACCGGGCGTT 179 CGCCCGGUACCGUGGAGGGTT 180 AD-
    15198
     782-800 CACGGUACCGGGCGGAUGATsT 181 UCAUCCGCCCGGUACCGUGTsT 182 AD-
    9609
     782-800 cAcGGuAccGGGcGGAuGATsT 183 UcAUCCGCCCGGuACCGUGTsT 184 AD-
    9735
     783-801 ACGGUACCGGGCGGAUGAATsT 185 UUCAUCCGCCCGGUACCGUTsT 186 AD-
    9537
     783-801 AcGGuAccGGGcGGAuGAATsT 187 UUcAUCCGCCCGGuACCGUTsT 188 AD-
    9663
     784-802 CGGUACCGGGCGGAUGAAUTsT 189 AUUCAUCCGCCCGGUACCGTsT 190 AD-
    9528
     784-802 cGGuAccGGGcGGAuGAAuTsT 191 AUUcAUCCGCCCGGuACCGTsT 192 AD-
    9654
     785-803 GGUACCGGGCGGAUGAAUATsT 193 UAUUCAUCCGCCCGGUACCTsT 194 AD-
    9515
     785-803 GGuAccGGGcGGAuGAAuATsT 195 uAUUcAUCCGCCCGGuACCTsT 196 AD-
    9641
     786-804 GUACCGGGCGGAUGAAUACTsT 197 GUAUUCAUCCGCCCGGUACTsT 198 AD-
    9514
     786-804 GuAccGGGcGGAuGAAuAcTsT 199 GuAUUcAUCCGCCCGGuACTsT 200 AD-
    9640
     788-806 ACCGGGCGGAUGAAUACCATsT 201 UGGUAUUCAUCCGCCCGGUTsT 202 AD-
    9530
     788-806 AccGGGcGGAuGAAuAccATsT 203 UGGuAUUcAUCCGCCCGGUTsT 204 AD-
    9656
     789-807 CCGGGCGGAUGAAUACCAGTsT 205 CUGGUAUUCAUCCGCCCGGTsT 206 AD-
    9538
     789-807 ccGGGcGGAuGAAuAccAGTsT 207 CUGGuAUUcAUCCGCCCGGTsT 208 AD-
    9664
     825-843 CCUGGUGGAGGUGUAUCUCTsT 209 GAGAUACACCUCCACCAGGTsT 210 AD-
    9598
     825-843 ccuGGuGGAGGuGuAucucTsT 211 GAGAuAcACCUCcACcAGGTsT 212 AD-
    9724
     826-844 CUGGUGGAGGUGUAUCUCCTsT 213 GGAGAUACACCUCCACCAGTsT 214 AD-
    9625
     826-844 cuGGuGGAGGuGuAucuccTsT 215 GGAGAuAcACCUCcACcAGTsT 216 AD-
    9751
     827-845 UGGUGGAGGUGUAUCUCCUTsT 217 AGGAGAUACACCUCCACCATsT 218 AD-
    9556
     827-845 uGGuGGAGGuGuAucuccuTsT 219 AGGAGAuAcACCUCcACcATsT 220 AD-
    9682
     828-846 GGUGGAGGUGUAUCUCCUATsT 221 UAGGAGAUACACCUCCACCTsT 222 AD-
    9539
     828-846 GGuGGAGGuGuAucuccuATsT 223 uAGGAGAuAcACCUCcACCTsT 224 AD-
    9665
     831-849 GGAGGUGUAUCUCCUAGACTsT 225 GUCUAGGAGAUACACCUCCTsT 226 AD-
    9517
     831-849 GGAGGuGuAucuccuAGAcTsT 227 GUCuAGGAGAuAcACCUCCTsT 228 AD-
    9643
     833-851 AGGUGUAUCUCCUAGACACTsT 229 GUGUCUAGGAGAUACACCUTsT 230 AD-
    9610
     833-851 AGGuGuAucuccuAGAcAcTsT 231 GUGUCuAGGAGAuAcACCUTsT 232 AD-
    9736
     833-851 AfgGfuGfuAfuCfuCfcUfaGfaCfaC 233 p- 234 AD-
    fTsT gUfgUfcUfaGfgAfgAfuAfcAfcCfuTsT 14681
     833-851 AGGUfGUfAUfCfUfCfCfUfAGACfAC 235 GUfGUfCfUfAGGAGAUfACfACfCfUfTsT 236 AD-
    fTsT 14691
     833-851 AgGuGuAuCuCcUaGaCaCTsT 237 p- 238 AD-
    gUfgUfcUfaGfgAfgAfuAfcAfcCfuTsT 14701
     833-851 AgGuGuAuCuCcUaGaCaCTsT 239 GUfGUfCfUfAGGAGAUfACfACfCfUfTsT 240 AD-
    14711
     833-851 AfgGfuGfuAfuCfuCfcUfaGfaCfaC 241 GUGUCuaGGagAUACAccuTsT 242 AD-
    fTsT 14721
     833-851 AGGUfGUfAUfCfUfCfCfUfAGACfAC 243 GUGUCuaGGagAUACAccuTsT 244 AD-
    fTsT 14731
     833-851 AgGuGuAuCuCcUaGaCaCTsT 245 GUGUCuaGGagAUACAccuTsT 246 AD-
    14741
     833-851 GfcAfcCfcUfcAfuAfgGfcCfuGfgA 247 p- 248 AD-
    fTsT uCfcAfgGfcCfuAfuGfaGfgGfuGfcTsT 15087
     833-851 GCfACfCfCfUfCfAUfAGGCfCfUfGG 249 UfCfCfAGGCfCfUfAUfGAGGGUfGCfTsT 250 AD-
    ATsT 15097
     833-851 GcAcCcUcAuAgGcCuGgATsT 251 p- 252 AD-
    uCfcAfgGfcCfuAfuGfaGfgGfuGfcTsT 15107
     833-851 GcAcCcUcAuAgGcCuGgATsT 253 UfCfCfAGGCfCfUfAUfGAGGGUfGCfTsT 254 AD-
    15117
     833-851 GfcAfcCfcUfcAfuAfgGfcCfuGfgA 255 UCCAGgcCUauGAGGGugcTsT 256 AD-
    fTsT 15127
     833-851 GCfACfCfCfUfCfAUfAGGCfCfUfGG 257 UCCAGgcCUauGAGGGugcTsT 258 AD-
    ATsT 15137
     833-851 GcAcCcUcAuAgGcCuGgATsT 259 UCCAGgcCUauGAGGGugcTsT 260 AD-
    15147
     836-854 UGUAUCUCCUAGACACCAGTsT 261 CUGGUGUCUAGGAGAUACATsT 262 AD-
    9516
     836-854 uGuAucuccuAGAcAccAGTsT 263 CUGGUGUCuAGGAGAuAcATsT 264 AD-
    9642
     840-858 UCUCCUAGACACCAGCAUATsT 265 UAUGCUGGUGUCUAGGAGATsT 266 AD-
    9562
     840-858 ucuccuAGAcAccAGcAuATsT 267 uAUGCUGGUGUCuAGGAGATsT 268 AD-
    9688
     840-858 UfcUfcCfuAfgAfcAfcCfaGfcAfuA 269 p- 270 AD-
    fTsT uAfuGfcUfgGfuGfuCfuAfgGfaGfaTsT 14677
     840-858 UfCfUfCfCfUfAGACfACfCfAGCfAU 271 UfAUfGCfUfGGUfGUfCfUfAGGAGATsT 272 AD-
    fATsT 14687
     840-858 UcUcCuAgAcAcCaGcAuATsT 273 p- 274 AD-
    uAfuGfcUfgGfuGfuCfuAfgGfaGfaTsT 14697
     840-858 UcUcCuAgAcAcCaGcAuATsT 275 UfAUfGCfUfGGUfGUfCfUfAGGAGATsT 276 AD-
    14707
     840-858 UfcUfcCfuAafAfcAfcCfaGfcAfuA 277 UAUGCugGUguCUAGGagaTsT 278 AD-
    fTsT 14717
     840-858 UfCfUfCfCfUfAGACfACfCfAGCfAU 279 UAUGCugGUguCUAGGagaTsT 280 AD-
    fATsT 14727
     840-858 UcUcCuAgAcAcCaGcAuATsT 281 UAUGCugGUguCUAGGagaTsT 282 AD-
    14737
     840-858 AfgGfcCfuGfgAfgUfuUfaUfuCfgG 283 p- 284 AD-
    fTsT cCfgAfaUfaAfaCfuCfcAfgGfcCfuTsT 15083
     840-858 AGGCfCfUfGGAGUfUfUfAUfUfCfGG 285 CfCfGAAUfAAACfUfCfCfAGGCfCfUfTs 286 AD-
    TsT T 15093
     840-858 AgGcCuGgAgUuUaUuCgGTsT 287 p- 288 AD-
    cCfgAfaUfaAfaCfuCfcAfgGfcCfuTsT 15103
     840-858 AgGcCuGgAgUuUaUuCgGTsT 289 CfCfGAAUfAAACfUfCfCfAGGCfCfUfTs 290 AD-
    T 15113
     840-858 AfgGfcCfuGfgAfgUfuUfaUfuCfgG 291 CCGAAuaAAcuCCAGGccuTsT 292 AD-
    fTsT 15123
     840-858 AGGCfCfUfGGAGUfUfUfAUfUfCfGG 293 CCGAAuaAAcuCCAGGccuTsT 294 AD-
    TsT 15133
     840-858 AgGcCuGgAgUuUaUuCgGTsT 295 CCGAAuaAAcuCCAGGccuTsT 296 AD-
    15143
     841-859 CUCCUAGACACCAGCAUACTsT 297 GUAUGCUGGUGUCUAGGAGTsT 298 AD-
    9521
     841-859 cuccuAGAcAccAGcAuAcTsT 299 GuAUGCUGGUGUCuAGGAGTsT 300 AD-
    9647
     842-860 UCCUAGACACCAGCAUACATsT 301 UGUAUGCUGGUGUCUAGGATsT 302 AD-
    9611
     842-860 uccuAGAcAccAGcAuAcATsT 303 UGuAUGCUGGUGUCuAGGATsT 304 AD-
    9737
     843-861 CCUAGACACCAGCAUACAGTsT 305 CUGUAUGCUGGUGUCUAGGTsT 306 AD-
    9592
     843-861 ccuAGAcAccAGcAuAcAGTsT 307 CUGuAUGCUGGUGUCuAGGTsT 308 AD-
    9718
     847-865 GACACCAGCAUACAGAGUGTsT 309 CACUCUGUAUGCUGGUGUCTsT 310 AD-
    9561
     847-865 GAcAccAGcAuAcAGAGuGTsT 311 cACUCUGuAUGCUGGUGUCTsT 312 AD-
    9687
     855-873 CAUACAGAGUGACCACCGGTsT 313 CCGGUGGUCACUCUGUAUGTsT 314 AD-
    9636
     855-873 cAuAcAGAGuGAccAccGGTsT 315 CCGGUGGUcACUCUGuAUGTsT 316 AD-
    9762
     860-878 AGAGUGACCACCGGGAAAUTsT 317 AUUUCCCGGUGGUCACUCUTsT 318 AD-
    9540
     860-878 AGAGuGAccAccGGGAAAuTsT 319 AUUUCCCGGUGGUcACUCUTsT 320 AD-
    9666
     861-879 GAGUGACCACCGGGAAAUCTsT 321 GAUUUCCCGGUGGUCACUCTsT 322 AD-
    9535
     861-879 GAGuGAccAccGGGAAAucTsT 323 GAUUUCCCGGUGGUcACUCTsT 324 AD-
    9661
     863-881 GUGACCACCGGGAAAUCGATsT 325 UCGAUUUCCCGGUGGUCACTsT 326 AD-
    9559
     863-881 GuGAccAccGGGAAAucGATsT 327 UCGAUUUCCCGGUGGUcACTsT 328 AD-
    9685
     865-883 GACCACCGGGAAAUCGAGGTsT 329 CCUCGAUUUCCCGGUGGUCTsT 330 AD-
    9533
     865-883 GAccAccGGGAAAucGAGGTsT 331 CCUCGAUUUCCCGGUGGUCTsT 332 AD-
    9659
     866-884 ACCACCGGGAAAUCGAGGGTsT 333 CCCUCGAUUUCCCGGUGGUTsT 334 AD-
    9612
     866-884 AccAccGGGAAAucGAGGGTsT 335 CCCUCGAUUUCCCGGUGGUTsT 336 AD-
    9738
     867-885 CCACCGGGAAAUCGAGGGCTsT 337 GCCCUCGAUUUCCCGGUGGTsT 338 AD-
    9557
     867-885 ccAccGGGAAAucGAGGGcTsT 339 GCCCUCGAUUUCCCGGUGGTsT 340 AD-
    9683
     875-893 AAAUCGAGGGCAGGGUCAUTsT 341 AUGACCCUGCCCUCGAUUUTsT 342 AD-
    9531
     875-893 AAAucGAGGGcAGGGucAuTsT 343 AUGACCCUGCCCUCGAUUUTsT 344 AD-
    9657
     875-893 AfaAfuCfgAfgGfgCfaGfgGfuCfaU 345 p- 346 AD-
    fTsT aUfgAfcCfcUfgCfcCfuCfgAfuUfuTsT 14673
     875-893 AAAUfCfGAGGGCfAGGGUfCfAUfTsT 347 AUfGACfCfCfUfGCfCfCfUfCfGAUfUfU 348 AD-
    fTsT 14683
     875-893 AaAuCgAgGgCaGgGuCaUTsT 349 p- 350 AD-
    aUfgAfcCfcUfgCfcCfuCfgAfuUfuTsT 14693
     875-893 AaAuCgAgGgCaGgGuCaUTsT 351 AUfGACfCfCfUfGCfCfCfUfCfGAUfUfU 352 AD-
    fTsT 14703
     875-893 AfaAfuCfgAfgGfgCfaGfgGfuCfaU 353 AUGACccUGccCUCGAuuuTsT 354 AD-
    fTsT 14713
     875-893 AAAUfCfGAGGGCfAGGGUfCfAUfTsT 355 AUGACccUGccCUCGAuuuTsT 356 AD-
    14723
     875-893 AaAuCgAgGgCaGgGuCaUTsT 357 AUGACccUGccCUCGAuuuTsT 358 AD-
    14733
     875-893 CfgGfcAfcCfcUfcAfuAfgGfcCfuG 359 p- 360 AD-
    fTsT cAfgGfcCfuAfuGfaGfgGfuGfcCfgTsT 15079
     875-893 CfGGCfACfCfCfUfCfAUfAGGCfCfU 361 CfAGGCfCfUfAUfGAGGGUfGCfCfGTsT 362 AD-
    fGTsT 15089
     875-893 CgGcAcCcUcAuAgGcCuGTsT 363 p- 364 AD-
    cAfgGfcCfuAfuGfaGfgGfuGfcCfgTsT 15099
     875-893 CgGcAcCcUcAuAgGcCuGTsT 365 CfAGGCfCfUfAUfGAGGGUfGCfCfGTsT 366 AD-
    15109
     875-893 CfgGfcAfcCfcUfcAfuAfgGfcCfuG 367 CAGGCcuAUgaGGGUGccgTsT 368 AD-
    fTsT 15119
     875-893 CfGGCfACfCfCfUfCfAUfAGGCfCfU 369 CAGGCcuAUgaGGGUGccgTsT 370 AD-
    fGTsT 15129
     875-893 CgGcAcCcUcAuAgGcCuGTsT 371 CAGGCcuAUgaGGGUGccgTsT 372 AD-
    15139
     877-895 AUCGAGGGCAGGGUCAUGGTsT 373 CCAUGACCCUGCCCUCGAUTsT 374 AD-
    9542
     877-895 AucGAGGGcAGGGucAuGGTsT 375 CcAUGACCCUGCCCUCGAUTsT 376 AD-
    9668
     878-896 cGAGGGcAGGGucAuGGucTsT 377 GACcAUGACCCUGCCCUCGTsT 378 AD-
    9739
     880-898 GAGGGCAGGGUCAUGGUCATsT 379 UGACCAUGACCCUGCCCUCTsT 380 AD-
    9637
     880-898 GAGGGcAGGGucAuGGucATsT 381 UGACcAUGACCCUGCCCUCTsT 382 AD-
    9763
     882-900 GGGCAGGGUCAUGGUCACCTsT 383 GGUGACCAUGACCCUGCCCTsT 384 AD-
    9630
     882-900 GGGcAGGGucAuGGucAccTsT 385 GGUGACcAUGACCCUGCCCTsT 386 AD-
    9756
     885-903 CAGGGUCAUGGUCACCGACTsT 387 GUCGGUGACCAUGACCCUGTsT 388 AD-
    9593
     885-903 cAGGGucAuGGucAccGAcTsT 389 GUCGGUGACcAUGACCCUGTsT 390 AD-
    9719
     886-904 AGGGUCAUGGUCACCGACUTsT 391 AGUCGGUGACCAUGACCCUTsT 392 AD-
    9601
     886-904 AGGGucAuGGucAccGAcuTsT 393 AGUCGGUGACcAUGACCCUTsT 394 AD-
    9727
     892-910 AUGGUCACCGACUUCGAGATsT 395 UCUCGAAGUCGGUGACCAUTsT 396 AD-
    9573
     892-910 AuGGucAccGAcuucGAGATsT 397 UCUCGAAGUCGGUGACcAUTsT 398 AD-
    9699
     899-917 CCGACUUCGAGAAUGUGCCTT 399 GGCACAUUCUCGAAGUCGGTT 400 AD-
    15228
     921-939 GGAGGACGGGACCCGCUUCTT 401 GAAGCGGGUCCCGUCCUCCTT 402 AD-
    15395
     993- CAGCGGCCGGGAUGCCGGCTsT 403 GCCGGCAUCCCGGCCGCUGTsT 404 AD-
    1011 9602
     993- cAGcGGccGGGAuGccGGcTsT 405 GCCGGcAUCCCGGCCGCUGTsT 406 AD-
    1011 9728
    1020- GGGUGCCAGCAUGCGCAGCTT 407 GCUGCGCAUGCUGGCACCCTT 408 AD-
    1038 15386
    1038- CCUGCGCGUGCUCAACUGCTsT 409 GCAGUUGAGCACGCGCAGGTsT 410 AD-
    1056 9580
    1038- ccuGcGcGuGcucAAcuGcTsT 411 GcAGUUGAGcACGCGcAGGTsT 412 AD-
    1056 9706
    1040- UGCGCGUGCUCAACUGCCATsT 413 UGGCAGUUGAGCACGCGCATsT 414 AD-
    1058 9581
    1040- uGcGcGuGcucAAcuGccATsT 415 UGGcAGUUGAGcACGCGcATsT 416 AD-
    1058 9707
    1042- CGCGUGCUCAACUGCCAAGTsT 417 CUUGGCAGUUGAGCACGCGTsT 418 AD-
    1060 9543
    1042- cGcGuGcucAAcuGccAAGTsT 419 CUUGGcAGUUGAGcACGCGTsT 420 AD-
    1060 9669
    1053- CUGCCAAGGGAAGGGCACGTsT 421 CGUGCCCUUCCCUUGGCAGTsT 422 AD-
    1071 9574
    1053- cuGccAAGGGAAGGGcAcGTsT 423 CGUGCCCUUCCCUUGGcAGTsT 424 AD-
    1071 9700
    1057- CAAGGGAAGGGCACGGUUATT 425 UAACCGUGCCCUUCCCUUGTT 426 AD-
    1075 15320
    1058- AAGGGAAGGGCACGGUUAGTT 427 CUAACCGUGCCCUUCCCUUTT 428 AD-
    1076 15321
    1059- AGGGAAGGGCACGGUUAGCTT 429 GCUAACCGUGCCCUUCCCUTT 430 AD-
    1077 15199
    1060- GGGAAGGGCACGGUUAGCGTT 431 CGCUAACCGUGCCCUUCCCTT 432 AD-
    1078 15167
    1061- GGAAGGGCACGGUUAGCGGTT 433 CCGCUAACCGUGCCCUUCCTT 434 AD-
    1079 15164
    1062- GAAGGGCACGGUUAGCGGCTT 435 GCCGCUAACCGUGCCCUUCTT 436 AD-
    1080 15166
    1063- AAGGGCACGGUUAGCGGCATT 437 UGCCGCUAACCGUGCCCUUTT 438 AD-
    1081 15322
    1064- AGGGCACGGUUAGCGGCACTT 439 GUGCCGCUAACCGUGCCCUTT 440 AD-
    1082 15200
    1068- CACGGUUAGCGGCACCCUCTT 441 GAGGGUGCCGCUAACCGUGTT 442 AD-
    1086 15213
    1069- ACGGUUAGCGGCACCCUCATT 443 UGAGGGUGCCGCUAACCGUTT 444 AD-
    1087 151229
    1072- GUUAGCGGCACCCUCAUAGTT 445 CUAUGAGGGUGCCGCUAACTT 446 AD-
    1090 152215
    1073- UUAGCGGCACCCUCAUAGGTT 447 CCUAUGAGGGUGCCGCUAATT 448 AD-
    1091 15214
    1076- GCGGCACCCUCAUAGGCCUTsT 449 AGGCCUAUGAGGGUGCCGCTsT 450 AD-
    1094 9315
    1079- GCACCCUCAUAGGCCUGGATsT 451 UCCAGGCCUAUGAGGGUGCTsT 452 AD-
    1097 9326
    1085- UCAUAGGCCUGGAGUUUAUTsT 453 AUAAACUCCAGGCCUAUGATsT 454 AD-
    1103 9318
    1090- GGCCUGGAGUUUAUUCGGATsT 455 UCCGAAUAAACUCCAGGCCTsT 456 AD-
    1108 9323
    1091- GCCUGGAGUUUAUUCGGAATsT 457 UUCCGAAUAAACUCCAGGCTsT 458 AD-
    1109 9314
    1091- GccuGGAGuuuAuucGGAATsT 459 UUCCGAAuAAACUCcAGGCTsT 460 AD-
    1109 10792
    1091- GccuGGAGuuuAuucGGAATsT 461 UUCCGAAUAACUCCAGGCTsT 462 AD-
    1109 10796
    1093- CUGGAGUUUAUUCGGAAAATsT 463 UUUUCCGAAUAAACUCCAGTsT 464 AD-
    1111 9638
    1093- cuGGAGuuuAuucGGAAAATsT 465 UUUUCCGAAuAAACUCcAGTsT 466 AD-
    1111 9764
    1095- GGAGUUUAUUCGGAAAAGCTsT 467 GCUUUUCCGAAUAAACUCCTsT 468 AD-
    1113 9525
    1095- GGAGuuuAuucGGAAAAGcTsT 469 GCUUUUCCGAAuAAACUCCTsT 470 AD-
    1113 9651
    1096- GAGUUUAUUCGGAAAAGCCTsT 471 GGCUUUUCCGAAUAAACUCTsT 472 AD-
    1114 9560
    1096- GAGuuuAuucGGAAAAGccTsT 473 GGCUUUUCCGAAuAAACUCTsT 474 AD-
    1114 9686
    1100- UUAUUCGGAAAAGCCAGCUTsT 475 AGCUGGCUUUUCCGAAUAATsT 476 AD-
    1118 9536
    1100- uuAuucGGAAAAGccAGcuTsT 477 AGCUGGCUUUUCCGAAuAATsT 478 AD-
    1118 9662
    1154- CCCUGGCGGGUGGGUACAGTsT 479 CUGUACCCACCCGCCAGGGTsT 480 AD-
    1172 9584
    1154- cccuGGcGGGuGGGuAcAGTsT 481 CUGuACCcACCCGCcAGGGTsT 482 AD-
    1172 9710
    1155- CCUGGCGGGUGGGUACAGCTT 483 GCUGUACCCACCCGCCAGGTT 484 AD-
    1173 15323
    1157- UGGCGGGUGGGUACAGCCGTsT 485 CGGCUGUACCCACCCGCCATsT 486 AD-
    1175 9551
    1157- uGGcGGGuGGGuAcAGccGTsT 487 CGGCUGuACCcACCCGCcATsT 488 AD-
    1175 9677
    1158- GGCGGGUGGGUACAGCCGCTT 489 GCGGCUGUACCCACCCGCCTT 490 AD-
    1176 15230
    1162- GGUGGGUACAGCCGCGUCCTT 491 GGACGCGGCUGUACCCACCTT 492 AD-
    1180 15231
    1164- UGGGUACAGCCGCGUCCUCTT 493 GAGGACGCGGCUGUACCCATT 494 AD-
    1182 15285
    1172- GCCGCGUCCUCAACGCCGCTT 495 GCGGCGUUGAGGACGCGGCTT 496 AD-
    1190 15396
    1173- CCGCGUCCUCAACGCCGCCTT 497 GGCGGCGUUGAGGACGCGGTT 498 AD-
    1191 15397
    1216- GUCGUGCUGGUCACCGCUGTsT 499 CAGCGGUGACCAGCACGACTsT 500 AD-
    1234 9600
    1216- GucGuGcuGGucAccGcuGTsT 501 cAGCGGUGACcAGcACGACTsT 502 AD-
    1234 9726
    1217- UCGUGCUGGUCACCGCUGCTsT 503 GCAGCGGUGACCAGCACGATsT 504 AD-
    1235 9606
    1217- ucGuGcuGGucAccGcuGcTsT 505 GcAGCGGUGACcAGcACGATsT 506 AD-
    1235 9732
    1223- UGGUCACCGCUGCCGGCAATsT 507 UUGCCGGCAGCGGUGACCATsT 508 AD-
    1241 9633
    1223- uGGucAccGcuGccGGcAATsT 509 UUGCCGGcAGCGGUGACcATsT 510 AD-
    1241 9759
    1224- GGUCACCGCUGCCGGCAACTsT 511 GUUGCCGGCAGCGGUGACCTsT 512 AD-
    1242 9588
    1224- GGucAccGcuGccGGcAAcTsT 513 GUUGCCGGcAGCGGUGACCTsT 514 AD-
    1242 9714
    1227- CACCGCUGCCGGCAACUUCTsT 515 GAAGUUGCCGGCAGCGGUGTsT 516 AD-
    1245 9589
    1227- cAccGcuGccGGcAAcuucTsT 517 GAAGUUGCCGGcAGCGGUGTsT 518 AD-
    1245 9715
    1229 CCGCUGCCGGCAACUUCCGTsT 519 CGGAAGUUGCCGGCAGCGGTsT 520 AD-
    1247 9575
    1229- ccGcuGccGGcAAcuuccGTsT 521 CGGAAGUUGCCGGcAGCGGTsT 522 AD-
    1247 9701
    1230- CGCUGCCGGCAACUUCCGGTsT 523 CCGGAAGUUGCCGGCAGCGTsT 524 AD-
    1248 9563
    1230- cGcuGccGGcAAcuuccGGTsT 525 CCGGAAGUUGCCGGcAGCGTsT 526 AD-
    1248 9689
    1231- GCUGCCGGCAACUUCCGGGTsT 527 CCCGGAAGUUGCCGGCAGCTsT 528 AD-
    1249 9594
    1231- GcuGccGGcAAcuuccGGGTsT 529 CCCGGAAGUUGCCGGcAGCTsT 530 AD-
    1249 9720
    1236- CGGCAACUUCCGGGACGAUTsT 531 AUCGUCCCGGAAGUUGCCGTsT 532 AD-
    1254 9585
    1236- cGGcAAcuuccGGGAcGAuTsT 533 AUCGUCCCGGAAGUUGCCGTsT 534 AD-
    1254 9711
    1237- GGCAACUUCCGGGACGAUGTsT 535 CAUCGUCCCGGAAGUUGCCTsT 536 AD-
    1255 9614
    1237- GGcAAcuuccGGGAcGAuGTsT 537 cAUCGUCCCGGAAGUUGCCTsT 538 AD-
    1255 9740
    1243- UUCCGGGACGAUGCCUGCCTsT 539 GGCAGGCAUCGUCCCGGAATsT 540 AD-
    1261 9615
    1243- uuccGGGAcGAuGccuGccTsT 541 GGcAGGcAUCGUCCCGGAATsT 542 AD-
    1261 9741
    1248- GGACGAUGCCUGCCUCUACTsT 543 GUAGAGGCAGGCAUCGUCCTsT 544 AD-
    1266 9534
    1248- GGACGAUGCCUGCCUCUACTsT 545 GUAGAGGCAGGCAUCGUCCTsT 546 AD-
    1266 9534
    1248- GGAcGAuGccuGccucuAcTsT 547 GuAGAGGcAGGcAUCGUCCTsT 548 AD-
    1266 9660
    1279 GCUCCCGAGGUCAUCACAGTT 549 CUGUGAUGACCUCGGGAGCTT 550 AD-
    1297 15324
    1280- CUCCCGAGGUCAUCACAGUTT 551 ACUGUGAUGACCUCGGGAGTT 552 AD-
    1298 15232
    1281- UCCCGAGGUCAUCACAGUUTT 553 AACUGUGAUGACCUCGGGATT 554 AD-
    1299 15233
    1314- CCAAGACCAGCCGGUGACCTT 555 GGUCACCGGCUGGUCUUGGTT 556 AD-
    1332 15234
    1315- CAAGACCAGCCGGUGACCCTT 557 GGGUCACCGGCUGGUCUUGTT 558 AD-
    1333 15286
    1348- ACCAACUUUGGCCGCUGUGTsT 559 CACAGCGGCCAAAGUUGGUTsT 560 AD-
    1366 9590
    1348- AccAAcuuuGGccGcuGuGTsT 561 cAcAGCGGCcAAAGUUGGUTsT 562 AD-
    1366 9716
    1350- CAACUUUGGCCGCUGUGUGTsT 563 CACACAGCGGCCAAAGUUGTsT 564 AD-
    1368 9632
    1350- cAAcuuuGGccGcuGuGuGTsT 565 cAcAcAGCGGCcAAAGUUGTsT 566 AD-
    1368 9758
    1360- CGCUGUGUGGACCUCUUUGTsT 567 CAAAGAGGUCCACACAGCGTsT 568 AD-
    1378 9567
    1360- cGcuGuGuGGAccucuuuGTsT 569 cAAAGAGGUCcAcAcAGCGTsT 570 AD-
    1378 9693
    1390- GACAUCAUUGGUGCCUCCATsT 571 UGGAGGCACCAAUGAUGUCTsT 572 AD-
    1408 9586
    1390- GAcAucAuuGGuGccuccATsT 573 UGGAGGcACcAAUGAUGUCTsT 574 AD-
    1408 9712
    1394- UCAUUGGUGCCUCCAGCGATsT 575 UCGCUGGAGGCACCAAUGATsT 576 AD-
    1412 9564
    1394- ucAuuGGuGccuccAGcGATsT 577 UCGCUGGAGGcACcAAUGATsT 578 AD-
    1412 9690
    1417- AGCACCUGCUUUGUGUCACTsT 579 GUGACACAAAGCAGGUGCUTsT 580 AD-
    1435 9616
    1417- AGcAccuGcuuuGuGucAcTsT 581 GUGAcAcAAAGcAGGUGCUTsT 582 AD-
    1435 9742
    1433- CACAGAGUGGGACAUCACATT 583 UGUGAUGUCCCACUCUGUGTT 584 AD-
    1451 15398
    1486- AUGCUGUCUGCCGAGCCGGTsT 585 CCGGCUCGGCAGACAGCAUTsT 586 AD-
    1504 9617
    1486- AuGcuGucuGccGAGccGGTsT 587 CCGGCUCGGcAGAcAGcAUTsT 588 AD-
    1504 9743
    1491- GUCUGCCGAGCCGGAGCUCTsT 589 GAGCUCCGGCUCGGCAGACTsT 590 AD-
    1509 9635
    1491- GucuGccGAGccGGAGcucTsT 591 GAGCUCCGGCUCGGcAGACTsT 592 AD-
    1509 9761
    1521- GUUGAGGCAGAGACUGAUCTsT 593 GAUCAGUCUCUGCCUCAACTsT 594 AD-
    1539 9568
    1521- GuuGAGGcAGAGAcuGAucTsT 595 GAUcAGUCUCUGCCUcAACTsT 596 AD-
    1539 9694
    1527- GCAGAGACUGAUCCACUUCTsT 597 GAAGUGGAUCAGUCUCUGCTsT 598 AD-
    1545 9576
    1527- GcAGAGAcuGAuccAcuucTsT 599 GAAGUGGAUcAGUCUCUGCTsT 600 AD-
    1545 9702
    1529- AGAGACUGAUCCACUUCUCTsT 601 GAGAAGUGGAUCAGUCUCUTsT 602 AD-
    1547 9627
    1529- AGAGAcuGAuccAcuucucTsT 603 GAGAAGUGGAUcAGUCUCUTsT 604 AD-
    1547 9753
    1543- UUCUCUGCCAAAGAUGUCATsT 605 UGACAUCUUUGGCAGAGAATsT 606 AD-
    1561 9628
    1543- uucucuGccAAAGAuGucATsT 607 UGAcAUCUUUGGcAGAGAATsT 608 AD-
    1561 9754
    1545- CUCUGCCAAAGAUGUCAUCTsT 609 GAUGACAUCUUUGGCAGAGTsT 610 AD-
    1563 9631
    1545- cucuGccAAAGAuGucAucTsT 611 GAUGAcAUCUUUGGcAGAGTsT 612 AD-
    1563 9757
    1580- CUGAGGACCAGCGGGUACUTsT 613 AGUACCCGCUGGUCCUCAGTsT 614 AD-
    1598 9595
    1580- cuGAGGAccAGcGGGuAcuTsT 615 AGuACCCGCUGGUCCUcAGTsT 616 AD-
    1598 9721
    1581- UGAGGACCAGCGGGUACUGTsT 617 CAGUACCCGCUGGUCCUCATsT 618 AD-
    1599 9544
    1581- uGAGGAccAGcGGGuAcuGTsT 619 cAGuACCCGCUGGUCCUcATsT 620 AD-
    1599 9670
    1666- ACUGUAUGGUCAGCACACUTT 621 AGUGUGCUGACCAUACAGUTT 622 AD-
    1684 15235
    1668- UGUAUGGUCAGCACACUCGTT 623 CGAGUGUGCUGACCAUACATT 624 AD-
    1686 15236
    1669- GUAUGGUCAGCACACUCGGTT 625 CCGAGUGUGCUGACCAUACTT 626 AD-
    1687 15168
    1697 GGAUGGCCACAGCCGUCGCTT 627 GCGACGGCUGUGGCCAUCCTT 628 AD-
    1715 15174
    1698 GAUGGCCACAGCCGUCGCCTT 629 GGCGACGGCUGUGGCCAUCTT 630 AD-
    1716 15325
    1806 CAAGCUGGUCUGCCGGGCCTT 631 GGCCCGGCAGACCAGCUUGTT 632 AD-
    1824 15326
    1815- CUGCCGGGCCCACAACGCUTsT 633 AGCGUUGUGGGCCCGGCAGTsT 634 AD-
    1833 9570
    1815- cuGccGGGcccAcAAcGcuTsT 635 AGCGUUGUGGGCCCGGcAGTsT 636 AD-
    1833 9696
    1816- UGCCGGGCCCACAACGCUUTsT 637 AAGCGUUGUGGGCCCGGCATsT 638 AD-
    1834 9566
    1816- uGccGGGcccAcAAcGcuuTsT 639 AAGCGUUGUGGGCCCGGcATsT 640 AD-
    1834 9692
    1818- CCGGGCCCACAACGCUUUUTsT 641 AAAAGCGUUGUGGGCCCGGTsT 642 AD-
    1836 9532
    1818- ccGGGcccAcAAcGcuuuuTsT 643 AAAAGCGUUGUGGGCCCGGTsT 644 AD-
    1836 9658
    1820- GGGCCCACAACGCUUUUGGTsT 645 CCAAAAGCGUUGUGGGCCCTsT 646 AD-
    1838 9549
    1820- GGGcccAcAAcGcuuuuGGTsT 647 CcAAAAGCGUUGUGGGCCCTsT 648 AD-
    1838 9675
    1840- GGUGAGGGUGUCUACGCCATsT 649 UGGCGUAGACACCCUCACCTsT 650 AD-
    1858 9541
    1840- GGuGAGGGuGucuAcGccATsT 651 UGGCGuAGAcACCCUcACCTsT 652 AD-
    1858 9667
    1843- GAGGGUGUCUACGCCAUUGTsT 653 CAAUGGCGUAGACACCCUCTsT 654 AD-
    1861 9550
    1843- GAGGGuGucuAcGccAuuGTsT 655 cAAUGGCGuAGAcACCCUCTsT 656 AD-
    1861 9676
    1861- GCCAGGUGCUGCCUGCUACTsT 657 GUAGCAGGCAGCACCUGGCTsT 658 AD-
    1879 9571
    1861- GccAGGuGcuGccuGcuAcTsT 659 GuAGcAGGcAGcACCUGGCTsT 660 AD-
    1879 9697
    1862- CCAGGUGCUGCCUGCUACCTsT 661 GGUAGCAGGCAGCACCUGGTsT 662 AD-
    1880 9572
    1862- ccAGGuGcuGccuGcuAccTsT 663 GGuAGcAGGcAGcACCUGGTsT 664 AD-
    1880 9698
    2008- ACCCACAAGCCGCCUGUGCTT 665 GCACAGGCGGCUUGUGGGUTT 666 AD-
    2026 15327
    2023- GUGCUGAGGCCACGAGGUCTsT 667 GACCUCGUGGCCUCAGCACTsT 668 AD-
    2041 9639
    2023- GuGcuGAGGccAcGAGGucTsT 669 GACCUCGUGGCCUcAGcACTsT 670 AD-
    2041 9765
    2024- UGCUGAGGCCACGAGGUCATsT 671 UGACCUCGUGGCCUCAGCATsT 672 AD-
    2042 9518
    2024- UGCUGAGGCCACGAGGUCATsT 673 UGACCUCGUGGCCUCAGCATsT 674 AD-
    2042 9518
    2024- uGcuGAGGccAcGAGGucATsT 675 UGACCUCGUGGCCUcAGcATsT 676 AD-
    2042 9644
    2024- UfgCfuGfaGfgCfcAfcGfaGfgUfcA 677 p- 678 AD-
    2042 fTsT uGfaCfcUfcGfuGfgCfcUfcAfgCfaTsT 14672
    2024- UfGCfUfGAGGCfCfACfGAGGUfCfAT 679 UfGACfCfUfCfGUfGGCfCfUfCfAGCfAT 680 AD-
    2042 sT sT 14682
    2024- UgCuGaGgCcAcGaGgUcATsT 681 p- 682 AD-
    2042 uGfaCfcUfcGfuGfgCfcUfcAfgCfaTsT 14692
    2024- UgCuGaGgCcAcGaGgUcATsT 683 UfGACfCfUfCfGUfGGCfCfUfCfAGCfAT 684 AD-
    2042 sT 14702
    2024- UfgCfuGfaGfgCfcAfcGfaGfgUfcA 685 UGACCucGUggCCUCAgcaTsT 686 AD-
    2042 fTsT 14712
    2024- UfGCfUfGAGGCfCfACfGAGGUfCfAT 687 UGACCucGUggCCUCAgcaTsT 688 AD-
    2042 sT 14722
    2024- UgCuGaGgCcAcGaGgUcATsT 689 UGACCucGUggCCUCAgcaTsT 690 AD-
    2042 14732
    2024- GfuGfgUfcAfgCfgGfcCfgGfgAfuG 691 p- 692 AD-
    2042 fTsT cAfuCfcCfgGfcCfgCfuGfaCfcAfcTsT 15078
    2024- GUfGGUfCfAGCfGGCfCfGGGAUfGTs 693 CfAUfCfCfCfGGCfCfGCfUfGACfCfACf 694 AD-
    2042 T TsT 15088
    2024- GuGgUcAgCgGcCgGgAuGTsT 695 p- 696 AD-
    2042 cAfuCfcCfgGfcCfgCfuGfaCfcAfcTsT 15098
    2024- GuGgUcAgCgGcCgGgAuGTsT 697 CfAUfCfCfCfGGCfCfGCfUfGACfCfACf 698 AD-
    2042 TsT 15108
    2024- GfuGfgUfcAfgCfgGfcCfgGfgAfuG 699 CAUCCcgGCcgCUGACcacTsT 700 AD-
    2042 fTsT 15118
    2024- GUfGGUfCfAGCfGGCfCfGGGAUfGTs 701 CAUCCcgGCcgCUGACcacTsT 702 AD-
    2042 T 15128
    2024- GuGgUcAgCgGcCgGgAuGTsT 703 CAUCCcgGCcgCUGACcacTsT 704 AD-
    2042 15138
    2030- GGCCACGAGGUCAGCCCAATT 705 UUGGGCUGACCUCGUGGCCTT 706 AD-
    2048 15237
    2035- CGAGGUCAGCCCAACCAGUTT 707 ACUGGUUGGGCUGACCUCGTT 708 AD-
    2053 15287
    2039- GUCAGCCCAACCAGUGCGUTT 709 ACGCACUGGUUGGGCUGACTT 710 AD-
    2057 15238
    2041- CAGCCCAACCAGUGCGUGGTT 711 CCACGCACUGGUUGGGCUGTT 712 AD-
    2059 15328
    2062- CACAGGGAGGCCAGCAUCCTT 713 GGAUGCUGGCCUCCCUGUGTT 714 AD-
    2080 15399
    2072- CCAGCAUCCACGCUUCCUGTsT 715 CAGGAAGCGUGGAUGCUGGTsT 716 A D-
    2090 9582
    2072- ccAGcAuccAcGcuuccuGTsT 717 cAGGAAGCGUGGAUGCUGGTsT 718 AD-
    2090 9708
    2118- AGUCAAGGAGCAUGGAAUCTsT 719 GAUUCCAUGCUCCUUGACUTsT 720 AD-
    2136 9545
    2118- AGucAAGGAGcAuGGAAucTsT 721 GAUUCcAUGCUCCUUGACUTsT 722 AD-
    2136 9671
    2118- AfgUfcAfaGfgAfgCfaUfgGfaAfuC 723 p- 724 AD-
    2136 fTsT gAfuUfcCfaUfgCfuCfcUfuGfaCfuTsT 14674
    2118- AGUfCfAAGGAGCfAUfGGAAUfCfTsT 725 GAUfUfCfCfAUfGCfUfCfCfUfUfGACfU 726 AD-
    2136 fTsT 14684
    2118- AgUcAaGgAgCaUgGaAuCTsT 727 p- 728 AD-
    2136 gAfuUfcCfaUfgCfuCfcUfuGfaCfuTsT 14694
    2118- AgUcAaGgAgCaUgGaAuCTsT 729 GAUfUfCfCfAUfGCfUfCfCfUfUfGACfU 730 AD-
    2136 fTsT 14704
    2118- AfgUfcAfaGfgAfgCfaUfgGfaAfuC 731 GAUUCcaUGcuCCUUGacuTsT 732 AD-
    2136 fTsT 14714
    2118- AGUfCfAAGGAGCfAUfGGAAUfCfTsT 733 GAUUCcaUGcuCCUUGacuTsT 734 AD-
    2136 14724
    2118- AgUcAaGgAgCaUgGaAuCTsT 735 GAUUCcaUGcuCCUUGacuTsT 736 AD-
    2136 14734
    2118- GfcGfgCfaCfcCfuCfaUfaGfgCfcU 737 p- 738 AD-
    2136 fTsT aGfgCfcUfaUfgAfgGfgUfgCfcGfcTsT 15080
    2118- GCfGGCfACfCfCfUfCfAUfAGGCfCf 739 AGGCfCfUfAUfGAGGGUfGCfCfGCfTsT 740 AD-
    2136 UfTsT 15090
    2118- GcGgCaCcCuCaUaGgCcUTsT 741 P- 742 AD-
    2136 aGfgCfcUfaUfgAfgGfgUfgCfcGfcTsT 15100
    2118- GcGgCaCcCuCaUaGgCcUTsT 743 AGGCfCfUfAUfGAGGGUfGCfCfGCfTsT 744 AD-
    2136 15110
    2118- GfcGfgCfaCfcCfuCfaUfaGfgCfcU 745 AGGCCuaUGagGGUGCcgcTsT 746 AD-
    2136 fTsT 15120
    2118- GCfGGCfACfCfCfUfCfAUfAGGCfCf 747 AGGCCuaUGagGGUGCcgcTsT 748 AD-
    2136 UfTsT 15130
    2118- GcGgCaCcCuCaUaGgCcUTsT 749 AGGCCuaUGagGGUGCcgcTsT 750 AD-
    2136 15140
    2122- AAGGAGCAUGGAAUCCCGGTsT 751 CCGGGAUUCCAUGCUCCUUTsT 752 AD-
    2140 9522
    2122- AAGGAGcAuGGAAucccGGTsT 753 CCGGGAUUCcAUGCUCCUUTsT 754 AD-
    2140 9648
    2123- AGGAGCAUGGAAUCCCGGCTsT 755 GCCGGGAUUCCAUGCUCCUTsT 756 AD-
    2141 9552
    2123- AGGAGcAuGGAAucccGGcTsT 757 GCCGGGAUUCcAUGCUCCUTsT 758 AD-
    2141 9678
    2125- GAGCAUGGAAUCCCGGCCCTsT 759 GGGCCGGGAUUCCAUGCUCTsT 760 AD-
    2143 9618
    2125- GAGcAuGGAAucccGGcccTsT 761 GGGCCGGGAUUCcAUGCUCTsT 762 AD-
    2143 9744
    2230- GCCUACGCCGUAGACAACATT 763 UGUUGUCUACGGCGUAGGCTT 764 AD-
    2248 15239
    2231- CCUACGCCGUAGACAACACTT 765 GUGUUGUCUACGGCGUAGGTT 766 AD-
    2249 15212
    2232- CUACGCCGUAGACAACACGTT 767 CGUGUUGUCUACGGCGUAGTT 768 AD-
    2250 15240
    2233- UACGCCGUAGACAACACGUTT 769 ACGUGUUGUCUACGGCGUATT 770 AD-
    2251 15177
    2235- CGCCGUAGACAACACGUGUTT 771 ACACGUGUUGUCUACGGCGTT 772 AD-
    2253 15179
    2236- GCCGUAGACAACACGUGUGTT 773 CACACGUGUUGUCUACGGCTT 774 AD-
    2254 15180
    2237- CCGUAGACAACACGUGUGUTT 775 ACACACGUGUUGUCUACGGTT 776 AD-
    2255 15241
    2238- CGUAGACAACACGUGUGUATT 777 UACACACGUGUUGUCUACGTT 778 AD-
    2256 15268
    2240- UAGACAACACGUGUGUAGUTT 779 ACUACACACGUGUUGUCUATT 780 AD-
    2258 15242
    2241- AGACAACACGUGUGUAGUCTT 781 GACUACACACGUGUUGUCUTT 782 AD-
    2259 15216
    2242- GACAACACGUGUGUAGUCATT 783 UGACUACACACGUGUUGUCTT 784 AD-
    2260 15176
    2243- ACAACACGUGUGUAGUCAGTT 785 CUGACUACACACGUGUUGUTT 786 AD-
    2261 15181
    2244 CAACACGUGUGUAGUCAGGTT 787 CCUGACUACACACGUGUUGTT 788 AD-
    2262 15243
    2247- CACGUGUGUAGUCAGGAGCTT 789 GCUCCUGACUACACACGUGTT 790 AD-
    2265 15182
    2248- ACGUGUGUAGUCAGGAGCCTT 791 GGCUCCUGACUACACACGUTT 792 AD-
    2266 15244
    2249 CGUGUGUAGUCAGGAGCCGTT 793 CGGCUCCUGACUACACACGTT 794 AD-
    2267 15387
    2251- UGUGUAGUCAGGAGCCGGGTT 795 CCCGGCUCCUGACUACACATT 796 AD-
    2269 15245
    2257- GUCAGGAGCCGGGACGUCATsT 797 UGACGUCCCGGCUCCUGACTsT 798 AD-
    2275 9555
    2257- GucAGGAGccGGGAcGucATsT 799 UGACGUCCCGGCUCCUGACTsT 800 AD-
    2275 9681
    2258- UCAGGAGCCGGGACGUCAGTsT 801 CUGACGUCCCGGCUCCUGATsT 802 AD-
    2276 9619
    2258- ucAGGAGccGGGAcGucAGTsT 803 CUGACGUCCCGGCUCCUGATsT 804 AD-
    2276 9745
    2259- CAGGAGCCGGGACGUCAGCTsT 805 GCUGACGUCCCGGCUCCUGTsT 806 AD-
    2277 9620
    2259- cAGGAGccGGGAcGucAGcTsT 807 GCUGACGUCCCGGCUCCUGTsT 808 AD-
    2277 9746
    2263- AGCCGGGACGUCAGCACUATT 809 UAGUGCUGACGUCCCGGCUTT 810 AD-
    2281 15288
    2265- CCGGGACGUCAGCACUACATT 811 UGUAGUGCUGACGUCCCGGTT 812 AD-
    2283 15246
    2303- CCGUGACAGCCGUUGCCAUTT 813 AUGGCAACGGCUGUCACGGTT 814 AD-
    2321 15289
    2317- GCCAUCUGCUGCCGGAGCCTsT 815 GGCUCCGGCAGCAGAUGGCTsT 816 AD-
    2335 9324
    2375- CCCAUCCCAGGAUGGGUGUTT 817 ACACCCAUCCUGGGAUGGGTT 818 AD-
    2393 15329
    2377- CAUCCCAGGAUGGGUGUCUTT 819 AGACACCCAUCCUGGGAUGTT 820 AD-
    2395 15330
    2420- AGCUUUAAAAUGGUUCCGATT 821 UCGGAACCAUUUUAAAGCUTT 822 AD-
    2438 15169
    2421- GCUUUAAAAUGGUUCCGACTT 823 GUCGGAACCAUUUUAAAGCTT 824 AD-
    2439 15201
    2422- CUUUAAAAUGGUUCCGACUTT 825 AGUCGGAACCAUUUUAAAGTT 826 AD-
    2440 15331
    2423- UUUAAAAUGGUUCCGACUUTT 827 AAGUCGGAACCAUUUUAAATT 828 AD-
    2441 15190
    2424- UUAAAAUGGUUCCGACUUGTT 829 CAAGUCGGAACCAUUUUAATT 830 AD-
    2442 15247
    2425- UAAAAUGGUUCCGACUUGUTT 831 ACAAGUCGGAACCAUUUUATT 832 AD-
    2443 15248
    2426- AAAAUGGUUCCGACUUGUCTT 833 GACAAGUCGGAACCAUUUUTT 834 AD-
    2444 15175
    2427- AAAUGGUUCCGACUUGUCCTT 835 GGACAAGUCGGAACCAUUUTT 836 AD-
    2445 15249
    2428- AAUGGUUCCGACUUGUCCCTT 837 GGGACAAGUCGGAACCAUUTT 838 AD-
    2446 15250
    2431- GGUUCCGACUUGUCCCUCUTT 839 AGAGGGACAAGUCGGAACCTT 840 AD-
    2449 15400
    2457- CUCCAUGGCCUGGCACGAGTT 841 CUCGUGCCAGGCCAUGGAGTT 842 AD-
    2475 15332
    2459- CCAUGGCCUGGCACGAGGGTT 843 CCCUCGUGCCAGGCCAUGGTT 844 AD-
    2477 15388
    2545- GAACUCACUCACUCUGGGUTT 845 ACCCAGAGUGAGUGAGUUCTT 846 AD-
    2563 15333
    2549- UCACUCACUCUGGGUGCCUTT 847 AGGCACCCAGAGUGAGUGATT 848 AD-
    2567 15334
    2616- UUUCACCAUUCAAACAGGUTT 849 ACCUGUUUGAAUGGUGAAATT 850 AD-
    2634 15335
    2622- CAUUCAAACAGGUCGAGCUTT 851 AGCUCGACCUGUUUGAAUGTT 852 AD-
    2640 15183
    2623- AUUCAAACAGGUCGAGCUGTT 853 CAGCUCGACCUGUUUGAAUTT 854 AD-
    2641 15202
    2624- UUCAAACAGGUCGAGCUGUTT 855 ACAGCUCGACCUGUUUGAATT 856 AD-
    2642 15203
    2625- UCAAACAGGUCGAGCUGUGTT 857 CACAGCUCGACCUGUUUGATT 858 AD-
    2643 15272
    2626- CAAACAGGUCGAGCUGUGCTT 859 GCACAGCUCGACCUGUUUGTT 860 AD-
    2644 15217
    2627- AAACAGGUCGAGCUGUGCUTT 861 AGCACAGCUCGACCUGUUUTT 862 AD-
    2645 15290
    2628- AACAGGUCGAGCUGUGCUCTT 863 GAGCACAGCUCGACCUGUUTT 864 AD-
    2646 15218
    2630- CAGGUCGAGCUGUGCUCGGTT 865 CCGAGCACAGCUCGACCUGTT 866 AD-
    2648 15389
    2631- AGGUCGAGCUGUGCUCGGGTT 867 CCCGAGCACAGCUCGACCUTT 868 AD-
    2649 15336
    2633- GUCGAGCUGUGCUCGGGUGTT 869 CACCCGAGCACAGCUCGACTT 870 AD-
    2651 15337
    2634- UCGAGCUGUGCUCGGGUGCTT 871 GCACCCGAGCACAGCUCGATT 872 AD-
    2652 15191
    2657- AGCUGCUCCCAAUGUGCCGTT 873 CGGCACAUUGGGAGCAGCUTT 874 AD-
    2675 15390
    2658- GCUGCUCCCAAUGUGCCGATT 875 UCGGCACAUUGGGAGCAGCTT 876 AD-
    2676 15338
    2660- UGCUCCCAAUGUGCCGAUGTT 877 CAUCGGCACAUUGGGAGCATT 878 AD-
    2678 15204
    2663- UCCCAAUGUGCCGAUGUCCTT 879 GGACAUCGGCACAUUGGGATT 880 AD-
    2681 15251
    2665- CCAAUGUGCCGAUGUCCGUTT 881 ACGGACAUCGGCACAUUGGTT 882 AD-
    2683 15205
    2666- CAAUGUGCCGAUGUCCGUGTT 883 CACGGACAUCGGCACAUUGTT 884 AD-
    2684 15171
    2667- AAUGUGCCGAUGUCCGUGGTT 885 CCACGGACAUCGGCACAUUTT 886 AD-
    2685 15252
    2673- CCGAUGUCCGUGGGCAGAATT 887 UUCUGCCCACGGACAUCGGTT 888 AD-
    2691 15339
    2675- GAUGUCCGUGGGCAGAAUGTT 889 CAUUCUGCCCACGGACAUCTT 890 AD-
    2693 15253
    2678- GUCCGUGGGCAGAAUGACUTT 891 AGUCAUUCUGCCCACGGACTT 892 AD-
    2696 15340
    2679- UCCGUGGGCAGAAUGACUUTT 893 AAGUCAUUCUGCCCACGGATT 894 AD-
    2697 15291
    2683- UGGGCAGAAUGACUUUUAUTT 895 AUAAAAGUCAUUCUGCCCATT 896 AD-
    2701 15341
    2694- ACUUUUAUUGAGCUCUUGUTT 897 ACAAGAGCUCAAUAAAAGUTT 898 AD-
    2712 15401
    2700- AUUGAGCUCUUGUUCCGUGTT 899 CACGGAACAAGAGCUCAAUTT 900 AD-
    2718 15342
    2704- AGCUCUUGUUCCGUGCCAGTT 901 CUGGCACGGAACAAGAGCUTT 902 AD-
    2722 15343
    2705- GCUCUUGUUCCGUGCCAGGTT 903 CCUGGCACGGAACAAGAGCTT 904 AD-
    2723 15292
    2710- UGUUCCGUGCCAGGCAUUCTT 905 GAAUGCCUGGCACGGAACATT 906 AD-
    2728 15344
    2711- GUUCCGUGCCAGGCAUUCATT 907 UGAAUGCCUGGCACGGAACTT 908 AD-
    2729 15254
    2712- UUCCGUGCCAGGCAUUCAATT 909 UUGAAUGCCUGGCACGGAATT 910 AD-
    2730 15345
    2715- CGUGCCAGGCAUUCAAUCCTT 911 GGAUUGAAUGCCUGGCACGTT 912 AD-
    2733 15206
    2716- GUGCCAGGCAUUCAAUCCUTT 913 AGGAUUGAAUGCCUGGCACTT 914 AD-
    2734 15346
    2728- CAAUCCUCAGGUCUCCACCTT 915 GGUGGAGACCUGAGGAUUGTT 916 AD-
    2746 15347
    2743- CACCAAGGAGGCAGGAUUCTsT 917 GAAUCCUGCCUCCUUGGUGTsT 918 AD-
    2761 9577
    2743- cAccAAGGAGGcAGGAuucTsT 919 GAAUCCUGCCUCCUUGGUGTsT 920 AD-
    2761 9703
    2743- CfaCfcAfaGfgAfgGfcAfgGfaUfuC 921 p- 922 AD-
    2761 fTsT gAfaUfcCfuGfcCfuCfcUfuGfgUfgTsT 14678
    2743- CfACfCfAAGGAGGCfAGGAUfUfCfTs 923 GAAUfCfCfUfGCfCfUfCfCfUfUfGGUfG 924 AD-
    2761 T TsT 14688
    2743- CaCcAaGgAgGcAgGaUuCTsT 925 p- 926 AD-
    2761 gAfaUfcCfuGfcCfuCfcUfuGfgUfgTsT 14698
    2743- CaCcAaGgAgGcAgGaUuCTsT 927 GAAUfCfCfUfGCfCfUfCfCfUfUfGGUfG 928 AD-
    2761 TsT 14708
    2743- CfaCfcAfaGfgAfgGfcAfgGfaUfuC 929 GAAUCcuGCcuCCUUGgugTsT 930 AD-
    2761 fTsT 14718
    2743- CfACfCfAAGGAGGCfAGGAUfUfCfTs 931 GAAUCcuGCcuCCUUGgugTsT 932 AD-
    2761 T 14728
    2743- CaCcAaGgAgGcAgGaUuCTsT 933 GAAUCcuGCcuCCUUGgugTsT 934 AD-
    2761 14738
    2743- GfgCfcUfgGfaGfuUfuAfuUfcGfgA 935 p- 936 AD-
    2761 fTsT uCfcGfaAfuAfaAfcUfcCfaGfgCfcTsT 15084
    2743- GGCfCfUfGGAGUfUfUfAUfUfCfGGA 937 UfCfCfGAAUfAAACfUfCfCfAGGCfCfTs 938 AD-
    2761 TsT T 15094
    2743- GgCcUgGaGuUuAuUcGgATsT 939 p- 940 AD-
    2761 uCfcGfaAfuAfaAfcUfcCfaGfgCfcTsT 15104
    2743- GgCcUgGaGuUuAuUcGgATsT 941 UfCfCfGAAUfAAACfUfCfCfAGGCfCfTs 942 AD-
    2761 T 15114
    2743- GfgCfcUfgGfaGfuUfuAfuUfcGfgA 943 UCCGAauAAacUCCAGgccTsT 944 AD-
    2761 fTsT 15124
    2743- GGCfCfUfGGAGUfUfUfAUfUfCfGGA 945 UCCGAauAAacUCCAGgccTsT 946 AD-
    2761 TsT 15134
    2743- Gg CcUgGaGuUuAuUcGgATsT 947 UCCGAauAAacUCCAGgccTsT 948 AD-
    2761 15144
    2753- GCAGGAUUCUUCCCAUGGATT 949 UCCAUGGGAAGAAUCCUGCTT 950 AD-
    2771 15391
    2794- UGCAGGGACAAACAUCGUUTT 951 AACGAUGUUUGUCCCUGCATT 952 AD-
    2812 15348
    2795- GCAGGGACAAACAUCGUUGTT 953 CAACGAUGUUUGUCCCUGCTT 954 AD-
    2813 15349
    2797- AGGGACAAACAUCGUUGGGTT 955 CCCAACGAUGUUUGUCCCUTT 956 AD-
    2815 15170
    2841- CCCUCAUCUCCAGCUAACUTT 957 AGUUAGCUGGAGAUGAGGGTT 958 AD-
    2859 15350
    2845- CAUCUCCAGCUAACUGUGGTT 959 CCACAGUUAGCUGGAGAUGTT 960 AD-
    2863 15402
    2878- GCUCCCUGAUUAAUGGAGGTT 961 CCUCCAUUAAUCAGGGAGCTT 962 AD-
    2896 15293
    2881- CCCUGAUUAAUGGAGGCUUTT 963 AAGCCUCCAUUAAUCAGGGTT 964 AD-
    2899 15351
    2882- CCUGAUUAAUGGAGGCUUATT 965 UAAGCCUCCAUUAAUCAGGTT 966 AD-
    2900 15403
    2884- UGAUUAAUGGAGGCUUAGCTT 967 GCUAAGCCUCCAUUAAUCATT 968 AD-
    2902 15404
    2885- GAUUAAUGGAGGCUUAGCUTT 969 AGCUAAGCCUCCAUUAAUCTT 970 AD-
    2903 15207
    2886- AUUAAUGGAGGCUUAGCUUTT 971 AAGCUAAGCCUCCAUUAAUTT 972 AD-
    2904 15352
    2887- UUAAUGGAGGCUUAGCUUUTT 973 AAAGCUAAGCCUCCAUUAATT 974 AD-
    2905 15255
    2903- UUUCUGGAUGGCAUCUAGCTsT 975 GCUAGAUGCCAUCCAGAAATsT 976 AD-
    2921 9603
    2903- uuucuGGAuGGcAucuAGcTsT 977 GCuAGAUGCcAUCcAGAAATsT 978 AD-
    2921 9729
    2904- UUCUGGAUGGCAUCUAGCCTsT 979 GGCUAGAUGCCAUCCAGAATsT 980 AD-
    2922 9599
    2904- uucuGGAuGGcAucuAGccTsT 981 GGCuAGAUGCcAUCcAGAATsT 982 AD-
    2922 9725
    2905- UCUGGAUGGCAUCUAGCCATsT 983 UGGCUAGAUGCCAUCCAGATsT 984 AD-
    2923 9621
    2905- ucuGGAuGGcAucuAGccATsT 985 UGGCuAGAUGCcAUCcAGATsT 986 AD-
    2923 9747
    2925- AGGCUGGAGACAGGUGCGCTT 987 GCGCACCUGUCUCCAGCCUTT 988 AD-
    2943 15405
    2926- GGCUGGAGACAGGUGCGCCTT 989 GGCGCACCUGUCUCCAGCCTT 990 AD-
    2944 15353
    2927- GCUGGAGACAGGUGCGCCCTT 991 GGGCGCACCUGUCUCCAGCTT 992 AD-
    2945 15354
    2972- UUCCUGAGCCACCUUUACUTT 993 AGUAAAGGUGGCUCAGGAATT 994 AD-
    2990 15406
    2973- UCCUGAGCCACCUUUACUCTT 995 GAGUAAAGGUGGCUCAGGATT 996 AD-
    2991 15407
    2974- CCUGAGCCACCUUUACUCUTT 997 AGAGUAAAGGUGGCUCAGGTT 998 AD-
    2992 15355
    2976- UGAGCCACCUUUACUCUGCTT 999 GCAGAGUAAAGGUGGCUCATT 1000 AD-
    2994 15356
    2978- AGCCACCUUUACUCUGCUCTT 1001 GAGCAGAGUAAAGGUGGCUTT 1002 AD-
    2996 15357
    2981- CACCUUUACUCUGCUCUAUTT 1003 AUAGAGCAGAGUAAAGGUGTT 1004 AD-
    2999 15269
    2987- UACUCUGCUCUAUGCCAGGTsT 1005 CCUGGCAUAGAGCAGAGUATsT 1006 AD-
    3005 9565
    2987- uAcucuGcucuAuGccAGGTsT 1007 CCUGGcAuAGAGcAGAGuATsT 1008 AD-
    3005 9691
    2998- AUGCCAGGCUGUGCUAGCATT 1009 UGCUAGCACAGCCUGGCAUTT 1010 AD-
    3016 15358
    3003- AGGCUGUGCUAGCAACACCTT 1011 GGUGUUGCUAGCACAGCCUTT 1012 AD-
    3021 15359
    3006- CUGUGCUAGCAACACCCAATT 1013 UUGGGUGUUGCUAGCACAGTT 1014 AD-
    3024 15360
    3010- GCUAGCAACACCCAAAGGUTT 1015 ACCUUUGGGUGUUGCUAGCTT 1016 AD-
    3028 15219
    3038- GGAGCCAUCACCUAGGACUTT 1017 AGUCCUAGGUGAUGGCUCCTT 1018 AD-
    3056 15361
    3046- CACCUAGGACUGACUCGGCTT 1019 GCCGAGUCAGUCCUAGGUGTT 1020 AD-
    3064 15273
    3051- AGGACUGACUCGGCAGUGUTT 1021 ACACUGCCGAGUCAGUCCUTT 1022 AD-
    3069 15362
    3052- GGACUGACUCGGCAGUGUGTT 1023 CACACUGCCGAGUCAGUCCTT 1024 AD-
    3070 15192
    3074- UGGUGCAUGCACUGUCUCATT 1025 UGAGACAGUGCAUGCACCATT 1026 AD-
    3092 15256
    3080- AUGCACUGUCUCAGCCAACTT 1027 GUUGGCUGAGACAGUGCAUTT 1028 AD-
    3098 15363
    3085- CUGUCUCAGCCAACCCGCUTT 1029 AGCGGGUUGGCUGAGACAGTT 1030 AD-
    3103 15364
    3089- CUCAGCCAACCCGCUCCACTsT 1031 GUGGAGCGGGUUGGCUGAGTsT 1032 AD-
    3107 9604
    3089- cucAGccAAcccGcuccAcTsT 1033 GUGGAGCGGGUUGGCUGAGTsT 1034 AD-
    3107 9730
    3093- GCCAACCCGCUCCACUACCTsT 1035 GGUAGUGGAGCGGGUUGGCTsT 1036 AD-
    3111 9527
    3093- GccAAcccGcuccAcuAccTsT 1037 GGuAGUGGAGCGGGUUGGCTsT 1038 AD-
    3111 9653
    3096- AACCCGCUCCACUACCCGGTT 1039 CCGGGUAGUGGAGCGGGUUTT 1040 AD-
    3114 15365
    3099- CCGCUCCACUACCCGGCAGTT 1041 CUGCCGGGUAGUGGAGCGGTT 1042 AD-
    3117 15294
    3107- CUACCCGGCAGGGUACACATT 1043 UGUGUACCCUGCCGGGUAGTT 1044 AD-
    3125 15173
    3108- UACCCGGCAGGGUACACAUTT 1045 AUGUGUACCCUGCCGGGUATT 1046 AD-
    3126 15366
    3109- ACCCGGCAGGGUACACAUUTT 1047 AAUGUGUACCCUGCCGGGUTT 1048 AD-
    3127 15367
    3110- CCCGGCAGGGUACACAUUCTT 1049 GAAUGUGUACCCUGCCGGGTT 1050 AD-
    3128 15257
    3112- CGGCAGGGUACACAUUCGCTT 1051 GCGAAUGUGUACCCUGCCGTT 1052 AD-
    3130 15184
    3114- GCAGGGUACACAUUCGCACTT 1053 GUGCGAAUGUGUACCCUGCTT 1054 AD-
    3132 15185
    3115- CAGGGUACACAUUCGCACCTT 1055 GGUGCGAAUGUGUACCCUGTT 1056 AD-
    3133 15258
    3116- AGGGUACACAUUCGCACCCTT 1057 GGGUGCGAAUGUGUACCCUTT 1058 AD-
    3134 15186
    3196- GGAACUGAGCCAGAAACGCTT 1059 GCGUUUCUGGCUCAGUUCCTT 1060 AD-
    3214 15274
    3197- GAACUGAGCCAGAAACGCATT 1061 UGCGUUUCUGGCUCAGUUCTT 1062 AD-
    3215 15368
    3198- AACUGAGCCAGAAACGCAGTT 1063 CUGCGUUUCUGGCUCAGUUTT 1064 AD-
    3216 15369
    3201- UGAGCCAGAAACGCAGAUUTT 1065 AAUCUGCGUUUCUGGCUCATT 1066 AD-
    3219 15370
    3207- AGAAACGCAGAUUGGGCUGTT 1067 CAGCCCAAUCUGCGUUUCUTT 1068 AD-
    3225 15259
    3210- AACGCAGAUUGGGCUGGCUTT 1069 AGCCAGCCCAAUCUGCGUUTT 1070 AD-
    3228 15408
    3233- AGCCAAGCCUCUUCUUACUTsT 1071 AGUAAGAAGAGGCUUGGCUTsT 1072 AD-
    3251 9597
    3233- AGccAAGccucuucuuAcuTsT 1073 AGuAAGAAGAGGCUUGGCUTsT 1074 AD-
    3251 9723
    3233- AfgCfcAfaGfcCfuCfuUfcUfuAfcU 1075 p- 1076 AD-
    3251 fTsT aGfuAfaGfaAfgAfgGfcUfuGfgCfuTsT 14680
    3233- AGCfCfAAGCfCfUfCfUfUfCfUfUfA 1077 AGUfAAGAAGAGGCfUfUfGGCfUfTsT 1078 AD-
    3251 CfUfTsT 14690
    3233- AgCcAaGcCuCuUcUuAcUTsT 1079 p- 1080 AD-
    3251 aGfuAfaGfaAfgAfgGfcUfuGfgCfuTsT 14700
    3233- AgCcAaGcCuCuUcUuAcUTsT 1081 AGUfAAGAAGAGGCfUfUfGGCfUfTsT 1082 AD-
    3251 14710
    3233- AfgCfcAfaGfcCfuCfuUfcUfuAfcU 1083 AGUAAgaAGagGCUUGgcuTsT 1084 AD-
    3251 fTsT 14720
    3233- AGCfCfAAGCfCfUfCfUfUfCfUfUfA 1085 AGUAAgaAGagGCUUGgcuTsT 1086 AD-
    3251 CfUfTsT 14730
    3233- AgCcAaGcCuCuUcUuAcUTsT 1087 AGUAAgaAGagGCUUGgcuTsT 1088 AD-
    3251 14740
    3233- UfgGfuUfcCfcUfgAfgGfaCfcAfgC 1089 p- 1090 AD-
    3251 fTsT gCfuGfgUfcCfuCfaGfgGfaAfcCfaTsT 15086
    3233- UfGGUfUfCfCfCfUfGAGGACfCfAGC 1091 GCfUfGGUfCfCfUfCfAGGGAACfCfATsT 1092 AD-
    3251 fTsT 15096
    3233- UgGuUcCcUgAgGaCcAgCTsT 1093 p- 1094 AD-
    3251 gCfuGfgUfcCfuCfaGfgGfaAfcCfaTsT 15106
    3233- UgGuUcCcUgAgGaCcAgCTsT 1095 GCfUfGGUfCfCfUfCfAGGGAACfCfATsT 1096 AD-
    3251 15116
    3233- UfgGfuUfcCfcUfgAfgGfaCfcAfgC 1097 GCUGGucCUcaGGGAAccaTsT 1098 AD-
    3251 fTsT 15126
    3233- UfGGUfUfCfCfCfUfGAGGACfCfAGC 1099 GCUGGucCUcaGGGAAccaTsT 1100 AD-
    3251 fTsT 15136
    3233- UgGuUcCcUgAgGaCcAgCTsT 1101 GCUGGucCUcaGGGAAccaTsT 1102 AD-
    3251 15146
    3242- UCUUCUUACUUCACCCGGCTT 1103 GCCGGGUGAAGUAAGAAGATT 1104 AD-
    3260 15260
    3243- CUUCUUACUUCACCCGGCUTT 1105 AGCCGGGUGAAGUAAGAAGTT 1106 AD-
    3261 15371
    3244- UUCUUACUUCACCCGGCUGTT 1107 CAGCCGGGUGAAGUAAGAATT 1108 AD-
    3262 15372
    3262- GGGCUCCUCAUUUUUACGGTT 1109 CCGUAAAAAUGAGGAGCCCTT 1110 AD-
    3280 15172
    3263- GGCUCCUCAUUUUUACGGGTT 1111 CCCGUAAAAAUGAGGAGCCTT 1112 AD-
    3281 15295
    3264- GCUCCUCAUUUUUACGGGUTT 1113 ACCCGUAAAAAUGAGGAGCTT 1114 AD-
    3282 15373
    3265- CUCCUCAUUUUUACGGGUATT 1115 UACCCGUAAAAAUGAGGAGTT 1116 AD-
    3283 15163
    3266- UCCUCAUUUUUACGGGUAATT 1117 UUACCCGUAAAAAUGAGGATT 1118 AD-
    3284 15165
    3267- CCUCAUUUUUACGGGUAACTT 1119 GUUACCCGUAAAAAUGAGGTT 1120 AD-
    3285 15374
    3268- CUCAUUUUUACGGGUAACATT 1121 UGUUACCCGUAAAAAUGAGTT 1122 AD-
    3286 15296
    3270- CAUUUUUACGGGUAACAGUTT 1123 ACUGUUACCCGUAAAAAUGTT 1124 AD-
    3288 15261
    3271- AUUUUUACGGGUAACAGUGTT 1125 CACUGUUACCCGUAAAAAUTT 1126 AD-
    3289 15375
    3274- UUUACGGGUAACAGUGAGGTT 1127 CCUCACUGUUACCCGUAAATT 1128 AD-
    3292 15262
    3308- CAGACCAGGAAGCUCGGUGTT 1129 CACCGAGCUUCCUGGUCUGTT 1130 AD-
    3326 15376
    3310- GACCAGGAAGCUCGGUGAGTT 1131 CUCACCGAGCUUCCUGGUCTT 1132 AD-
    3328 15377
    3312- CCAGGAAGCUCGGUGAGUGTT 1133 CACUCACCGAGCUUCCUGGTT 1134 AD-
    3330 15409
    3315- GGAAGCUCGGUGAGUGAUGTT 1135 CAUCACUCACCGAGCUUCCTT 1136 AD-
    3333 15378
    3324- GUGAGUGAUGGCAGAACGATT 1137 UCGUUCUGCCAUCACUCACTT 1138 AD-
    3342 15410
    3326- GAGUGAUGGCAGAACGAUGTT 1139 CAUCGUUCUGCCAUCACUCTT 1140 AD-
    3344 15379
    3330- GAUGGCAGAACGAUGCCUGTT 1141 CAGGCAUCGUUCUGCCAUCTT 1142 AD-
    3348 15187
    3336- AGAACGAUGCCUGCAGGCATT 1143 UGCCUGCAGGCAUCGUUCUTT 1144 AD-
    3354 15263
    3339- ACGAUGCCUGCAGGCAUGGTT 1145 CCAUGCCUGCAGGCAUCGUTT 1146 AD-
    3357 15264
    3348- GCAGGCAUGGAACUUUUUCTT 1147 GAAAAAGUUCCAUGCCUGCTT 1148 AD-
    3366 15297
    3356- GGAACUUUUUCCGUUAUCATT 1149 UGAUAACGGAAAAAGUUCCTT 1150 AD-
    3374 15208
    3357- GAACUUUUUCCGUUAUCACTT 1151 GUGAUAACGGAAAAAGUUCTT 1152 AD-
    3375 15209
    3358- AACUUUUUCCGUUAUCACCTT 1153 GGUGAUAACGGAAAAAGUUTT 1154 AD-
    3376 15193
    3370- UAUCACCCAGGCCUGAUUCTT 1155 GAAUCAGGCCUGGGUGAUATT 1156 AD-
    3388 15380
    3378- AGGCCUGAUUCACUGGCCUTT 1157 AGGCCAGUGAAUCAGGCCUTT 1158 AD-
    3396 15298
    3383- UGAUUCACUGGCCUGGCGGTT 1159 CCGCCAGGCCAGUGAAUCATT 1160 AD-
    3401 15299
    3385- AUUCACUGGCCUGGCGGAGTT 1161 CUCCGCCAGGCCAGUGAAUTT 1162 AD-
    3403 15265
    3406- GCUUCUAAGGCAUGGUCGGTT 1163 CCGACCAUGCCUUAGAAGCTT 1164 AD-
    3424 15381
    3407- CUUCUAAGGCAUGGUCGGGTT 1165 CCCGACCAUGCCUUAGAAGTT 1166 AD-
    3425 15210
    3429- GAGGGCCAACAACUGUCCCTT 1167 GGGACAGUUGUUGGCCCUCTT 1168 AD-
    3447 15270
    3440- ACUGUCCCUCCUUGAGCACTsT 1169 GUGCUCAAGGAGGGACAGUTsT 1170 AD-
    3458 9591
    3440- AcuGucccuccuuGAGcAcTsT 1171 GUGCUcAAGGAGGGAcAGUTsT 1172 AD-
    3458 9717
    3441- CUGUCCCUCCUUGAGCACCTsT 1173 GGUGCUCAAGGAGGGACAGTsT 1174 AD-
    3459 9622
    3441- cuGucccuccuuGAGcAccTsT 1175 GGUGCUcAAGGAGGGAcAGTsT 1176 AD-
    3459 9748
    3480- ACAUUUAUCUUUUGGGUCUTsT 1177 AGACCCAAAAGAUAAAUGUTsT 1178 AD-
    3498 9587
    3480- AcAuuuAucuuuuGGGucuTsT 1179 AGACCcAAAAGAuAAAUGUTsT 1180 AD-
    3498 9713
    3480- AfcAfuUfuAfuCfuUfuUfgGfgUfcU 1181 p- 1182 AD-
    3498 fTsT aGfaCfcCfaAfaAfgAfuAfaAfuGfuTsT 14679
    3480- ACfAUfUfUfAUfCfUfUfUfUfGGGUf 1183 AGACfCfCfAAAAGAUfAAAUfGUfTsT 1184 AD-
    3498 CfUfTsT 14689
    3480- 1185 p- 1186 AD-
    3498 AcAuUuAuCuUuUgGgUcUTsT aGfaCfcCfaAfaAfgAfuAfaAfuGfuTsT 14699
    3480- AcAuUuAuCuUuUgGgUcUTsT 1187 AGACfCfCfAAAAGAUfAAAUfGUfTsT 1188 AD-
    3498 14709
    3480- AfcAfuUfuAfuCfuUfuUfgGfgUfcU 1189 AGACCcaAAagAUAAAuguTsT 1190 AD-
    3498 fTsT 14719
    3480- ACfAUfUfUfAUfCfUfUfUfUfGGGUf 1191 AGACCcaAAagAUAAAuguTsT 1192 AD-
    3498 CfUfTsT 14729
    3480- AcAuUuAuCuUuUgGgUcUTsT 1193 AGACCcaAAagAUAAAuguTsT 1194 AD-
    3498 14739
    3480- GfcCfaUfcUfgCfuGfcCfgGfaGfcC 1195 p- 1196 AD-
    3498 fTsT gGfcUfcCfgGfcAfgCfaGfaUfgGfcTsT 15085
    3480- GCfCfAUfCfUfGCfUfGCfCfGGAGCf 1197 GGCfUfCfCfGGCfAGCfAGAUfGGCfTsT 1198 AD-
    3498 CfTsT 15095
    3480- GcCaUcUgCuGcCgGaGcCTsT 1199 p- 1200 AD-
    3498 gGfcUfcCfgGfcAfgCfaGfaUfgGfcTsT 15105
    3480- GcCaUcUgCuGcCgGaGcCTsT 1201 GGCfUfCfCfGGCfAGCfAGAUfGGCfTsT 1202 AD-
    3498 15115
    3480- GfcCfaUfcUfgCfuGfcCfgGfaGfcC 1203 GGCUCauGCagCAGAUggcTsT 1204 AD-
    3498 fTsT 15125
    3480- GCfCfAUfCfUfGCfUfGCfCfGGAGCf 1205 GGCUCauGCagCAGAUggcTsT 1206 AD-
    3498 CfTsT 15135
    3480- GcCaUcUgCuGcCgGaGcCTsT 1207 GGCUCauGCagCAGAUggcTsT 1208 AD-
    3498 15145
    3481- CAUUUAUCUUUUGGGUCUGTsT 1209 CAGACCCAAAAGAUAAAUGTsT 1210 AD-
    3499 9578
    3481- cAuuuAucuuuuGGGucuGTsT 1211 cAGACCcAAAAGAuAAAUGTsT 1212 AD-
    3499 9704
    3485- UAUCUUUUGGGUCUGUCCUTsT 1213 AGGACAGACCCAAAAGAUATsT 1214 AD-
    3503 9558
    3485- uAucuuuuGGGucuGuccuTsT 1215 AGGAcAGACCcAAAAGAuATsT 1216 AD-
    3503 9684
    3504- CUCUGUUGCCUUUUUACAGTsT 1217 CUGUAAAAAGGCAACAGAGTsT 1218 AD-
    3522 9634
    3504- cucuGuuGccuuuuuAcAGTsT 1219 CUGuAAAAAGGcAAcAGAGTsT 1220 AD-
    3522 9760
    3512- CCUUUUUACAGCCAACUUUTT 1221 AAAGUUGGCUGUAAAAAGGTT 1222 AD-
    3530 15411
    3521- AGCCAACUUUUCUAGACCUTT 1223 AGGUCUAGAAAAGUUGGCUTT 1224 AD-
    3539 15266
    3526- ACUUUUCUAGACCUGUUUUTT 1225 AAAACAGGUCUAGAAAAGUTT 1226 AD-
    3544 15382
    3530- UUCUAGACCUGUUUUGCUUTsT 1227 AAGCAAAACAGGUCUAGAATsT 1228 AD-
    3548 9554
    3530- uucuAGAccuGuuuuGcuuTsT 1229 AAGcAAAAcAGGUCuAGAATsT 1230 AD-
    3548 9680
    3530- UfuCfuAfgAfcCfuGfuUfuUfgCfuU 1231 p- 1232 AD-
    3548 fTsT aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT 14676
    3530- UfUfCfUfAGACfCfUfGUfUfUfUfGC 1233 AAGCfAAAACfAGGUfCfUfAGAATsT 1234 AD-
    3548 fUfUfTsT 14686
    3530- UuCuAgAcCuGuUuUgCuUTsT 1235 p- 1236 AD-
    3548 aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT 14696
    3530- UuCuAgAcCuGuUuUgCuUTsT 1237 AAGCfAAAACfAGGUfCfUfAGAATsT 1238 AD-
    3548 14706
    3530- UfuCfuAfgAfcCfuGfuUfuUffCfuU 1239 AAGcAaaACagGUCUAgaaTsT 1240 AD-
    3548 fTsT 14716
    3530- UfUfCfUfAGACfCfUfGUfUfUfUfGC 1241 AAGcAaaACagGUCUAgaaTsT 1242 AD-
    3548 fUfUfTsT 14726
    3530- UuCuAgAcCuGuUuUgCuUTsT 1243 AAGcAaaACagGUCUAgaaTsT 1244 AD-
    3548 14736
    3530- CfaUfaGfgCfcUfgGfaGfuUfuAfuU 1245 p- 1246 AD-
    3548 fTsT aAfuAfaAfcUfcCfaGfgCfcUfaUfgTsT 15082
    3530- CfAUfAGGCfCfUfGGAGUfUfUfAUfU 1247 AAUfAAACfUfCfCfAGGCfCfUfAUfGTsT 1248 AD-
    3548 fTsT 15092
    3530- CaUaGgCcUgGaGuUuAuUTsT 1249 p- 1250 AD-
    3548 aAfuAfaAfcUfcCfaGfgCfcUfaUfgTsT 15102
    3530- CaUaGgCcUgGaGuUuAuUTsT 1251 AAUfAAACfUfCfCfAGGCfCfUfAUfGTsT 1252 AD-
    3548 15112
    3530- CfaUfaGfgCfcUfgGfaGfuUfuAfuU 1253 AAUAAacUCcaGGCCUaugTsT 1254 AD-
    3548 fTsT 15122
    3530- CfAUfAGGCfCfUfGGAGUfUfUfAUfU 1255 AAUAAacUCcaGGCCUaugTsT 1256 AD-
    3548 fTsT 15132
    3530- CaUaGgCcUgGaGuUuAuUTsT 1257 AAUAAacUCcaGGCCUaugTsT 1258 AD-
    3548 15142
    3531- UCUAGACCUGUUUUGCUUUTsT 1259 AAAGCAAAACAGGUCUAGATsT 1260 AD-
    3549 9553
    3531- ucuAGAccuGuuuuGcuuuTsT 1261 AAAGcAAAAcAGGUCuAGATsT 1262 AD-
    3549 9679
    3531- UfcUfaGfaCfcUfgUfuUfuGfcUfuU 1263 p- 1264 AD-
    3549 fTsT aAfaGfcAfaAfaCfaGfgUfcUfaGfaTsT 14675
    3531- UfCfUfAGACfCfUfGUfUfUfUfGCfU 1265 AAAGCfAAAACfAGGUfCfUfAGATsT 1266 AD-
    3549 fUfUfTsT 14685
    3531- UcUaGaCcUgUuUuGcUuUTsT 1267 p- 1268 AD-
    3549 aAfaGfcAfaAfaCfaGfgUfcUfaGfaTsT 14695
    3531- UcUaGaCcUgUuUuGcUuUTsT 1269 AAAGCfAAAACfAGGUfCfUfAGATsT 1270 AD-
    3549 14705
    3531- UfcUfaGfaCfcUfgUfuUfuGfcUfuU 1271 AAAGCaaAAcaGGUCUagaTsT 1272 AD-
    3549 fTsT 14715
    3531- UfCfUfAGACfCfUfGUfUfUfUfGCfU 1273 AAAGCaaAAcaGGUCUagaTsT 1274 AD-
    3549 fUfUfTsT 14725
    3531- UcUaGaCcUgUuUuGcUuUTsT 1275 AAAGCaaAAcaGGUCUagaTsT 1276 AD-
    3549 14735
    3531- UfcAfuAfgGfcCfuGfgAfgUfuUfaU 1277 p- 1278 AD-
    3549 fTsT aUfaAfaCfuCfcAfgGfcCfuAfuGfaTsT 15081
    3531- UfCfAUfAGGCfCfUfGGAGUfUfUfAU 1279 AUfAAACfUfCfCfAGGCfCfUfAUfGATsT 1280 AD-
    3549 fTsT 15091
    3531- UcAuAgGcCuGgAgUuUaUTsT 1281 p- 1282 AD-
    3549 aUfaAfaCfuCfcAfgGfcCfuAfuGfaTsT 15101
    3531- UcAuAgGcCuGgAgUuUaUTsT 1283 AUfAAACfUfCfCfAGGCfCfUfAUfGATsT 1284 AD-
    3549 15111
    3531- UfcAfuAfgGfcCfuGfgAfgUfuUfaU 1285 AUAAAcuCCagGCCUAugaTsT 1286 AD-
    3549 fTsT 15121
    3531- UfCfAUfAGGCfCfUfGGAGUfUfUfAU 1287 AUAAAcuCCagGCCUAugaTsT 1288 AD-
    3549 fTsT 15131
    3531- UcAuAgGcCuGgAgUuUaUTsT 1289 AUAAAcuCCagGCCUAugaTsT 1290 AD-
    3549 15141
    3557- UGAAGAUAUUUAUUCUGGGTsT 1291 CCCAGAAUAAAUAUCUUCATsT 1292 AD-
    3575 9626
    3557- uGAAGAuAuuuAuucuGGGTsT 1293 CCcAGAAuAAAuAUCUUcATsT 1294 AD-
    3575 9752
    3570- UCUGGGUUUUGUAGCAUUUTsT 1295 AAAUGCUACAAAACCCAGATsT 1296 AD-
    3588 9629
    3570- ucuGGGuuuuGuAGcAuuuTsT 1297 AAAUGCuAcAAAACCcAGATsT 1298 AD-
    3588 9755
    3613- AUAAAAACAAACAAACGUUTT 1299 AACGUUUGUUUGUUUUUAUTT 1300 AD-
    3631 15412
    3617- AAACAAACAAACGUUGUCCTT 1301 GGACAACGUUUGUUUGUUUTT 1302 AD-
    3635 15211
    3618- AACAAACAAACGUUGUCCUTT 1303 AGGACAACGUUUGUUUGUUTT 1304 AD-
    3636 15300
    U, C, A, G: corresponding ribonucleotide; T: deoxythymidine; u, c, a, g: corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding 2′-deoxy-2′-fluoro ribonucleotide; where nucleotides are written in sequence, they are connected by 3′-5′ phosphodiester groups; nucleotides with interjected “s” are connected by 3′-O-5′-O phosphorothiodiester groups; unless denoted by prefix “p-”, oligonucleotides are devoid of a 5′-phosphate group on the 5′-most nucleotide; all oligonucleotides bear 3′-OH on the 3′-most nucleotide
  • TABLE 1b
    Screening of siRNAs targeted to PCSK9
    Mean percent remaining
    mRNA transcript at IC50 in
    siRNA concentration/in cell type Cynomolgous
    100 30 IC50 in monkey
    Duplex nM/ 30 nM/ 3 nM/ nM/ HepG2 Hepatocyte
    name HepG2 HepG2 HepG2 HeLa [nM] [nM]s
    AD-15220 35
    AD-15275 56
    AD-15301 70
    AD-15276 42
    AD-15302 32
    AD-15303 37
    AD-15221 30
    AD-15413 61
    AD-15304 70
    AD-15305 36
    AD-15306 20
    AD-15307 38
    AD-15277 50
    AD-9526 74 89
    AD-9652 97
    AD-9519 78
    AD-9645 66
    AD-9523 55
    AD-9649 60
    AD-9569 112
    AD-9695 102
    AD-15222 75
    AD-15278 78
    AD-15178 83
    AD-15308 84
    AD-15223 67
    AD-15309 34
    AD-15279 44
    AD-15194 63
    AD-15310 42
    AD-15311 30
    AD-15392 18
    AD-15312 21
    AD-15313 19
    AD-15280 81
    AD-15267 82
    AD-15314 32
    AD-15315 74
    AD-9624 94
    AD-9750 96
    AD-9623 43 66
    AD-9749 105
    AD-15384 48
    AD-9607 32 28 0.20
    AD-9733 78 73
    AD-9524 23 28 0.07
    AD-9650 91 90
    AD-9520 23 32
    AD-9520 23
    AD-9646 97 108
    AD-9608 37
    AD-9734 91
    AD-9546 32
    AD-9672 57
    AD-15385 54
    AD-15393 31
    AD-15316 37
    AD-15317 37
    AD-15318 63
    AD-15195 45
    AD-15224 57
    AD-15188 42
    AD-15225 51
    AD-15281 89
    AD-15282 75
    AD-15319 61
    AD-15226 56
    AD-15271 25
    AD-15283 25
    AD-15284 64
    AD-15189 17
    AD-15227 62
    AD-9547 31 29 0.20
    AD-9673 56 57
    AD-9548 54 60
    AD-9674 36 57
    AD-9529 60
    AD-9655 140
    AD-9605 27 31 0.27
    AD-9731 31 31 0.32
    AD-9596 37
    AD-9722 76
    AD-9583 42
    AD-9709 104
    AD-9579 113
    AD-9705 81
    AD-15394 32
    AD-15196 72
    AD-15197 85
    AD-15198 71
    AD-9609 66 71
    AD-9735 115
    AD-9537 145
    AD-9663 102
    AD-9528 113
    AD-9654 107
    AD-9515 49
    AD-9641 92
    AD-9514 57
    AD-9640 89
    AD-9530 75
    AD-9656 77
    AD-9538 79 80
    AD-9664 53
    AD-9598 69 83
    AD-9724 127
    AD-9625 58 88
    AD-9751 60
    AD-9556 46
    AD-9682 38
    AD-9539 56 63
    AD-9665 83
    AD-9517 36
    AD-9643 40
    AD-9610 36 34 0.04
    AD-9736 22 29 0.04 0.5
    AD-14681 33
    AD-14691 27
    AD-14701 32
    AD-14711 33
    AD-14721 22
    AD-14731 21
    AD-14741 22
    AD-15087 37
    AD-15097 51
    AD-15107 26
    AD-15117 28
    AD-15127 33
    AD-15137 54
    AD-15147 52
    AD-9516 94
    AD-9642 105
    AD-9562 46 51
    AD-9688 26 34 4.20
    AD-14677 38
    AD-14687 52
    AD-14697 35
    AD-14707 58
    AD-14717 42
    AD-14727 50
    AD-14737 32
    AD-15083 16
    AD-15093 24
    AD-15103 11
    AD-15113 34
    AD-15123 19
    AD-15133 15
    AD-15143 16
    AD-9521 50
    AD-9647 62
    AD-9611 48
    AD-9737 68
    AD-9592 46 55
    AD-9718 78
    AD-9561 64
    AD-9687 84
    AD-9636 42 41 2.10
    AD-9762 9 28 0.40 0.5
    AD-9540 45
    AD-9666 81
    AD-9535 48 73
    AD-9661 83
    AD-9559 35
    AD-9685 77
    AD-9533 100
    AD-9659 88
    AD-9612 122
    AD-9738 83
    AD-9557 75 96
    AD-9683 48
    AD-9531 31 32 0.53
    AD-9657 23 29 0.66 0.5
    AD-14673 81
    AD-14683 56
    AD-14693 56
    AD-14703 68
    AD-14713 55
    AD-14723 24
    AD-14733 34
    AD-15079 85
    AD-15089 54
    AD-15099 70
    AD-15109 67
    AD-15119 67
    AD-15129 57
    AD-15139 69
    AD-9542 160
    AD-9668 92
    AD-9739 109
    AD-9637 56 83
    AD-9763 79
    AD-9630 82
    AD-9756 63
    AD-9593 55
    AD-9719 115
    AD-9601 111
    AD-9727 118
    AD-9573 36 42 1.60
    AD-9699 32 36 2.50
    AD-15228 26
    AD-15395 53
    AD-9602 126
    AD-9728 94
    AD-15386 45
    AD-9580 112
    AD-9706 86
    AD-9581 35
    AD-9707 81
    AD-9543 51
    AD-9669 97
    AD-9574 74
    AD-9700
    AD-15320 26
    AD-15321 34
    AD-15199 64
    AD-15167 86
    AD-15164 41
    AD-15166 43
    AD-15322 64
    AD-15200 46
    AD-15213 27
    AD-15229 44
    AD-15215 49
    AD-15214 101
    AD-9315 15 32 0.98
    AD-9326 35 51
    AD-9318 14 37 0.40
    AD-9323 14 33
    AD-9314 11 22 0.04
    AD-10792 0.10 0.10
    AD-10796 0.1 0.1
    AD-9638 101
    AD-9764 112
    AD-9525 53
    AD-9651 58
    AD-9560 97
    AD-9686 111
    AD-9536 157
    AD-9662 81
    AD-9584 52 68
    AD-9710 111
    AD-15323 62
    AD-9551 91
    AD-9677 62
    AD-15230 52
    AD-15231 25
    AD-15285 36
    AD-15396 27
    AD-15397 56
    AD-9600 112
    AD-9726 95
    AD-9606 107
    AD-9732 105
    AD-9633 56 75
    AD-9759 111
    AD-9588 66
    AD-9714 106
    AD-9589 67 85
    AD-9715 113
    AD-9575 120
    AD-9701 100
    AD-9563 103
    AD-9689 81
    AD-9594 80 95
    AD-9720 92
    AD-9585 83
    AD-9711 122
    AD-9614 100
    AD-9740 198
    AD-9615 116
    AD-9741 130
    AD-9534 32 30
    AD-9534 32
    AD-9660 89 79
    AD-15324 46
    AD-15232 19
    AD-15233 25
    AD-15234 59
    AD-15286 109
    AD-9590 122
    AD-9716 114
    AD-9632 34
    AD-9758 96
    AD-9567 41
    AD-9693 50
    AD-9586 81 104
    AD-9712 107
    AD-9564 120
    AD-9690 92
    AD-9616 74 84
    AD-9742 127
    AD-15398 24
    AD-9617 111
    AD-9743 104
    AD-9635 73 90
    AD-9761 15 33 0.5
    AD-9568 76
    AD-9694 52
    AD-9576 47
    AD-9702 79
    AD-9627 69
    AD-9753 127
    AD-9628 141
    AD-9754 89
    AD-9631 80
    AD-9757 78
    AD-9595 31 32
    AD-9721 87 70
    AD-9544 68
    AD-9670 67
    AD-15235 25
    AD-15236 73
    AD-15168 100
    AD-15174 92
    AD-15325 81
    AD-15326 65
    AD-9570 35 42
    AD-9696 77
    AD-9566 38
    AD-9692 78
    AD-9532 100
    AD-9658 102
    AD-9549 50
    AD-9675 78
    AD-9541 43
    AD-9667 73
    AD-9550 36
    AD-9676 100
    AD-9571 27 32
    AD-9697 74 89
    AD-9572 47 53
    AD-9698 73
    AD-15327 82
    AD-9639 30 35
    AD-9765 82 74
    AD-9518 31 35 0.60
    AD-9518 31
    AD-9644 35 37 2.60 0.5
    AD-14672 26
    AD-14682 27
    AD-14692 22
    AD-14702 19
    AD-14712 25
    AD-14722 18
    AD-14732 32
    AD-15078 86
    AD-15088 97
    AD-15098 74
    AD-15108 67
    AD-15118 76
    AD-15128 86
    AD-15138 74
    AD-15237 30
    AD-15287 30
    AD-15238 36
    AD-15328 35
    AD-15399 47
    AD-9582 37
    AD-9708 81
    AD-9545 31 43
    AD-9671 15 33 2.50
    AD-14674 16
    AD-14684 26
    AD-14694 18
    AD-14704 27
    AD-14714 20
    AD-14724 18
    AD-14734 18
    AD-15080 29
    AD-15090 23
    AD-15100 26
    AD-15110 23
    AD-15120 20
    AD-15130 20
    AD-15140 19
    AD-9522 59
    AD-9648 78
    AD-9552 80
    AD-9678 76
    AD-9618 90
    AD-9744 91
    AD-15239 38
    AD-15212 19
    AD-15240 43
    AD-15177 59
    AD-15179 13
    AD-15180 15
    AD-15241 14
    AD-15268 42
    AD-15242 21
    AD-15216 28
    AD-15176 35
    AD-15181 35
    AD-15243 22
    AD-15182 42
    AD-15244 31
    AD-15387 23
    AD-15245 18
    AD-9555 34
    AD-9681 55
    AD-9619 42 61
    AD-9745 56
    AD-9620 44 77
    AD-9746 89
    AD-15288 19
    AD-15246 16
    AD-15289 37
    AD-9324 59 67
    AD-15329 103
    AD-15330 62
    AD-15169 22
    AD-15201 6
    AD-15331 14
    AD-15190 47
    AD-15247 61
    AD-15248 22
    AD-15175 45
    AD-15249 51
    AD-15250 96
    AD-15400 12
    AD-15332 22
    AD-15388 30
    AD-15333 20
    AD-15334 96
    AD-15335 75
    AD-15183 16
    AD-15202 41
    AD-15203 39
    AD-15272 49
    AD-15217 16
    AD-15290 15
    AD-15218 13
    AD-15389 13
    AD-15336 40
    AD-15337 19
    AD-15191 33
    AD-15390 25
    AD-15338 9
    AD-15204 33
    AD-15251 76
    AD-15205 14
    AD-15171 16
    AD-15252 58
    AD-15339 20
    AD-15253 15
    AD-15340 18
    AD-15291 17
    AD-15341 11
    AD-15401 13
    AD-15342 30
    AD-15343 21
    AD-15292 16
    AD-15344 20
    AD-15254 18
    AD-15345 18
    AD-15206 15
    AD-15346 16
    AD-15347 62
    AD-9577 33 31
    AD-9703 17 26 1
    AD-14678 22
    AD-14688 23
    AD-14698 23
    AD-14708 14
    AD-14718 31
    AD-14728 25
    AD-14738 31
    AD-15084 19
    AD-15094 11
    AD-15104 16
    AD-15114 15
    AD-15124 11
    AD-15134 12
    AD-15144 9
    AD-15391 7
    AD-15348 13
    AD-15349 8
    AD-15170 40
    AD-15350 14
    AD-15402 27
    AD-15293 27
    AD-15351 14
    AD-15403 11
    AD-15404 38
    AD-15207 15
    AD-15352 23
    AD-15255 31
    AD-9603 123
    AD-9729 56
    AD-9599 139
    AD-9725 38
    AD-9621 77
    AD-9747 63
    AD-15405 32
    AD-15353 39
    AD-15354 49
    AD-15406 35
    AD-15407 39
    AD-15355 18
    AD-15356 50
    AD-15357 54
    AD-15269 23
    AD-9565 74
    AD-9691 49
    AD-15358 12
    AD-15359 24
    AD-15360 13
    AD-15219 19
    AD-15361 24
    AD-15273 36
    AD-15362 31
    AD-15192 20
    AD-15256 19
    AD-15363 33
    AD-15364 24
    AD-9604 35 49
    AD-9730 85
    AD-9527 45
    AD-9653 86
    AD-15365 62
    AD-15294 30
    AD-15173 12
    AD-15366 21
    AD-15367 11
    AD-15257 18
    AD-15184 50
    AD-15185 12
    AD-15258 73
    AD-15186 36
    AD-15274 19
    AD-15368 7
    AD-15369 17
    AD-15370 19
    AD-15259 38
    AD-15408 52
    AD-9597 23 21 0.04
    AD-9723 12 26 0.5
    AD-14680 15
    AD-14690 18
    AD-14700 15
    AD-14710 15
    AD-14720 18
    AD-14730 18
    AD-14740 17
    AD-15086 85
    AD-15096 70
    AD-15106 71
    AD-15116 73
    AD-15126 71
    AD-15136 56
    AD-15146 72
    AD-15260 79
    AD-15371 24
    AD-15372 52
    AD-15172 27
    AD-15295 22
    AD-15373 11
    AD-15163 18
    AD-15165 13
    AD-15374 23
    AD-15296 13
    AD-15261 20
    AD-15375 90
    AD-15262 72
    AD-15376 14
    AD-15377 19
    AD-15409 17
    AD-15378 18
    AD-15410 8
    AD-15379 11
    AD-15187 36
    AD-15263 18
    AD-15264 75
    AD-15297 21
    AD-15208 6
    AD-15209 28
    AD-15193 131
    AD-15380 88
    AD-15298 43
    AD-15299 99
    AD-15265 95
    AD-15381 18
    AD-15210 40
    AD-15270 83
    AD-9591 75 95
    AD-9717 105
    AD-9622 94
    AD-9748 103
    AD-9587 63 49
    AD-9713 22 25 0.5
    AD-14679 19
    AD-14689 24
    AD-14699 19
    AD-14709 21
    AD-14719 24
    AD-14729 23
    AD-14739 24
    AD-15085 74
    AD-15095 60
    AD-15105 33
    AD-15115 30
    AD-15125 54
    AD-15135 51
    AD-15145 49
    AD-9578 49 61
    AD-9704 111
    AD-9558 66
    AD-9684 63
    AD-9634 29 30
    AD-9760 14 27
    AD-15411 5
    AD-15266 23
    AD-15382 12
    AD-9554 23 24
    AD-9680 12 22 0.1 0.1
    AD-14676 12 .1
    AD-14686 13
    AD-14696 12 .1
    AD-14706 18 .1
    AD-14716 17 .1
    AD-14726 16 .1
    AD-14736 9 .1
    AD-15082 27
    AD-15092 28
    AD-15102 19
    AD-15112 17
    AD-15122 56
    AD-15132 39
    AD-15142 46
    AD-9553 27 22 0.02
    AD-9679 17 21 0.1
    AD-14675 11
    AD-14685 19
    AD-14695 12
    AD-14705 16
    AD-14715 19
    AD-14725 19
    AD-14735 19
    AD-15081 30
    AD-15091 16
    AD-15101 16
    AD-15111 11
    AD-15121 19
    AD-15131 17
    AD-15141 18
    AD-9626 97 68
    AD-9752 28 33
    AD-9629 23 24
    AD-9755 28 29 0.5
    AD-15412 21
    AD-15211 73
    AD-15300 41
  • TABLE 2a
    Sequences of modified dsRNA targeted to PCSK9
    SEQ SEQ
    Duplex ID ID
    number Sense strand sequence (5′-3′)1 NO: Antisense-strand sequence (5′-3′)1 NO:
    AD-10792 GccuGGAGuuuAuucGGAATsT 1305 UUCCGAAuAAACUCcAGGCTsT 1306
    AD-10793 GccuGGAGuuuAuucGGAATsT 1307 uUcCGAAuAAACUccAGGCTsT 1308
    AD-10796 GccuGGAGuuuAuucGGAATsT 1309 UUCCGAAUAAACUCCAGGCTsT 1310
    AD-12038 GccuGGAGuuuAuucGGAATsT 1311 uUCCGAAUAAACUCCAGGCTsT 1312
    AD-12039 GccuGGAGuuuAuucGGAATsT 1313 UuCCGAAUAAACUCCAGGCTsT 1314
    AD-12040 GccuGGAGuuuAuucGGAATsT 1315 UUcCGAAUAAACUCCAGGCTsT 1316
    AD-12041 GccuGGAGuuuAuucGGAATsT 1317 UUCCGAAUAAACUCCAGGCTsT 1318
    AD-12042 GCCUGGAGUUUAUUCGGAATsT 1319 uUCCGAAUAAACUCCAGGCTsT 1320
    AD-12043 GCCUGGAGUUUAUUCGGAATsT 1321 UuCCGAAUAAACUCCAGGCTsT 1322
    AD-12044 GCCUGGAGUUUAUUCGGAATsT 1323 UUcCGAAUAAACUCCAGGCTsT 1324
    AD-12045 GCCUGGAGUUUAUUCGGAATsT 1325 UUCCGAAUAAACUCCAGGCTsT 1326
    AD-12046 GccuGGAGuuuAuucGGAA 1327 UUCCGAAUAAACUCCAGGCscsu 1328
    AD-12047 GccuGGAGuuuAuucGGAAA 1329 UUUCCGAAUAAACUCCAGGCscsu 1330
    AD-12048 GccuGGAGuuuAuucGGAAAA 1331 UUUUCCGAAUAAACUCCAGGCscsu 1332
    AD-12049 GccuGGAGuuuAuucGGAAAAG 1333 CUUUUCCGAAUAAACUCCAGGCscsu 1334
    AD-12050 GccuGGAGuuuAuucGGAATTab 1335 UUCCGAAUAAACUCCAGGCTTab 1336
    AD-12051 GccuGGAGuuuAuucGGAAATTab 1337 UUUCCGAAuAAACUCCAGGCTTab 1338
    AD-12052 GccuGGAGuuuAuucGGAAAATTab 1339 UUUUCCGAAUAAACUCCAGGCTTab 1340
    AD-12053 GccuGGAGuuuAuucGGAAAAGTTab 1341 CUUUUCCGAAUAAACUCCAGGCTTab 1342
    AD-12054 GCCUGGAGUUUAUUCGGAATsT 1343 UUCCGAAUAAACUCCAGGCscsu 1344
    AD-12055 GccuGGAGuuuAuucGGAATsT 1345 UUCCGAAUAAACUCCAGGCscsu 1346
    AD-12056 GcCuGgAgUuUaUuCgGaA 1347 UUCCGAAUAAACUCCAGGCTTab 1348
    AD-12057 GcCuGgAgUuUaUuCgGaA 1349 UUCCGAAUAAACUCCAGGCTsT 1350
    AD-12058 GcCuGgAgUuUaUuCgGaA 1351 UUCCGAAuAAACUCcAGGCTsT 1352
    AD-12059 GcCuGgAgUuUaUuCgGaA 1353 uUcCGAAuAAACUccAGGCTsT 1354
    AD-12060 GcCuGgAgUuUaUuCgGaA 1355 UUCCGaaUAaaCUCCAggc 1356
    AD-12061 GcCuGgnAgUuUaUuCgGaATsT 1357 UUCCGaaUAaaCUCCAggcTsT 1358
    AD-12062 GcCuGgAgUuUaUuCgGaATTab 1359 UUCCGaaUAaaCUCCAggcTTab 1360
    AD-12063 GcCuGgAgUuUaUuCgGaA 1361 UUCCGaaUAaaCUCCAggcscsu 1362
    AD-12064 GcCuGgnAgUuUaUuCgGaATsT 1363 UUCCGAAuAAACUCcAGGCTsT 1364
    AD-12065 GcCuGgAgUuUaUuCgGaATTab 1365 UUCCGAAuAAACUCcAGGCTTab 1366
    AD-12066 GcCuGgAgUuUaUuCgGaA 1367 UUCCGAAuAAACUCcAGGCscsu 1368
    AD-12067 GcCuGgnAgUuUaUuCgGaATsT 1369 UUCCGAAUAAACUCCAGGCTsT 1370
    AD-12068 GcCuGgAgUuUaUuCgGaATTab 1371 UUCCGAAUAAACUCCAGGCTTab 1372
    AD-12069 GcCuGgAgUuUaUuCgGaA 1373 UUCCGAAUAAACUCCAGGCscsu 1374
    AD-12338 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1375 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1376
    AD-12339 GcCuGgAgUuUaUuCgGaA 1377 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1378
    AD-12340 GccuGGAGuuuAuucGGAA 1379 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1380
    AD-12341 GfcCfuGfgAfgUfuUfaUfuCfgGfaAffsT 1381 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcTsT 1382
    AD-12342 GfcCfuGfgAfgUfuUfaUfuCfgGfaAffsT 1383 UUCCGAAuAAACUCcAGGCTsT 1384
    AD-12343 GfcCfuGfgAfgUfuUfaUfuCfgGfaAffsT 1385 uUcCGAAuAAACUccAGGCTsT 1386
    AD-12344 GfcCfuGfgAfgUfuUfaUfuCfgGfaAffsT 1387 UUCCGAAUAAACUCCAGGCTsT 1388
    AD-12345 GfcCfuGfgAfgUfuUfaUfuCfgGfaAffsT 1389 UUCCGAAUAAACUCCAGGCscsu 1390
    AD-12346 GfcCfuGfgAfgUfuUfaUfuCfgGfaAffsT 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
    AD-12357 GmocCmouGmogAm02gUmouUmoaUmouCm 1411 UUCCGaaUAaaCUCCAggc 1412
    ogGmoaA
    AD-12358 GmocCmouGmogAm02gUmouUmoaUmouCm 1413 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1414
    ogGmoaA
    AD-12359 GmocCmouGmogAm02gUmouUmoaUmouCm 1415 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcsCfsu 1416
    ogGmoaA
    AD-12360 GmocCmouGmogAm02gUmouUmoaUmouCm 1417 UUCCGAAUAAACUCCAGGCscsu 1418
    ogGmoaA
    AD-12361 GmocCmouGmogAm02gUmouUmoaUmouCm 1419 UUCCGAAuAAACUCcAGGCTsT 1420
    ogGmoaA
    AD-12362 GmocCmouGmogAm02gUmouUmoaUmouCm 1421 uUcCGAAuAAACUccAGGCTsT 1422
    ogGmoaA
    AD-12363 GmocCmouGmogAm02gUmouUmoaUmouCm 1423 UUCCGaaUAaaCUCCAggcscsu 1424
    ogGmoaA
    AD-12364 GmocCmouGmogAmogUmouUmoaUmouCmo 1425 UUCCGaaUAaaCUCCAggcTsT 1426
    gGmoaATsT
    AD-12365 GmocCmouGmogAmogUmouUmoaUmouCmo 1427 UUCCGAAuAAACUCcAGGCTsT 1428
    gGmoaATsT
    AD-12366 GmocCmouGmogAmogUmouUmoaUmouCmo 1429 UUCCGAAUAAACUCCAGGCTsT 1430
    gGmoaATsT
    AD-12367 GmocmocmouGGAGmoumoumouAmoumoum 1431 UUCCGaaUAaaCUCCAggcTsT 1432
    ocGGAATsT
    AD-12368 GmocmocmouGGAGmoumoumouAmoumoum 1433 UUCCGAAuAAACUCcAGGCTsT 1434
    ocGGAATsT
    AD-12369 GmocmocmouGGAGmoumoumouAmoumoum 1435 UUCCGAAUAAACUCCAGGCTsT 1436
    ocGGAATsT
    AD-12370 GmocmocmouGGAGmoumoumouAmoumoum 1437 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT 1438
    ocGGAATsT
    AD-12371 GmocmocmouGGAGmoumoumouAmoumoum 1439 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1440
    ocGGAATsT
    AD-12372 GmocmocmouGGAGmoumoumouAmoumoum 1441 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcsCfsu 1442
    ocGGAATsT
    AD-12373 GmocmocmouGGAGmoumoumouAmoumoum 1443 UUCCGAAUAAACUCCAGGCTsT 1444
    ocGGAATsT
    AD-12374 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1445 UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT 1446
    AD-12375 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1447 UUCCGAAUAAACUCCAGGCTsT 1448
    AD-12377 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1449 uUcCGAAuAAACUccAGGCTsT 1450
    AD-12378 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1451 UUCCGaaUAaaCUCCAggcscsu 1452
    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
    AD-12395 GmocCmouGmogAmogUmouUmoaUmouCmo 1485 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1486
    gGmoaATsT
    AD-12396 GmocCmouGmogAm02gUmouUmoaUmouCm 1487 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1488
    ogGmoaA
    AD-12397 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1489 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1490
    AD-12398 GfcCfuGfgAfgUfuUfaUfuCfgGfaAffsT 1491 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1492
    AD-12399 GcCuGgnAgUuUaUuCgGaATsT 1493 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1494
    AD-12400 GCCUGGAGUUUAUUCGGAATsT 1495 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1496
    AD-12401 GccuGGAGuuuAuucGGAATsT 1497 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1498
    AD-12402 GccuGGAGuuuAuucGGAA 1499 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1500
    AD-12403 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1501 P-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
    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; moc, mou, mog, moa: corresponding 2′-MOE nucleotide; where nucleotides are written in sequence, they are connected by 3′-5′ phosphodiester groups; ab: 3′-terminal abasic nucleotide; nucleotides with interjected “s” are connected by 3′-O-5′-O phosphorothiodiester groups; unless denoted by prefix “p-”, oligonucleotides are devoid of a 5′-phosphate group on the 5′-most nucleotide; all oligonucleotides bear 3′-OH on the 3′-most nucleotide
  • TABLE 2b
    Screening of dsRNAs targeted to PCSK9
    Remaining mRNA in
    % of controls at
    Duplex number siRNA conc. of 30 nM
    AD-10792 15
    AD-10793 32
    AD-10796 13
    AD-12038 13
    AD-12039 29
    AD-12040 10
    AD-12041 11
    AD-12042 12
    AD-12043 13
    AD-12044 7
    AD-12045 8
    AD-12046 13
    AD-12047 17
    AD-12048 43
    AD-12049 34
    AD-12050 16
    AD-12051 31
    AD-12052 81
    AD-12053 46
    AD-12054 8
    AD-12055 13
    AD-12056 11
    AD-12057 8
    AD-12058 9
    AD-12059 23
    AD-12060 10
    AD-12061 7
    AD-12062 10
    AD-12063 19
    AD-12064 15
    AD-12065 16
    AD-12066 20
    AD-12067 17
    AD-12068 18
    AD-12069 13
    AD-12338 15
    AD-12339 14
    AD-12340 19
    AD-12341 12
    AD-12342 13
    AD-12343 24
    AD-12344 9
    AD-12345 12
    AD-12346 13
    AD-12347 11
    AD-12348 8
    AD-12349 11
    AD-12350 17
    AD-12351 11
    AD-12352 11
    AD-12354 11
    AD-12355 9
    AD-12356 25
    AD-12357 56
    AD-12358 29
    AD-12359 30
    AD-12360 15
    AD-12361 20
    AD-12362 51
    AD-12363 11
    AD-12364 25
    AD-12365 18
    AD-12366 23
    AD-12367 42
    AD-12368 40
    AD-12369 26
    AD-12370 68
    AD-12371 60
    AD-12372 60
    AD-12373 55
    AD-12374 9
    AD-12375 16
    AD-12377 88
    AD-12378 6
    AD-12379 6
    AD-12380 8
    AD-12381 10
    AD-12382 7
    AD-12383 7
    AD-12384 8
    AD-12385 8
    AD-12386 11
    AD-12387 13
    AD-12388 19
    AD-12389 16
    AD-12390 17
    AD-12391 21
    AD-12392 28
    AD-12393 17
    AD-12394 75
    AD-12395 55
    AD-12396 59
    AD-12397 20
    AD-12398 11
    AD-12399 13
    AD-12400 12
    AD-12401 13
    AD-12402 14
    AD-12403 4
    AD-9314 9
  • TABLE 3
    Cholesterol levels of rats treated with LNP01-10792
    Dosage of 5 mg/kg, n = 6 rats per group
    Day Total serum cholesterol (relative to PBS control)
    2 0.329 ± 0.035
    4 0.350 ± 0.055
    7 0.402 ± 0.09 
    9 0.381 ± 0.061
    11 0.487 ± 0.028
    14 0.587 ± 0.049
    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 pre-dose)
    Day 3 Day 4 Day 5 Day 7 Day 14 Day 21
    PBS 1.053 ± 0.158 0.965 ± 0.074 1.033 ± 0.085 1.033 ± 0.157 1.009 ± 0.034
    n = 3
    LNP01-1955 1.027 ± 0.068 1.104 ± 0.114
    n = 3
    LNP01-10792 0.503 ± 0.055 0.596 ± 0.111 0.674 ± 0.139 0.644 ± 0.121 0.958 ± 0.165 1.111 ± 0.172
    n = 5
    LNP01-9680 0.542 ± 0.155 0.437 ± 0.076 0.505 ± 0.071 0.469 ± 0.066 0.596 ± 0.080 0.787 ± 0.138
    n = 4
  • TABLE 5a
    Modified dsRNA targeted to PCSK9
    Position SEQ
    in human ID
    Name access.# Sense Antisense Sequence  5′-3′ NO:
    AD- 1091 unmodified unmodified GCCUGGAGUUUAUUCGGAAdTdT 1505
    1a1 UUCCGAAUAAACUCCAGGCdTsdT 1506
    AD- 1091 2′OMe 2′OMe GccuGGAGuuuAuucGGAAdTsdT 1507
    1a2 UUCCGAAuAAACUCcAGGCdTsdT 1508
    AD- 1091 Alt 2′F, Alt 2′F, GfcCfuGfgAfgUfuUfaUfuCfgGfaAfdTdT 1509
    1a3 2′OMe 2′OMe puUfcCfgAfaUfaAfaCfuCfcAfgGfcdTsdT 1510
    AD- 1091 2′OMe 2′F all Py, GccuGGAGuuuAuucGGAAdTsdT 1511
    1a4 5′Phosphate PUfUfCfCfGAAUfAAACfUfCfCfAGGCfdTsdT 1512
    AD- 1091 2′F 2′F all Py, GCfCfUfGGAGUfUfUfAUfUfCfGGAAdTsdT 1513
    1a5 5′Phosphate PUfUfCfCfGAAUfAAACfUfCfCfAGGCfdTsdT 1514
    AD-2a1 3530 2′OMe 2′OMe uucuAGAccuGuuuuGcuudTsdT 1515
    (3′UTR) AAGcAAAAcAGGUCuAGAAdTsdT 1516
    AD-3a1  833 2′OMe 2′OMe AGGuGuAucuccuAGAcAcdTsdT 1517
    GUGUCuAGGAGAuAcACCUdTsdT 1518
    AD- N/A 2′OMe 2′OMe cuuAcGcuGAGuAcuucGAdTsdT 1519
    ctrl UCGAAGuACUcAGCGuAAGdTsdT 1520
    (Luc.)
    U, C, A, G: corresponding ribonucleotide; T: deoxythymidine; u, c, a, g: corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding 2′-deoxy-2′-fluoro ribonucleotide; where nucleotides are written in sequence, they are connected by 3′-5′ phosphodiester groups; nucleotides with interjected “s” are connected by 3′-O-5′-O phosphorothiodiester
    groups; unless denoted by prefix “p-”, oligonucleotides are devoid of a 5′-phosphate group on the 5′-most nucleotide; all oligonucleotides bear 3′-OH on the 3′-most nucleotide.
  • TABLE 5b
    Silencing activity of modified dsRNA in monkey hepatocytes
    Primary
    Position in IFN-α/ Cynomolgus Monkey
    human TNF-α Hepatocytes
    Name access.# Induction Sense Antisense ~IC50, nM
    AD-1a1 1091 Yes/Yes unmodified unmodified 0.07-0.2
    AD-1a2 1091 No/No 2′OMe 2′OMe 0.07-0.2
    AD-1a3 1091 No/No Alt 2′F, Alt 2′F, 2′OMe 0.07-0.2
    2′OMe
    AD-1a4 1091 No/No 2′OMe 2′F all Py, 0.07-0.2
    5′Phosphate
    AD-1a5 1091 No/No 2′F 2′F all Py, 0.07-0.2
    5′Phosphate
    AD-2a1 3530 No/No 2′OMe 2′OMe 0.07-0.2
    (3′UTR)
    AD-3a1  833 No/No 2′OMe 2′OMe  0.1-0.3
    AD-ctrl N/A No/No 2′OMe 2′OMe N/A
    (Luc.)
  • TABLE 6
    dsRNA targeted to PCSK9: mismatches and
    modifications
    SEQ
    Duplex ID
    # Strand NO: Sequence 5′ to 3′
    AD-9680 S 1531 uucuAGAccuGuuuuGcuudTsdT
    AS 1532 AAGcAAAAcAGGUCuAGAAdTsdT
    AD-3267 S 1535 uucuAGAcCuGuuuuGcuuTsT
    AS 1536 AAGcAAAAcAGGUCuAGAATsT
    AD-3268 S 1537 uucuAGAccUGuuuuGcuuTsT
    AS 1538 AAGcAAAAcAGGUCuAGAATsT
    AD-3269 S 1539 uucuAGAcCUGuuuuGcuuTsT
    AS 1540 AAGcAAAAcAGGUCuAGAATsT
    AD-3270 S 1541 uucuAGAcY1uGuuuuGcuuTsT
    AS 1542 AAGcAAAAcAGGUCuAGAATsT
    AD-3271 S 1543 uucuAGAcY1UGuuuuGcuuTsT
    AS 1544 AAGcAAAAcAGGUCuAGAATsT
    AD-3272 S 1545 uucuAGAccY1GuuuuGcuuTsT
    AS 1546 AAGcAAAAcAGGUCuAGAATsT
    AD-3273 S 1547 uucuAGAcCY1GuuuuGcuuTsT
    AS 1548 AAGcAAAAcAGGUCuAGAATsT
    AD-3274 S 1549 uucuAGAccuY1uuuuGcuuTsT
    AS 1550 AAGcAAAAcAGGUCuAGAATsT
    AD-3275 S 1551 uucuAGAcCUY1uuuuGcuuTsT
    AS 1552 AAGcAAAAcAGGUCuAGAATsT
    AD-14676 S 1553 UfuCfuAfgAfcCfuGfuUfuUfgCfuUfTsT
    AS 1554 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-3276 S 1555 UfuCfuAfgAfcCuGfuUfuUfgCfuUfTsT
    AS 1556 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-3277 S 1557 UfuCfuAfgAfcCfUGfuUfuUfgCfuUfTsT
    AS 1558 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-3278 S 1559 UfuCfuAfgAfcCUGfuUfuUfgCfuUfTsT
    AS 1560 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-3279 S 1561 UfuCfuAfgAfcY1uGfuUfuUfgCfuUfTsT
    AS 1562 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-3280 S 1563 UfuCfuAfgAfcY1UGfuUfuUfgCfuUfTsT
    AS 1564 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-3281 S 1565 UfuCfuAfgAfcCfY1GfuUfuUfgCfuUfTsT
    AS 1566 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-3282 S 1567 UfuCfuAfgAfcCY1GfuUfuUfgCfuUfTsT
    AS 1568 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-3283 S 1569 UfuCfuAfgAfcCfuY1uUfuUfgCfuUfTsT
    AS 1570 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-3284 S 1571 UfuCfuAfgAfcCUY1uUfuUfgCfuUfTsT
    AS 1572 p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT
    AD-10792 S 459 GccuGGAGuuuAuucGGAATsT
    AS 460 UUCCGAAuAAACUCcAGGCTsT
    AD-3254 S 1573 GccuGGAGuY1uAuucGGAATsT
    AS 1574 UUCCGAAuAAACUCcAGGCTsT
    AD-3255 S 1575 GccuGGAGUY1uAuucGGAATsT
    AS 1576 UUCCGAAuAAACUCcAGGCTsT
    Strand: S/Sense; AS/Antisense
    U, C, A, G: corresponding ribonucleotide; T: deoxythymidine; u, c, a, g: corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding 2′-deoxy-2′-fluoro ribonucleotide; Y1 corresponds to DFT difluorotoluyl ribo(or deoxyribo)nucleotide; where nucleotides are written in sequence, they are connected by 3′-5′ phosphodiester groups; nucleotides with
    interjected “s” are connected by 3′-O-5′-O phosphorothiodiester groups; unless denoted by prefix “p-”, oligonucleotides are devoid of a 5′-phosphate group on the 5′-most nucleotide; all oligonucleotides bear 3′-OH on the 3′-most nucleotide
  • TABLE 7
    Sequences of unmodified siRNA flanking AD-9680
    Target
    Duplex Type Sequence (5′ to 3′) site SEQ ID NO:
    AD-22169-b1 sense CAGCCAACUUUUCUAGACCdTsdT 3520 1577
    antis GGUCUAGAAAAGUUGGCUGdTsdT 3520 1578
    AD-22170-b1 sense AGCCAACUUUUCUAGACCUdTsdT 3521 1579
    antis AGGUCUAGAAAAGUUGGCUdTsdT 3521 1580
    AD-22171-b1 sense GCCAACUUUUCUAGACCUGdTsdT 3522 1581
    antis CAGGUCUAGAAAAGUUGGCdTsdT 3522 1582
    AD-22172-b1 sense CCAACUUUUCUAGACCUGUdTsdT 3523 1583
    antis ACAGGUCUAGAAAAGUUGGdTsdT 3523 1584
    AD-22173-b1 sense CAACUUUUCUAGACCUGUUdTsdT 3524 1585
    antis AACAGGUCUAGAAAAGUUGdTsdT 3524 1586
    AD-22174-b1 sense AACUUUUCUAGACCUGUUUdTsdT 3525 1587
    antis AAACAGGUCUAGAAAAGUUdTsdT 3525 1588
    AD-22175-b1 sense ACUUUUCUAGACCUGUUUUdTsdT 3526 1589
    antis AAAACAGGUCUAGAAAAGUdTsdT 3526 1590
    AD-22176-b1 sense CUUUUCUAGACCUGUUUUGdTsdT 3527 1591
    antis CAAAACAGGUCUAGAAAAGdTsdT 3527 1592
    AD-22177-b1 sense UUUUCUAGACCUGUUUUGCdTsdT 3528 1593
    antis GCAAAACAGGUCUAGAAAAdTsdT 3528 1594
    AD-22178-b1 sense UUUCUAGACCUGUUUUGCUdTsdT 3529 1595
    antis AGCAAAACAGGUCUAGAAAdTsdT 3529 1596
    AD-22179-b1 sense UCUAGACCUGUUUUGCUUUdTsdT 3531 1597
    antis AAAGCAAAACAGGUCUAGAdTsdT 3531 1598
    AD-22180-b1 sense CUAGACCUGUUUUGCUUUUdTsdT 3532 1599
    antis AAAAGCAAAACAGGUCUAGdTsdT 3532 1600
    AD-22181-b1 sense UAGACCUGUUUUGCUUUUGdTsdT 3533 1601
    antis CAAAAGCAAAACAGGUCUAdTsdT 3533 1602
    AD-22182-b1 sense AGACCUGUUUUGCUUUUGUdTsdT 3534 1603
    antis ACAAAAGCAAAACAGGUCUdTsdT 3534 1604
    AD-22183-b1 sense GACCUGUUUUGCUUUUGUAdTsdT 3535 1605
    antis UACAAAAGCAAAACAGGUCdTsdT 3535 1606
    AD-22184-b1 sense ACCUGUUUUGCUUUUGUAAdTsdT 3536 1607
    antis UUACAAAAGCAAAACAGGUdTsdT 3536 1608
    AD-22185-b1 sense CCUGUUUUGCUUUUGUAACdTsdT 3537 1609
    antis GUUACAAAAGCAAAACAGGdTsdT 3537 1610
    AD-22186-b1 sense CUGUUUUGCUUUUGUAACUdTsdT 3538 1611
    antis AGUUACAAAAGCAAAACAGdTsdT 3538 1612
    AD-22187-b1 sense UGUUUUGCUUUUGUAACUUdTsdT 3539 1613
    antis AAGUUACAAAAGCAAAACAdTsdT 3539 1614
    AD-22188-b1 sense GUUUUGCUUUUGUAACUUGdTsdT 3540 1615
    antis CAAGUUACAAAAGCAAAACdTsdT 3540 1616
    AD-22189-b1 sense UUUUGCUUUUGUAACUUGAdTsdT 3541 1617
    antis UCAAGUUACAAAAGCAAAAdTsdT 3541 1618
    AD-22190-b1 sense UUUGCUUUUGUAACUUGAAdTsdT 3542 1619
    antis UUCAAGUUACAAAAGCAAAdTsdT 3542 1620
    AD-22191-b1 sense UUGCUUUUGUAACUUGAAGdTsdT 3543 1621
    antis CUUCAAGUUACAAAAGCAAdTsdT 3543 1622
    AD-22192-b1 sense UGCUUUUGUAACUUGAAGAdTsdT 3544 1623
    antis UCUUCAAGUUACAAAAGCAdTsdT 3544 1624
    AD-22193-b1 sense GCUUUUGUAACUUGAAGAUdTsdT 3545 1625
    antis AUCUUCAAGUUACAAAAGCdTsdT 3545 1626
    AD-22194-b1 sense CUUUUGUAACUUGAAGAUAdTsdT 3546 1627
    antis UAUCUUCAAGUUACAAAAGdTsdT 3546 1628
    AD-22195-b1 sense UUUUGUAACUUGAAGAUAUdTsdT 3547 1629
    antis AUAUCUUCAAGUUACAAAAdTsdT 3547 1630
    AD-22196-b1 sense UUUGUAACUUGAAGAUAUUdTsdT 3548 1631
    antis AAUAUCUUCAAGUUACAAAdTsdT 3548 1632
    AD-22197-b1 sense UUGUAACUUGAAGAUAUUUdTsdT 3549 1633
    antis AAAUAUCUUCAAGUUACAAdTsdT 3549 1634
    AD-22198-b1 sense UGUAACUUGAAGAUAUUUAdTsdT 3550 1635
    antis UAAAUAUCUUCAAGUUACAdTsdT 3550 1636
    AD-22199-b1 sense GUAACUUGAAGAUAUUUAUdTsdT 3551 1637
    antis AUAAAUAUCUUCAAGUUACdTsdT 3551 1638
    AD-22200-b1 sense UAACUUGAAGAUAUUUAUUdTsdT 3552 1639
    antis AAUAAAUAUCUUCAAGUUAdTsdT 3552 1640
    AD-22201-b1 sense AACUUGAAGAUAUUUAUUCdTsdT 3553 1641
    antis GAAUAAAUAUCUUCAAGUUdTsdT 3553 1642
    AD-22202-b1 sense ACUUGAAGAUAUUUAUUCUdTsdT 3554 1643
    antis AGAAUAAAUAUCUUCAAGUdTsdT 3554 1644
    AD-22203-b1 sense CUUGAAGAUAUUUAUUCUGdTsdT 3555 1645
    antis CAGAAUAAAUAUCUUCAAGdTsdT 3555 1646
    AD-22204-b1 sense UUGAAGAUAUUUAUUCUGGdTsdT 3556 1647
    antis CCAGAAUAAAUAUCUUCAAdTsdT 3556 1648
    AD-22205-b1 sense UGAAGAUAUUUAUUCUGGGdTsdT 3557 1649
    antis CCCAGAAUAAAUAUCUUCAdTsdT 3557 1650
    AD-22206-b1 sense GAAGAUAUUUAUUCUGGGUdTsdT 3558 1651
    antis ACCCAGAAUAAAUAUCUUCdTsdT 3558 1652
  • TABLE 8
    Sequences of modified siRNA flanking AD-9680
    Duplex Type Sequence (5′ to 3′) Target SEQ ID NO:
    AD-22098-b1 sense cAGccAAcuuuucuAGAccdTsdT 3520 1653
    antis GGUCuAGAAAAGUUGGCUGdTsdT 3520 1654
    AD-22099-b1 sense AGccAAcuuuucuAGAccudTsdT 3521 1655
    antis AGGUCuAGAAAAGUUGGCUdTsdT 3521 1656
    AD-22100-b1 sense GccAAcuuuucuAGAccuGdTsdT 3522 1657
    antis cAGGUCuAGAAAAGUUGGCdTsdT 3522 1658
    AD-22101-b1 sense ccAAcuuuucuAGAccuGudTsdT 3523 1659
    antis AcAGGUCuAGAAAAGUUGGdTsdT 3523 1660
    AD-22102-b1 sense cAAcuuuucuAGAccuGuudTsdT 3524 1661
    antis AAcAGGUCuAGAAAAGUUGdTsdT 3524 1662
    AD-22103-b1 sense AAcuuuucuAGAccuGuuudTsdT 3525 1663
    antis AAAcAGGUCuAGAAAAGUUdTsdT 3525 1664
    AD-22104-b1 sense AcuuuucuAGAccuGuuuudTsdT 3526 1665
    antis AAAAcAGGUCuAGAAAAGUdTsdT 3526 1666
    AD-22105-b1 sense cuuuucuAGAccuGuuuuGdTsdT 3527 1667
    antis cAAAAcAGGUCuAGAAAAGdTsdT 3527 1668
    AD-22106-b1 sense uuuucuAGAccuGuuuuGcdTsdT 3528 1669
    antis GcAAAAcAGGUCuAGAAAAdTsdT 3528 1670
    AD-22107-b1 sense uuucuAGAccuGuuuuGcudTsdT 3529 1671
    antis AGcAAAAcAGGUCuAGAAAdTsdT 3529 1672
    AD-22108-b1 sense ucuAGAccuGuuuuGcuuudTsdT 3531 1673
    antis AAAGcAAAAcAGGUCuAGAdTsdT 3531 1674
    AD-22109-b1 sense cuAGAccuGuuuuGcuuuudTsdT 3532 1675
    antis AAAAGcAAAAcAGGUCuAGdTsdT 3532 1676
    AD-22110-b1 sense uAGAccuGuuuuGcuuuuGdTsdT 3533 1677
    antis cAAAAGcAAAAcAGGUCuAdTsdT 3533 1678
    AD-22111-b1 sense AGAccuGuuuuGcuuuuGudTsdT 3534 1679
    antis AcAAAAGcAAAAcAGGUCUdTsdT 3534 1680
    AD-22112-b1 sense GAccuGuuuuGcuuuuGuAdTsdT 3535 1681
    antis uAcAAAAGcAAAAcAGGUCdTsdT 3535 1682
    AD-22113-b1 sense AccuGuuuuGcuuuuGuAAdTsdT 3536 1683
    antis UuAcAAAAGcAAAAcAGGUdTsdT 3536 1684
    AD-22114-b1 sense ccuGuuuuGcuuuuGuAAcdTsdT 3537 1685
    antis GUuAcAAAAGcAAAAcAGGdTsdT 3537 1686
    AD-22115-b1 sense cuGuuuuGcuuuuGuAAcudTsdT 3538 1687
    antis AGUuAcAAAAGcAAAAcAGdTsdT 3538 1688
    sense uGuuuuGcuuuuGuAAcuudTsdT 3539 1689
    antis AAGUuAcAAAAGcAAAAcAdTsdT 3539 1690
    AD-22116-b1 sense GuuuuGcuuuuGuAAcuuGdTsdT 3540 1691
    antis cAAGUuAcAAAAGcAAAACdTsdT 3540 1692
    AD-22117-b1 sense uuuuGcuuuuGuAAcuuGAdTsdT 3541 1693
    antis UcAAGUuAcAAAAGcAAAAdTsdT 3541 1694
    AD-22118-b1 sense uuuGcuuuuGuAAcuuGAAdTsdT 3542 1695
    antis UUcAAGUuAcAAAAGcAAAdTsdT 3542 1696
    AD-22119-b1 sense uuGcuuuuGuAAcuuGAAGdTsdT 3543 1697
    antis CUUcAAGUuAcAAAAGcAAdTsdT 3543 1698
    AD-22120-b1 sense uGcuuuuGuAAcuuGAAGAdTsdT 3544 1699
    antis UCUUcAAGUuAcAAAAGcAdTsdT 3544 1700
    AD-22121-b1 sense GcuuuuGuAAcuuGAAGAudTsdT 3545 1701
    antis AUCUUcAAGUuAcAAAAGCdTsdT 3545 1702
    AD-22122-b1 sense cuuuuGuAAcuuGAAGAuAdTsdT 3546 1703
    antis uAUCUUcAAGUuAcAAAAGdTsdT 3546 1704
    AD-22123-b1 sense uuuuGuAAcuuGAAGAuAudTsdT 3547 1705
    antis AuAUCUUcAAGUuAcAAAAdTsdT 3547 1706
    AD-22124-b1 sense uuuGuAAcuuGAAGAuAuudTsdT 3548 1707
    antis AAuAUCUUcAAGUuAcAAAdTsdT 3548 1708
    AD-22125-b1 sense uuGuAAcuuGAAGAuAuuudTsdT 3549 1709
    antis AAAuAUCUUcAAGUuAcAAdTsdT 3549 1710
    AD-22126-b1 sense uGuAAcuuGAAGAuAuuuAdTsdT 3550 1711
    antis uAAAuAUCUUcAAGUuAcAdTsdT 3550 1712
    AD-22127-b1 sense GuAAcuuGAAGAuAuuuAudTsdT 3551 1713
    antis AuAAAuAUCUUcAAGUuACdTsdT 3551 1714
    AD-22128-b1 sense uAAcuuGAAGAuAuuuAuudTsdT 3552 1715
    antis AAuAAAuAUCUUcAAGUuAdTsdT 3552 1716
    AD-22129-b1 sense AAcuuGAAGAuAuuuAuucdTsdT 3553 1717
    antis GAAuAAAuAUCUUcAAGUUdTsdT 3553 1718
    AD-22130-b1 sense AcuuGAAGAuAuuuAuucudTsdT 3554 1719
    antis AGAAuAAAuAUCUUcAAGUdTsdT 3554 1720
    AD-22131-b1 sense cuuGAAGAuAuuuAuucuGdTsdT 3555 1721
    antis cAGAAuAAAuAUCUUcAAGdTsdT 3555 1722
    AD-22132-b1 sense uuGAAGAuAuuuAuucuGGdTsdT 3556 1723
    antis CcAGAAuAAAuAUCUUcAAdTsdT 3556 1724
    AD-22133-b1 sense uGAAGAuAuuuAuucuGGGdTsdT 3557 1725
    antis CCcAGAAuAAAuAUCUUcAdTsdT 3557 1726
    AD-22134-b1 sense GAAGAuAuuuAuucuGGGudTsdT 3558 1727
    antis ACCcAGAAuAAAuAUCUUCdTsdT 3558 1728
  • TABLE 9
    Single dose treatment of HeLa cells with siRNA flanking AD-9680
    % message % message
    remaining remaining
    Duplex ID 0.1 nM SD 0.1 nM 10 nM SD 10 nM
    AD-22098-b1 10.6 1.9 9.2 3.7
    AD-22098-b1 7.7 1.7 7.9 0.7
    AD-22099-b1 21.3 4.5 27.4 7.2
    AD-22099-b1 25.9 2.4 29.6 9.1
    AD-22100-b1 58.6 9.6 35.8 11.1
    AD-22100-b1 62.5 0.3 27.4 3.5
    AD-22101-b1 21.9 3.8 12.9 1.4
    AD-22101-b1 19.3 0.3 9.7 1.3
    AD-22102-b1 6.6 0.1 7.7 3.3
    AD-22103-b1 8.7 0.0 8.2 1.3
    AD-22104-b1 7.6 0.2 8.5 2.8
    AD-22105-b1 13.4 1.0 8.1 2.3
    AD-22106-b1 59.1 0.4 35.4 4.6
    AD-22107-b1 9.1 0.8 8.4 3.7
    AD-22108-b1 8.8 0.9 6.2 1.7
    AD-22109-b1 9.8 0.9 8.2 1.7
    AD-22110-b1 24.8 1.7 15.3 5.9
    AD-22111-b1 8.3 0.7 6.2 1.7
    AD-22112-b1 15.1 0.0 10.3 2.9
    AD-22113-b1 10.9 0.6 10.0 2.0
    AD-22114-b1 8.9 1.1 7.3 1.3
    AD-22115-b1 5.3 0.8 3.7 0.7
    AD-22116-b1 58.1 0.4 34.5 7.3
    AD-22117-b1 19.9 0.9 12.2 2.9
    AD-22118-b1 5.3 0.0 4.4 1.0
    AD-22119-b1 8.6 1.9 5.8 2.3
    AD-22120-b1 7.2 0.8 5.8 2.4
    AD-22121-b1 7.3 0.9 6.4 2.1
    AD-22122-b1 32.5 2.5 18.1 6.3
    AD-22123-b1 14.7 0.8 16.7 7.0
    AD-22124-b1 12.8 1.9 10.5 5.3
    AD-22125-b1 7.4 0.6 9.0 4.6
    AD-22126-b1 12.8 0.4 16.4 7.3
    AD-22127-b1 8.8 0.5 9.6 5.0
    AD-22128-b1 9.9 0.2 12.4 5.9
    AD-22129-b1 85.9 10.3 94.9 49.8
    AD-22130-b1 5.6 1.0 6.2 4.1
    AD-22131-b1 26.9 8.4 12.9 7.3
    AD-22132-b1 78.5 18.5 67.5 34.1
    AD-22133-b1 26.4 7.1 15.0 6.7
    AD-22134-b1 26.9 0.1 22.4 6.5
    AD-22169-b1 7.3 0.6 6.0 1.5
    AD-22169-b1 7.0 1.1 6.1 1.3
    AD-22170-b1 9.3 1.6 7.2 1.8
    AD-22170-b1 9.7 1.1 11.2 1.0
    AD-22171-b1 7.1 2.3 4.5 0.2
    AD-22171-b1 6.5 1.9 4.4 2.8
    AD-22172-b1 7.2 1.1 7.6 3.7
    AD-22172-b1 7.0 0.4 7.0 2.4
    AD-22173-b1 15.7 12.5 5.9 0.1
    AD-22174-b1 8.9 2.7 6.4 0.9
    AD-22175-b1 10.7 4.3 7.9 2.4
    AD-22176-b1 9.6 0.8 8.4 3.1
    AD-22177-b1 38.9 5.9 21.4 1.2
    AD-22178-b1 6.5 0.5 5.6 0.9
    AD-22179-b1 7.0 0.8 5.9 0.1
    AD-22180-b1 7.3 3.7 7.2 1.6
    AD-22181-b1 11.1 0.9 10.0 1.0
    AD-22182-b1 5.4 1.4 4.0 1.5
    AD-22183-b1 3.8 0.4 2.9 0.4
    AD-22184-b1 5.1 0.2 3.7 0.7
    AD-22185-b1 5.7 0.6 5.0 1.5
    AD-22186-b1 5.3 0.3 5.7 1.0
    AD-22187-b1 5.3 1.2 5.3 1.4
    AD-22188-b1 12.6 2.6 11.6 0.2
    AD-22189-b1 5.2 0.5 4.5 1.8
    AD-22190-b1 4.7 1.3 3.4 1.1
    AD-22191-b1 10.5 0.6 7.9 0.9
    AD-22192-b1 6.9 2.2 5.8 3.5
    AD-22193-b1 7.5 1.5 5.2 0.6
    AD-22194-b1 8.0 1.4 6.5 1.9
    AD-22195-b1 7.0 1.9 4.9 2.3
    AD-22196-b1 5.4 0.0 3.8 0.9
    AD-22197-b1 6.6 0.4 5.2 1.2
    AD-22198-b1 7.3 0.8 8.5 2.4
    AD-22199-b1 5.5 0.7 4.2 1.2
    AD-22200-b1 11.0 0.5 12.5 3.1
    AD-22201-b1 44.0 3.1 47.3 8.3
    AD-22202-b1 9.0 1.2 7.2 0.9
    AD-22203-b1 12.5 0.0 12.7 2.2
    AD-22204-b1 57.1 5.2 50.2 10.2
    AD-22205-b1 27.0 0.4 24.5 0.0
    AD-22206-b1 13.9 1.1 11.4 1.3
    AD-9680 7.1 ND 9.3 ND
  • TABLE 10
    IC50 in HeLa cells using siRNA flanking AD-9680
    Rep1 Rep2
    IC50 IC50 Average IC50
    Duplex Name (pM) (pM) (pM)
    AD-22098 6.0 6.7 6.4
    AD-22099 25.0 37.8 31.4
    AD-22101 66.5 81.9 74.2
    AD-22102 2.3 1.5 1.9
    AD-22103 6.3 1.2 3.8
    AD-22104 2.2 1.4 1.8
    AD-22105 13.3 0.1 6.7
    AD-22107 2.2 0.9 1.6
    AD-22108 2.3 2.0 2.1
    AD-22109 5.5 6.3 5.9
    AD-22110 59.1 42.2 50.7
    AD-22111 9.1 8.2 8.7
    AD-22112 25.8 31.0 28.4
    AD-22113 4.2 4.4 4.3
    AD-22114 6.9 4.0 5.5
    AD-22115 3.0 2.2 2.6
    AD-22117 56.0 37.6 46.8
    AD-22118 2.9 1.7 2.3
    AD-22119 6.7 0.0 3.4
    AD-22120 2.0 1.2 1.6
    AD-22121 2.1 4.1 3.1
    AD-22122 203.3 156.3 179.8
    AD-22123 33.1 50.7 41.9
    AD-22124 18.8 13.1 15.9
    AD-22125 3.3 2.6 3.0
    AD-22126 17.9 18.5 18.2
    AD-22127 11.1 4.3 7.7
    AD-22128 14.6 3.3 8.9
    AD-22130 1.7 0.3 1.0
    AD-22131 172.5 59.6 116.0
    AD-22133 94.6 57.2 75.9
    AD-22134 113.0 81.3 97.2
    AD-9680 3.8 2.4 3.1

Claims (25)

1. A composition comprising a nucleic acid lipid particle comprising a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human PCSK9 gene in a cell, wherein:
the nucleic acid lipid particle comprises a lipid formulation comprising 45-65 mol % of a cationic lipid, 5 mol % to about 10 mol %, of a non-cationic lipid, 25-40 mol % of a sterol, and 0.5-5 mol % of a PEG or PEG-modified lipid,
the dsRNA consists of a sense strand and an antisense strand, and the sense strand comprises a first sequence and the antisense strand comprises a second sequence complementary to at least 15 contiguous nucleotides of a nucleotide sequence of a target sequence of a dsRNA found in Table 1a, Table 2a, Table 5a, Table 6, Table 7, Table 8,
wherein the first sequence is complementary to the second sequence and wherein the dsRNA is between 15 and 30 base pairs in length.
2. The composition of claim 1, wherein the cationic lipid comprises MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate).
3. The composition of claim 2, wherein the cationic lipid comprises MC3 and the lipid formulation is selected from the group consisting of:
LNP11 MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 LNP14 MC3/DSPC/Cholesterol/PEG-DMG 40/15/40/5 LNP15 MC3/DSPC/Cholesterol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 LNP16 MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 LNP17 MC3/DSPC/Cholesterol/PEG-DSG 50/10/38.5/1.5 LNP18 MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 LNP19 MC3/DSPC/Cholesterol/PEG-DMG 50/10/35/5 LNP20 MC3/DSPC/Cholesterol/PEG-DPG 50/10/38.5/1.5
4.-9. (canceled)
10. The composition of claim 1, wherein the sense strand comprises SEQ ID NO:1227 and the antisense strand comprises SEQ ID NO:1228.
11. (canceled)
12. (canceled)
13. The composition of claim 1, wherein the dsRNA comprises at least one modified nucleotide.
14. The composition of claim 13, wherein the modified nucleotide is chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.
15. The composition of claim 13, wherein the modified nucleotide is chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
16. The composition of claim 1, wherein the dsRNA comprises at least one 2′-O-methyl modified ribonucleotide and at least one nucleotide comprising a 5′-phosphorothioate group.
17. The composition of claim 1, wherein each strand of the dsRNA is 19-23 bases in length.
18. The composition of claim 1, wherein each strand of the dsRNA is 21-23 bases in length.
19. The composition of claim 1, wherein each strand of the dsRNA is 21 bases in length.
20. The composition of claim 1, further comprising a lipoprotein.
21. The composition of claim 1, further comprising apolipoprotein E (ApoE).
22. The composition of claim 21, wherein the dsRNA is conjugated to a lipophile.
23. The composition of claim 22, wherein the lipophile is cholesterol.
24. The composition of claim 21, wherein the ApoE is reconstituted with at least one amphiphilic agent.
25. The composition of claim 24, wherein the amphiphilic agent is a phospholipid.
26. The composition of claim 24, wherein the amphilic agent is a phospholipid selected from the group consisting of dimyristoyl phosphatidyl choline (DMPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), -phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), and combinations thereof.
27. The composition of claim 25, wherein the ApoE is reconstituted high density lipoprotein (rHDL).
28.-33. (canceled)
34. A method for inhibiting the expression of PCSK9 in a cell comprising administering the composition of claim 1 to the cell.
35.-43. (canceled)
US14/058,052 2009-06-15 2013-10-18 Lipid formulated dsrna targeting the pcsk9 gene Abandoned US20140121263A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US14/058,052 US20140121263A1 (en) 2009-06-15 2013-10-18 Lipid formulated dsrna targeting the pcsk9 gene
US15/060,492 US20170000815A1 (en) 2009-06-15 2016-03-03 Lipid formulated dsrna targeting the pcsk9 gene

Applications Claiming Priority (9)

Application Number Priority Date Filing Date Title
US18716909P 2009-06-15 2009-06-15
US21835009P 2009-06-18 2009-06-18
US24479009P 2009-09-22 2009-09-22
US28559809P 2009-12-11 2009-12-11
US29347410P 2010-01-08 2010-01-08
US31357810P 2010-03-12 2010-03-12
US12/816,207 US8273869B2 (en) 2009-06-15 2010-06-15 Lipid formulated dsRNA targeting the PCSK9 gene
US13/568,898 US8598139B2 (en) 2009-06-15 2012-08-07 Lipid formulated dsRNA targeting the PCSK9 gene
US14/058,052 US20140121263A1 (en) 2009-06-15 2013-10-18 Lipid formulated dsrna targeting the pcsk9 gene

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US13/568,898 Continuation US8598139B2 (en) 2009-06-15 2012-08-07 Lipid formulated dsRNA targeting the PCSK9 gene

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US15/060,492 Continuation US20170000815A1 (en) 2009-06-15 2016-03-03 Lipid formulated dsrna targeting the pcsk9 gene

Publications (1)

Publication Number Publication Date
US20140121263A1 true US20140121263A1 (en) 2014-05-01

Family

ID=43357015

Family Applications (4)

Application Number Title Priority Date Filing Date
US12/816,207 Expired - Fee Related US8273869B2 (en) 2009-06-15 2010-06-15 Lipid formulated dsRNA targeting the PCSK9 gene
US13/568,898 Expired - Fee Related US8598139B2 (en) 2009-06-15 2012-08-07 Lipid formulated dsRNA targeting the PCSK9 gene
US14/058,052 Abandoned US20140121263A1 (en) 2009-06-15 2013-10-18 Lipid formulated dsrna targeting the pcsk9 gene
US15/060,492 Abandoned US20170000815A1 (en) 2009-06-15 2016-03-03 Lipid formulated dsrna targeting the pcsk9 gene

Family Applications Before (2)

Application Number Title Priority Date Filing Date
US12/816,207 Expired - Fee Related US8273869B2 (en) 2009-06-15 2010-06-15 Lipid formulated dsRNA targeting the PCSK9 gene
US13/568,898 Expired - Fee Related US8598139B2 (en) 2009-06-15 2012-08-07 Lipid formulated dsRNA targeting the PCSK9 gene

Family Applications After (1)

Application Number Title Priority Date Filing Date
US15/060,492 Abandoned US20170000815A1 (en) 2009-06-15 2016-03-03 Lipid formulated dsrna targeting the pcsk9 gene

Country Status (13)

Country Link
US (4) US8273869B2 (en)
EP (1) EP2442792A4 (en)
JP (2) JP5894913B2 (en)
KR (1) KR20120050429A (en)
CN (2) CN104651408A (en)
AU (1) AU2010260148A1 (en)
BR (1) BRPI1010689A2 (en)
CA (1) CA2764832A1 (en)
EA (1) EA201270019A1 (en)
IL (1) IL216886A0 (en)
MX (1) MX2011013421A (en)
NZ (1) NZ597504A (en)
WO (1) WO2010148013A2 (en)

Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
US8999380B2 (en) 2012-04-02 2015-04-07 Moderna Therapeutics, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
US9107886B2 (en) 2012-04-02 2015-08-18 Moderna Therapeutics, Inc. Modified polynucleotides encoding basic helix-loop-helix family member E41
US9181319B2 (en) 2010-08-06 2015-11-10 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9186372B2 (en) 2011-12-16 2015-11-17 Moderna Therapeutics, Inc. Split dose administration
US9255154B2 (en) 2012-05-08 2016-02-09 Alderbio Holdings, Llc Anti-PCSK9 antibodies and use thereof
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US9334328B2 (en) 2010-10-01 2016-05-10 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9409869B1 (en) * 2014-09-04 2016-08-09 Preceres Inc. Hydrazinyl lipidoids and uses thereof
US9428535B2 (en) 2011-10-03 2016-08-30 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
WO2016154544A1 (en) * 2015-03-25 2016-09-29 The Regents Of The University Of Michigan Compositions and methods for delivery of biomacromolecule agents
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9533047B2 (en) 2011-03-31 2017-01-03 Modernatx, Inc. Delivery and formulation of engineered nucleic acids
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9597380B2 (en) 2012-11-26 2017-03-21 Modernatx, Inc. Terminally modified RNA
WO2017127750A1 (en) 2016-01-22 2017-07-27 Modernatx, Inc. Messenger ribonucleic acids for the production of intracellular binding polypeptides and methods of use thereof
WO2017223085A3 (en) * 2016-06-20 2018-02-15 The Regents Of The University Of Michigan Compositions and methods for delivery of biomacromolecule agents
US10125369B2 (en) 2012-12-05 2018-11-13 Alnylam Pharmaceuticals, Inc. PCSK9 iRNA compositions and methods of use thereof
WO2018213731A1 (en) 2017-05-18 2018-11-22 Modernatx, Inc. Polynucleotides encoding tethered interleukin-12 (il12) polypeptides and uses thereof
US10195156B2 (en) 2015-12-22 2019-02-05 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
US10207010B2 (en) 2015-12-10 2019-02-19 Modernatx, Inc. Compositions and methods for delivery of agents
US10266485B2 (en) 2015-09-17 2019-04-23 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US10307491B2 (en) 2015-01-30 2019-06-04 The Regents Of The University Of Michigan Liposomal particles comprising biological molecules and uses thereof
US10323076B2 (en) 2013-10-03 2019-06-18 Modernatx, Inc. Polynucleotides encoding low density lipoprotein receptor
US10815291B2 (en) 2013-09-30 2020-10-27 Modernatx, Inc. Polynucleotides encoding immune modulating polypeptides
US10851377B2 (en) 2015-08-25 2020-12-01 Alnylam Pharmaceuticals, Inc. Methods and compositions for treating a proprotein convertase subtilisin kexin (PCSK9) gene-associated disorder
US10857105B2 (en) 2017-03-15 2020-12-08 MordernaTX, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US11066355B2 (en) 2019-09-19 2021-07-20 Modernatx, Inc. Branched tail lipid compounds and compositions for intracellular delivery of therapeutic agents
US11203569B2 (en) 2017-03-15 2021-12-21 Modernatx, Inc. Crystal forms of amino lipids
US11524023B2 (en) 2021-02-19 2022-12-13 Modernatx, Inc. Lipid nanoparticle compositions and methods of formulating the same
US11583504B2 (en) 2016-11-08 2023-02-21 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
US11969506B2 (en) 2017-03-15 2024-04-30 Modernatx, Inc. Lipid nanoparticle formulation
US12077501B2 (en) 2017-06-14 2024-09-03 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
US12151029B2 (en) 2018-09-19 2024-11-26 Modernatx, Inc. PEG lipids and uses thereof
US12263248B2 (en) 2018-09-19 2025-04-01 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US12383508B2 (en) 2018-09-19 2025-08-12 Modernatx, Inc. High-purity peg lipids and uses thereof
US12552738B2 (en) 2021-11-12 2026-02-17 Modernatx, Inc. Crystal forms of amino lipids

Families Citing this family (146)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2584047B1 (en) 2006-05-11 2014-11-19 Alnylam Pharmaceuticals Inc. Compositions and methods for inhibiting expression of the PCSK9 gene
AU2009241591A1 (en) * 2008-01-31 2009-11-05 Alnylam Pharmaceuticals, Inc. Optimized methods for delivery of DSRNA targeting the PCSK9 gene
IL302142B2 (en) 2008-10-20 2024-07-01 Alnylam Pharmaceuticals Inc Compositions and Methods for Inhabiting Expression of TRANSTHYRETIN
CA3033577A1 (en) 2008-11-10 2010-05-14 Arbutus Biopharma Corporation Novel lipids and compositions for the delivery of therapeutics
JO3672B1 (en) 2008-12-15 2020-08-27 Regeneron Pharma High Affinity Human Antibodies to PCSK9
NZ597504A (en) 2009-06-15 2013-10-25 Alnylam Pharmaceuticals Inc Lipid formulated dsrna targeting the pcsk9 gene
US9051567B2 (en) 2009-06-15 2015-06-09 Tekmira Pharmaceuticals Corporation Methods for increasing efficacy of lipid formulated siRNA
WO2011038031A1 (en) 2009-09-22 2011-03-31 Alnylam Pharmaceuticals, Inc. Dual targeting sirna agents
HUE038039T2 (en) 2009-12-01 2018-09-28 Translate Bio Inc Delivery of mrna for the augmentation of proteins and enzymes in human genetic diseases
CA2812046A1 (en) 2010-09-15 2012-03-22 Alnylam Pharmaceuticals, Inc. Modified irna agents
JP2013545736A (en) * 2010-10-29 2013-12-26 アルナイラム ファーマシューティカルズ， インコーポレイテッド Compositions and methods for inhibition of the PCSK9 gene
EP3470395A1 (en) 2010-11-15 2019-04-17 Life Technologies Corporation Amine-containing transfection reagents and methods for making and using same
BR122020024388B1 (en) * 2010-12-29 2021-09-21 F. Hoffmann-La Roche Ag OLIGONUCLEOTIDE AND PHARMACEUTICAL COMPOSITION
BR112013018877A2 (en) 2011-01-28 2016-10-04 Sanofi Sa pharmaceutical compositions comprising human antibodies to pcsk9
US9175290B2 (en) 2011-03-29 2015-11-03 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of TMPRSS6 gene
WO2012149386A1 (en) * 2011-04-27 2012-11-01 Isis Pharmaceuticals, Inc. Modulation of cideb expression
ME03491B (en) 2011-06-08 2020-01-20 Translate Bio Inc Lipid nanoparticle compositions and methods for mrna delivery
US9315813B2 (en) * 2011-06-21 2016-04-19 Alnylam Pharmaceuticals, Inc Compositions and methods for inhibition of expression of apolipoprotein C-III (APOC3) genes
EP4092120A1 (en) * 2011-06-21 2022-11-23 Alnylam Pharmaceuticals, Inc. Angiopoietin-like 3 (anglptl3) irna compositions and methods of use thereof
EP3388068A1 (en) 2011-06-21 2018-10-17 Alnylam Pharmaceuticals, Inc. Composition and methods for inhibition of expression of protein c (proc) genes
AR087305A1 (en) 2011-07-28 2014-03-12 Regeneron Pharma STABILIZED FORMULATIONS CONTAINING ANTI-PCSK9 ANTIBODIES, PREPARATION METHOD AND KIT
EP3453761A1 (en) 2011-08-29 2019-03-13 Ionis Pharmaceuticals, Inc. Oligomer-conjugate complexes and their use
RU2014117690A (en) * 2011-10-05 2015-11-10 Протива Байотерапьютикс Инк. COMPOSITIONS AND METHODS FOR ALDEHYDE-DEHYDROGENASE SILENCING
WO2013075035A1 (en) * 2011-11-18 2013-05-23 Alnylam Pharmaceuticals Rnai agents, compositions and methods of use thereof for treating transthyretin (ttr) associated diseases
JP2015502931A (en) * 2011-11-18 2015-01-29 アルナイラム ファーマシューティカルズ， インコーポレイテッドＡｌｎｙｌａｍ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． Modified RNAi agent
US9133461B2 (en) * 2012-04-10 2015-09-15 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of the ALAS1 gene
US9127274B2 (en) * 2012-04-26 2015-09-08 Alnylam Pharmaceuticals, Inc. Serpinc1 iRNA compositions and methods of use thereof
EP2859102A4 (en) 2012-06-08 2016-05-11 Shire Human Genetic Therapies Nuclease resistant polynucleotides and uses thereof
WO2014062736A1 (en) 2012-10-15 2014-04-24 Isis Pharmaceuticals, Inc. Methods for monitoring c9orf72 expression
US10058630B2 (en) * 2012-10-22 2018-08-28 Concievalve, Llc Methods for inhibiting stenosis, obstruction, or calcification of a stented heart valve or bioprosthesis
US9958448B2 (en) 2012-10-23 2018-05-01 Caris Life Sciences Switzerland Holdings Gmbh Aptamers and uses thereof
US10942184B2 (en) 2012-10-23 2021-03-09 Caris Science, Inc. Aptamers and uses thereof
US9939443B2 (en) 2012-12-19 2018-04-10 Caris Life Sciences Switzerland Holdings Gmbh Compositions and methods for aptamer screening
EA037922B1 (en) 2013-03-14 2021-06-07 Шир Хьюман Дженетик Терапис, Инк. CFTR mRNA AND COMPOSITIONS FOR TREATING CYSTIC FIBROSIS IN A MAMMAL
EP3467108B1 (en) 2013-03-14 2024-05-22 Translate Bio, Inc. Methods for purification of messenger rna
WO2014160129A2 (en) * 2013-03-14 2014-10-02 Alnylam Pharmaceuticals, Inc. Complement component c5 irna compositions and methods of use thereof
KR20160002977A (en) 2013-05-01 2016-01-08 아이시스 파마수티컬즈 인코포레이티드 Compositions and methods
TW201515650A (en) 2013-05-06 2015-05-01 艾爾妮蘭製藥公司 Dosages and methods for delivering lipid formulated nucleic acid molecules
PT2999785T (en) * 2013-05-22 2018-07-09 Alnylam Pharmaceuticals Inc Serpina1 irna compositions and methods of use thereof
AU2014268529C1 (en) 2013-05-22 2020-10-01 Alnylam Pharmaceuticals, Inc. TMPRSS6 iRNA compositions and methods of use thereof
EP2810955A1 (en) 2013-06-07 2014-12-10 Sanofi Methods for inhibiting atherosclerosis by administering an inhibitor of PCSK9
HK1222865A1 (en) 2013-06-07 2017-07-14 Regeneron Pharmaceuticals, Inc. Methods for inhibiting atherosclerosis by administering an inhibitor of pcsk9
EP2862877A1 (en) 2013-10-18 2015-04-22 Sanofi Methods for inhibiting atherosclerosis by administering an inhibitor of PCSK9
EP2853595A1 (en) * 2013-09-30 2015-04-01 Soluventis GmbH NOTCH 1 specific siRNA molecules
JP6613227B2 (en) * 2013-10-04 2019-11-27 アルナイラム ファーマシューティカルズ， インコーポレイテッド Composition and method for inhibiting the expression of ALAS1 gene
MY192689A (en) 2013-10-11 2022-09-01 Ionis Pharmaceuticals Inc Compositions for modulating c9orf72 expression
EA202090893A3 (en) * 2013-10-17 2021-09-30 Элнилэм Фармасьютикалз, Инк. COMPOSITIONS WITH IRNA K PCSK9 AND METHODS OF THEIR APPLICATION
EP3060258A1 (en) 2013-10-22 2016-08-31 Shire Human Genetic Therapies, Inc. Mrna therapy for phenylketonuria
AU2014340092B2 (en) 2013-10-22 2019-09-19 Translate Bio, Inc. mRNA therapy for Argininosuccinate Synthetase Deficiency
MX362066B (en) * 2013-11-04 2019-01-07 Dow Agrosciences Llc Optimal soybean loci.
KR101647178B1 (en) * 2013-11-28 2016-08-23 충남대학교산학협력단 Stabilized Plasmid-Lipid Particles Using polyethylen glycol-lipid composed of an Enzymatically Cleavable Linker or oligo-lysines
CN118845818A (en) 2014-02-11 2024-10-29 阿尔尼拉姆医药品有限公司 Ketohexokinase (KHK) iRNA compositions and methods of use thereof
EP3978610A3 (en) * 2014-03-19 2022-08-24 Ionis Pharmaceuticals, Inc. Compositions for modulating ataxin 2 expression
AU2015249553B2 (en) * 2014-04-23 2021-03-04 Modernatx, Inc. Nucleic acid vaccines
EP3636742B1 (en) 2014-04-25 2025-11-05 Translate Bio, Inc. Methods for purification of messenger rna
EP3647318B1 (en) 2014-04-28 2021-06-30 Ionis Pharmaceuticals, Inc. Linkage modified oligomeric compounds
JP2017521045A (en) 2014-05-01 2017-08-03 アイオーニス ファーマシューティカルズ， インコーポレーテッドＩｏｎｉｓ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ，Ｉｎｃ． Compositions and methods for modulating angiopoietin-like factor 3 expression
RU2724527C2 (en) 2014-05-01 2020-06-23 Ионис Фармасьютикалз, Инк. Compositions and methods for modulating growth hormone receptor expression
CN110724687B (en) 2014-05-01 2023-10-13 Ionis制药公司 Compositions and methods for modulating complement factor B expression
AU2015252917B2 (en) 2014-05-01 2019-09-26 Ionis Pharmaceuticals, Inc. Compositions and methods for modulating PKK expression
CN112852809A (en) 2014-05-22 2021-05-28 阿尔尼拉姆医药品有限公司 Angiotensinogen (AGT) iRNA compositions and methods of use thereof
US10570169B2 (en) 2014-05-22 2020-02-25 Ionis Pharmaceuticals, Inc. Conjugated antisense compounds and their use
KR20230074283A (en) 2014-07-16 2023-05-26 사노피 바이오테크놀로지 METHODS FOR TREATING PATIENTS WITH HETEROZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA(heFH)
WO2016030863A1 (en) 2014-08-29 2016-03-03 Glaxosmithkline Intellectual Property Development Limited Compounds and methods for treating viral infections
US9950194B2 (en) 2014-09-09 2018-04-24 Mevion Medical Systems, Inc. Patient positioning system
WO2016040748A1 (en) 2014-09-12 2016-03-17 Ionis Pharmaceuticals, Inc. Compositions and methods for detection of smn protein in a subject and treatment of a subject
WO2016085852A1 (en) 2014-11-24 2016-06-02 Alnylam Pharmaceuticals, Inc. Tmprss6 irna compositions and methods of use thereof
WO2016168286A1 (en) 2015-04-13 2016-10-20 Alnylam Pharmaceuticals, Inc. Angiopoietin-like 3 (angptl3) irna compositions and methods of use thereof
KR102104163B1 (en) 2015-04-16 2020-04-23 아이오니스 파마수티컬즈, 인코포레이티드 Composition for modulating C9ORF72 expression
GB2537614A (en) * 2015-04-20 2016-10-26 Heart Biotech Ltd Formulations for inhibition of PCSK9 for the treatment of hypercholesterolemia
US20180312845A1 (en) 2015-07-10 2018-11-01 Ionis Pharmaceuticals, Inc. Modulators of diacyglycerol acyltransferase 2 (dgat2)
SI3329002T1 (en) 2015-07-31 2021-02-26 Alnylam Pharmaceuticals, Inc. Transthyretin (ttr) irna compositions and methods of use thereof for treating or preventing ttr-associated diseases
KR20180031025A (en) 2015-07-31 2018-03-27 아크투루스 쎄라퓨틱스, 인크. Multiple ligand agents for drug delivery
ES3052976T3 (en) 2015-08-18 2026-01-16 Regeneron Pharma Anti-pcsk9 inhibitory antibodies for treating patients with hyperlipidemia undergoing lipoprotein apheresis
KR20180051626A (en) 2015-09-24 2018-05-16 아이오니스 파마수티컬즈, 인코포레이티드 Modulator of KRAS expression
US11260073B2 (en) 2015-11-02 2022-03-01 Ionis Pharmaceuticals, Inc. Compounds and methods for modulating C90RF72
US20190046555A1 (en) 2015-11-06 2019-02-14 Ionis Pharmaceuticals, Inc. Conjugated antisense compounds for use in therapy
CA2999341A1 (en) 2015-11-06 2017-05-11 Ionis Pharmaceuticals, Inc. Modulating apolipoprotein (a) expression
CN106810609A (en) * 2015-11-27 2017-06-09 苏州君盟生物医药科技有限公司 Anti- PCSK9 antibody and its application
MA45295A (en) * 2016-04-19 2019-02-27 Alnylam Pharmaceuticals Inc HIGH DENSITY LIPOPROTEIN BINDING PROTEIN (HDLBP / VIGILINE) RNA COMPOSITION AND METHODS FOR USING THEM
JP7066632B2 (en) * 2016-05-09 2022-05-13 アストラゼネカ・アクチエボラーグ Lipid nanoparticles containing lipophilic anti-inflammatory agents and how to use them
CN107441506A (en) * 2016-05-30 2017-12-08 上海交通大学 Gene delivery carrier and its preparation and application
HUE060831T2 (en) 2016-07-15 2023-04-28 Ionis Pharmaceuticals Inc Compounds and methods for modulation of smn2
WO2018067900A1 (en) 2016-10-06 2018-04-12 Ionis Pharmaceuticals, Inc. Method of conjugating oligomeric compounds
WO2018089851A2 (en) 2016-11-11 2018-05-17 Modernatx, Inc. Influenza vaccine
JOP20190112A1 (en) 2016-11-14 2019-05-14 Amgen Inc Combined therapies for atherosclerosis, including atherosclerotic cardiovascular disease
TW202313978A (en) 2016-11-23 2023-04-01 美商阿尼拉製藥公司 Serpina1 irna compositions and methods of use thereof
WO2018102397A1 (en) 2016-11-29 2018-06-07 PureTech Health LLC Exosomes for delivery of therapeutic agents
CN108265052B (en) * 2016-12-30 2021-05-28 苏州瑞博生物技术股份有限公司 Small interfering nucleic acid, pharmaceutical composition and application thereof
EP3585417B1 (en) 2017-02-27 2023-02-22 Translate Bio, Inc. Method of making a codon-optimized cftr mrna
JOP20190215A1 (en) 2017-03-24 2019-09-19 Ionis Pharmaceuticals Inc Modulators of pcsk9 expression
US12487152B2 (en) 2017-05-09 2025-12-02 Ultragenyx Pharmaceutical Inc. Scalable method for producing transfection reagents
CA3063531A1 (en) 2017-05-16 2018-11-22 Translate Bio, Inc. Treatment of cystic fibrosis by delivery of codon-optimized mrna encoding cftr
WO2019006455A1 (en) 2017-06-30 2019-01-03 Solstice Biologics, Ltd. Chiral phosphoramidite auxiliaries and methods of their use
KR20250046363A (en) 2017-09-19 2025-04-02 알닐람 파마슈티칼스 인코포레이티드 Compositions and methods for treating transthyretin (ttr) mediated amyloidosis
JP7261494B2 (en) 2017-12-01 2023-04-20 スーチョウ リボ ライフ サイエンス カンパニー、リミテッド Nucleic acids, compositions and complexes containing said nucleic acids, preparation methods and uses
KR20250107294A (en) 2017-12-01 2025-07-11 쑤저우 리보 라이프 사이언스 컴퍼니, 리미티드 Nucleic acid, composition and conjugate comprising the same, and preparation method and use thereof
EP3719128B1 (en) 2017-12-01 2025-01-15 Suzhou Ribo Life Science Co., Ltd. Double-stranded oligonucleotide, composition and conjugate comprising double-stranded oligonucleotide, preparation method therefor and use thereof
CN118291456A (en) 2017-12-01 2024-07-05 苏州瑞博生物技术股份有限公司 Nucleic acid, composition containing nucleic acid, conjugate, preparation method and application
JP7273417B2 (en) 2017-12-01 2023-05-15 スーチョウ リボ ライフ サイエンス カンパニー、リミテッド Nucleic acids, compositions and complexes containing such nucleic acids, and preparation methods and uses
US20230139322A1 (en) * 2017-12-26 2023-05-04 Guangzhou Ribobio Co., Ltd. SiRNA molecule inhibiting the expression of the PCSK9 gene and use thereof
US11633482B2 (en) 2017-12-29 2023-04-25 Suzhou Ribo Life Science Co., Ltd. Conjugates and preparation and use thereof
KR20200113214A (en) 2018-01-15 2020-10-06 아이오니스 파마수티컬즈, 인코포레이티드 DNM2 expression regulator
AU2019218987B2 (en) 2018-02-12 2025-04-24 Ionis Pharmaceuticals, Inc. Modified compounds and uses thereof
MX2020011006A (en) 2018-04-18 2021-01-20 Dicerna Pharmaceuticals Inc Pcsk9 targeting oligonucleotides for treating hypercholesterolemia and related conditions.
BR112020020957B1 (en) 2018-05-09 2022-05-10 Ionis Pharmaceuticals, Inc Oligomeric compounds, population and pharmaceutical composition thereof and their uses
TWI851574B (en) 2018-05-14 2024-08-11 美商阿尼拉製藥公司 ANGIOTENSINOGEN (AGT) iRNA COMPOSITIONS AND METHODS OF USE THEREOF
EP3823725A4 (en) 2018-07-17 2023-05-10 Aronora, Inc. METHODS OF SAFELY REDUCING THROMBOPOIETIN
EP3833397A4 (en) 2018-08-08 2023-06-14 Arcturus Therapeutics, Inc. COMPOSITIONS AND AGENTS AGAINST NON-ALCOHOLIC STEATOHEPATITIS
CN111655849B (en) 2018-08-21 2024-05-10 苏州瑞博生物技术股份有限公司 Nucleic acid, pharmaceutical composition and conjugate containing the nucleic acid and use thereof
CN118421617A (en) 2018-08-24 2024-08-02 川斯勒佰尔公司 Method for purifying messenger RNA
TWI856973B (en) 2018-09-19 2024-10-01 美商Ionis製藥公司 Modulators of pnpla3 expression
JP7376952B2 (en) 2018-09-30 2023-11-09 スーチョウ リボ ライフ サイエンス カンパニー、リミテッド siRNA complex and its preparation method and use
MX2021005969A (en) 2018-11-21 2021-09-14 Translate Bio Inc TREATMENT OF CYSTIC FIBROSIS BY DELIVERY OF NEBULIZED mRNA ENCODING CFTR.
AU2019403447B2 (en) 2018-12-21 2023-07-27 Ionis Pharmaceuticals, Inc. Modulators of HSD17B13 expression
JP7507495B2 (en) 2018-12-28 2024-06-28 スーチョウ リボ ライフ サイエンス カンパニー、リミテッド Nucleic acids, compositions and complexes containing the same, and methods of preparation and use
KR20220018960A (en) * 2019-03-19 2022-02-15 아크투루스 쎄라퓨틱스, 인크. Methods for making lipid-encapsulated RNA nanoparticles
AU2020279101B2 (en) * 2019-05-17 2025-07-24 Alnylam Pharmaceuticals, Inc. Oral delivery of oligonucleotides
US12540323B2 (en) 2019-05-22 2026-02-03 Suzhou Ribo Life Science Co., Ltd. Nucleic acid, pharmaceutical composition, conjugate, preparation method, and use
CN113227376B (en) 2019-05-22 2024-04-09 苏州瑞博生物技术股份有限公司 Nucleic acid, pharmaceutical composition and conjugate, preparation method and use thereof
US20220233459A1 (en) * 2019-06-05 2022-07-28 Guide Therapeutics, Inc. Analysis of materials for tissue delivery
US20220290156A1 (en) * 2019-08-27 2022-09-15 Sanofi Compositions and methods for inhibiting pcsk9
EP4045062A1 (en) 2019-10-14 2022-08-24 Astrazeneca AB Modulators of pnpla3 expression
CN110638788A (en) * 2019-10-25 2020-01-03 广州医科大学 siRNA capable of silencing Pcsk9 protein, nano delivery system and application thereof
KR102269256B1 (en) * 2019-11-04 2021-06-24 건국대학교 산학협력단 Probiotic yeast Kluyveromyces marxianus A5 from kefir and uses thereof
CN111087332B (en) * 2019-12-11 2021-07-06 东南大学 A kind of cationic amino lipid and its synthesis method and application
WO2021167841A1 (en) 2020-02-18 2021-08-26 Alnylam Pharmaceuticals, Inc. Apolipoprotein c3 (apoc3) irna compositions and methods of use thereof
AR121446A1 (en) 2020-02-28 2022-06-08 Ionis Pharmaceuticals Inc COMPOUNDS AND METHODS FOR MODULATING SMN2
EP4081642A1 (en) * 2020-03-16 2022-11-02 Argonaute Rna Limited Antagonist of pcsk9
IL297483A (en) 2020-04-21 2022-12-01 Flagship Pioneering Inc Bifunctional molecules and methods of using thereof
EP4488371A3 (en) 2020-11-18 2025-04-09 Ionis Pharmaceuticals, Inc. Compounds and methods for modulating angiotensinogen expression
CN114657136A (en) * 2020-12-22 2022-06-24 未来智人再生医学研究院(广州)有限公司 Pluripotent stem cell expressing shRNA and/or shRNA-miR of target PCSK9 or derivative thereof
BR112023016645A2 (en) 2021-02-26 2023-11-14 Alnylam Pharmaceuticals Inc KETOHEXOKINASE (KHK) IRNA COMPOSITIONS AND METHODS OF USE THEREOF
AU2022231003A1 (en) 2021-03-04 2023-09-14 Alnylam Pharmaceuticals, Inc. Angiopoietin-like 3 (angptl3) irna compositions and methods of use thereof
WO2022231999A1 (en) 2021-04-26 2022-11-03 Alnylam Pharmaceuticals, Inc. Transmembrane protease, serine 6 (tmprss6) irna compositions and methods of use thereof
JP2024530018A (en) 2021-08-03 2024-08-14 アルナイラム ファーマシューティカルズ， インコーポレイテッド Transthyretin (TTR) iRNA Compositions and Methods of Use Thereof
WO2023051822A1 (en) * 2021-09-30 2023-04-06 北京安龙生物医药有限公司 Targeting oligonucleotide for treating diseases associated with pcsk9
AU2022357561A1 (en) 2021-10-01 2024-04-18 Adarx Pharmaceuticals, Inc. Prekallikrein-modulating compositions and methods of use thereof
KR20240167063A (en) 2022-04-01 2024-11-26 나노베이션 테라퓨틱스 인크. mRNA delivery method and composition thereof
EP4608382A1 (en) * 2022-10-25 2025-09-03 Nanovation Therapeutics Inc. Lipid nanoparticle formulations for anti-sense oligonucleotide delivery
CN121285630A (en) 2023-04-20 2026-01-06 阿达尔克斯制药有限公司 MAPT modulating compositions and methods of use thereof
AU2024273075A1 (en) 2023-05-12 2025-11-20 Adarx Pharmaceuticals, Inc. Nmda ligand conjugated compounds and uses thereof
CN118086311B (en) * 2023-05-25 2024-08-09 苏州时安生物技术有限公司 SiRNA for inhibiting PCSK9 gene expression, conjugate, pharmaceutical composition and application thereof
WO2024249328A2 (en) 2023-05-26 2024-12-05 Adarx Pharmaceuticals, Inc. Sod1-modulating compositions and methods of use thereof
AU2024313301A1 (en) 2023-06-20 2025-12-11 Adarx Pharmaceuticals, Inc. Lrrk2-modulating compositions and methods of use thereof
WO2025187760A1 (en) * 2024-03-07 2025-09-12 日東電工株式会社 Lipid nanoparticle

Family Cites Families (60)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6054299A (en) 1994-04-29 2000-04-25 Conrad; Charles A. Stem-loop cloning vector and method
JP4335310B2 (en) 1995-06-07 2009-09-30 ザ ユニバーシティ オブ ブリティッシュ コロンビア Lipid-nucleic acid particles prepared through hydrophobic lipid-nucleic acid complex intermediates and use for gene transfer
US6506559B1 (en) 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
CA2325344C (en) 1998-04-08 2017-12-05 Commonwealth Scientific And Industrial Research Organisation Methods and means for obtaining modified phenotypes
AR020078A1 (en) 1998-05-26 2002-04-10 Syngenta Participations Ag METHOD FOR CHANGING THE EXPRESSION OF AN OBJECTIVE GENE IN A PLANT CELL
BR9914773A (en) 1998-10-09 2002-02-05 Ingene Inc Set of generic elements, method for the production of single-stranded DNA, mrna transcription, nucleic acid construction, ssdna transcription, vector, vector system, host cell, set for the production of a single-stranded nucleic acid sequence, method for in vivo or in vitro production of a single-stranded nucleic acid sequence, transcription of single-stranded cdna, inhibitory nucleic acid, heteroduplex molecule, and pharmaceutical composition
CA2346155A1 (en) 1998-10-09 2000-04-20 Ingene, Inc. Enzymatic synthesis of ssdna
DE19956568A1 (en) 1999-01-30 2000-08-17 Roland Kreutzer Method and medicament for inhibiting the expression of a given gene
US6271359B1 (en) 1999-04-14 2001-08-07 Musc Foundation For Research Development Tissue-specific and pathogen-specific toxic agents and ribozymes
DE10100586C1 (en) 2001-01-09 2002-04-11 Ribopharma Ag Inhibiting gene expression in cells, useful for e.g. treating tumors, by introducing double-stranded complementary oligoRNA having unpaired terminal bases
AU2001247951A1 (en) * 2000-02-07 2001-08-14 Roche Diagnostics Corporation Novel cationic amphiphiles
WO2003070918A2 (en) 2002-02-20 2003-08-28 Ribozyme Pharmaceuticals, Incorporated Rna interference by modified short interfering nucleic acid
WO2002081628A2 (en) 2001-04-05 2002-10-17 Ribozyme Pharmaceuticals, Incorporated Modulation of gene expression associated with inflammation proliferation and neurite outgrowth, using nucleic acid based technologies
US20040259247A1 (en) 2000-12-01 2004-12-23 Thomas Tuschl Rna interference mediating small rna molecules
US20080249040A1 (en) 2001-05-18 2008-10-09 Sirna Therapeutics, Inc. RNA interference mediated inhibition of sterol regulatory element binding protein 1 (SREBP1) gene expression using short interfering nucleic acid (siNA)
US20070173473A1 (en) 2001-05-18 2007-07-26 Sirna Therapeutics, Inc. RNA interference mediated inhibition of proprotein convertase subtilisin Kexin 9 (PCSK9) gene expression using short interfering nucleic acid (siNA)
US20030170891A1 (en) 2001-06-06 2003-09-11 Mcswiggen James A. RNA interference mediated inhibition of epidermal growth factor receptor gene expression using short interfering nucleic acid (siNA)
US20040009216A1 (en) * 2002-04-05 2004-01-15 Rodrigueza Wendi V. Compositions and methods for dosing liposomes of certain sizes to treat or prevent disease
US7923547B2 (en) 2002-09-05 2011-04-12 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
AU2003295600A1 (en) 2002-11-14 2004-06-15 Dharmacon, Inc. Functional and hyperfunctional sirna
DE10302421A1 (en) 2003-01-21 2004-07-29 Ribopharma Ag New double-stranded interfering RNA, useful for inhibiting hepatitis C virus, has one strand linked to a lipophilic group to improve activity and eliminate the need for transfection auxiliaries
WO2004080406A2 (en) 2003-03-07 2004-09-23 Alnylam Pharmaceuticals Therapeutic compositions
CA2488224A1 (en) 2003-04-03 2004-10-21 Alnylam Pharmaceuticals, Inc. Irna conjugates
EP1471152A1 (en) 2003-04-25 2004-10-27 Institut National De La Sante Et De La Recherche Medicale (Inserm) Mutations in the human PCSK9 gene associated to hypercholesterolemia
DK3604537T3 (en) 2003-06-13 2022-02-28 Alnylam Europe Ag Double-stranded ribonucleic acid with increased efficiency in an organism
US20050064595A1 (en) 2003-07-16 2005-03-24 Protiva Biotherapeutics, Inc. Lipid encapsulated interfering RNA
CA2546616A1 (en) 2003-11-21 2005-06-09 Alza Corporation Gene delivery mediated by liposome-dna complex with cleavable peg surface modification
WO2006073419A2 (en) * 2004-04-01 2006-07-13 Gang Zheng Lipoprotein nanoplatforms
EP1766035B1 (en) 2004-06-07 2011-12-07 Protiva Biotherapeutics Inc. Lipid encapsulated interfering rna
US7745651B2 (en) 2004-06-07 2010-06-29 Protiva Biotherapeutics, Inc. Cationic lipids and methods of use
AU2005306533B2 (en) 2004-11-17 2012-05-31 Arbutus Biopharma Corporation siRNA silencing of apolipoprotein B
CA2597724A1 (en) * 2005-02-14 2007-08-02 Sirna Therapeutics, Inc. Cationic lipids and formulated molecular compositions containing them
TWI335352B (en) 2005-03-31 2011-01-01 Calando Pharmaceuticals Inc Inhibitors of ribonucleotide reductase subunit 2 and uses thereof
US7915230B2 (en) 2005-05-17 2011-03-29 Molecular Transfer, Inc. Reagents for transfection of eukaryotic cells
CN103989633A (en) 2005-07-27 2014-08-20 普洛体维生物治疗公司 Systems and methods for manufacturing liposomes
WO2007051303A1 (en) 2005-11-02 2007-05-10 Protiva Biotherapeutics, Inc. MODIFIED siRNA MOLECULES AND USES THEREOF
JP5704741B2 (en) 2006-03-31 2015-04-22 アルナイラム ファーマシューティカルズ， インコーポレイテッドＡｌｎｙｌａｍ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． Compositions and methods for suppression of Eg5 gene expression
EP2584047B1 (en) 2006-05-11 2014-11-19 Alnylam Pharmaceuticals Inc. Compositions and methods for inhibiting expression of the PCSK9 gene
US8598333B2 (en) 2006-05-26 2013-12-03 Alnylam Pharmaceuticals, Inc. SiRNA silencing of genes expressed in cancer
WO2008011431A2 (en) * 2006-07-17 2008-01-24 Sirna Therapeutics Inc. Rna interference mediated inhibition of proprotein convertase subtilisin kexin 9 (pcsk9) gene expression using short interfering nucleic acid (sina)
CA2848238C (en) 2006-10-03 2016-07-19 Tekmira Pharmaceuticals Corporation Lipid containing formulations
TW200838551A (en) 2006-11-27 2008-10-01 Isis Pharmaceuticals Inc Methods for treating hypercholesterolemia
WO2008109369A2 (en) * 2007-03-02 2008-09-12 Mdrna, Inc. Nucleic acid compounds for inhibiting tnf gene expression and uses thereof
JOP20080381B1 (en) 2007-08-23 2023-03-28 Amgen Inc Antigen Binding Proteins to Proprotein Convertase subtillisin Kexin type 9 (pcsk9)
CA2930393C (en) * 2007-12-04 2022-11-29 Alnylam Pharmaceuticals, Inc. Carbohydrate conjugates as delivery agents for oligonucleotides
CA2710713C (en) 2007-12-27 2017-09-19 Protiva Biotherapeutics, Inc. Silencing of polo-like kinase expression using interfering rna
EP3100718B1 (en) 2008-01-02 2019-11-27 Arbutus Biopharma Corporation Improved compositions and methods for the delivery of nucleic acids
AU2009241591A1 (en) * 2008-01-31 2009-11-05 Alnylam Pharmaceuticals, Inc. Optimized methods for delivery of DSRNA targeting the PCSK9 gene
KR101397407B1 (en) 2008-03-05 2014-06-19 알닐람 파마슈티칼스 인코포레이티드 Compositions and methods for inhibiting expression of Eg5 and VEGF genes
HUE034483T2 (en) 2008-04-15 2018-02-28 Protiva Biotherapeutics Inc Novel lipid formulations for nucleic acid delivery
AU2009303345B2 (en) 2008-10-09 2015-08-20 Arbutus Biopharma Corporation Improved amino lipids and methods for the delivery of nucleic acids
IL302142B2 (en) * 2008-10-20 2024-07-01 Alnylam Pharmaceuticals Inc Compositions and Methods for Inhabiting Expression of TRANSTHYRETIN
CA3033577A1 (en) * 2008-11-10 2010-05-14 Arbutus Biopharma Corporation Novel lipids and compositions for the delivery of therapeutics
NZ593618A (en) 2008-12-10 2013-02-22 Alnylam Pharmaceuticals Inc Gnaq targeted dsrna compositions and methods for inhibiting expression
WO2010088537A2 (en) 2009-01-29 2010-08-05 Alnylam Pharmaceuticals, Inc. Improved lipid formulation
WO2010129709A1 (en) 2009-05-05 2010-11-11 Alnylam Pharmaceuticals, Inc. Lipid compositions
SMT201800499T1 (en) 2009-06-10 2018-11-09 Arbutus Biopharma Corp Improved lipid formulation
NZ597504A (en) 2009-06-15 2013-10-25 Alnylam Pharmaceuticals Inc Lipid formulated dsrna targeting the pcsk9 gene
US9051567B2 (en) 2009-06-15 2015-06-09 Tekmira Pharmaceuticals Corporation Methods for increasing efficacy of lipid formulated siRNA
JP2013545736A (en) 2010-10-29 2013-12-26 アルナイラム ファーマシューティカルズ， インコーポレイテッド Compositions and methods for inhibition of the PCSK9 gene

Cited By (97)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9447164B2 (en) 2010-08-06 2016-09-20 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9181319B2 (en) 2010-08-06 2015-11-10 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9937233B2 (en) 2010-08-06 2018-04-10 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US9657295B2 (en) 2010-10-01 2017-05-23 Modernatx, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9334328B2 (en) 2010-10-01 2016-05-10 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US10064959B2 (en) 2010-10-01 2018-09-04 Modernatx, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9701965B2 (en) 2010-10-01 2017-07-11 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US9950068B2 (en) 2011-03-31 2018-04-24 Modernatx, Inc. Delivery and formulation of engineered nucleic acids
US9533047B2 (en) 2011-03-31 2017-01-03 Modernatx, Inc. Delivery and formulation of engineered nucleic acids
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US10022425B2 (en) 2011-09-12 2018-07-17 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US10751386B2 (en) 2011-09-12 2020-08-25 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US9428535B2 (en) 2011-10-03 2016-08-30 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9271996B2 (en) 2011-12-16 2016-03-01 Moderna Therapeutics, Inc. Formulation and delivery of PLGA microspheres
US9186372B2 (en) 2011-12-16 2015-11-17 Moderna Therapeutics, Inc. Split dose administration
US9295689B2 (en) 2011-12-16 2016-03-29 Moderna Therapeutics, Inc. Formulation and delivery of PLGA microspheres
US9303079B2 (en) 2012-04-02 2016-04-05 Moderna Therapeutics, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US8999380B2 (en) 2012-04-02 2015-04-07 Moderna Therapeutics, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
US9255129B2 (en) 2012-04-02 2016-02-09 Moderna Therapeutics, Inc. Modified polynucleotides encoding SIAH E3 ubiquitin protein ligase 1
US9254311B2 (en) 2012-04-02 2016-02-09 Moderna Therapeutics, Inc. Modified polynucleotides for the production of proteins
US9233141B2 (en) 2012-04-02 2016-01-12 Moderna Therapeutics, Inc. Modified polynucleotides for the production of proteins associated with blood and lymphatic disorders
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US9221891B2 (en) 2012-04-02 2015-12-29 Moderna Therapeutics, Inc. In vivo production of proteins
US10501512B2 (en) 2012-04-02 2019-12-10 Modernatx, Inc. Modified polynucleotides
US9301993B2 (en) 2012-04-02 2016-04-05 Moderna Therapeutics, Inc. Modified polynucleotides encoding apoptosis inducing factor 1
US9220792B2 (en) 2012-04-02 2015-12-29 Moderna Therapeutics, Inc. Modified polynucleotides encoding aquaporin-5
US9050297B2 (en) 2012-04-02 2015-06-09 Moderna Therapeutics, Inc. Modified polynucleotides encoding aryl hydrocarbon receptor nuclear translocator
US9220755B2 (en) 2012-04-02 2015-12-29 Moderna Therapeutics, Inc. Modified polynucleotides for the production of proteins associated with blood and lymphatic disorders
US9216205B2 (en) 2012-04-02 2015-12-22 Moderna Therapeutics, Inc. Modified polynucleotides encoding granulysin
US9061059B2 (en) 2012-04-02 2015-06-23 Moderna Therapeutics, Inc. Modified polynucleotides for treating protein deficiency
US9192651B2 (en) 2012-04-02 2015-11-24 Moderna Therapeutics, Inc. Modified polynucleotides for the production of secreted proteins
US9149506B2 (en) 2012-04-02 2015-10-06 Moderna Therapeutics, Inc. Modified polynucleotides encoding septin-4
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9587003B2 (en) 2012-04-02 2017-03-07 Modernatx, Inc. Modified polynucleotides for the production of oncology-related proteins and peptides
US9089604B2 (en) 2012-04-02 2015-07-28 Moderna Therapeutics, Inc. Modified polynucleotides for treating galactosylceramidase protein deficiency
US9114113B2 (en) 2012-04-02 2015-08-25 Moderna Therapeutics, Inc. Modified polynucleotides encoding citeD4
US9675668B2 (en) 2012-04-02 2017-06-13 Moderna Therapeutics, Inc. Modified polynucleotides encoding hepatitis A virus cellular receptor 2
US9107886B2 (en) 2012-04-02 2015-08-18 Moderna Therapeutics, Inc. Modified polynucleotides encoding basic helix-loop-helix family member E41
US9095552B2 (en) 2012-04-02 2015-08-04 Moderna Therapeutics, Inc. Modified polynucleotides encoding copper metabolism (MURR1) domain containing 1
US9782462B2 (en) 2012-04-02 2017-10-10 Modernatx, Inc. Modified polynucleotides for the production of proteins associated with human disease
US9814760B2 (en) 2012-04-02 2017-11-14 Modernatx, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
US9827332B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of proteins
US9828416B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of secreted proteins
US9878056B2 (en) 2012-04-02 2018-01-30 Modernatx, Inc. Modified polynucleotides for the production of cosmetic proteins and peptides
US9255154B2 (en) 2012-05-08 2016-02-09 Alderbio Holdings, Llc Anti-PCSK9 antibodies and use thereof
US10259885B2 (en) 2012-05-08 2019-04-16 Alderbio Holdings Llc Anti-PCSK9 antibodies and use thereof
US9597380B2 (en) 2012-11-26 2017-03-21 Modernatx, Inc. Terminally modified RNA
US12460206B2 (en) 2012-12-05 2025-11-04 Alnylam Pharmaceuticals, Inc. PCSK9 iRNA compositions and methods of use thereof
US10125369B2 (en) 2012-12-05 2018-11-13 Alnylam Pharmaceuticals, Inc. PCSK9 iRNA compositions and methods of use thereof
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
US10815291B2 (en) 2013-09-30 2020-10-27 Modernatx, Inc. Polynucleotides encoding immune modulating polypeptides
US10323076B2 (en) 2013-10-03 2019-06-18 Modernatx, Inc. Polynucleotides encoding low density lipoprotein receptor
US9409869B1 (en) * 2014-09-04 2016-08-09 Preceres Inc. Hydrazinyl lipidoids and uses thereof
US10307491B2 (en) 2015-01-30 2019-06-04 The Regents Of The University Of Michigan Liposomal particles comprising biological molecules and uses thereof
AU2016238290B2 (en) * 2015-03-25 2019-06-06 The Regents Of The University Of Michigan Compositions and methods for delivery of biomacromolecule agents
WO2016154544A1 (en) * 2015-03-25 2016-09-29 The Regents Of The University Of Michigan Compositions and methods for delivery of biomacromolecule agents
US11219673B2 (en) 2015-03-25 2022-01-11 The Regents Of The University Of Michigan Compositions and methods for delivery of biomacromolecule agents
AU2016238290B9 (en) * 2015-03-25 2019-06-13 The Regents Of The University Of Michigan Compositions and methods for delivery of biomacromolecule agents
US12324843B2 (en) 2015-03-25 2025-06-10 The Regents Of The University Of Michigan Compositions and methods for delivery of biomacromolecule agents
US11833196B2 (en) 2015-03-25 2023-12-05 The Regents Of The University Of Michigan Compositions and methods for delivery of biomacromolecule agents
US10851377B2 (en) 2015-08-25 2020-12-01 Alnylam Pharmaceuticals, Inc. Methods and compositions for treating a proprotein convertase subtilisin kexin (PCSK9) gene-associated disorder
US10442756B2 (en) 2015-09-17 2019-10-15 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US10392341B2 (en) 2015-09-17 2019-08-27 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US12151995B2 (en) 2015-09-17 2024-11-26 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US11220476B2 (en) 2015-09-17 2022-01-11 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US12404232B2 (en) 2015-09-17 2025-09-02 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US10266485B2 (en) 2015-09-17 2019-04-23 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US12491260B2 (en) 2015-12-10 2025-12-09 Modernatx, Inc. Compositions and methods for delivery of agents
US10207010B2 (en) 2015-12-10 2019-02-19 Modernatx, Inc. Compositions and methods for delivery of agents
US10485885B2 (en) 2015-12-10 2019-11-26 Modernatx, Inc. Compositions and methods for delivery of agents
US11285222B2 (en) 2015-12-10 2022-03-29 Modernatx, Inc. Compositions and methods for delivery of agents
US10556018B2 (en) 2015-12-10 2020-02-11 Modernatx, Inc. Compositions and methods for delivery of agents
US12396961B2 (en) 2015-12-22 2025-08-26 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
US10195156B2 (en) 2015-12-22 2019-02-05 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
US10799463B2 (en) 2015-12-22 2020-10-13 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
WO2017127750A1 (en) 2016-01-22 2017-07-27 Modernatx, Inc. Messenger ribonucleic acids for the production of intracellular binding polypeptides and methods of use thereof
US12257352B2 (en) 2016-06-20 2025-03-25 The Regents Of The University Of Michigan Compositions and methods for delivery of biomacromolecule agents
WO2017223085A3 (en) * 2016-06-20 2018-02-15 The Regents Of The University Of Michigan Compositions and methods for delivery of biomacromolecule agents
EP3471778A4 (en) * 2016-06-20 2020-02-19 The Regents of The University of Michigan COMPOSITIONS AND METHOD FOR DELIVERING BIOMACROMOLECOLIC ACTIVE SUBSTANCES
US12144895B2 (en) 2016-11-08 2024-11-19 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
US11583504B2 (en) 2016-11-08 2023-02-21 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
US11203569B2 (en) 2017-03-15 2021-12-21 Modernatx, Inc. Crystal forms of amino lipids
US10857105B2 (en) 2017-03-15 2020-12-08 MordernaTX, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US11969506B2 (en) 2017-03-15 2024-04-30 Modernatx, Inc. Lipid nanoparticle formulation
US12324859B2 (en) 2017-03-15 2025-06-10 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
WO2018213731A1 (en) 2017-05-18 2018-11-22 Modernatx, Inc. Polynucleotides encoding tethered interleukin-12 (il12) polypeptides and uses thereof
US12077501B2 (en) 2017-06-14 2024-09-03 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
US12383508B2 (en) 2018-09-19 2025-08-12 Modernatx, Inc. High-purity peg lipids and uses thereof
US12151029B2 (en) 2018-09-19 2024-11-26 Modernatx, Inc. PEG lipids and uses thereof
US12263248B2 (en) 2018-09-19 2025-04-01 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
US11066355B2 (en) 2019-09-19 2021-07-20 Modernatx, Inc. Branched tail lipid compounds and compositions for intracellular delivery of therapeutic agents
US12312293B2 (en) 2019-09-19 2025-05-27 Modernatx, Inc. Branched tail lipid compounds and compositions for intracellular delivery of therapeutic agents
US11597698B2 (en) 2019-09-19 2023-03-07 Modernatx, Inc. Branched tail lipid compounds and compositions for intracellular delivery of therapeutic agents
US11524023B2 (en) 2021-02-19 2022-12-13 Modernatx, Inc. Lipid nanoparticle compositions and methods of formulating the same
US11622972B2 (en) 2021-02-19 2023-04-11 Modernatx, Inc. Lipid nanoparticle compositions and methods of formulating the same
US12508278B2 (en) 2021-02-19 2025-12-30 Modernatx, Inc. Lipid nanoparticle compositions and methods of formulating the same
US12552738B2 (en) 2021-11-12 2026-02-17 Modernatx, Inc. Crystal forms of amino lipids

Also Published As

Publication number Publication date
JP2012530143A (en) 2012-11-29
US8273869B2 (en) 2012-09-25
WO2010148013A2 (en) 2010-12-23
CA2764832A1 (en) 2010-12-23
EA201270019A1 (en) 2012-06-29
EP2442792A2 (en) 2012-04-25
US20110015252A1 (en) 2011-01-20
JP2016074685A (en) 2016-05-12
CN102458366A (en) 2012-05-16
AU2010260148A1 (en) 2012-02-02
JP5894913B2 (en) 2016-03-30
IL216886A0 (en) 2012-02-29
BRPI1010689A2 (en) 2016-03-15
US8598139B2 (en) 2013-12-03
CN104651408A (en) 2015-05-27
US20130035371A1 (en) 2013-02-07
US20170000815A1 (en) 2017-01-05
NZ597504A (en) 2013-10-25
MX2011013421A (en) 2012-03-16
EP2442792A4 (en) 2015-12-23
CN102458366B (en) 2015-02-11
KR20120050429A (en) 2012-05-18
WO2010148013A3 (en) 2011-02-24

Similar Documents

Publication Publication Date Title
US8598139B2 (en) Lipid formulated dsRNA targeting the PCSK9 gene
US10053689B2 (en) Methods for increasing efficacy of lipid formulated siRNA
AU2010223967B2 (en) Lipid formulated compositions and methods for inhibiting expression of Eg5 and VEGF genes
US9029338B2 (en) Lipid formulated compositions and methods for inhibiting expression of a gene from the ebola virus
AU2009221775B2 (en) Compositions and methods for inhibiting expression of Eg5 and VEGF genes
US20100010066A1 (en) Optimized Methods For Delivery Of DSRNA Targeting The PCSK9 Gene
WO2011028938A1 (en) Methods for lowering serum cholestrol in a subject using inhibition of pcsk9
WO2011017548A1 (en) Lipid formulated compositions and methods for inhibiting expression of eg5 and vegf genes

Legal Events

Date Code Title Description
AS Assignment

Owner name: ALNYLAM PHARMACEUTICALS, INC., MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:FITZGERALD, KEVIN;HINKLE, GREGORY;AKINC, AKIN;AND OTHERS;SIGNING DATES FROM 20100803 TO 20100811;REEL/FRAME:031439/0549

AS Assignment

Owner name: ALNYLAM PHARMACEUTICALS, INC., MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:FITZGERALD, KEVIN;HINKLE, GREGORY;AKINC, AKIN;AND OTHERS;SIGNING DATES FROM 20100803 TO 20100811;REEL/FRAME:032148/0473

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION