WO2016071857A1 - Compositions et méthodes pour le silençage de l'expression du virus ebola - Google Patents

Compositions et méthodes pour le silençage de l'expression du virus ebola Download PDF

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WO2016071857A1
WO2016071857A1 PCT/IB2015/058539 IB2015058539W WO2016071857A1 WO 2016071857 A1 WO2016071857 A1 WO 2016071857A1 IB 2015058539 W IB2015058539 W IB 2015058539W WO 2016071857 A1 WO2016071857 A1 WO 2016071857A1
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lipid
mol
nucleic acid
particle
peg
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Amy C. H. Lee
Ian Maclachlan
Emily P. THI
Zhe Zhou
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Protiva Biotherapeutics, Inc.
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    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • C12N15/1131—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 viruses
    • 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
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • Filoviruses e.g., Ebolavirus and Marburg virus (MA V)
  • MA V Marburg virus
  • Filoviruses are of particular concern as possible biological weapons since they have the potential for aerosol dissemination and weaponization.
  • the incubation period for Filovirus infection ranges from 2 to 21 days.
  • the onset of illness is abrupt and is characterized by high fever, headaches, joint and muscle aches, sore throat, fatigue, diarrhea, vomiting, and stomach pain.
  • a rash, red eyes, hiccups and internal and external bleeding may be seen in some patients.
  • Within one week of becoming infected with the virus most patients experience chest pains and multiple organ failure, go into shock, and die. Some patients also experience blindness and extensive bleeding before dying.
  • Filoviridae are a family of RNA viruses. Two members of the Filoviridae family have been identified: ebolavirus and Marburg virus. There is one identified species of MARV and four identified species of ebolavirus: Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV), Tai Forest ebolavirus (TAFV), and Reston ebolavirus (RESTV). The exact origin, locations, and natural habitat of Filoviridae are unknown. However, on the basis of available evidence and the nature of similar viruses, it is postulated that Filoviridae are zoonotic (i.e., animal-borne) and are normally maintained in an animal host that is native to the African continent.
  • MARV Zaire ebolavirus
  • SUDV Sudan ebolavirus
  • TAFV Tai Forest ebolavirus
  • RESTV Reston ebolavirus
  • ebolaviruses have been associated with periodic episodes of hemorrhagic fever in Central Africa that produce severe disease in infected patients. Mortality rates in outbreaks have ranged from 50% for the Sudan species of ebolavirus (SUDV) to up to 90% for the Zaire species of ebolavirus (EBOV) (Sanchez et al, Filoviridae: Marburg and Ebola Viruses, in Fields Virology (eds. JCnipe, D.M. & Howley, P.M.) 1409-1448 (Lippincott Williams & Wilkins, Philadelphia)).
  • SUDV Sudan species of ebolavirus
  • EBOV Zaire species of ebolavirus
  • Bundibugyo ebolavirus in Kenya resulted in a fatality rate of about 25% (Towner et al, PLoS Pathog, 4:e 1000212 (2008)).
  • EBOV has also decimated populations of wild apes in this same region of Africa (Walsh et al, Nature, 422:61 1-614 (2003)).
  • the World Health Organization was notified of an outbreak of a communicable disease characterized by fever, severe diarrhea, vomiting, and a high fatality rate in Guinea, which disease has been determined to be caused by a strain of EBOV (see Baize et al, New England Journal of Medicine, 371, 1418-1425 (2014)).
  • compositions and methods for treating and preventing ebolavirus infections e.g., by specifically modulating ebolavirus gene expression.
  • the present invention addresses these and other needs.
  • the present invention provides isolated, double stranded, siRNA molecules that each include a sense strand and an antisense strand that is hybridized to the sense strand.
  • the siRNA of this aspect of the invention target one or more genes and/or transcripts of the EBOV genome.
  • Examples of siRNA molecules of this aspect of the invention are the siRNA molecules set forth in Tables A and B herein.
  • the siRNA molecules of the invention are useful, for example, for the treatment of EBOV infection when administered in a therapeutic amount to a human subject infected with EBOV. More generally, the invention provides siRNA molecules that are capable of inhibiting or silencing EBOV gene expression in vitro and in vivo.
  • the siRNAs of the present invention are effective against EBOV, and in certain embodiments, particularly effective against the EBOV Guinea strain.
  • the present invention provides isolated, single stranded, nucleic acid molecules, such as the isolated sense and antisense strands of the siRNA molecules set forth in Tables A or B (see Tables C and D).
  • the siRNA and single stranded nucleic acid molecules of the invention are modified and include one or more 2'O-methyl modifications.
  • compositions such as pharmaceutical
  • compositions that include one or more siRNA molecules of the invention (see, e.g., the siRNA molecules described in Tables A and B).
  • the present invention provides compositions that include two different siRNA molecules of the invention (e.g., two different siRNA molecules selected from the siRNA molecules disclosed in Tables A and B herein, e.g., 1 and 2 or lm and 2m).
  • the present invention provides compositions (e.g., pharmaceutical compositions) that include one of the aforementioned two-way combinations of the siRNAs set forth in Tables A and B.
  • the present invention also provides nucleic acid-lipid particles, and formulations thereof, wherein the lipid particles each include one or more (e.g., a cocktail) of the siRNA described herein, a cationic lipid, and a non-cationic lipid, and optionally a conjugated lipid that inhibits aggregation of particles.
  • siRNA molecules that can be included in the lipid particles of the invention are the siRNA molecules set forth in Tables A and B, and combinations of the foregoing siRNA (e.g., the two way combinations described herein, e.g., 1 and 2 or lm and 2m).
  • the siRNA is fully encapsulated within the lipid particle.
  • the lipid particles of the invention are useful, for example, for delivering a therapeutically effective amount of siRNA into cells of a human body infected with EBOV, thereby treating the EBOV infection and/or ameliorating one or more symptoms of EBOV infection.
  • the present invention also provides a pharmaceutical composition comprising one or a cocktail of siRNA molecules that target EBOV gene expression, and a pharmaceutically acceptable carrier.
  • the present invention provides pharmaceutical compositions that each include one or two of the siRNA molecules set forth in Tables A and B that target EBOV gene expression.
  • the different siRNA molecules may be co-encapsulated in the same lipid particle, or each type of siRNA species present in the cocktail may be encapsulated in its own particle, or some siRNA species may be coencapsulated in the same particle while other siRNA species are encapsulated in different particles within the formulation.
  • the siRNA molecules of the invention are fully encapsulated in the lipid particle.
  • the nucleic acid-lipid particles of the invention are useful for the prophylactic or therapeutic delivery, into a human infected with EBOV, of siRNA molecules that silence the expression of one or more EBOV genes, thereby ameliorating at least one symptom of EBOV infection in the human.
  • one or more of the siRNA molecules described herein are formulated into nucleic acid-lipid particles, and the particles are administered to a mammal (e.g., a human) requiring such treatment.
  • a therapeutically effective amount of the nucleic acid-lipid particle can be administered to the mammal, (e.g., for treating EBOV infection in a human being).
  • nucleic acid-lipid particle can be by any route known in the art, such as, e.g., oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, or intradermal.
  • the nucleic acid-lipid particle is administered systemically, e.g., via enteral or parenteral routes of administration.
  • downregulation of EBOV gene expression is determined by detecting EBOV RNA or protein levels in a biological sample from a mammal after nucleic acid-lipid particle administration. In other embodiments, downregulation of EBOV gene expression is determined by detecting EBOV mRNA or protein levels in a biological sample from a mammal after nucleic acid-lipid particle administration. In certain embodiments, downregulation of EBOV or EBOV gene expression is detected by monitoring symptoms associated with EBOV infection in a mammal after particle administration.
  • the present invention provides methods for introducing an siRNA that silences EBOV gene expression into a living cell, the method comprising the step of contacting the cell with a nucleic acid-lipid particle of the invention, wherein the nucleic acid- lipid particle includes an siRNA that targets EBOV, under conditions whereby the siRNA enters the cell and silences the expression of an EBOV gene within the cell.
  • the present invention provides a method for ameliorating one or more symptoms associated with EBOV infection in a human, the method including the step of administering to the human a therapeutically effective amount of a nucleic acid-lipid particle of the present invention.
  • the nucleic acid-lipid particles used in the methods of this aspect of the invention include one or two different siRNA independently selected from the siRNAs set forth in Tables A and B.
  • the present invention provides methods for silencing EBOV gene expression in a mammal (e.g. , a human) in need thereof, wherein the methods each include the step of administering to the mammal a nucleic acid-lipid particle of the present invention.
  • the present invention provides methods for treating and/or ameliorating one or more symptoms associated with EBOV infection in a human, wherein the methods each include the step of administering to the human a therapeutically effective amount of a nucleic acid-lipid particle of the present invention.
  • the present invention provides methods for inhibiting the expression of EBOV in a mammal in need thereof (e.g. , a human infected with EBOV), wherein the methods each include the step of administering to the mammal a therapeutically effective amount of a nucleic acid-lipid particle of the present invention.
  • the present invention provides methods for treating EBOV infection in a human, wherein the methods each include the step of administering to the human a therapeutically effective amount of a nucleic acid-lipid particle of the present invention.
  • the present invention provides for use of a siRNA molecule of the present invention for inhibiting EBOV gene expression in a living cell.
  • the present invention provides for use of a pharmaceutical composition of the present invention for inhibiting EBOV gene expression in a living cell.
  • compositions of the invention are also useful, for example, in biological assays (e.g. , in vivo or in vitro assays) for inhibiting the expression of one or more EBOV genes and/or transcripts to investigate EBOV replication and biology, and/or to investigate or modulate the function of one or more EBOV genes or transcripts.
  • biological assays e.g. , in vivo or in vitro assays
  • the siRNA molecules of the invention can be screened using a biological assay to identify siRNA molecules that inhibit replication of EBOV and that are candidate therapeutic agents for the treatment of EBOV infection in humans, and/or the amelioration of at least one symptom associated with EBOV infection in a human.
  • siRNA drug therapy described herein advantageously provides significant new compositions and methods for treating EBOV infection in human beings and the symptoms associated therewith.
  • Embodiments of the present invention can be administered, for example, once per day, once per week, or once every several weeks (e.g., once every two, three, four, five or six weeks).
  • nucleic acid-lipid particles described herein enable the effective delivery of a nucleic acid drug such as a siRNA into target tissues and cells within the body.
  • a nucleic acid drug such as a siRNA
  • the presence of the lipid particle confers protection from nuclease degradation in the bloodstream, allows preferential accumulation in target tissue and provides a means of drug entry into the cellular cytoplasm where the siRNAs can perform their intended function of RNA interference.
  • the present invention provides siRNA molecules that target the expression of the Guinea/Sierra Leone (SL) variant of Ebola virus genes, nucleic acid-lipid particles comprising one or more of the siRNAs (e.g., a cocktail), and methods of delivering and/or administering the nucleic acid-lipid particles (e.g., for the treatment of Ebola virus infection in humans).
  • SL Guinea/Sierra Leone
  • the present invention provides siRNA molecules that target expression of one or more Guinea/Sierra Leone (SL) variant of Ebola virus genes.
  • the present invention provides compositions comprising a combination (e.g., a cocktail, pool, or mixture) of siRNAs that target different regions of the Guinea/Sierra Leone (SL) variant of
  • the siRNA molecules of the invention are capable of inhibiting the replication of Guinea/Sierra Leone (SL) variant of Ebola virus genes in vitro or in vivo.
  • the siRNA molecules of the invention may also be capable of inhibiting the replication of other Ebola virus variants in addition to the Guinea/Sierra Leone variant.
  • the term “combination” means that the combined siRNA molecules are present together in the same composition of matter (e.g., dissolved together within the same solution; or present together within the same nucleic acid-lipid particle; or present together in the same pharmaceutical formulation of nucleic acid-lipid particles, although each nucleic acid-lipid particle within the pharmaceutical formulation may or may not include each different siRNA of the siRNA combination).
  • the combined siRNA molecules usually are not covalently linked together.
  • lm and 2m are used as a combination.
  • 1 and 2 are used as a combination.
  • the present invention provides the siRNA molecules shown in Tables A and B, wherein the top strand of each double-stranded siRNA molecule is the sense strand running in a 5' to 3' direction from left to right; and the lower strand of each double- stranded siRNA molecule is the antisense strand running in a 5' to 3' direction from right to left.
  • the siRNA molecules may comprise ribonucleotides with a 2'-0-methyl modification as shown in Table B. Table A.
  • the present invention provides the isolated sense strands and antisense strands (i.e., isolated single stranded nucleic acid molecules) of the siRNA molecules set forth in Tables A and B, respectively.
  • the nucleic acid sequences set forth in Table C and Table D are arranged as pairs of sequences, wherein each pair includes a sense strand and its complementary antisense strand.
  • Each pair of sequences (sense plus antisense strand) is identified with a particular name, which corresponds to the names shown in Tables A and B.
  • isolated sense and antisense strands are useful, for example, for making siRNA molecules that are useful to reduce the expression of one or more Guinea/Sierra Leone (SL) variant, as well as other variants, of Ebola virus genes in vivo or in vitro.
  • These isolated sense and antisense strands are also useful, for example, as hybridization probes for identifying and measuring the amount of Guinea/Sierra Leone (SL) variant of Ebola virus genome in a biological material, such as a tissue or blood sample from a human being infected with ebolavirus.
  • an oligonucleotide (such as the sense and antisense RNA strands set forth in Tables C and D) of the invention specifically hybridizes to or is
  • oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable.
  • an oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target sequence interferes with the normal function of the target sequence to cause a loss of utility or expression therefrom, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or, in the case of in vitro assays, under conditions in which the assays are conducted.
  • the oligonucleotide may include 1, 2, 3, or more base substitutions as compared to the region of a gene or mRNA sequence that it is targeting or to which it specifically hybridizes.
  • small-interfering RNA or "siRNA” as used herein refers to double stranded RNA (i.e., duplex RNA) that is capable of reducing or inhibiting the expression of a target gene or sequence (e.g., by mediating the degradation or inhibiting the translation of mRNAs which are complementary to the siRNA sequence) when the siRNA is in the same cell as the target gene or sequence.
  • the siRNA may have substantial or complete identity to the target gene or sequence, or may comprise a region of mismatch (i.e., a mismatch motif). In certain
  • the siRNAs may be about 19-25 (duplex) nucleotides in length, and is preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length.
  • siRNA duplexes may comprise 3' overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and 5' phosphate termini.
  • Examples of siRNA include, without limitation, a double-stranded polynucleotide molecule assembled from two separate stranded molecules, wherein one strand is the sense strand and the other is the complementary antisense strand.
  • siRNA are chemically synthesized.
  • a target gene refers to the ability of a siRNA of the invention to silence, reduce, or inhibit expression of a target gene (e.g., a gene within the EBOV genome).
  • a test sample e.g., a biological sample from an organism of interest expressing the target gene or a sample of cells in culture expressing the target gene
  • a siRNA that silences, reduces, or inhibits expression of the target gene.
  • Expression of the target gene in the test sample is compared to expression of the target gene in a control sample (e.g., a biological sample from an organism of interest expressing the target gene or a sample of cells in culture expressing the target gene) that is not contacted with the siRNA.
  • Control samples e.g., samples expressing the target gene
  • silencing, inhibition, or reduction of expression of a target gene is achieved when the value of the test sample relative to the control sample (e.g., buffer only, an siRNA sequence that targets a different gene, a scrambled siRNA sequence, etc.) is about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%.
  • the control sample e.g., buffer only, an siRNA sequence that targets a different gene, a scrambled siRNA sequence, etc.
  • Suitable assays include, without limitation, examination of protein or mRNA levels using techniques known to those of skill in the art, such as, e.g., dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.
  • An "effective amount” or “therapeutically effective amount” of a therapeutic nucleic acid such as a siRNA is an amount sufficient to produce the desired effect, e.g., an inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of a siRNA.
  • inhibition of expression of a target gene or target sequence is achieved when the value obtained with a siR A relative to the control (e.g., buffer only, an siRNA sequence that targets a different gene, a scrambled siRNA sequence, etc.) is about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%.
  • Suitable assays for measuring the expression of a target gene or target sequence include, but are not limited to, examination of protein or mRNA levels using techniques known to those of skill in the art, such as, e.g., dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.
  • nucleic acid refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single- or double-stranded form and includes DNA and RNA.
  • nucleotides contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups.
  • Bases include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols,
  • Nucleic acids include nucleic acids containing nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs and/or modified residues include, without limitation,
  • Nucleic acids can include one or more UNA moieties.
  • nucleic acid includes any oligonucleotide or polynucleotide, with fragments containing up to 60 nucleotides generally termed oligonucleotides, and longer fragments termed polynucleotides.
  • a deoxyribooligonucleotide consists of a 5-carbon sugar called deoxyribose joined covalently to phosphate at the 5' and 3' carbons of this sugar to form an alternating, unbranched polymer.
  • DNA may be in the form of, e.g. , antisense molecules, plasmid DNA, pre-condensed DNA, a PCR product, vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups.
  • ribooligonucleotide consists of a similar repeating structure where the 5-carbon sugar is ribose.
  • RNA may be in the form, for example, of small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof.
  • polynucleotide and oligonucleotide refer to a polymer or oligomer of nucleotide or nucleoside monomers consisting of naturally- occurring bases, sugars and intersugar (backbone) linkages.
  • polynucleotide and oligonucleotide also include polymers or oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly.
  • modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al, Mol. Cell. Probes, 8:91-98 (1994)).
  • an "isolated” or “purified” DNA molecule or RNA molecule is a DNA molecule or RNA molecule that exists apart from its native environment.
  • An isolated DNA molecule or RNA molecule may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.
  • an "isolated” or “purified” nucleic acid molecule or biologically active portion thereof is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e. , sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • gene refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises partial length or entire length coding sequences necessary for the production of a polypeptide or precursor polypeptide.
  • Gene product refers to a product of a gene such as an RNA transcript or a polypeptide.
  • the term “unlocked nucleobase analogue” (abbreviated as “UNA”) refers to an acyclic nucleobase in which the C2' and C3' atoms of the ribose ring are not covalently linked.
  • unlocked nucleobase analogue includes nucleobase analogues having the following structure identified as Structure A:
  • Base is any natural or unnatural base such as, for example, adenine (A), cytosine (C), guanine (G) and thymine (T).
  • UNA useful in the practice of the present invention include the molecules identified as acyclic 2'-3'-seco-nucleotide monomers in U.S. patent serial number 8,314,227 which is incorporated by reference herein in its entirety.
  • lipid refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include
  • phospholipids and glycolipids include phospholipids and glycolipids; and (3) "derived lipids” such as steroids.
  • lipid particle includes a lipid formulation that can be used to deliver a therapeutic nucleic acid (e.g., siRNA) to a target site of interest (e.g., cell, tissue, organ, and the like).
  • a therapeutic nucleic acid e.g., siRNA
  • the lipid particle of the invention is typically formed from a cationic lipid, a non-cationic lipid, and optionally a conjugated lipid that prevents aggregation of the particle.
  • a lipid particle that includes a nucleic acid molecule e.g., siRNA molecule
  • the nucleic acid is fully encapsulated within the lipid particle, thereby protecting the nucleic acid from enzymatic degradation.
  • nucleic acid-lipid particles are extremely useful for systemic applications, as they can exhibit extended circulation lifetimes following intravenous (i.v.) injection, they can accumulate at distal sites (e.g., sites physically separated from the
  • the nucleic acid may be complexed with a condensing agent and encapsulated within a lipid particle as set forth in PCT Publication No. WO 00/03683, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • the lipid particles of the invention typically have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 1 10 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 1 10 nm, 1 15 nm, 120 nm
  • nucleic acids when present in the lipid particles of the present invention, are resistant in aqueous solution to degradation with a nuclease.
  • Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Patent Publication Nos. 20040142025 and 20070042031, the disclosures of which are herein incorporated by reference in their entirety for all purposes.
  • lipid encapsulated can refer to a lipid particle that provides a therapeutic nucleic acid such as a siRNA, with full encapsulation, partial encapsulation, or both.
  • the nucleic acid e.g., siRNA
  • the nucleic acid is fully encapsulated in the lipid particle (e.g., to form a nucleic acid-lipid particle).
  • lipid conjugate refers to a conjugated lipid that inhibits aggregation of lipid particles.
  • lipid conjugates include, but are not limited to, PEG-lipid conjugates such as, e.g., PEG coupled to dialkyloxypropyls (e.g., PEG-DAA conjugates), PEG coupled to diacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides (see, e.g., U.S. Patent No.
  • cationic PEG lipids cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugates), polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof.
  • POZ polyoxazoline
  • DAA polyoxazoline conjugates
  • polyamide oligomers e.g., ATTA-lipid conjugates
  • PEG or POZ can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety.
  • Any linker moiety suitable for coupling the PEG or the POZ to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties.
  • non-ester containing linker moieties such as amides or carbamates, are used.
  • amphipathic lipid refers, in part, 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.
  • Hydrophilic characteristics derive from the presence of polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxyl, and other like groups. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.
  • phospholipids include, but are not limited to,
  • phosphatidylcholine phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine,
  • amphipathic lipids can be mixed with other lipids including triglycerides and sterols.
  • neutral lipid refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH.
  • lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.
  • non-cationic lipid refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid.
  • anionic lipid refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerols, cardiolipins,
  • diacylphosphatidylserines diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines,
  • lysylphosphatidylglycerols palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.
  • POPG palmitoyloleyolphosphatidylglycerol
  • hydrophobic lipid refers to compounds having apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups optionally substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N- N-dialkylamino, l,2-diacyloxy-3-aminopropane, and l,2-dialkyl-3-aminopropane.
  • cationic lipid and “amino lipid” are used interchangeably herein to include those lipids and salts thereof having one, two, three, or more fatty acid or fatty alkyl chains and a pH-titratable amino head group (e.g., an alkylamino or dialkylamino head group).
  • the cationic lipid is typically protonated (i.e., positively charged) at a pH below the pK a of the cationic lipid and is substantially neutral at a pH above the pK a .
  • the cationic lipids of the invention may also be termed titratable cationic lipids.
  • the cationic lipids comprise: a protonatable tertiary amine (e.g., pH-titratable) head group; Cj alkyl chains, wherein each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds; and ether, ester, or ketal linkages between the head group and alkyl chains.
  • a protonatable tertiary amine e.g., pH-titratable
  • Cj alkyl chains wherein each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds
  • ether, ester, or ketal linkages between the head group and alkyl chains e.g., 1, 2, or 3
  • Such cationic lipids include, but are not limited to, DSDMA, DODMA, DLinDMA, DLenDMA, ⁇ -DLenDMA, DLin-K-DMA, DLin-K- C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K), DLin-K-C3 -DMA, DLin-K-C4- DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, DLin-M-C2-DMA (also known as MC2), and DLin-M-C3 -DMA (also known as MC3).
  • salts includes any anionic and cationic complex, such as the complex formed between a cationic lipid and one or more anions.
  • anions include inorganic and organic anions, e.g., hydride, fluoride, chloride, bromide, iodide, oxalate (e.g., hemioxalate), phosphate, phosphonate, hydrogen phosphate, dihydrogen phosphate, oxide, carbonate, bicarbonate, nitrate, nitrite, nitride, bisulfite, sulfide, sulfite, bisulfate, sulfate, thiosulfate, hydrogen sulfate, borate, formate, acetate, benzoate, citrate, tartrate, lactate, acrylate, polyacrylate, fumarate, maleate, itaconate, glycolate, gluconate, malate, mandelate, tiglate, ascorbate,
  • alkyl includes a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.
  • Representative saturated straight chain alkyls include, but are not limited to, methyl, ethyl, ⁇ -propyl, w-butyl, «-pentyl, «-hexyl, and the like, while saturated branched alkyls include, without limitation, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.
  • saturated cyclic alkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like, while unsaturated cyclic alkyls include, without limitation, cyclopentenyl, cyclohexenyl, and the like.
  • alkenyl includes 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, but are not limited to, ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-l -butenyl, 2- methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.
  • alkynyl includes 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, without limitation, acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1- pentynyl, 2-pentynyl, 3-methyl-l butynyl, and the like.
  • heterocycle includes 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 quatemized, 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, but are not limited to, heteroaryls as defined below, as well as morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
  • substituents include, but are not limited to, oxo, halogen, heterocycle, -CN, -OR x ,
  • the term "optionally substituted,” when used before a list of substituents, means that each of the substituents in the list may be optionally substituted as described herein.
  • halogen includes fluoro, chloro, bromo, and iodo.
  • the term "fusogenic” refers to the ability of a lipid particle to fuse with the membranes of a cell.
  • the membranes can be either the plasma membrane or membranes surrounding organelles, e.g. , endosome, nucleus, etc.
  • aqueous solution refers to a composition comprising in whole, or in part, water.
  • organic lipid solution refers to a composition comprising in whole, or in part, an organic solvent having a lipid.
  • organic lipid solution refers to a composition comprising in whole, or in part, an organic solvent having a lipid.
  • electroconductive core when used to describe a lipid particle of the present invention, refers to the dark appearance of the interior portion of a lipid particle when visualized using cryo transmission electron microscopy ("cyroTEM").
  • Some lipid particles of the present invention have an electron dense core and lack a lipid bilayer structure.
  • Some lipid particles of the present invention have an elctron dense core, lack a lipid bilayer structure, and have an inverse Hexagonal or Cubic phase structure.
  • the non-bilayer lipid packing provides a 3-dimensional network of lipid cylinders with water and nucleic acid on the inside, i.e., essentially a lipid droplet interpenetrated with aqueous channels containing the nucleic acid.
  • Distal site refers to a physically separated site, which is not limited to an adjacent capillary bed, but includes sites broadly distributed throughout an organism.
  • “Serum-stable” in relation to nucleic acid-lipid particles means that the particle is not significantly degraded after exposure to a serum or nuclease assay that would significantly degrade free DNA or RNA.
  • Suitable assays include, for example, a standard serum assay, a DNAse assay, or an RNAse assay.
  • Systemic delivery refers to delivery of lipid particles that leads to a broad biodistribution of an active agent such as a siRNA within an organism. Some techniques of administration can lead to the systemic delivery of certain agents, but not others. Systemic delivery means that a useful, preferably therapeutic, amount of an agent is exposed to most parts of the body. To obtain broad biodistribution generally requires a blood lifetime such that the agent is not rapidly degraded or cleared (such as by first pass organs (liver, lung, etc.) or by rapid, nonspecific cell binding) before reaching a disease site distal to the site of administration.
  • Systemic delivery of lipid particles can be by any means known in the art including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment, systemic delivery of lipid particles is by intravenous delivery.
  • “Local delivery,” as used herein, refers to delivery of an active agent such as a siRNA directly to a target site within an organism.
  • an agent can be locally delivered by direct injection into a disease site, other target site, or a target organ such as the liver, heart, pancreas, kidney, and the like.
  • virus particle load refers to a measure of the number of virus particles (e.g., EBOV) present in a bodily fluid, such as blood.
  • particle load may be expressed as the number of virus particles per milliliter of, e.g., blood.
  • Particle load testing may be performed using nucleic acid amplification based tests, as well as non-nucleic acid-based tests ⁇ see, e.g., Puren et al., The Journal of Infectious Diseases, 201 :S27-36 (2010)).
  • mamammal refers to any mammalian species such as a human, mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and the like.
  • the present invention provides siRNA molecules that target the expression of one or more EBOV genes, nucleic acid-lipid particles comprising one or more (e.g., a cocktail) of the siRNAs, and methods of delivering and/or administering the nucleic acid-lipid particles (e.g., for the treatment of EBOV infection in humans).
  • the present invention provides siRNA molecules that target expression of one or more EBOV genes.
  • the present invention provides compositions comprising a combination (e.g., a cocktail, pool, or mixture) of siRNAs that target different regions of the EBOV genome.
  • the siRNA molecules of the invention are capable of inhibiting the replication of EBOV in vitro or in vivo.
  • the present invention provides the siRNA molecules shown in Table A, wherein the top strand of each double-stranded siRNA molecule is the sense strand running in a 5' to 3' direction from left to right; and the lower strand of each double-stranded siRNA molecule is the antisense strand running in a 5' to 3' direction from right to left.
  • the siRNA molecules in certain embodiments may comprise one or more ribonucleotides with a 2'-0-methyl modifications.
  • the present invention provides the isolated sense strands and antisense strands (i.e. , isolated single stranded nucleic acid molecules) of the siRNA molecules set forth in Tables A and B.
  • the nucleic acid sequences set forth in Tablse C and D are arranged as pairs of sequences, wherein each pair includes a sense strand and its complementary antisense strand.
  • Each pair of sequences (sense plus antisense strand) is identified with a particular name, which correspond to the names shown in Table A or B.
  • isolated sense and antisense strands are useful, for example, for making siRNA molecules that are useful to reduce the expression of one or more EBOV genes in vivo or in vitro.
  • isolated sense and antisense strands are also useful, for example, as hybridization probes for identifying and measuring the amount of EBOV genome in a biological material, such as a tissue or blood sample from a human being infected with EBOV.
  • an oligonucleotide (such as the sense and antisense RNA strands set forth in Tables C and D) of the invention specifically hybridizes to or is
  • oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable.
  • an oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target sequence interferes with the normal function of the target sequence to cause a loss of utility or expression therefrom, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or, in the case of in vitro assays, under conditions in which the assays are conducted.
  • the oligonucleotide may include 1 , 2, 3, or more base substitutions as compared to the region of a gene or mR A sequence that it is targeting or to which it specifically hybridizes.
  • certain embodiments of the invention provide a double-stranded, siRNA molecule (e.g., and isolated molecule) selected from the group consisting of 1 (SEQ ID NO:l and 2), 2 (SEQ ID NO:3 and 4), lm (SEQ ID NO:5 and 6) and 2m (SEQ ID NO:7 and 8).
  • a double-stranded, siRNA molecule selected from the group consisting of 1 (SEQ ID NO:l and 2), 2 (SEQ ID NO:3 and 4), lm (SEQ ID NO:5 and 6) and 2m (SEQ ID NO:7 and 8).
  • nucleic acid molecule e.g., an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7.
  • nucleic acid molecule e.g. , an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8.
  • the present invention also provides a composition comprising one or more double stranded siRNA molecules described herein.
  • the composition comprises two different double stranded siRNA molecules selected from the group consisting of 1 (SEQ ID NO:l and 2), 2 (SEQ ID NO:3 and 4), lm (SEQ ID NO:5 and 6) and 2m (SEQ ID NO:7 and 8).
  • the composition comprises the four double stranded siRNA molecules 1 (SEQ ID NO:l and 2), 2 (SEQ ID NO:3 and 4), lm (SEQ ID NO:5 and 6) and 2m (SEQ ID NO:7 and 8).
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising one or more (e.g., a cocktail) of the siRNAs described herein and a pharmaceutically acceptable carrier.
  • composition described herein comprises one or more siRNA molecules, which silences expression of an EBOV gene.
  • the present invention provides a nucleic acid-lipid particle that targets EBOV gene expression.
  • the nucleic acid-lipid particles typically comprise one or more (e.g., a cocktail) of the double-stranded siRNA molecules described herein (e.g., as described in Tables A and B), a cationic lipid, and a non-cationic lipid.
  • the nucleic acid- lipid particles further comprise a conjugated lipid that inhibits aggregation of particles.
  • the nucleic acid-lipid particles comprise one or more (e.g., a cocktail) of the double- stranded siRNA molecules described herein, a cationic lipid, a non-cationic lipid, and a conjugated lipid that inhibits aggregation of particles.
  • the nucleic acid-lipid particle comprises two different double stranded siRNA molecules selected from the group consisting of 1 (SEQ ID NO: l and 2), 2 (SEQ ID NO:3 and 4), lm (SEQ ID NO:5 and 6) and 2m (SEQ ID NO:7 and 8).
  • the siRNAs of the present invention are fully encapsulated in the nucleic acid-lipid particle.
  • the different types of siRNA species present in the cocktail e.g., siRNA compounds with different sequences
  • each type of siRNA species present in the cocktail may be encapsulated in a separate particle.
  • the siRNA cocktail may be formulated in the particles described herein using a mixture of two or more individual siRNAs (each having a unique sequence) at identical, similar, or different concentrations or molar ratios.
  • a cocktail of siRNAs (corresponding to a plurality of siRNAs with different sequences) is formulated using identical, similar, or different concentrations or molar ratios of each siRNA species, and the different types of siRNAs are co-encapsulated in the same particle.
  • each type of siRNA species present in the cocktail is encapsulated in different particles at identical, similar, or different siRNA concentrations or molar ratios, and the particles thus formed (each containing a different siRNA payload) are administered separately (e.g., at different times in accordance with a therapeutic regimen), or are combined and administered together as a single unit dose (e.g., with a pharmaceutically acceptable carrier).
  • the particles described herein are serum-stable, are resistant to nuclease degradation, and are substantially non-toxic to mammals such as humans.
  • the cationic lipid in the nucleic acid-lipid particles of the invention may comprise, e.g., one or more cationic lipids of Formula I-III described herein or any other cationic lipid species.
  • cationic lipid is a dialkyl lipid.
  • the cationic lipid is a trialkyl lipid.
  • the cationic lipid is selected from the group consisting of 1 ,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1 ,2- dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1 ,2-di-y-linolenyloxy-N,N- dimethylaminopropane ( ⁇ -DLenDMA; Compound (15)), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l ,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4- dimethylaminomethyl-[l ,3]-dioxolane (DLin-K-DMA), dilinoleylmethyl-3- dimethylaminopropionate (DLin-M-C2-DMA), (6Z,9Z,28Z,3 lZ
  • the cationic lipid is selected from the group consisting of l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy- N,N-dimethylaminopropane (DLenDMA), 1 ,2-di-y-linolenyloxy-N,N-dimethylaminopropane ( ⁇ -DLenDMA; Compound (15)) , 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19- yloxy)-N,N-dimethylpropan-l -amine (DLin-MP-DMA; Compound (8)), (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) (Com
  • the cationic lipid comprises from about 48 mol % to about 62 mol % of the total lipid present in the particle.
  • the non-cationic lipid in the nucleic acid-lipid particles of the present invention may comprise, e.g., one or more anionic lipids and/or neutral lipids.
  • the non- cationic lipid comprises one of the following neutral lipid components: (1) a mixture of a phospholipid and cholesterol or a derivative thereof; (2) cholesterol or a derivative thereof; or (3) a phospholipid.
  • the phospholipid comprises
  • the non-cationic lipid is a mixture of DPPC and cholesterol. In a preferred embodiment, the non-cationic lipid is a mixture of DSPC and cholesterol.
  • the non-cationic lipid comprises a mixture of a phospholipid and cholesterol or a derivative thereof, wherein the phospholipid comprises from about 7 mol % to about 17 mol % of the total lipid present in the particle and the cholesterol or derivative thereof comprises from about 25 mol % to about 40 mol % of the total lipid present in the particle.
  • the lipid conjugate in the nucleic acid-lipid particles of the invention inhibits aggregation of particles and may comprise, e.g., one or more of the lipid conjugates described herein.
  • the lipid conjugate comprises a PEG-lipid conjugate.
  • PEG-lipid conjugates include, but are not limited to, PEG-DAG conjugates, PEG- DAA conjugates, and mixtures thereof.
  • the PEG-lipid conjugate is selected from the group consisting of a PEG-diacylglycerol (PEG-DAG) conjugate, a PEG- dialkyloxypropyl (PEG-DAA) conjugate, a PEG-phospholipid conjugate, a PEG-ceramide (PEG-Cer) conjugate, and a mixture thereof.
  • PEG-lipid conjugate is a PEG-DAA conjugate.
  • the PEG-DAA conjugate in the lipid particle may comprise a PEG-didecyloxypropyl (C 10 ) conjugate, a PEG-dilauryloxypropyl (C) 2 ) conjugate, a PEG-dimyristyloxypropyl (C 14 ) conjugate, a PEG-dipalmityloxypropyl (C 16 ) conjugate, a PEG-distearyloxypropyl (Ci ) conjugate, or mixtures thereof.
  • a PEG-didecyloxypropyl (C 10 ) conjugate conjugate
  • PEG-dilauryloxypropyl (C) 2 ) conjugate conjugate
  • PEG-dimyristyloxypropyl (C 14 ) conjugate a PEG-dipalmityloxypropyl (C 16 ) conjugate
  • a PEG-distearyloxypropyl (Ci ) conjugate or mixtures thereof.
  • the PEG-DAA conjugate is a PEG-dimyristyloxypropyl (C 14 ) conjugate.
  • the PEG-DAA conjugate is a compound (66) (PEG-C-DMA) conjugate.
  • the lipid conjugate comprises a POZ-lipid conjugate such as a POZ- DAA conjugate.
  • the conjugated lipid that inhibits aggregation of particles comprises from about 0.5 mol % to about 3 mol % of the total lipid present in the particle.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of from about 5 : 1 to about 15: 1.
  • the nucleic acid-lipid particle has a median diameter of from about 30 nra to about 150 nm.
  • the nucleic acid-lipid particle has an electron dense core.
  • the present invention provides nucleic acid-lipid particles comprising: (a) one or more (e.g., a cocktail) siRNA molecules described herein (e.g., see,
  • the nucleic acid-lipid particle comprises: (a) one or more (e.g., a cocktail) siRNA molecules described herein (e.g., see, Tables A and B); (b) a cationic lipid or a salt thereof comprising from about 52 mol % to about 62 mol % of the total lipid present in the particle; (c) a mixture of a phospholipid and cholesterol or a derivative thereof comprising from about 36 mol % to about 47 mol % of the total lipid present in the particle; and (d) a PEG-lipid conjugate comprising from about 1 mol % to about 2 mol % of the total lipid present in the particle.
  • siRNA molecules described herein e.g., see, Tables A and B
  • a cationic lipid or a salt thereof comprising from about 52 mol % to about 62 mol % of the total lipid present in the particle
  • a mixture of a phospholipid and cholesterol or a derivative thereof comprising from about
  • the formulation is a four- component system comprising about 1.4 mol % PEG-lipid conjugate (e.g., PEG2000-C-DMA), about 57.1 mol % cationic lipid (e.g., DLin- -C2-DMA) or a salt thereof, about 7.1 mol % DPPC (or DSPC), and about 34.3 mol % cholesterol (or derivative thereof).
  • PEG-lipid conjugate e.g., PEG2000-C-DMA
  • 57.1 mol % cationic lipid e.g., DLin- -C2-DMA
  • a salt thereof e.g., DLin- -C2-DMA
  • DPPC or DSPC
  • 34.3 mol % cholesterol or derivative thereof.
  • the nucleic acid-lipid particle comprises: (a) one or more (e.g., a cocktail) siRNA molecules described herein (e.g., see, Tables A and B); (b) a cationic lipid or a salt thereof comprising from about 56.5 mol % to about 66.5 mol % of the total lipid present in the particle; (c) cholesterol or a derivative thereof comprising from about 31.5 mol % to about 42.5 mol % of the total lipid present in the particle; and (d) a PEG-lipid conjugate comprising from about 1 mol % to about 2 mol % of the total lipid present in the particle.
  • siRNA molecules described herein e.g., see, Tables A and B
  • a cationic lipid or a salt thereof comprising from about 56.5 mol % to about 66.5 mol % of the total lipid present in the particle
  • cholesterol or a derivative thereof comprising from about 31.5 mol % to about 42.5 mol % of the
  • the formulation is a three-component system which is phospholipid-free and comprises about 1.5 mol % PEG-lipid conjugate (e.g., PEG2000-C- DMA), about 61.5 mol % cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, and about 36.9 mol % cholesterol (or derivative thereof).
  • PEG-lipid conjugate e.g., PEG2000-C- DMA
  • 61.5 mol % cationic lipid e.g., DLin-K-C2-DMA
  • a salt thereof e.g., DLin-K-C2-DMA
  • nucleic acid-lipid particles comprising: (a) one or more (e.g., a cocktail) siRNA molecules described herein (e.g., see,
  • the nucleic acid-lipid particle comprises: (a) one or more (e.g., a cocktail) siRNA molecules described herein (e.g., see, Tables A and B); (b) a cationic lipid or a salt thereof comprising from about 30 mol % to about 50 mol % of the total lipid present in the particle; (c) a mixture of a phospholipid and cholesterol or a derivative thereof comprising from about 47 mol % to about 69 mol % of the total lipid present in the particle; and (d) a PEG-lipid conjugate comprising from about 1 mol % to about 3 mol % of the total lipid present in the particle.
  • siRNA molecules described herein e.g., see, Tables A and B
  • a cationic lipid or a salt thereof comprising from about 30 mol % to about 50 mol % of the total lipid present in the particle
  • a mixture of a phospholipid and cholesterol or a derivative thereof comprising from about 47
  • the formulation is a four- component system which comprises about 2 mol % PEG-lipid conjugate (e.g., PEG2000-C- DMA), about 40 mol % cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, about 10 mol % DPPC (or DSPC), and about 48 mol % cholesterol (or derivative thereof).
  • PEG-lipid conjugate e.g., PEG2000-C- DMA
  • 40 mol % cationic lipid e.g., DLin-K-C2-DMA
  • a salt thereof e.g., DLin-K-C2-DMA
  • 10 mol % DPPC or DSPC
  • 48 mol % cholesterol or derivative thereof.
  • the present invention provides nucleic acid-lipid particles comprising: (a) one or more (e.g., a cocktail) siRNA molecules described herein (e.g., see, Tables A and B); (b) one or more cationic lipids or salts thereof comprising from about 50 mol % to about 65 mol % of the total lipid present in the particle; (c) one or more non-cationic lipids comprising from about 25 mol % to about 45 mol % of the total lipid present in the particle; and (d) one or more conjugated lipids that inhibit aggregation of particles comprising from about 5 mol % to about 10 mol % of the total lipid present in the particle.
  • a cocktail siRNA molecules described herein
  • the nucleic acid-lipid particle comprises: (a) one or more (e.g., a cocktail) siRNA molecules described herein (e.g., see, Tables A and B); (b) a cationic lipid or a salt thereof comprising from about 50 mol % to about 60 mol % of the total lipid present in the particle; (c) a mixture of a phospholipid and cholesterol or a derivative thereof comprising from about 35 mol % to about 45 mol % of the total lipid present in the particle; and (d) a PEG-lipid conjugate comprising from about 5 mol % to about 10 mol % of the total lipid present in the particle.
  • siRNA molecules described herein e.g., see, Tables A and B
  • a cationic lipid or a salt thereof comprising from about 50 mol % to about 60 mol % of the total lipid present in the particle
  • a mixture of a phospholipid and cholesterol or a derivative thereof comprising from about 35
  • the non-cationic lipid mixture in the formulation comprises: (i) a phospholipid of from about 5 mol % to about 10 mol % of the total lipid present in the particle; and (ii) cholesterol or a derivative thereof of from about 25 mol % to about 35 mol % of the total lipid present in the particle.
  • the formulation is a four-component system which comprises about 7 mol % PEG-lipid conjugate (e.g., PEG750-C-DMA), about 54 mol % cationic lipid (e.g., DLin- -C2-DMA) or a salt thereof, about 7 mol % DPPC (or DSPC), and about 32 mol % cholesterol (or derivative thereof).
  • the nucleic acid-lipid particle comprises: (a) one or more (e.g., a cocktail) siRNA molecules described herein (e.g., see, Tables A and B); (b) a cationic lipid or a salt thereof comprising from about 55 mol % to about 65 mol % of the total lipid present in the particle; (c) cholesterol or a derivative thereof comprising from about 30 mol % to about 40 mol % of the total lipid present in the particle; and (d) a PEG-lipid conjugate comprising from about 5 mol % to about 10 mol % of the total lipid present in the particle.
  • siRNA molecules described herein e.g., see, Tables A and B
  • a cationic lipid or a salt thereof comprising from about 55 mol % to about 65 mol % of the total lipid present in the particle
  • cholesterol or a derivative thereof comprising from about 30 mol % to about 40 mol % of the total lipid present in the particle
  • the formulation is a three-component system which is phospholipid- free and comprises about 7 mol % PEG-lipid conjugate (e.g., PEG750-C-DMA), about 58 mol % cationic lipid (e.g., DLin-K-C2-DMA) or a salt thereof, and about 35 mol % cholesterol (or derivative thereof).
  • PEG-lipid conjugate e.g., PEG750-C-DMA
  • 58 mol % cationic lipid e.g., DLin-K-C2-DMA
  • a salt thereof e.g., DLin-K-C2-DMA
  • the nucleic acid-lipid particle comprises: (a) one or more (e.g., a cocktail) siRNA molecules described herein (e.g., see, Tables A and B); (b) a cationic lipid or a salt thereof comprising from about 48 mol % to about 62 mol % of the total lipid present in the particle; (c) a mixture of a phospholipid and cholesterol or a derivative thereof, wherein the phospholipid comprises about 7 mol % to about 17 mol % of the total lipid present in the particle, and wherein the cholesterol or derivative thereof comprises about 25 mol % to about 40 mol % of the total lipid present in the particle; and (d) a PEG-lipid conjugate comprising from about 0.5 mol % to about 3.0 mol % of the total lipid present in the particle.
  • Exemplary lipid formulations A-Z of this aspect of the invention are included below.
  • Exemplary lipid formulation A includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (1.2%), cationic lipid (53.2%), phospholipid (9.3%), cholesterol (36.4%), wherein the actual amounts of the lipids present may vary by, e.g. , ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DMA (compound (66)) (1.2%)
  • the cationic lipid is 1 ,2- dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) (53.2%)
  • the phospholipid is DPPC
  • nucleic acid-lipid particle based on formulation A which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation A may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of from about 5: 1 to about 15:1, or about 5: 1, 6: 1 , 7: 1 , 8:1, 9: 1, 10:1 , 11 :1, 12: 1, 13:1, 14:1 , or 15:1, or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9: 1 (e.g., a lipid:drug ratio of from 8.5: 1 to 10:1, or from 8.9: 1 to 10: 1, or from 9:1 to 9.9:1 , including 9.1 :1, 9.2: 1 , 9.3: 1, 9.4:1, 9.5:1 , 9.6: 1, 9.7:1, and 9.8: 1).
  • a lipid:drug ratio of from 8.5: 1 to 10:1, or from 8.9: 1 to 10: 1, or from 9:1 to 9.9:1 , including 9.1 :1, 9.2: 1 , 9.3: 1, 9.4:1, 9.5:1 , 9.6: 1, 9.7:1, and 9.8: 1).
  • Exemplary lipid formulation B which includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (0.8%), cationic lipid (59.7%), phospholipid (14.2%), cholesterol (25.3%), wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g. , ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DOMG (compound (67)) (0.8%)
  • the cationic lipid is 1,2- dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) (59.7%)
  • the phospholipid is DSPC (14.2%)
  • cholesterol is present at 25.3%, wherein the actual amounts of the lipids present may vary by, e.g. , ⁇ 5 % (or e.g.
  • nucleic acid-lipid particle based on formulation B which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation B may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of from about 5: 1 to about 15: 1 , or about 5:1, 6: 1, 7:1, 8:1, 9:1, 10: 1, 1 1 : 1 , 12:1, 13:1 , 14: 1, or 15:1 , or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9: 1 (e.g., a lipid:drug ratio of from 8.5:1 to 10: 1, or from 8.9:1 to 10:1, or from 9:1 to 9.9: 1, including 9.1 :1, 9.2:1, 9.3:1, 9.4:1 , 9.5: 1 , 9.6: 1 , 9.7:1 , and 9.8:1).
  • a lipid:drug ratio of from 8.5:1 to 10: 1, or from 8.9:1 to 10:1, or from 9:1 to 9.9: 1, including 9.1 :1, 9.2:1, 9.3:1, 9.4:1 , 9.5: 1 , 9.6: 1 , 9.7:1 , and 9.8:1.
  • Exemplary lipid formulation C includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (1.9%), cationic lipid (52.5%), phospholipid (14.8%), cholesterol (30.8%), wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DOMG (compound (67)) (1.9%)
  • the cationic lipid is 1,2- di-y-linolenyloxy-N,N-dimethylaminopropane ( ⁇ -DLenDMA; Compound (15)) (52.5%)
  • the phospholipid is DSPC (14.8%)
  • cholesterol is present at 30.8%, wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • nucleic acid-lipid particle based on formulation C which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation C may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid.siRNA mass ratio of from about 5: 1 to about 15: 1, or about 5: 1, 6: 1, 7:1, 8: 1, 9: 1, 10:1, 11 : 1, 12: 1, 13: 1, 14:1 , or 15:1, or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9:1 (e.g., a lipid:drug ratio of from 8.5: 1 to 10: 1, or from 8.9:1 to 10: 1 , or from 9:1 to 9.9: 1, including 9.1 :1, 9.2: 1, 9.3: 1, 9.4:1, 9.5:1, 9.6: 1, 9.7:1, and 9.8:1).
  • a lipid:drug ratio of from 8.5: 1 to 10: 1, or from 8.9:1 to 10: 1 , or from 9:1 to 9.9: 1, including 9.1 :1, 9.2: 1, 9.3: 1, 9.4:1, 9.5:1, 9.6: 1, 9.7:1, and 9.8:1.
  • Exemplary lipid formulation D includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (0.7%), cationic lipid (60.3%), phospholipid (8.4%), cholesterol (30.5%), wherein the actual amounts of the lipids present may vary by, e.g. , ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DMA (compound (66)) (0.7%)
  • the cationic lipid is 3- ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-l -amine (DLin-MP-DMA; Compound (8) (60.3%)
  • the phospholipid is DSPC (8.4%)
  • cholesterol is present at 30.5%, wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • nucleic acid-lipid particle based on formulation D which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation D may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of from about 5 : 1 to about 15 : 1 , or about 5: 1, 6:1 , 7: 1, 8:1, 9:1, 10: 1, 1 1 : 1, 12:1, 13: 1, 14: 1, or 15: 1, or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9:1 (e.g., a lipid:drug ratio of from 8.5:1 to 10:1, or from 8.9:1 to 10: 1, or from 9: 1 to 9.9: 1 , including 9.1 : 1, 9.2:1, 9.3:1, 9.4: 1, 9.5: 1, 9.6:1, 9.7:1, and 9.8:1).
  • 9:1 e.g., a lipid:drug ratio of from 8.5:1 to 10:1, or from 8.9:1 to 10: 1, or from 9: 1 to 9.9: 1 , including 9.1 : 1, 9.2:1, 9.3:1, 9.4: 1, 9.5: 1, 9.6:1, 9.7:1, and 9.8:1).
  • Exemplary lipid formulation E includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (1.8%), cationic lipid (52.1%), phospholipid (7.5%), cholesterol (38.5%), wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g. , ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DMA (compound (66)) (1.8%)
  • the cationic lipid is (6Z,9Z,28Z,31 Z)-heptatriaconta-6,9,28,31 -tetraen- 19-yl 4-(dimethylamino)butanoate)
  • composition (7) (Compound (7)) (52.1%), the phospholipid is DPPC (7.5%), and cholesterol is present at 38.5%, wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g. , ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • formulation E which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation E may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid:siR A mass ratio of from about 5:1 to about 15:1, or about 5:1 , 6:1, 7:1 , 8:1 , 9:1 , 10: 1, 1 1 : 1, 12: 1, 13:1, 14:1, or 15:1, or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9:1 (e.g., a lipid:drug ratio of from 8.5 : 1 to 10:1, or from 8.9:1 to 10:1 , or from 9:1 to 9.9:1, including 9.1 :1 , 9.2: 1 , 9.3:1, 9.4:1, 9.5: 1, 9.6:1, 9.7:1 , and 9.8:1).
  • a lipid:drug ratio of from 8.5 : 1 to 10:1, or from 8.9:1 to 10:1 , or from 9:1 to 9.9:1, including 9.1 :1 , 9.2: 1 , 9.3:1, 9.4:1, 9.5: 1, 9.6:1, 9.7:1 , and 9.8:1.
  • Exemplary formulation F includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (0.9%), cationic lipid (57.1%), phospholipid (8.1%), cholesterol (33.8%), wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DOMG (compound (67)) (0.9%)
  • the cationic lipid is 1 ,2-dilinolenyloxy- ⁇ , ⁇ -dimethylaminopropane (DLenDMA), 1 ,2-di-y-linolenyloxy-N,N-dimethylaminopropane ( ⁇ -DLenDMA; Compound (15)) (57.1 %)
  • the phospholipid is DSPC (8.1%)
  • cholesterol is present at 33.8%, wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • nucleic acid-lipid particle based on formulation F which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation F may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of from about 5:1 to about 15:1, or about 5: 1, 6: 1, 7:1, 8:1, 9:1, 10:1, 11 :1, 12:1, 13:1, 14:1 , or 15:1 , or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid: siRNA mass ratio of about 9: 1 (e.g., a lipid:drug ratio of from 8.5:1 to 10: 1, or from 8.9: 1 to 10:1, or from 9: 1 to 9.9:1 , including 9.1 :1, 9.2:1, 9.3:1, 9.4: 1, 9.5:1, 9.6:1 , 9.7:1, and 9.8:1).
  • a lipid:drug ratio of from 8.5:1 to 10: 1, or from 8.9: 1 to 10:1, or from 9: 1 to 9.9:1 , including 9.1 :1, 9.2:1, 9.3:1, 9.4: 1, 9.5:1, 9.6:1 , 9.7:1, and 9.8:1.
  • Exemplary lipid formulation G includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (1.7%), cationic lipid (61.6%), phospholipid (1 1.2%), cholesterol (25.5%), wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DOMG (compound (67)) (1.7%)
  • the cationic lipid is 1,2- dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1 ,2-di-y-linolenyloxy-N,N- dimethylaminopropane ( ⁇ -DLenDMA; Compound (15)) (61.6%)
  • the phospholipid is DPPC (11.2%)
  • cholesterol is present at 25.5%, wherein the actual amounts of the lipids present may vary by, e.g. , ⁇ 5 % (or e.g.
  • nucleic acid-lipid particle based on formulation G which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation G may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid: siRNA mass ratio of from about 5:1 to about 15: 1, or about 5: 1, 6: 1, 7:1, 8:1, 9:1, 10:1, 11 :1, 12: 1, 13: 1, 14:1 , or 15: 1, or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9:1 (e.g., a lipid:drug ratio of from 8.5:1 to 10: 1, or from 8.9:1 to 10: 1, or from 9: 1 to 9.9:1, including 9.1 :1 , 9.2: 1, 9.3:1, 9.4:1, 9.5:1, 9.6:1, 9.7:1, and 9.8:1).
  • a lipid:drug ratio of from 8.5:1 to 10: 1, or from 8.9:1 to 10: 1, or from 9: 1 to 9.9:1, including 9.1 :1 , 9.2: 1, 9.3:1, 9.4:1, 9.5:1, 9.6:1, 9.7:1, and 9.8:1.
  • Exemplary lipid formulation H includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (1.1%), cationic lipid (55.0%), phospholipid (1 1.0%), cholesterol (33.0%), wherein the actual amounts of the lipids present may vary by, e.g. , ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DMA (compound (66)) (1.1%)
  • the cationic lipid is (6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-l 1-yl 5-(dimethylamino)pentanoate (Compound (13)) (55.0%)
  • the phospholipid is DSPC (11.0%)
  • cholesterol is present at 33.0%, wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • nucleic acid-lipid particle based on formulation H which comprises one or more siR A molecules described herein.
  • the nucleic acid lipid particle based on formulation H may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of from about 5: 1 to about 15:1, or about 5:1, 6:1, 7:1, 8: 1, 9: 1, 10:1, 11 :1, 12: 1, 13: 1, 14 : 1 , or 15 : 1 , or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9:1 (e.g., a lipid:drug ratio of from 8.5: 1 to 10:1 , or from 8.9: 1 to 10: 1 , or from 9:1 to 9.9:1 , including 9.1 :1 , 9.2:1 , 9.3: 1, 9.4: 1 , 9.5:1, 9.6:1, 9.7:1 , and 9.8: 1).
  • a lipid:drug ratio of from 8.5: 1 to 10:1 , or from 8.9: 1 to 10: 1 , or from 9:1 to 9.9:1 , including 9.1 :1 , 9.2:1 , 9.3: 1, 9.4: 1 , 9.5:1, 9.6:1, 9.7:1 , and 9.8: 1).
  • Exemplary lipid formulation I includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (2.6%), cationic lipid (53.1%), phospholipid (9.4%), cholesterol (35.0%), wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DMA (compound (66)) (2.6%)
  • the cationic lipid is
  • nucleic acid-lipid particle based on formulation I, which comprises one or more siRNA molecules described herein.
  • formulation I which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation I may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid.siRNA mass ratio of from about 5:1 to about 15: 1, or about 5:1 , 6:1, 7:1 , 8:1 , 9:1, 10:1, 1 1 :1, 12:1, 13: 1, 14:1, or 15:1 , or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9:1 (e.g., a lipid:drug ratio of from 8.5:1 to 10:1, or from 8.9: 1 to 10:1, or from 9:1 to 9.9:1 , including 9.1 :1, 9.2: 1, 9.3:1 , 9.4:1, 9.5: 1 , 9.6:1 , 9.7:1 , and 9.8:1).
  • a lipid:drug ratio of from 8.5:1 to 10:1, or from 8.9: 1 to 10:1, or from 9:1 to 9.9:1 , including 9.1 :1, 9.2: 1, 9.3:1 , 9.4:1, 9.5: 1 , 9.6:1 , 9.7:1 , and 9.8:1.
  • Exemplary lipid formulation J includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (0.6%), cationic lipid
  • lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DMA (compound (66)) (0.6%)
  • the cationic lipid is 1,2- dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) (59.4%)
  • the phospholipid is DPPC (10.2%)
  • cholesterol is present at 29.8%, wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • nucleic acid-lipid particle based on formulation J which comprises one or more siR A molecules described herein.
  • the nucleic acid lipid particle based on formulation J may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of from about 5:1 to about 15:1, or about 5:l, 6:l , 7:l, 8: l , 9:l, 10: 1, 1 1 : 1, 12: 1, 13: 1, 14: 1, or 15:1, or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid: siRNA mass ratio of about 9: 1 (e.g., a lipid:drug ratio of from 8.5: 1 to 10:1, or from 8.9: 1 to 10: 1, or from 9: 1 to 9.9: 1, including 9.1 :1, 9.2: 1 , 9.3:1, 9.4: 1, 9.5: 1 , 9.6: 1, 9.7: 1 , and 9.8: 1).
  • a lipid:drug ratio of from 8.5: 1 to 10:1, or from 8.9: 1 to 10: 1, or from 9: 1 to 9.9: 1, including 9.1 :1, 9.2: 1 , 9.3:1, 9.4: 1, 9.5: 1 , 9.6: 1, 9.7: 1 , and 9.8: 1).
  • Exemplary lipid formulation K includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (0.5%), cationic lipid (56.7%), phospholipid (13.1%), cholesterol (29.7%), wherein the actual amounts of the lipids present may vary by, e.g. , ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DOMG (compound (67)) (0.5%)
  • the cationic lipid is (6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31 -tetraen-19-yl 4-(dimethylamino)butanoate)
  • composition K (Compound (7)) (56.7%), the phospholipid is DSPC (13.1%), and cholesterol is present at 29.7%), wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g. , ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • formulation K which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation K may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of from about 5:1 to about 15:1, or about 5: 1 , 6:1, 7:1, 8: 1, 9: 1, 10:1, 1 1 : 1, 12:1, 13:1, 14:1, or 15:1, or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9: 1 (e.g., a lipid:drug ratio of from 8.5:1 to 10: 1 , or from 8.9: 1 to 10:1, or from 9: 1 to 9.9:1, including 9.1 :1 , 9.2: 1, 9.3:1 , 9.4: 1 , 9.5:1, 9.6:1 , 9.7: 1 , and 9.8:1).
  • a lipid:drug ratio of from 8.5:1 to 10: 1 , or from 8.9: 1 to 10:1, or from 9: 1 to 9.9:1, including 9.1 :1 , 9.2: 1, 9.3:1 , 9.4: 1 , 9.5:1, 9.6:1 , 9.7: 1 , and 9.8:1.
  • Exemplary lipid formulation L includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (2.2%), cationic lipid (52.0%), phospholipid (9.7%), cholesterol (36.2%), wherein the actual amounts of the lipids present may vary by, e.g. , ⁇ 5 % (or e.g. , ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DOMG (compound (67)) (2.2%)
  • the cationic lipid is 1,2- di-y-linolenyloxy-N,N-dimethylaminopropane ( ⁇ -DLenDMA; Compound (15)) (52.0%)
  • the phospholipid is DSPC (9.7%)
  • cholesterol is present at 36.2%, wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • nucleic acid-lipid particle based on formulation L which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation L may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipidrsiRNA mass ratio of from about 5:1 to about 15: 1, or about 5:1 , 6: 1 , 7: 1, 8:1, 9:1 , 10: 1, 1 1 :1, 12:1 , 13:1, 14: 1, or 15: 1, or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9: 1 (e.g., a lipid:drug ratio of from 8.5:1 to 10: 1 , or from 8.9:1 to 10:1 , or from 9: 1 to 9.9: 1, including 9.1 : 1, 9.2:1 , 9.3: 1, 9.4:1 , 9.5: 1, 9.6:1, 9.7: 1 , and 9.8: 1).
  • 9: 1 e.g., a lipid:drug ratio of from 8.5:1 to 10: 1 , or from 8.9:1 to 10:1 , or from 9: 1 to 9.9: 1, including 9.1 : 1, 9.2:1 , 9.3: 1, 9.4:1 , 9.5: 1, 9.6:1, 9.7: 1 , and 9.8: 1).
  • Exemplary lipid formulation M includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (2.7%), cationic lipid (58.4%o), phospholipid (13.1%>), cholesterol (25.7%), wherein the actual amounts of the lipids present may vary by by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DMA (compound (66)) (2.7%)
  • the cationic lipid is 1,2- dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) (58.4%>)
  • the phospholipid is DPPC (13.1%)
  • cholesterol is present at 25.7%, wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • nucleic acid-lipid particle based on formulation M which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation M may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of from about 5:1 to about 15:1, or about 5: 1, 6:1, 7:1, 8:1, 9: 1, 10:1, 1 1 : 1, 12:1, 13:1, 14: 1, or 15: 1, or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9: 1 (e.g., a lipid:drug ratio of from 8.5:1 to 10:1, or from 8.9: 1 to 10:1, or from 9: 1 to 9.9:1, including 9.1 :1 , 9.2: 1, 9.3:1, 9.4:1, 9.5: 1, 9.6:1, 9.7:1 , and 9.8:1).
  • a lipid:drug ratio of from 8.5:1 to 10:1, or from 8.9: 1 to 10:1, or from 9: 1 to 9.9:1, including 9.1 :1 , 9.2: 1, 9.3:1, 9.4:1, 9.5: 1, 9.6:1, 9.7:1 , and 9.8:1.
  • Exemplary lipid formulation N includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (3.0%), cationic lipid (53.3%o), phospholipid (12.1%), cholesterol (31.5%), wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DMA (compound (66)) (3.0%)
  • the cationic lipid is 1,2- dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) (53.3%)
  • the phospholipid is DPPC (12.1 %)
  • cholesterol is present at 31.5%, wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • nucleic acid-lipid particle based on formulation N which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation N may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid: siRNA mass ratio of from about 5:1 to about 15: 1, or about 5: 1, 6: 1, 7: 1, 8: 1, 9:1, 10: 1, 1 1 : 1, 12: 1, 13: 1, 14: 1, or 15:1, or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9: 1 (e.g., a lipid:drug ratio of from 8.5: 1 to 10:1, or from 8.9: 1 to 10: 1, or from 9: 1 to 9.9:1 , including 9.1 :1, 9.2:1, 9.3: 1, 9.4: 1, 9.5: 1, 9.6:1, 9.7:1, and 9.8:1).
  • a lipid:drug ratio of from 8.5: 1 to 10:1, or from 8.9: 1 to 10: 1, or from 9: 1 to 9.9:1 , including 9.1 :1, 9.2:1, 9.3: 1, 9.4: 1, 9.5: 1, 9.6:1, 9.7:1, and 9.8:1.
  • Exemplary lipid formulation O includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (1.5%), cationic lipid (56.2%), phospholipid (7.8%), cholesterol (34.7%), wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DMA (compound (66)) (1.5%)
  • the cationic lipid is 1,2- dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) (56.2%)
  • the phospholipid is DPPC (7.8%)
  • cholesterol is present at 34.7%, wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • nucleic acid-lipid particle based on formulation O which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation O may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of from about 5:1 to about 15: 1, or about 5:1 , 6:1, 7:1, 8:1, 9:1, 10: 1, 11 :1, 12: 1 , 13: 1, 14: 1, or 15: 1, or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9:1 (e.g., a lipid:drug ratio of from 8.5: 1 to 10:1, or from 8.9:1 to 10:1, or from 9:1 to 9.9:1 , including 9.1 : 1, 9.2: 1, 9.3:1, 9.4: 1, 9.5: 1 , 9.6:1, 9.7:1, and 9.8:1).
  • a lipid:drug ratio of from 8.5: 1 to 10:1, or from 8.9:1 to 10:1, or from 9:1 to 9.9:1 , including 9.1 : 1, 9.2: 1, 9.3:1, 9.4: 1, 9.5: 1 , 9.6:1, 9.7:1, and 9.8:1.
  • Exemplary lipid formulation P includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (2.1%), cationic lipid (48.6%), phospholipid (15.5%), cholesterol (33.8%), wherein the actual amounts of the lipids present may vary by, e.g. , ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DOMG (compound (67)) (2.1%)
  • the cationic lipid is 3- ((6Z,9Z,28Z,31 Z)-heptatriaconta-6,9,28,31 -tetraen- 19-yloxy)-N,N-dimethylpropan- 1 -amine (DLin-MP-DMA; Compound (8)) (48.6%)
  • the phospholipid is DSPC (15.5%)
  • cholesterol is present at 33.8%, wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g.
  • nucleic acid-lipid particle based on formulation P which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation P may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid: siRNA mass ratio of from about 5:1 to about 15:1, or about 5: 1, 6:1, 7: 1 , 8: 1, 9: 1, 10: 1, 1 1 : 1, 12:1, 13:1, 14: 1, or 15: 1, or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9: 1 (e.g., a lipid:drug ratio of from 8.5:1 to 10: 1, or from 8.9: 1 to 10:1, or from 9: 1 to 9.9:1, including 9.1 : 1, 9.2:1, 9.3: 1, 9.4: 1, 9.5: 1, 9.6: 1, 9.7: 1, and 9.8: 1).
  • a lipid:drug ratio of from 8.5:1 to 10: 1, or from 8.9: 1 to 10:1, or from 9: 1 to 9.9:1, including 9.1 : 1, 9.2:1, 9.3: 1, 9.4: 1, 9.5: 1, 9.6: 1, 9.7: 1, and 9.8: 1).
  • Exemplary lipid formulation Q includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (2.5%), cationic lipid (57.9%), phospholipid (9.2%), cholesterol (30.3%), wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DMA (compound (66)) (2.5%)
  • the cationic lipid is (6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-l 1-yl 5-(dimethylamino)pentanoate (Compound (13)) (57.9%)
  • the phospholipid is DSPC (9.2%)
  • cholesterol is present at 30.3%, wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • nucleic acid-lipid particle based on formulation Q which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation Q may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9:1 (e.g., a lipid:drug ratio of from 8.5:1 to 10:1, or from 8.9:1 to 10: 1, or from 9:1 to 9.9:1, including 9.1 : 1, 9.2: 1, 9.3:1 , 9.4:1, 9.5:1, 9.6:1, 9.7:1, and 9.8:1).
  • 9:1 e.g., a lipid:drug ratio of from 8.5:1 to 10:1, or from 8.9:1 to 10: 1, or from 9:1 to 9.9:1, including 9.1 : 1, 9.2: 1, 9.3:1 , 9.4:1, 9.5:1, 9.6:1, 9.7:1, and 9.8:1).
  • Exemplary lipid formulation R includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (1.6%), cationic lipid (54.6%), phospholipid (10.9%), cholesterol (32.8%), wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DMA (compound (66)) (1.6%)
  • the cationic lipid is 3- ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-l -amine (Compound (8)) (54.6%)
  • the phospholipid is DSPC (10.9%)
  • cholesterol is present at 32.8%, wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • nucleic acid-lipid particle based on formulation R which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation R may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of from about 5:1 to about 15: 1, or about 5:1 , 6:1, 7: 1, 8:1, 9:1 , 10:1, 11 :1, 12: 1, 13:1 , 14: 1 , or 15: 1 , or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9: 1 (e.g., a lipid:drug ratio of from 8.5: 1 to 10:1, or from 8.9: 1 to 10:1, or from 9:1 to 9.9: 1, including 9.1 :1, 9.2:1, 9.3: 1, 9.4:1, 9.5:1 , 9.6:1 , 9.7:1, and 9.8:1).
  • a lipid:drug ratio of from 8.5: 1 to 10:1, or from 8.9: 1 to 10:1, or from 9:1 to 9.9: 1, including 9.1 :1, 9.2:1, 9.3: 1, 9.4:1, 9.5:1 , 9.6:1 , 9.7:1, and 9.8:1).
  • Exemplary lipid formulation S includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (2.9%), cationic lipid (49.6%), phospholipid (16.3%), cholesterol (31.3%), wherein the actual amounts of the lipids present may vary by, e.g. , ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DMA (compound (66)) (2.9%)
  • the cationic lipid is (6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-l 1-yl 5-(dimethylamino)pentanoate (Compound (13)) (49.6%)
  • the phospholipid is DPPC (16.3%)
  • cholesterol is present at 31.3%, wherein the actual amounts of the lipids present may vary by, e.g. , ⁇ 5 % (or e.g.
  • nucleic acid-lipid particle based on formulation S which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation S may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of from about 5:1 to about 15: 1 , or about 5: 1 , 6:1, 7: 1 , 8:1, 9:1, 10:1, 1 1 :1, 12: 1, 13: 1, 14 : 1 , or 15 : 1 , or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9:1 (e.g., a lipid:drug ratio of from 8.5: 1 to 10: 1, or from 8.9: 1 to 10:1 , or from 9: 1 to 9.9: 1, including 9.1 :1, 9.2: 1, 9.3:1 , 9.4:1, 9.5: 1, 9.6: 1, 9.7:1, and 9.8:1).
  • a lipid:drug ratio of from 8.5: 1 to 10: 1, or from 8.9: 1 to 10:1 , or from 9: 1 to 9.9: 1, including 9.1 :1, 9.2: 1, 9.3:1 , 9.4:1, 9.5: 1, 9.6: 1, 9.7:1, and 9.8:1.
  • Exemplary lipid formulation T includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (0.7%), cationic lipid (50.5%), phospholipid (8.9%), cholesterol (40.0%), wherein the actual amounts of the lipids present may vary by, e.g. , ⁇ 5 % (or e.g. , ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DOMG (compound (67)) (0.7%)
  • the cationic lipid is 1,2- dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) (50.5%)
  • the phospholipid is DPPC (8.9%)
  • cholesterol is present at 40.0%, wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • nucleic acid-lipid particle based on formulation T which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation T may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of from about 5:1 to about 15: 1 , or about 5:1, 6:1, 7:1, 8:1, 9:1, 10: 1, 1 1 : 1, 12:1, 13:1, 14: 1 , or 15:1 , or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9: 1 (e.g., a lipid:drug ratio of from 8.5:1 to 10: 1, or from 8.9: 1 to 10: 1, or from 9: 1 to 9.9: 1 , including 9.1 :1, 9.2:1 , 9.3:1 , 9.4:1, 9.5:1 , 9.6:1, 9.7:1 , and 9.8: 1).
  • 9: 1 e.g., a lipid:drug ratio of from 8.5:1 to 10: 1, or from 8.9: 1 to 10: 1, or from 9: 1 to 9.9: 1 , including 9.1 :1, 9.2:1 , 9.3:1 , 9.4:1, 9.5:1 , 9.6:1, 9.7:1 , and 9.8: 1.
  • Exemplary lipid formulation U includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (1.0%), cationic lipid
  • phospholipid (15.0%), cholesterol (32.6%), wherein the actual amounts of the lipids present may vary by, e.g. , ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DOMG (compound (67)) (1.0%)
  • the cationic lipid is 1,2- dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) (51.4%)
  • the phospholipid is DSPC (15.0%)
  • cholesterol is present at 32.6%, wherein the actual amounts of the lipids present may vary by, e.g.
  • nucleic acid-lipid particle based on formulation U which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation U may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of from about 5: 1 to about 15: 1, or about 5:1, 6: 1, 7:1, 8:1, 9:1, 10:1, 1 1 : 1, 12:1, 13: 1, 14:1, or 15: 1, or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9:1 (e.g., a lipid:drug ratio of from 8.5:1 to 10: 1, or from 8.9: 1 to 10:1, or from 9:1 to 9.9:1, including 9.1 : 1, 9.2: 1, 9.3: 1 , 9.4: 1, 9.5: 1, 9.6:1, 9.7:1, and 9.8:1).
  • a lipid:drug ratio of from 8.5:1 to 10: 1, or from 8.9: 1 to 10:1, or from 9:1 to 9.9:1, including 9.1 : 1, 9.2: 1, 9.3: 1 , 9.4: 1, 9.5: 1, 9.6:1, 9.7:1, and 9.8:1.
  • Exemplary lipid formulation V includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (1.3%), cationic lipid (60.0%), phospholipid (7.2%), cholesterol (31.5%), wherein the actual amounts of the lipids present may vary by, e.g. , ⁇ 5 % (or e.g. , ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DOMG (compound (67)) (1.3%)
  • the cationic lipid is 1,2- dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA) (60.0%)
  • the phospholipid is DSPC (7.2%)
  • cholesterol is present at 31.5%, wherein the actual amounts of the lipids present may vary by, e.g. , ⁇ 5 % (or e.g.
  • nucleic acid-lipid particle based on formulation V which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation V may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of from about 5 : 1 to about 15:1, or about 5:1, 6: 1, 7: 1, 8:1, 9:1, 10:1, 1 1 : 1 , 12: 1, 13: 1, 14: 1, or 15:1, or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9: 1 (e.g., a lipid:drug ratio of from 8.5: 1 to 10: 1, or from 8.9:1 to 10:1, or from 9:1 to 9.9:1, including 9.1 : 1, 9.2: 1, 9.3:1, 9.4: 1, 9.5: 1, 9.6:1, 9.7: 1, and 9.8: 1).
  • a lipid:drug ratio of from 8.5: 1 to 10: 1, or from 8.9:1 to 10:1, or from 9:1 to 9.9:1, including 9.1 : 1, 9.2: 1, 9.3:1, 9.4: 1, 9.5: 1, 9.6:1, 9.7: 1, and 9.8: 1).
  • Exemplary lipid formulation W includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (1.8%), cationic lipid (51.6%), phospholipid (8.4%), cholesterol (38.3%), wherein the actual amounts of the lipids present may vary by, e.g. , ⁇ 5 % (or e.g. , ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DMA (compound (66)) (1.8%)
  • the cationic lipid is 1 ,2- dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) (51.6%)
  • the phospholipid is DSPC (8.4%)
  • cholesterol is present at 38.3%, wherein the actual amounts of the lipids present may vary by, e.g.
  • nucleic acid-lipid particle based on formulation W which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation W may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid:siR A mass ratio of from about 5:1 to about 15:1 , or about 5:l , 6: l, 7:l, 8: l, 9:l, 10:1, 1 1 :1, 12:1, 13: 1, 14: 1, or 15: 1, or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9: 1 (e.g., a lipid:drug ratio of from 8.5:1 to 10: 1, or from 8.9:1 to 10: 1, or from 9:1 to 9.9: 1, including 9.1 :1, 9.2: 1, 9.3: 1 , 9.4:1 , 9.5:1 , 9.6:1, 9.7: 1 , and 9.8:1).
  • a lipid:drug ratio of from 8.5:1 to 10: 1, or from 8.9:1 to 10: 1, or from 9:1 to 9.9: 1, including 9.1 :1, 9.2: 1, 9.3: 1 , 9.4:1 , 9.5:1 , 9.6:1, 9.7: 1 , and 9.8:1.
  • Exemplary lipid formulation X includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (2.4%), cationic lipid (48.5%), phospholipid (10.0%), cholesterol (39.2%), wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g. , ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DMA (compound (66)) (2.4%)
  • the cationic lipid is 1,2- di-y-linolenyloxy-N,N-dimethylaminopropane ( ⁇ -DLenDMA; Compound (15)) (48.5%)
  • the phospholipid is DPPC (10.0%)
  • cholesterol is present at 39.2%, wherein the actual amounts of the lipids present may vary by, e.g.
  • nucleic acid-lipid particle based on formulation X which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation X may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of from about 5:1 to about 15: 1, or about 5:1 , 6:1 , 7:1, 8: 1, 9: 1 , 10:1, 11 : 1, 12:1 , 13: 1, 14: 1, or 15: 1 , or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total Hpid:siRNA mass ratio of about 9: 1 (e.g., a lipid:drug ratio of from 8.5:1 to 10: 1, or from 8.9: 1 to 10: 1, or from 9: 1 to 9.9: 1, including 9.1 :1 , 9.2: 1, 9.3: 1, 9.4: 1, 9.5: 1, 9.6:1, 9.7: 1 , and 9.8:1).
  • a lipid:drug ratio of from 8.5:1 to 10: 1, or from 8.9: 1 to 10: 1, or from 9: 1 to 9.9: 1, including 9.1 :1 , 9.2: 1, 9.3: 1, 9.4: 1, 9.5: 1, 9.6:1, 9.7: 1 , and 9.8:1).
  • Exemplary lipid formulation Y includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (2.6%), cationic lipid (61.2%), phospholipid (7.1%), cholesterol (29.2%), wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g. , ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DMA (compound (66)) (2.6%)
  • the cationic lipid is (6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-l 1-yl 5-(dimethylamino)pentanoate (Compound (13)) (61.2%)
  • the phospholipid is DSPC (7.1%)
  • cholesterol is present at 29.2%, wherein the actual amounts of the lipids present may vary by, e.g. , ⁇ 5 % (or e.g.
  • nucleic acid-lipid particle based on formulation Y which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation Y may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9:1 (e.g., a lipid:drug ratio of from 8.5: 1 to 10: 1, or from 8.9: 1 to 10:1 , or from 9: 1 to 9.9: 1 , including 9.1 : 1, 9.2: 1, 9.3: 1, 9.4:1 , 9.5: 1, 9.6: 1, 9.7:1, and 9.8:1).
  • 9:1 e.g., a lipid:drug ratio of from 8.5: 1 to 10: 1, or from 8.9: 1 to 10:1 , or from 9: 1 to 9.9: 1 , including 9.1 : 1, 9.2: 1, 9.3: 1, 9.4:1 , 9.5: 1, 9.6: 1, 9.7:1, and 9.8:1.
  • Exemplary lipid formulation Z includes the following components (wherein the percentage values of the components are mole percent): PEG-lipid (2.2%), cationic lipid (49.7%), phospholipid (12.1%), cholesterol (36.0%), wherein the actual amounts of the lipids present may vary by, e.g. , ⁇ 5 % (or e.g. , ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • the PEG-lipid is PEG-C-DOMG (compound (67)) (2.2%)
  • the cationic lipid is (6Z,9Z,28Z,31 Z)-heptatriaconta-6,9,28,31 -tetraen- 19-yl 4-(dimethylamino)butanoate)
  • the phospholipid is DPPC (12.1%), and cholesterol is present at 36.0%, wherein the actual amounts of the lipids present may vary by, e.g., ⁇ 5 % (or e.g., ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %).
  • a nucleic acid-lipid particle based on formulation Z, which comprises one or more siRNA molecules described herein.
  • the nucleic acid lipid particle based on formulation Z may comprise two different siRNA molecules.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of from about 5:1 to about 15:1, or about 5: 1, 6: 1, 7:1, 8:1, 9: 1, 10: 1, 11 :1, 12: 1 , 13: 1, 14: 1 , or 15: 1, or any fraction thereof or range therein.
  • the nucleic acid-lipid particle has a total lipid:siRNA mass ratio of about 9: 1 (e.g., a lipid:drug ratio of from 8.5:1 to 10: 1, or from 8.9:1 to 10:1, or from 9:1 to 9.9:1 , including 9.1 :1, 9.2:1, 9.3:1 , 9.4: 1, 9.5:1, 9.6:1, 9.7: 1, and 9.8:1).
  • 9: 1 e.g., a lipid:drug ratio of from 8.5:1 to 10: 1, or from 8.9:1 to 10:1, or from 9:1 to 9.9:1 , including 9.1 :1, 9.2:1, 9.3:1 , 9.4: 1, 9.5:1, 9.6:1, 9.7: 1, and 9.8:1).
  • certain embodiments of the invention provide a nucleic acid-lipid particle described herein, wherein the lipids are formulated as described in any one of formulations A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y or Z.
  • the present invention also provides pharmaceutical compositions comprising a nucleic acid-lipid particle and a pharmaceutically acceptable carrier.
  • the nucleic acid-lipid particles of the present invention are useful, for example, for the therapeutic delivery of siRNAs that silence the expression of one or more EBOV genes.
  • a cocktail of siRNAs that target different regions (e.g., overlapping and/or non-overlapping sequences) of an EBOV gene or transcript is formulated into the same or different nucleic acid-lipid particles, and the particles are administered to a mammal (e.g., a human) requiring such treatment.
  • a therapeutically effective amount of the nucleic acid-lipid particles can be administered to the mammal, e.g., for treating EBOV infection in a human.
  • the present invention provides a method for introducing one or more siRNA molecules described herein into a cell by contacting the cell with a nucleic acid- lipid particle described herein.
  • the present invention provides a method for introducing one or more siRNA molecules that silence expression of an EBOV gene into a cell by contacting the cell with a nucleic acid-lipid particle described herein under conditions whereby the siRNA enters the cell and silences the expression of the EBOV gene within the cell.
  • the cell is in a mammal, such as a human.
  • the human has been diagnosed with an EBOV infection.
  • silencing of the EBOV gene expression reduces EBOV particle load in the mammal by at least about 50% (e.g., about 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100%) relative to EBOV particle load in the absence of the nucleic acid-lipid particle.
  • the present invention provides a method for silencing expression of an EBOV gene in a cell, the method comprising the step of contacting a cell comprising an expressed EBOV gene with a nucleic acid-lipid particle or a composition (e.g., a pharmaceutical composition) described herein under conditions whereby the siRNA enters the cell and silences the expression of the EBOV gene within the cell.
  • the cell is in a mammal, such as a human.
  • the human has been diagnosed with an EBOV infection.
  • silencing of the EBOV gene expression reduces EBOV particle load in the mammal by at least about 50% (e.g., about 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100%) relative to EBOV particle load in the absence of the nucleic acid-lipid particle.
  • the nucleic acid-lipid particles or compositions (e.g., a pharmaceutical composition) described herein are administered by one of the following routes of administration: oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and intradermal.
  • the nucleic acid-lipid particles are administered systemically, e.g., via enteral or parenteral routes of administration.
  • the present invention provides methods for silencing EBOV gene expression in a mammal (e.g., human) in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a nucleic acid-lipid particle comprising one or more siRNAs described herein (e.g., one or more siRNAs shown in Tables A and B).
  • nucleic acid-lipid particles comprising one or more siRNAs described herein reduces EBOV RNA levels by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range therein) relative to EBOV RNA levels detected in the absence of the siRNA (e.g., buffer control or irrelevant non- EBOV targeting siRNA control).
  • siRNA e.g., buffer control or irrelevant non- EBOV targeting siRNA control
  • nucleic acid-lipid particles comprising one or more EBOV-targeting siRNAs reduces EBOV RNA levels for at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 days or more (or any range therein) relative to a negative control such as, e.g., a buffer control or an irrelevant non-EBOV targeting siRNA control.
  • a negative control such as, e.g., a buffer control or an irrelevant non-EBOV targeting siRNA control.
  • the present invention provides methods for silencing EBOV gene expression in a mammal (e.g., human) in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a nucleic acid-lipid particle comprising one or more siRNAs described herein (e.g., siRNAs described in Tables A and B).
  • a mammal e.g., human
  • a therapeutically effective amount of a nucleic acid-lipid particle comprising one or more siRNAs described herein (e.g., siRNAs described in Tables A and B).
  • administration of nucleic acid-lipid particles comprising one or more EBOV siRNAs reduces EBOV mRNA levels by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range therein) relative to EBOV mRNA levels detected in the absence of the siRNA (e.g., buffer control or irrelevant non-EBOV targeting siRNA control).
  • siRNA e.g., buffer control or irrelevant non-EBOV targeting siRNA control
  • nucleic acid-lipid particles comprising one or more EBOV-targeting siRNAs reduces EBOV mRNA levels for at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 days or more (or any range therein) relative to a negative control such as, e.g., a buffer control or an irrelevant non-EBOV targeting siRNA control.
  • a negative control such as, e.g., a buffer control or an irrelevant non-EBOV targeting siRNA control.
  • Certain embodiments of the invention provide a nucleic acid-lipid particle or a composition (e.g., a pharmaceutical composition) described herein for use in silencing expression of an EBOV gene in a cell in a mammal (e.g., a human). Certain embodiments of the invention provide the use of a nucleic acid-lipid particle or a composition (e.g., a pharmaceutical composition) described herein to prepare a medicament for silencing expression of an EBOV gene in a cell in a mammal (e.g., a human).
  • the present invention provides methods for treating, preventing, reducing the risk or likelihood of developing (e.g., reducing the susceptibility to), delaying the onset of, and/or ameliorating one or more symptoms associated with EBOV infection in a mammal (e.g., human) in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a nucleic acid-lipid particle comprising one or more siRNA molecules described herein (e.g., as described in Tables A and B) that target EBOV gene expression.
  • a nucleic acid-lipid particle comprising one or more siRNA molecules described herein (e.g., as described in Tables A and B) that target EBOV gene expression.
  • Certain embodiments of the invention provide a method for treating an EBOV infection in a mammal, the method comprising the step of administering to the mammal a therapeutically effective amount of a nucleic acid-lipid particle or a composition (e.g., a pharmaceutical composition) as described herein.
  • nucleic acid-lipid particle or a composition for use in treating an EBOV infection in a mammal (e.g., a human).
  • Certain embodiments of the invention provide the use of a nucleic acid-lipid particle or a composition (e.g., a pharmaceutical composition) to prepare a medicament for treating an EBOV infection in a mammal (e.g., a human).
  • a nucleic acid-lipid particle or a composition e.g., a pharmaceutical composition
  • Certain embodiments of the invention provide a method for ameliorating one or more symptoms associated with EBOV infection in a mammal, the method comprising the step of administering to the mammal a therapeutically effective amount of a nucleic acid-lipid particle or composition (e.g., a pharmaceutical composition) described herein, comprising one or more siRNA molecules described herein (e.g. , as described in Tables A and B).
  • a nucleic acid-lipid particle or composition e.g., a pharmaceutical composition
  • siRNA molecules described herein e.g. , as described in Tables A and B.
  • the particle is administered via a systemic route.
  • the siRNA of the nucleic acid-lipid particle inhibits expression of an EBOV gene in the mammal.
  • the mammal is a human.
  • nucleic acid-lipid particle or a composition as described herein for use in ameliorating one or more symptoms associated with an EBOV infection in a mammal (e.g., a human).
  • Certain embodiments of the invention provide the use of a nucleic acid-lipid particle or a composition (e.g., a pharmaceutical composition) as described herein to prepare a medicament for ameliorating one or more symptoms associated with an EBOV infection in a mammal (e.g., a human). Certain embodiments of the invention provide a nucleic acid-lipid particle or a composition (e.g., a pharmaceutical composition) as described herein for use in medical therapy.
  • a nucleic acid-lipid particle or a composition e.g., a pharmaceutical composition
  • the present invention provides a method for inactivating EBOV in a mammal (e.g., human) in need thereof (e.g., a human suffering from EBOV infection), the method comprising administering to the mammal a therapeutically effective amount of a nucleic acid-lipid particle comprising one or more siRNAs described herein that target EBOV gene expression.
  • nucleic acid-lipid particles comprising one or more EBOV-targeting siRNAs lowers, reduces, or decreases EBOV protein levels (e.g., EBOV surface antigen protein) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range therein) relative to the EBOV protein levels detected in the absence of the siRNA (e.g., buffer control or irrelevant non-EBOV targeting siRNA control).
  • siRNA e.g., buffer control or irrelevant non-EBOV targeting siRNA control
  • EBOV mRNA can be measured using a branched DNA assay (QuantiGene®; Affymetrix).
  • the branched DNA assay is a sandwich nucleic acid hybridization method that uses bDNA molecules to amplify signal from captured target RNA.
  • the siRNA described herein are also useful in research and development applications as well as diagnostic, prophylactic, prognostic, clinical, and other healthcare applications.
  • the siRNA can be used in target validation studies directed at testing whether a specific member of the EBOV gene family has the potential to be a therapeutic target.
  • siRNA can be provided in several forms including, e.g. , as one or more isolated small- interfering RNA (siRNA) duplexes, as longer double-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from a transcriptional cassette in a DNA plasmid.
  • siRNA may be produced enzymatically or by partial/total organic synthesis, and modified ribonucleotides can be introduced by in vitro enzymatic or organic synthesis.
  • each strand is prepared chemically. Methods of synthesizing RNA molecules are known in the art, e.g., the chemical synthesis methods as described in Verma and Eckstein (1998) or as described herein.
  • RNA, synthesizing RNA, hybridizing nucleic acids, making and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983); Sambrook et al, supra; Ausubel et al, supra), as are PCR methods (see, U.S. Patent Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)).
  • Expression libraries are also well known to those of skill in the art.
  • siRNA are chemically synthesized.
  • the oligonucleotides that comprise the siRNA molecules of the invention can be synthesized using any of a variety of techniques known in the art, such as those described in Usman et al., J. Am. Chem. Soc, 109:7845 (1987); Scaringe et al, Nucl. Acids Res., 18:5433 (1990); Wincott et al. , Nucl. Acids Res. , 23:2677-2684 (1995); and Wincott et al., Methods Mol. Bio., 74:59 (1997).
  • oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5 '-end and phosphoramidites at the 3' -end.
  • small scale syntheses can be conducted on an Applied Biosystems synthesizer using a 0.2 ⁇ scale protocol.
  • syntheses at the 0.2 ⁇ scale can be performed on a 96-well plate synthesizer from Protogene (Palo Alto, CA).
  • Protogene Protogene
  • a larger or smaller scale of synthesis is also within the scope of this invention.
  • Suitable reagents for oligonucleotide synthesis, methods for RNA deprotection, and methods for RNA purification are known to those of skill in the art.
  • siRNA molecules can be assembled from two distinct oligonucleotides, wherein one oligonucleotide comprises the sense strand and the other comprises the antisense strand of the siRNA.
  • each strand can be synthesized separately and joined together by hybridization or ligation following synthesis and/or deprotection.
  • the present invention provides lipid particles comprising one or more siRNA molecules (e.g., one or more siRNA molecules described in Tables A and B) and one or more of cationic (amino) lipids or salts thereof.
  • the lipid particles of the invention further comprise one or more non-cationic lipids.
  • the lipid particles further comprise one or more conjugated lipids capable of reducing or inhibiting particle aggregation.
  • the lipid particles of the invention preferably comprise one or more siRNA (e.g., an siRNA molecules described in Tables A and B), a cationic lipid, a non-cationic lipid, and a conjugated lipid that inhibits aggregation of particles.
  • siRNA e.g., an siRNA molecules described in Tables A and B
  • the siRNA molecule is fully encapsulated within the lipid portion of the lipid particle such that the siRNA molecule in the lipid particle is resistant in aqueous solution to nuclease degradation.
  • the lipid particles described herein are substantially non-toxic to mammals such as humans.
  • the lipid particles of the invention typically have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 1 10 nm, or from about 70 to about 90 nm. In certain embodiments, the lipid particles of the invention have a median diameter of from about 30 nm to about 150 nm.
  • the lipid particles of the invention also typically have a lipid:nucleic acid ratio (e.g., a lipid:siRNA ratio) (mass/mass ratio) of from about 1 : 1 to about 100: 1 , from about 1 : 1 to about 50: 1 , from about 2: 1 to about 25: 1, from about 3: 1 to about 20: 1 , from about 5 : 1 to about 15 : 1 , or from about 5 : 1 to about 10: 1.
  • the nucleic acid-lipid particle has a lipid:siRNA mass ratio of from about 5: 1 to about 15: 1.
  • the lipid particles of the invention are serum-stable nucleic acid-lipid particles which comprise one or more siRNA molecules (e.g., a siRNA molecule as described in Tables A and B), a cationic lipid (e.g., one or more cationic lipids of Formula I-III or salts thereof as set forth herein), a non-cationic lipid (e.g., mixtures of one or more phospholipids and cholesterol), and a conjugated lipid that inhibits aggregation of the particles (e.g., one or more PEG-lipid conjugates).
  • siRNA molecules e.g., a siRNA molecule as described in Tables A and B
  • a cationic lipid e.g., one or more cationic lipids of Formula I-III or salts thereof as set forth herein
  • a non-cationic lipid e.g., mixtures of one or more phospholipids and cholesterol
  • a conjugated lipid that inhibits aggregation of the particles
  • the lipid particle may comprise at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more siRNA molecules (e.g., siRNA molecules described in Tables A and B) that target one or more of the genes described herein.
  • siRNA molecules e.g., siRNA molecules described in Tables A and B
  • Nucleic acid-lipid particles and their method of preparation are described in, e.g., U.S. Patent Nos. 5,753,613; 5,785,992; 5,705,385; 5,976,567; 5,981 ,501 ; 6,1 10,745; and 6,320,017; and PCT Publication No. WO 96/40964, the disclosures of which are each herein incorporated by reference in their entirety for all purposes.
  • the one or more siRNA molecules may be fully encapsulated within the lipid portion of the particle, thereby protecting the siRNA from nuclease degradation.
  • the siRNA in the nucleic acid-lipid particle is not substantially degraded after exposure of the particle to a nuclease at 37°C for at least about 20, 30, 45, or 60 minutes.
  • the siRNA in the nucleic acid-lipid particle is not substantially degraded after incubation of the particle in serum at 37°C for at least about 30, 45, or 60 minutes or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours.
  • the siRNA is complexed with the lipid portion of the particle.
  • the nucleic acid-lipid particle compositions are substantially non-toxic to mammals such as humans.
  • the term "fully encapsulated” indicates that the siRNA (e.g., a siRNA molecule as described in Tables A and B) in the nucleic acid-lipid particle is not significantly degraded after exposure to serum or a nuclease assay that would significantly degrade free DNA or RNA.
  • the siRNA e.g., a siRNA molecule as described in Tables A and B
  • a fully encapsulated system preferably less than about 25% of the siRNA in the particle is degraded in a treatment that would normally degrade 100% of free siRNA, more preferably less than about 10%, and most preferably less than about 5% of the siRNA in the particle is degraded.
  • “Fully encapsulated” also indicates that the nucleic acid-lipid particles are serum- stable, that is, that they do not rapidly decompose into their component parts upon in vivo administration.
  • full encapsulation may be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that has enhanced fluorescence when associated with nucleic acid.
  • fluorescent dye exclusion assay which uses a dye that has enhanced fluorescence when associated with nucleic acid.
  • Specific dyes such as OliGreen ® and
  • RiboGreen (Invitrogen Corp.; Carlsbad, CA) are available for the quantitative determination of plasmid DNA, single-stranded deoxyribonucleotides, and/or single- or double-stranded ribonucleotides. Encapsulation is determined by adding the dye to a liposomal formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of nonionic detergent. Detergent-mediated disruption of the liposomal bilayer releases the encapsulated nucleic acid, allowing it to interact with the membrane-impermeable dye.
  • the present invention provides a nucleic acid-lipid particle composition comprising a plurality of nucleic acid-lipid particles.
  • the nucleic acid-lipid particle composition comprises a siRNA molecule that is fully encapsulated within the lipid portion of the particles, such that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about 60% to about 95%, from about 70% to about 95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
  • the nucleic acid-lipid particle composition comprises siRNA that is fully encapsulated within the lipid portion of the particles, such that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about 60% to about 95%, from about 70% to about 95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
  • the proportions of the components can be varied and the delivery efficiency of a particular formulation can be measured using, e.g., an endosomal release parameter (ERP) assay.
  • ERP endosomal release parameter
  • cationic lipids or salts thereof may be used in the lipid particles of the present invention either alone or in combination with one or more other cationic lipid species or non-cationic lipid species.
  • the cationic lipids include the (R) and/or (S) enantiomers thereof.
  • the cationic lipid is a dialkyl lipid.
  • dialkyl lipids may include lipids that comprise two saturated or unsaturated alkyl chains, wherein each of the alkyl chains may be substituted or unsubstituted.
  • each of the two alkyl chains comprise at least, e.g., 8 carbon atoms, 10 carbon atoms, 12 carbon atoms, 14 carbon atoms, 16 carbon atoms, 18 carbon atoms, 20 carbon atoms, 22 carbon atoms or 24 carbon atoms.
  • the cationic lipid is a trialkyl lipid.
  • trialkyl lipids may include lipids that comprise three saturated or unsaturated alkyl chains, wherein each of the alkyl chains may be substituted or unsubstituted.
  • each of the three alkyl chains comprise at least, e.g., 8 carbon atoms, 10 carbon atoms, 12 carbon atoms, 14 carbon atoms, 16 carbon atoms, 18 carbon atoms, 20 carbon atoms, 22 carbon atoms or 24 carbon atoms.
  • cationic lipids of Formula I having the following structure are useful in the present invention:
  • R 1 and R 2 are either the same or different and are independently hydrogen (H) or an
  • optionally substituted C]-C 6 alkyl, C 2 -C 6 alkenyl, or C 2 -C 6 alkynyl, or R and R may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and mixtures thereof;
  • R 3 is either absent or is hydrogen (H) or a Ci-C 6 alkyl to provide a quaternary amine
  • R 4 and R 5 are either the same or different and are independently an optionally substituted C 10 -C 2 4 alkyl, Ci 0 -C 24 alkenyl, Q0-C24 alkynyl, or C10-C24 acyl, wherein at least one of R 4 and R 5 comprises at least two sites of unsaturation;
  • n 0, 1 , 2, 3, or 4.
  • R and R are independently an optionally substituted Q-C4
  • R and R are both methyl groups.
  • n is 1 or 2.
  • R is absent when the pH is above the pK a of the cationic lipid and R 3 is hydrogen when the pH is below the pK a of the cationic lipid such that the amino head group is protonated.
  • R is an optionally substituted Q-C4 alkyl to provide a quaternary amine.
  • R 4 and R 5 are independently an optionally substituted Ci 2 -C 20 or C 14 -C 22 alkyl, C 12 -C 2 o or Ci4-C 2 2 alkenyl, Ci 2 -C 2 o or Ci4-C 22 alkynyl, or Ci 2 -C 2 o or Ci4-C2 2 acyl, wherein at least one of R 4 and R 5 comprises at least two sites of unsaturation.
  • R 4 and R 5 are independently selected from the group consisting of a dodecadienyl moiety, a tetradecadienyl moiety, a hexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl moiety, a dodecatrienyl moiety, a tetradectrienyl moiety, a hexadecatrienyl moiety, an octadecatrienyl moiety, an icosatrienyl moiety, an arachidonyl moiety, and a docosahexaenoyl moiety, as well as acyl derivatives thereof (e.g., linoleoyl, linolenoyl, ⁇ -linolenoyl, etc.).
  • acyl derivatives thereof e.g., linoleoyl, linolenoyl,
  • one of R 4 and R 5 comprises a branched alkyl group (e.g., a phytanyl moiety) or an acyl derivative thereof (e.g., a phytanoyl moiety).
  • the octadecadienyl moiety is a linoleyl moiety.
  • the octadecatrienyl moiety is a linolenyl moiety or a ⁇ -linolenyl moiety.
  • R' and R 5 are both linoleyl moieties, linolenyl moieties, or ⁇ -linolenyl moieties.
  • the cationic lipid of Formula I is l ,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l ,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1 ,2-dilinoleyloxy- (N,N-dimethyl)-butyl-4-amine (C2-DLinDMA), 1 ,2-dilinoleoyloxy-(N,N-dimethyl)-butyl-4- amine (C2-DLinDAP), or mixtures thereof.
  • DLinDMA l ,2-dilinoleyloxy-N,N-dimethylaminopropane
  • DLenDMA l ,2-dilinolenyloxy-N,N-dimethylaminopropane
  • C2-DLinDMA 1 ,2-dilinoleyloxy- (N,N-dimethyl)-
  • the cationic lipid of Formula I forms a salt (preferably a crystalline salt) with one or more anions.
  • the cationic lipid of Formula I is the oxalate (e.g., hemioxalate) salt thereof, which is preferably a crystalline salt.
  • cationic lipids such as DLinDMA and DLenDMA, as well as additional cationic lipids
  • U.S. Patent Publication No. 20060083780 the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • the synthesis of cationic lipids such as C2-DLinDMA and C2-DLinDAP, as well as additional cationic lipids, is described in international patent application number WO201 1/000106 the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • cationic lipids of Formula II having the following structure (or salts thereof) are useful in the present invention:
  • R and R are either the same or different and are independently an optionally substituted C 12 -C 24 alkyl, C 12 -C 24 alkenyl, Ci 2 -C 2 4 alkynyl, or C] 2 -C 24 acyl;
  • R 3 and R 4 are either the same or different and are independently an optionally substituted C C alkyl, C 2 -C alkenyl, or C 2 -C 6 alkynyl, or R 3 and R 4 may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen;
  • R 5 is either absent or is hydrogen (H) or a C!-C 6 alkyl to provide a quaternary amine;
  • m, n, and p are either the same or different and are independently either 0, 1 , or 2, with the proviso that m, n, and p are not simultaneously 0; q is 0, 1 , 2, 3, or 4; and
  • the cationic lipid of Formula II is 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l ,3]-dioxolane (DLin-K-C2-DMA; "XTC2" or “C2K”), 2,2-dilinoleyl-4- (3-dimethylaminopropyl)-[l ,3]-dioxolane (DLin-K-C3 -DMA; "C3K”), 2,2-dilinoleyl-4-(4- dimethylaminobutyl)-[l ,3]-dioxolane (DLin-K-C4-DMA; "C4K”), 2,2-dilinoleyl-5- dimethylaminomethyl-[l ,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino- [l ,3]-dioxo
  • the cationic lipid of Formula II forms a salt (preferably a crystalline salt) with one or more anions.
  • the cationic lipid of Formula II is the oxalate (e.g., hemioxalate) salt thereof, which is preferably a crystalline salt.
  • cationic lipids such as DLin-K-DMA, as well as additional cationic lipids, is described in PCT Publication No. WO 09/086558, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • cationic lipids such as DLin-K-C2-DMA, DLin-K-C3 -DM A, DLin-K-C4-DMA, DLin-K6-DMA, DLin-K-MPZ, DO- K-DMA, DS-K-DMA, DLin-K-MA, DLin-K-TMA.Cl, DLin-K 2 -DMA, and D-Lin-K-N- methylpiperzine, as well as additional cationic lipids, is described in PCT Application No.
  • cationic lipids of Formula III having the following structure are useful in the present invention:
  • R 1 and R 2 are either the same or different and are
  • Ci-C alkyl independently an optionally substituted Ci-C alkyl, C 2 -C 6 alkenyl, or C 2 -C 6 alkynyl, or R 1 and R may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and mixtures thereof;
  • R 3 is either absent or is hydrogen (H) or a C C 6 alkyl to provide a quaternary amine;
  • R 4 and R 5 are either absent or present and when present are either the same or different and are independently an optionally substituted Ci-C 10 alkyl or C 2 -C 10 alkenyl; and n is 0, 1 , 2, 3, or 4.
  • R and R are independently an optionally substituted C C 4 alkyl, C 2 -C 4 alkenyl, or C 2 -C 4 alkynyl.
  • R 1 and R 2 are both methyl groups.
  • R 4 and R 5 are both butyl groups.
  • n is 1.
  • R 3 is absent when the pH is above the pK a of the cationic lipid and R 3 is hydrogen when the pH is below the pK a of the cationic lipid such that the amino head group is protonated.
  • R 3 is an optionally substituted C1-C4 alkyl to provide a quaternary amine.
  • R 4 and R 5 are independently an optionally substituted C 2 -C 6 or C 2 -C 4 alkyl or C 2 -C or C 2 -C 4 alkenyl.
  • the cationic lipid of Formula III comprises ester linkages between the amino head group and one or both of the alkyl chains.
  • the cationic lipid of Formula III forms a salt (preferably a crystalline salt) with one or more anions.
  • the cationic lipid of Formula III is the oxalate (e.g., hemioxalate) salt thereof, which is preferably a crystalline salt.
  • each of the alkyl chains in Formula III contains cis double bonds at positions 6, 9, and 12 (i.e., cis, cis, cis- in an alternative embodiment, one, two, or three of these double bonds in one or both alkyl chains may be in the trans configuration.
  • the cationic lipid of Formula III has the structure:
  • MC3 cationic lipids
  • additional cationic lipids e.g., certain analogs of MC3
  • U.S. Provisional Application No. 61/185,800 entitled “Novel Lipids and Compositions for the Delivery of Therapeutics”
  • U.S. Provisional Application No. 61/287,995 entitled “Methods and Compositions for Delivery of Nucleic Acids,” filed December 18, 2009, the disclosures of which are herein incorporated by reference in their entirety for all purposes.
  • DLin-M-C2-DMA dilinoleylmethyl-3- dimethylaminopropionate
  • DLin-M-C2-DMA dilinoleylmethyl-3- dimethylaminopropionate
  • DLin-M-K-DMA dilin-M-K-DMA
  • DLin-M- DMA dilinoleylmethyl-3- dimethylaminopropionate
  • cationic lipids such as DLin-C-DAP, DLinDAC, DLinMA, DLinDAP, DLin-S-DMA, DLin-2-DMAP, DLinTMA.Cl, DLinTAP.Cl, DLinMPZ, DLinAP, DOAP, and DLin-EG-DMA, as well as additional cationic lipids, is described in PCT Publication No. WO 09/086558, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • cationic lipids such as DO-C-DAP, DMDAP, DOTAP.C1, DLin-M-C2-DMA, as well as additional cationic lipids, is described in PCT Application No. PCT/US2009/060251, entitled “Improved Amino Lipids and Methods for the Delivery of Nucleic Acids,” filed October 9, 2009, the disclosure of which is incorporated herein by reference in its entirety for all purposes.
  • the synthesis of a number of other cationic lipids and related analogs has been described in U.S. Patent Nos. 5,208,036;
  • LIPOFECTAMINE ® including DOSPA and DOPE, available from Invitrogen
  • TRANSFECTAM ® including DOGS, available from Promega Corp.
  • the cationic lipid comprises from about 50 mol % to about 90 mol %, from about 50 mol % to about 85 mol %, from about 50 mol % to about 80 mol %, from about 50 mol % to about 75 mol %, from about 50 mol % to about 70 mol %, from about 50 mol % to about 65 mol %, from about 50 mol % to about 60 mol %, from about 55 mol % to about 65 mol %, or from about 55 mol % to about 70 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the cationic lipid comprises about 50 mol %, 51 mol %, 52 mol %, 53 mol %, 54 mol %, 55 mol %, 56 mol %, 57 mol %, 58 mol %, 59 mol %, 60 mol %, 61 mol %, 62 mol %, 63 mol %, 64 mol %, or 65 mol % (or any fraction thereof) of the total lipid present in the particle.
  • the cationic lipid comprises from about 2 mol % to about 60 mol %, from about 5 mol % to about 50 mol %, from about 10 mol % to about 50 mol %, from about 20 mol % to about 50 mol %, from about 20 mol % to about 40 mol %, from about 30 mol % to about 40 mol %, or about 40 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the percentage of cationic lipid present in the lipid particles of the invention is a target amount, and that the actual amount of cationic lipid present in the formulation may vary, for example, by ⁇ 5 mol %.
  • the target amount of cationic lipid is 57.1 mol %, but the actual amount of cationic lipid may be ⁇ 5 mol %, ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol % of that target amount, with the balance of the formulation being made up of other lipid components (adding up to 100 mol % of total lipids present in the particle; however, one skilled in the art will understand that the total mol % may deviate slightly from 100% due to rounding, for example, 99.9 mol % or 100.1
  • cationic lipids useful for inclusion in lipid particles used present invention are shown below:
  • the non-cationic lipids used in the lipid particles of the invention can be any of a variety of neutral uncharged, zwitterionic, or anionic lipids capable of producing a stable complex.
  • Non-limiting examples of non-cationic lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC),
  • phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (
  • DOPG dioleoylphosphatidylglycerol
  • DPPG dipalmitoylphosphatidylglycerol
  • dioleoylphosphatidylethanolamine DOPE
  • palmitoyloleoyl-phosphatidylcholine POPC
  • palmitoyloleoyl-phosphatidylethanolamine POPE
  • palmitoyloleyol-phosphatidylglycerol POPG
  • dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 -carboxylate DOPE-mal
  • dipalmitoyl-phosphatidylethanolamine DPPE
  • dimyristoyl- phosphatidylethanolamine DMPE
  • distearoyl-phosphatidylethanolamine DSPE
  • monomethyl-phosphatidylethanolamine dimethyl-phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof.
  • DEPE dielaidoyl- phosphatidylethanolamine
  • SOPE stearoyloleoyl-phosphatidylethanolamine
  • lysophosphatidylcholine dilinoleoylphosphatidylcholine
  • Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used.
  • acyl groups in these lipids are preferably acyl groups derived from fatty acids having Ci 0 - C 2 4 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.
  • additional examples of non-cationic lipids include sterols such as cholesterol and derivatives thereof.
  • Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 5 -coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'- hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5P-cholestanone, and cholesteryl decanoate; and mixtures thereof.
  • the cholesterol derivative is a polar analogue such as cholesteryl-(4'-hydroxy)-butyl ether.
  • the synthesis of cholesteryl-(2'-hydroxy)-ethyl ether is described in PCT Publication No. WO 09/127060, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • the non-cationic lipid present in the lipid particles comprises or consists of a mixture of one or more phospholipids and cholesterol or a derivative thereof. In other embodiments, the non-cationic lipid present in the lipid particles comprises or consists of one or more phospholipids, e.g., a cholesterol-free lipid particle formulation. In yet other embodiments, the non-cationic lipid present in the lipid particles comprises or consists of cholesterol or a derivative thereof, e.g., a phospholipid-free lipid particle formulation.
  • non-cationic lipids suitable for use in the present invention include nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myri state, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, sphingomyelin, and the like.
  • nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate,
  • the non-cationic lipid comprises from about 10 mol % to about 60 mol %, from about 20 mol % to about 55 mol %, from about 20 mol % to about 45 mol %, from about 20 mol % to about 40 mol %, from about 25 mol % to about 50 mol %, from about 25 mol % to about 45 mol %, from about 30 mol % to about 50 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 35 mol % to about 45 mol %, from about 37 mol % to about 45 mol %, or about 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %, 43 mol %, 44 mol %, or 45 mol % (or any fraction thereof or range therein
  • the lipid particles contain a mixture of phospholipid and cholesterol or a cholesterol derivative
  • the mixture may comprise up to about 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % of the total lipid present in the particle.
  • the phospholipid component in the mixture may comprise from about 2 mol % to about 20 mol %, from about 2 mol % to about 15 mol %, from about 2 mol % to about 12 mol %, from about 4 mol % to about 15 mol %, or from about 4 mol % to about 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the phospholipid component in the mixture comprises from about 5 mol % to about 17 mol %, from about 7 mol % to about 17 mol %, from about 7 mol % to about 15 mol %, from about 8 mol % to about 15 mol %, or about 8 mol %, 9 mol %, 10 mol %, 1 1 mol %, 12 mol %, 13 mol %, 14 mol %, or 15 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • a lipid particle formulation comprising a mixture of phospholipid and cholesterol may comprise a phospholipid such as DPPC or DSPC at about 7 mol % (or any fraction thereof), e.g., in a mixture with cholesterol or a cholesterol derivative at about 34 mol % (or any fraction thereof) of the total lipid present in the particle.
  • a lipid particle formulation comprising a mixture of phospholipid and cholesterol may comprise a phospholipid such as DPPC or DSPC at about 7 mol % (or any fraction thereof), e.g., in a mixture with cholesterol or a cholesterol derivative at about 32 mol % (or any fraction thereof) of the total lipid present in the particle.
  • a lipid formulation useful in the practice of the invention has a lipid to drug (e.g. , siRNA) ratio of about 10: 1 (e.g. , a lipid:drug ratio of from 9.5: 1 to 1 1 : 1 , or from 9.9: 1 to 1 1 : 1 , or from 10: 1 to 10.9: 1 ).
  • a lipid to drug e.g. , siRNA
  • a lipid formulation useful in the practice of the invention has a lipid to drug (e.g., siRNA) ratio of about 9: 1 (e.g., a lipid:drug ratio of from 8.5: 1 to 10: 1 , or from 8.9: 1 to 10: 1 , or from 9: 1 to 9.9: 1 , including 9.1 : 1 , 9.2: 1 , 9.3: 1 , 9.4:1 , 9.5: 1 , 9.6: 1 , 9.7: 1 , and 9.8: 1).
  • a lipid to drug e.g., siRNA ratio of about 9: 1
  • a lipid:drug ratio of from 8.5: 1 to 10: 1 , or from 8.9: 1 to 10: 1 , or from 9: 1 to 9.9: 1 , including 9.1 : 1 , 9.2: 1 , 9.3: 1 , 9.4:1 , 9.5: 1 , 9.6: 1 , 9.7: 1 , and 9.8: 1).
  • the cholesterol component in the mixture may comprise from about 25 mol % to about 45 mol %, from about 25 mol % to about 40 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 27 mol % to about 37 mol %, from about 25 mol % to about 30 mol %, or from about 35 mol % to about 40 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the cholesterol component in the mixture comprises from about 25 mol % to about 35 mol %, from about 27 mol % to about 35 mol %, from about 29 mol % to about 35 mol %, from about 30 mol % to about 35 mol %, from about 30 mol % to about 34 mol %, from about 31 mol % to about 33 mol %, or about 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34 mol %, or 35 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the cholesterol or derivative thereof may comprise up to about 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % of the total lipid present in the particle.
  • the cholesterol or derivative thereof in the phospholipid-free lipid particle formulation may comprise from about 25 mol % to about 45 mol %, from about 25 mol % to about 40 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 31 mol % to about 39 mol %, from about 32 mol % to about 38 mol %, from about 33 mol % to about 37 mol %, from about 35 mol % to about 45 mol %, from about 30 mol % to about 35 mol %, from about 35 mol % to about 40 mol %, or about 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34 mol %, 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, or 40 mol % (or any fraction thereof or range therein) of the total mol %,
  • a lipid particle formulation may comprise cholesterol at about 37 mol % (or any fraction thereof) of the total lipid present in the particle.
  • a lipid particle formulation may comprise cholesterol at about 35 mol % (or any fraction thereof) of the total lipid present in the particle.
  • the non-cationic lipid comprises from about 5 mol % to about
  • the percentage of non-cationic lipid present in the lipid particles of the invention is a target amount, and that the actual amount of non-cationic lipid present in the formulation may vary, for example, by ⁇ 5 mol %, ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %.
  • the lipid particles of the invention may further comprise a lipid conjugate.
  • the conjugated lipid is useful in that it prevents the aggregation of particles.
  • Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, POZ-lipid conjugates, ATTA-lipid conjugates, cationic-polymer-lipid conjugates (CPLs), and mixtures thereof.
  • the particles comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate together with a CPL.
  • the lipid conjugate is a PEG-lipid.
  • PEG-lipids include, but are not limited to, PEG coupled to dialkyloxypropyls (PEG-DAA) as described in, e.g., PCT Publication No. WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in, e.g., U.S. Patent Publication Nos. 20030077829 and 2005008689, PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides as described in, e.g., U.S. Patent No. 5,885,613, PEG conjugated to cholesterol or a derivative thereof, and mixtures thereof.
  • PEG-lipids include, but are not limited to, PEG coupled to dialkyloxypropyls (PEG-DAA) as described in, e.g., PCT Publication No. WO 05/026372, PEG
  • Additional PEG-lipids suitable for use in the invention include, without limitation, mPEG2000-l,2-di-O-alky «3-carbomoylglyceride (PEG-C-DOMG).
  • PEG-C-DOMG mPEG2000-l,2-di-O-alky «3-carbomoylglyceride
  • PEG-C-DOMG mPEG2000-l,2-di-O-alky «3-carbomoylglyceride
  • PEG-C-DOMG mPEG2000-l,2-di-O-alky «3-carbomoylglyceride
  • PEG-C-DOMG mPEG2000-l,2-di-O-alky «3-carbomoylglyceride
  • PEG-C-DOMG mPEG2000-l,2-di-O-alky «3-carbomoylglyceride
  • PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified by their molecular weights; for example, PEG 2000 has an average molecular weight of about 2,000 daltons, and PEG 5000 has an average molecular weight of about 5,000 daltons. PEGs are commercially available from Sigma
  • PEGs such as those described in U.S. Patent Nos. 6,774,180 and 7,053,150 (e.g., mPEG (20 KDa) amine) are also useful for preparing the PEG-lipid conjugates of the present invention.
  • mPEG (20 KDa) amine e.g., mPEG (20 KDa) amine
  • the disclosures of these patents are herein incorporated by reference in their entirety for all purposes.
  • monomethoxypolyethyleneglycol-acetic acid MePEG-CH 2 COOH
  • PEG-DAA conjugates is particularly useful for preparing PEG-lipid conjugates including, e.g., PEG-DAA conjugates.
  • the PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain instances, the PEG moiety has an average molecular weight of from about 750 daltons to about 5,000 daltons (e.g., from about 1,000 daltons to about 5,000 daltons, from about 1,500 daltons to about 3,000 daltons, from about 750 daltons to about 3,000 daltons, from about 750 daltons to about 2,000 daltons, etc.). In preferred embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons.
  • the PEG can be optionally substituted by an alkyl, alkoxy, acyl, or aryl group.
  • the PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety.
  • Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties.
  • the linker moiety is a non-ester containing linker moiety.
  • non-ester containing linker moiety refers to a linker moiety that does not contain a carboxylic ester bond (-OC(O)-).
  • Suitable non-ester containing linker moieties include, but are not limited to, amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (- NHC(O)NH-), disulphide (-S-S-), ether (-0-), succinyl (-(0)CCH 2 CH 2 C(0)-), succinamidyl (- NHC(0)CH 2 CH 2 C(0)NH-), ether, disulphide, as well as combinations thereof (such as a linker containing both a carbamate linker moiety and an amido linker moiety).
  • a carbamate linker is used to couple the PEG to the lipid.
  • an ester containing linker moiety is used to couple the PEG to the lipid.
  • Suitable ester containing linker moieties include, e.g., carbonate (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonate esters, and combinations thereof.
  • Phosphatidylethanolamines having a variety of acyl chain groups of varying chain lengths and degrees of saturation can be conjugated to PEG to form the lipid conjugate.
  • Such phosphatidylethanolamines are commercially available, or can be isolated or synthesized using conventional techniques known to those of skill in the art.
  • Phosphatidyl-ethanolamines containing saturated or unsaturated fatty acids with carbon chain lengths in the range of C 10 to C20 are preferred.
  • Phosphatidylethanolamines with mono- or diunsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Suitable
  • phosphatidylethanolamines include, but are not limited to, dimyristoyl- phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).
  • DMPE dimyristoyl- phosphatidylethanolamine
  • DPPE dipalmitoyl-phosphatidylethanolamine
  • DOPE dioleoylphosphatidylethanolamine
  • DSPE distearoyl-phosphatidylethanolamine
  • ATT A or "polyamide” includes, without limitation, compounds described in U.S. Patent Nos. 6,320,017 and 6,586,559, the disclosures of which are herein incorporated by reference in their entirety for all purposes. These compounds include a compound having the formula:
  • R is a member selected from the group consisting of hydrogen, alkyl and acyl
  • R 1 is a member selected from the group consisting of hydrogen and alkyl; or optionally, R and R 1 and the nitrogen to which they are bound form an azido moiety
  • R is a member of the group selected from hydrogen, optionally substituted alkyl, optionally substituted aryl and a side chain of an amino acid
  • R is a member selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, mercapto, hydrazino, amino and NR 4 R 5 , wherein R 4 and R 5 are independently hydrogen or alkyl
  • n is 4 to 80
  • m is 2 to 6
  • p is 1 to 4
  • q is 0 or 1.
  • diacylglycerol or “DAG” includes a compound having 2 fatty acyl chains
  • R and R both of which have independently between 2 and 30 carbons bonded to the 1- and 2- position of glycerol by ester linkages.
  • the acyl groups can be saturated or have varying degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C 12 ), myristoyl (C 14 ), palmitoyl (Ci 6 ), stearoyl (C 18 ), and icosoyl (C 20 ).
  • R 1 and R 2 are examples of lauroyl (C 12 ), myristoyl (C 14 ), palmitoyl (Ci 6 ), stearoyl (C 18 ), and icosoyl (C 20 ).
  • R and R are both myristoyl (i.e., dimyristoyl), R and R are both stearoyl (i.e., distearoyl), etc.
  • Diacylglycerols have the following general formula:
  • dialkyloxypropyl includes a compound having 2 alkyl chains
  • R 1 and R 2 both of which have independently between 2 and 30 carbons.
  • the alkyl groups can be saturated or have varying degrees of unsaturation.
  • Dialkyloxypropyls have the following general formula:
  • the PEG-lipid is a PEG-DAA conjugate having the following formula:
  • R 1 and R 2 are independently selected and are long-chain alkyl groups having from about 10 to about 22 carbon atoms; PEG is a polyethyleneglycol; and L is a non-ester containing linker moiety or an ester containing linker moiety as described above.
  • the long-chain alkyl groups can be saturated or unsaturated. Suitable alkyl groups include, but are not limited to, decyl (Cio), lauryl (Ci 2 ), myristyl (C 14 ), palmityl (Cj ), stearyl (C] ), and icosyl (C 20 ).
  • R 1 and R 2 are the same, i.e., R 1 and R 2 are both myristyl (i.e.,
  • R and R are both stearyl (i.e., distearyl), etc.
  • the PEG has an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain instances, the PEG has an average molecular weight of from about 750 daltons to about 5,000 daltons (e.g., from about 1,000 daltons to about 5,000 daltons, from about 1,500 daltons to about 3,000 daltons, from about 750 daltons to about 3,000 daltons, from about 750 daltons to about 2,000 daltons, etc.). In preferred embodiments, the PEG has an average molecular weight of about 2,000 daltons or about 750 daltons.
  • the PEG can be optionally substituted with alkyl, alkoxy, acyl, or aryl groups. In certain embodiments, the terminal hydroxyl group is substituted with a methoxy or methyl group.
  • L is a non-ester containing linker moiety.
  • Suitable non- ester containing linkers include, but are not limited to, an amido linker moiety, an amino linker moiety, a carbonyl linker moiety, a carbamate linker moiety, a urea linker moiety, an ether linker moiety, a disulphide linker moiety, a succinamidyl linker moiety, and combinations thereof.
  • the non-ester containing linker moiety is a carbamate linker moiety (i.e., a PEG-C-DAA conjugate).
  • the non-ester containing linker moiety is an amido linker moiety (i.e., a PEG-,4-DAA conjugate). In yet another preferred embodiment, the non-ester containing linker moiety is a succinamidyl linker moiety (i.e., a PEG-S-DAA conjugate). ted from: (66) (PEG-C-DMA); and
  • the PEG-DAA conjugates are synthesized using standard techniques and reagents known to those of skill in the art. It will be recognized that the PEG-DAA conjugates will contain various amide, amine, ether, thio, carbamate, and urea linkages. Those of skill in the art will recognize that methods and reagents for forming these bonds are well known and readily available. See, e.g., March, ADVANCED ORGANIC CHEMISTRY (Wiley 1992); Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989); and Furniss, VOGEL'S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY, 5th ed. (Longman 1989).
  • the PEG-DAA conjugate is a PEG-didecyloxypropyl (C 10 ) conjugate, a PEG-dilauryloxypropyl (C 12 ) conjugate, a PEG-dimyristyloxypropyl (C 14 ) conjugate, a PEG- dipalmityloxypropyl (C ]6 ) conjugate, or a PEG-distearyloxypropyl (C 18 ) conjugate.
  • the PEG preferably has an average molecular weight of about 750 or about 2,000 daltons.
  • the PEG-lipid conjugate comprises
  • the PEG-lipid conjugate comprises PEG750-C- DMA, wherein the "750” denotes the average molecular weight of the PEG, the “C” denotes a carbamate linker moiety, and the "DMA” denotes dimyristyloxypropyl.
  • the terminal hydroxyl group of the PEG is substituted with a methyl group.
  • hydrophilic polymers can be used in place of PEG.
  • suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide,
  • polymethacrylamide and polydimethylacrylamide polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
  • the lipid particles of the present invention can further comprise cationic poly(ethylene glycol) (PEG) lipids or CPLs ⁇ see, e.g., Chen et al., Bioconj. Chem., 11 :433-437 (2000); U.S. Patent No. 6,852,334; PCT Publication No. WO 00/62813, the disclosures of which are herein incorporated by reference in their entirety for all purposes).
  • PEG poly(ethylene glycol)
  • Suitable CPLs include compounds of Formula VIII:
  • A is a lipid moiety such as an amphipathic lipid, a neutral lipid, or a hydrophobic lipid that acts as a lipid anchor.
  • Suitable lipid examples include, but are not limited to, diacylglycerolyls, dialkylglycerolyls, N-N-dialkylaminos, 1 ,2-diacyloxy- 3-aminopropanes, and l,2-dialkyl-3-aminopropanes.
  • W is a polymer or an oligomer such as a hydrophilic polymer or oligomer.
  • the hydrophilic polymer is a biocompatable polymer that is nonimmunogenic or possesses low inherent immunogenicity.
  • the hydrophilic polymer can be weakly antigenic if used with appropriate adjuvants.
  • Suitable nonimmunogenic polymers include, but are not limited to, PEG, polyamides, polylactic acid, polyglycolic acid, polylactic
  • the polymer has a molecular weight of from about 250 to about 7,000 daltons.
  • Y is a polycationic moiety.
  • polycationic moiety refers to a compound, derivative, or functional group having a positive charge, preferably at least 2 positive charges at a selected pH, preferably physiological pH.
  • Suitable polycationic moieties include basic amino acids and their derivatives such as arginine, asparagine, glutamine, lysine, and histidine;
  • polycationic moieties can be linear, such as linear tetralysine, branched or dendrimeric in structure.
  • Polycationic moieties have between about 2 to about 15 positive charges, preferably between about 2 to about 12 positive charges, and more preferably between about 2 to about 8 positive charges at selected pH values. The selection of which polycationic moiety to employ may be determined by the type of particle application which is desired.
  • the charges on the polycationic moieties can be either distributed around the entire particle moiety, or alternatively, they can be a discrete concentration of charge density in one particular area of the particle moiety e.g., a charge spike. If the charge density is distributed on the particle, the charge density can be equally distributed or unequally distributed. All variations of charge distribution of the polycationic moiety are encompassed by the present invention.
  • the lipid "A” and the nonimmunogenic polymer “W” can be attached by various methods and preferably by covalent attachment. Methods known to those of skill in the art can be used for the covalent attachment of "A” and “W.” Suitable linkages include, but are not limited to, amide, amine, carboxyl, carbonate, carbamate, ester, and hydrazone linkages. It will be apparent to those skilled in the art that "A” and “W” must have complementary functional groups to effectuate the linkage. The reaction of these two groups, one on the lipid and the other on the polymer, will provide the desired linkage.
  • the lipid is a diacylglycerol and the terminal hydroxyl is activated, for instance with NHS and DCC, to form an active ester, and is then reacted with a polymer which contains an amino group, such as with a polyamide (see, e.g., U.S. Patent Nos. 6,320,017 and 6,586,559, the disclosures of which are herein incorporated by reference in their entirety for all purposes), an amide bond will form between the two groups.
  • a polyamide see, e.g., U.S. Patent Nos. 6,320,017 and 6,586,559, the disclosures of which are herein incorporated by reference in their entirety for all purposes
  • the polycationic moiety can have a ligand attached, such as a targeting ligand or a chelating moiety for complexing calcium.
  • a ligand attached such as a targeting ligand or a chelating moiety for complexing calcium.
  • the cationic moiety maintains a positive charge.
  • the ligand that is attached has a positive charge.
  • Suitable ligands include, but are not limited to, a compound or device with a reactive functional group and include lipids, amphipathic lipids, carrier
  • bioaffinity compounds biomaterials, biopolymers, biomedical devices, analytically detectable compounds, therapeutically active compounds, enzymes, peptides, proteins, antibodies, immune stimulators, radiolabels, fluorogens, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes, micelles, immunoglobulins, functional groups, other targeting moieties, or toxins.
  • the lipid conjugate (e.g., PEG-lipid) comprises from about 0.1 mol % to about 3 mol %, from about 0.5 mol % to about 3 mol %, or about 0.6 mol %, 0.7 mol %, 0.8 mol %, 0.9 mol %, 1.0 mol %, 1.1 mol %, 1.2 mol %, 1.3 mol %, 1.4 mol %, 1.5 mol %, 1.6 mol %, 1.7 mol %, 1.8 mol %, 1.9 mol %, 2.0 mol %, 2.1 mol%, 2.2 mol%, 2.3 mol %, 2.4 mol %, 2.5 mol %, 2.6 mol %, 2.7 mol %, 2.8 mol %, 2.9 mol % or 3 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the lipid conjugate (e.g., PEG-lipid) comprises from about 0 mol % to about 20 mol %, from about 0.5 mol % to about 20 mol %, from about 2 mol % to about 20 mol %, from about 1.5 mol % to about 18 mol %, from about 2 mol % to about 15 mol %, from about 4 mol % to about 15 mol %, from about 2 mol % to about 12 mol %, from about 5 mol % to about 12 mol %, or about 2 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • PEG-lipid comprises from about 0 mol % to about 20 mol %, from about 0.5 mol % to about 20 mol %, from about 2 mol % to about 20 mol %, from about 1.5 mol % to about 18 mol %, from about 2 mol % to about 15 mol %, from about 4
  • the lipid conjugate (e.g., PEG-lipid) comprises from about 4 mol % to about 10 mol %, from about 5 mol % to about 10 mol %, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • PEG-lipid comprises from about 4 mol % to about 10 mol %, from about 5 mol % to about 10 mol %, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6 mol %
  • the percentage of lipid conjugate present in the lipid particles of the invention is a target amount, and that the actual amount of lipid conjugate present in the formulation may vary, for example, by ⁇ 5 mol %, ⁇ 4 mol %, ⁇ 3 mol %, ⁇ 2 mol %, ⁇ 1 mol %, ⁇ 0.75 mol %, ⁇ 0.5 mol %, ⁇ 0.25 mol %, or ⁇ 0.1 mol %.
  • concentration of the lipid conjugate can be varied depending on the lipid conjugate employed and the rate at which the lipid particle is to become fusogenic.
  • the rate at which the lipid conjugate exchanges out of the lipid particle and, in turn, the rate at which the lipid particle becomes fusogenic can be varied, for example, by varying the concentration of the lipid conjugate, by varying the molecular weight of the PEG, or by varying the chain length and degree of saturation of the alkyl groups on the PEG-DAA conjugate.
  • other variables including, for example, pH, temperature, ionic strength, etc. can be used to vary and/or control the rate at which the lipid particle becomes fusogenic. Other methods which can be used to control the rate at which the lipid particle becomes fusogenic will become apparent to those of skill in the art upon reading this disclosure.
  • the composition and concentration of the lipid conjugate one can control the lipid particle size.
  • Non-limiting examples of additional lipid-based carrier systems suitable for use in the present invention include lipoplexes (see, e.g., U.S. Patent Publication No. 20030203865; and Zhang et al., J. Control Release, 100: 165-180 (2004)), pH-sensitive lipoplexes (see, e.g., U.S. Patent Publication No. 20020192275), reversibly masked lipoplexes (see, e.g., U.S. Patent Publication Nos. 20030180950), cationic lipid-based compositions (see, e.g., U.S. Patent No. 6,756,054; and U.S. Patent Publication No. 20050234232), cationic liposomes (see, e.g., U.S. Patent Publication Nos. 20030229040, 20020160038, and 20020012998; U.S. Patent No.
  • anionic liposomes see, e.g., U.S. Patent Publication No. 20030026831
  • pH-sensitive liposomes see, e.g., U.S. Patent Publication No. 20020192274; and AU 2003210303
  • antibody-coated liposomes see, e.g., U.S. Patent
  • a nucleic acid e.g., a siRNA molecule, such as an siRNA molecule described in Tables A and B
  • a cationic polymer having a linear, branched, star, or dendritic polymeric structure that condenses the nucleic acid into positively charged particles capable of interacting with anionic proteoglycans at the cell surface and entering cells by endocytosis.
  • the polyplex comprises nucleic acid (e.g., a siRNA molecule, such as an siRNA molecule described in Tables A and B) complexed with a cationic polymer such as polyethylenimine (PEI) (see, e.g., U.S. Patent No. 6,013,240; commercially available from Qbiogene, Inc.
  • nucleic acid e.g., a siRNA molecule, such as an siRNA molecule described in Tables A and B
  • PEI polyethylenimine
  • porphyrin see, e.g., U.S. Patent No. 6,620,805
  • polyvinylether see, e.g., U.S. Patent Publication No. 20040156909
  • polycyclic amidinium see, e.g., U.S. Patent Publication No. 20030220289
  • other polymers comprising primary amine, imine, guanidine, and/or imidazole groups ⁇ see, e.g., U.S. Patent No. 6,013,240; PCT Publication No. WO/9602655; PCT Publication No. W095/21931; Zhang et al, J.
  • the polyplex comprises cationic polymer-nucleic acid complexes as described in U.S. Patent Publication Nos. 20060211643, 20050222064,
  • the siRNA may be complexed with cyclodextrin or a polymer thereof.
  • cyclodextrin-based carrier systems include the cyclodextrin- modified polymer-nucleic acid complexes described in U.S. Patent Publication No.
  • siRNA may be complexed with a peptide or polypeptide.
  • a protein-based carrier system includes, but is not limited to, the cationic oligopeptide-nucleic acid complex described in PCT Publication No. W095/21931.
  • nucleic acid-lipid particles of the present invention in which a nucleic acid ⁇ e.g., a siRNA as described in Tables A and/or B) is entrapped within the lipid portion of the particle and is protected from degradation, can be formed by any method known in the art including, but not limited to, a continuous mixing method, a direct dilution process, and an in-line dilution process.
  • the cationic lipids may comprise lipids of Formula I-III or salts thereof, alone or in combination with other cationic lipids.
  • the non- cationic lipids are egg sphingomyelin (ESM), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), l-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), dipalmitoyl-phosphatidylcholine (DPPC), monomethyl-phosphatidylethanolamine, dimethyl- phosphatidylethanolamine, 14:0 PE (1 ,2-dimyristoyl-phosphatidylethanolamine (DMPE)), 16:0 PE (1,2-dipalmitoyl-phosphatidylethanolamine (DPPE)), 18:0 PE (1 ,2-distearoyl- phosphatidylethanolamine (DSPE)), 18: 1 PE (1
  • the present invention provides nucleic acid-lipid particles produced via a continuous mixing method, e.g. , a process that includes providing an aqueous solution comprising a siRNA in a first reservoir, providing an organic lipid solution in a second reservoir (wherein the lipids present in the organic lipid solution are solubilized in an organic solvent, e.g., a lower alkanol such as ethanol), and mixing the aqueous solution with the organic lipid solution such that the organic lipid solution mixes with the aqueous solution so as to substantially instantaneously produce a lipid vesicle (e.g., liposome) encapsulating the siRNA within the lipid vesicle.
  • a lipid vesicle e.g., liposome
  • the action of continuously introducing lipid and buffer solutions into a mixing environment, such as in a mixing chamber, causes a continuous dilution of the lipid solution with the buffer solution, thereby producing a lipid vesicle substantially instantaneously upon mixing.
  • the phrase "continuously diluting a lipid solution with a buffer solution” generally means that the lipid solution is diluted sufficiently rapidly in a hydration process with sufficient force to effectuate vesicle generation.
  • the organic lipid solution undergoes a continuous stepwise dilution in the presence of the buffer solution (i. e. , aqueous solution) to produce a nucleic acid-lipid particle.
  • the nucleic acid-lipid particles formed using the continuous mixing method typically have a size of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, less than about 120 nm, 1 10 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90
  • the present invention provides nucleic acid-lipid particles produced via a direct dilution process that includes forming a lipid vesicle (e.g., liposome) solution and immediately and directly introducing the lipid vesicle solution into a collection vessel containing a controlled amount of dilution buffer.
  • the collection vessel includes one or more elements configured to stir the contents of the collection vessel to facilitate dilution.
  • the amount of dilution buffer present in the collection vessel is substantially equal to the volume of lipid vesicle solution introduced thereto.
  • a lipid vesicle solution in 45% ethanol when introduced into the collection vessel containing an equal volume of dilution buffer will advantageously yield smaller particles.
  • the present invention provides nucleic acid-lipid particles produced via an in-line dilution process in which a third reservoir containing dilution buffer is fluidly coupled to a second mixing region.
  • the lipid vesicle (e.g., liposome) solution formed in a first mixing region is immediately and directly mixed with dilution buffer in the second mixing region.
  • the second mixing region includes a T- connector arranged so that the lipid vesicle solution and the dilution buffer flows meet as opposing 180° flows; however, connectors providing shallower angles can be used, e.g., from about 27° to about 180° (e.g., about 90°).
  • a pump mechanism delivers a controllable flow of buffer to the second mixing region.
  • the flow rate of dilution buffer provided to the second mixing region is controlled to be substantially equal to the flow rate of lipid vesicle solution introduced thereto from the first mixing region.
  • This embodiment advantageously allows for more control of the flow of dilution buffer mixing with the lipid vesicle solution in the second mixing region, and therefore also the concentration of lipid vesicle solution in buffer throughout the second mixing process.
  • the nucleic acid-lipid particles formed using the direct dilution and in-line dilution processes typically have a size of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 1 10 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, less than about 120 nm, 1 10 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm,
  • the lipid particles of the invention can be sized by any of the methods available for sizing liposomes.
  • the sizing may be conducted in order to achieve a desired size range and relatively narrow distribution of particle sizes.
  • Extrusion of the particles through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing particle sizes to a relatively well-defined size distribution.
  • the suspension is cycled through the membrane one or more times until the desired particle size distribution is achieved.
  • the particles may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in size.
  • the nucleic acids present in the particles are precondensed as described in, e.g., U.S. Patent Application No. 09/744,103, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • the methods may further comprise adding non-lipid polycations which are useful to effect the lipofection of cells using the present compositions.
  • suitable non-lipid polycations include, hexadimethrine bromide (sold under the brand name POLYBRENE®, from Aldrich Chemical Co., Milwaukee, Wisconsin, USA) or other salts of hexadimethrine.
  • suitable polycations include, for example, salts of poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine, polyallylamine, and polyethyleneimine. Addition of these salts is preferably after the particles have been formed.
  • the nucleic acid (e.g., siRNA) to lipid ratios (mass/mass ratios) in a formed nucleic acid-lipid particle will range from about 0.01 to about 0.2, from about 0.05 to about 0.2, from about 0.02 to about 0.1 , from about 0.03 to about 0.1 , or from about 0.01 to about 0.08.
  • the ratio of the starting materials (input) also falls within this range.
  • the particle preparation uses about 400 ⁇ g nucleic acid per 10 mg total lipid or a nucleic acid to lipid mass ratio of about 0.01 to about 0.08 and, more preferably, about 0.04, which corresponds to 1.25 mg of total lipid per 50 ⁇ g of nucleic acid.
  • the particle has a nucleic acid:lipid mass ratio of about 0.08.
  • the lipid to nucleic acid (e.g., siRNA) ratios (mass/mass ratios) in a formed nucleic acid-lipid particle will range from about 1 (1:1) to about 100 (100:1), from about 5 (5:1) to about 100 (100:1), from about 1 (1:1) to about 50 (50:1), from about 2 (2:1) to about 50 (50:1), from about 3 (3:1) to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), from about 5 (5:1) to about 50 (50:1), from about 1 (1:1) to about 25 (25:1), from about 2 (2:1) to about 25 (25 : 1 ), from about 3 (3 : 1 ) to about 25 (25 : 1 ), from about 4 (4: 1 ) to about 25 (25 : 1 ), from about 5 (5:1) to about 25 (25:1), from about 5 (5:1) to about 20 (20:1), from about 5 (5:1) to about 15(15:1), from about 5
  • the conjugated lipid may further include a CPL.
  • CPL-containing lipid particles A variety of general methods for making lipid particle-CPLs (CPL-containing lipid particles) are discussed herein. Two general techniques include the "post-insertion” technique, that is, insertion of a CPL into, for example, a pre-formed lipid particle, and the "standard” technique, wherein the CPL is included in the lipid mixture during, for example, the lipid particle formation steps.
  • the post-insertion technique results in lipid particles having CPLs mainly in the external face of the lipid particle bilayer membrane, whereas standard techniques provide lipid particles having CPLs on both internal and external faces.
  • the method is especially useful for vesicles made from phospholipids (which can contain cholesterol) and also for vesicles containing PEG-lipids (such as PEG-DAAs and PEG-DAGs).
  • PEG-lipids such as PEG-DAAs and PEG-DAGs.
  • Methods of making lipid particle-CPLs are taught, for example, in U.S. Patent Nos.5,705,385; 6,586,410; 5,981,501; 6,534,484; and 6,852,334; U.S. Patent Publication No.20020072121; and PCT Publication No. WO 00/62813, the disclosures of which are herein incorporated by reference in their entirety for all purposes.
  • the kit comprises a container which is compartmentalized for holding the various elements of the lipid particles (e.g., the active agents, such as siRNA molecules and the individual lipid components of the particles).
  • the kit comprises a container (e.g., a vial or ampoule) which holds the lipid particles of the invention, wherein the particles are produced by one of the processes set forth herein.
  • the kit may further comprise an endosomal membrane destabilizer (e.g. , calcium ions).
  • the kit typically contains the particle compositions of the invention, either as a suspension in a pharmaceutically acceptable carrier or in dehydrated form, with instructions for their rehydration (if lyophilized) and administration.
  • the formulations of the present invention can be tailored to preferentially target particular cells, tissues, or organs of interest. Preferential targeting of a nucleic acid-lipid particle may be carried out by controlling the composition of the lipid particle itself.
  • the kits of the invention comprise these lipid particles, wherein the particles are present in a container as a suspension or in dehydrated form.
  • a targeting moiety attached to the surface of the lipid particle to further enhance the targeting of the particle.
  • Methods of attaching targeting moieties e.g. , antibodies, proteins, etc.
  • lipids such as those used in the present particles
  • the lipid particles of the invention are particularly useful for the introduction of a siRNA molecule (e.g., a siRNA molecule as described in Tables A or B) into cells.
  • a siRNA molecule e.g., a siRNA molecule as described in Tables A or B
  • the present invention also provides methods for introducing a siRNA molecule into a cell.
  • the siRNA molecule is introduced into an infected cell. The methods may be carried out in vitro or in vivo by first forming the particles as described above and then contacting the particles with the cells for a period of time sufficient for delivery of siRNA to the cells to occur.
  • the lipid particles of the invention can be adsorbed to almost any cell type with which they are mixed or contacted. Once adsorbed, the particles can either be endocytosed by a portion of the cells, exchange lipids with cell membranes, or fuse with the cells. Transfer or incorporation of the siRNA portion of the particle can take place via any one of these pathways. In particular, when fusion takes place, the particle membrane is integrated into the cell membrane and the contents of the particle combine with the intracellular fluid.
  • the lipid particles of the invention can be any suitable lipid particles of the invention.
  • nucleic acid-lipid particles can be any suitable lipid particles of the invention.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions,
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • the pharmaceutically acceptable carrier is generally added following lipid particle formation.
  • the particle can be diluted into
  • pharmaceutically acceptable carriers such as normal buffered saline.
  • the concentration of particles in the pharmaceutical formulations can vary widely, i.e. , from less than about 0.05%, usually at or at least about 2 to 5%, to as much as about 10 to 90% by weight, and will be selected primarily by fluid volumes, viscosities, etc. , in accordance with the particular mode of administration selected.
  • the concentration may be increased to lower the fluid load associated with treatment. This may be particularly desirable in patients having atherosclerosis-associated congestive heart failure or severe hypertension.
  • particles composed of irritating lipids may be diluted to low concentrations to lessen
  • compositions of the present invention may be sterilized by conventional, well-known sterilization techniques.
  • Aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • the compositions can contain
  • the particle suspension may include lipid-protective agents which protect lipids against free-radical and lipid-peroxidative damages on storage.
  • Lipophilic free-radical quenchers such as alphatocopherol, and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.
  • the lipid particles of the invention are particularly useful in methods for the therapeutic delivery of one or more siRNA molecules (e.g. , an siRNA molecule as described in Tables A and B).
  • siRNA molecules e.g. , an siRNA molecule as described in Tables A and B.
  • Systemic delivery for in vivo therapy e.g. , delivery of a siRNA molecule described herein, such as an siRNA described in Tables A or B, to a distal target cell via body systems such as the circulation, has been achieved using nucleic acid-lipid particles such as those described in PCT Publication Nos. WO 05/007196, WO 05/121348, WO 05/120152, and WO 04/002453, the disclosures of which are herein incorporated by reference in their entirety for all purposes.
  • the present invention also provides fully encapsulated lipid particles that protect the siRNA from nuclease degradation in serum, are non-immunogenic, are small in size, and are suitable for repeat dosing.
  • the one or more siRNA molecules may be administered alone in the lipid particles of the invention, or in combination ⁇ e.g., co-administered) with lipid particles comprising peptides, polypeptides, or small molecules such as conventional drugs.
  • administration can be in any manner known in the art, e.g., by injection, oral administration, inhalation ⁇ e.g., intransal or intratracheal), transdermal application, or rectal administration.
  • Administration can be accomplished via single or divided doses.
  • the pharmaceutical compositions can be administered parenterally, i.e., intraarticular ⁇ , intravenously, intraperitoneally, subcutaneously, or intramuscularly.
  • the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection ⁇ see, e.g., U.S. Patent No. 5,286,634).
  • Intracellular nucleic acid delivery has also been discussed in Straubringer et al, Methods Enzymol , 101 :512 (1983); Mannino et al,
  • the lipid particles can be administered by direct injection at the site of disease or by injection at a site distal from the site of disease ⁇ see, e.g., Culver, HUMAN GENE THERAPY, Mary A n Liebert, Inc., Publishers, New York. pp.70-71(1994)).
  • Culver HUMAN GENE THERAPY
  • Mary A n Liebert, Inc. Publishers, New York. pp.70-71(1994)
  • the disclosures of the above-described references are herein incorporated by reference in their entirety for all purposes.
  • the lipid particles of the present invention are administered intravenously, at least about 5%, 10%, 15%, 20%, or 25% of the total injected dose of the particles is present in plasma about 8, 12, 24, 36, or 48 hours after injection. In other embodiments, more than about 20%, 30%, 40% and as much as about 60%, 70% or 80%) of the total injected dose of the lipid particles is present in plasma about 8, 12, 24, 36, or 48 hours after injection. In certain instances, more than about 10% of a plurality of the particles is present in the plasma of a mammal about 1 hour after administration. In certain other instances, the presence of the lipid particles is detectable at least about 1 hour after administration of the particle.
  • the presence of a siRNA molecule is detectable in cells at about 8, 12, 24, 36, 48, 60, 72 or 96 hours after administration.
  • downregulation of expression of a target sequence, such as a viral or host sequence, by a siRNA molecule is detectable at about 8, 12, 24, 36, 48, 60, 72 or 96 hours after administration.
  • downregulation of expression of a target sequence, such as a viral or host sequence, by a siRNA molecule occurs preferentially in infected cells and/or cells capable of being infected.
  • the presence or effect of a siRNA molecule in cells at a site proximal or distal to the site of administration is detectable at about 12, 24, 48, 72, or 96 hours, or at about 6, 8, 10, 12, 14, 16, 18, 19, 20, 22, 24, 26, or 28 days after administration.
  • the lipid particles of the invention are administered parenterally or intraperitoneally.
  • compositions of the present invention can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation (e.g., intranasally or intratracheally) (see, Brigham et al., Am. J. Sci., 298:278 (1989)). Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • the pharmaceutical compositions may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles.
  • Methods for delivering nucleic acid compositions directly to the lungs via nasal aerosol sprays have been described, e.g., in U.S. Patent Nos. 5,756,353 and 5,804,212.
  • delivery of drugs using intranasal microparticle resins and lysophosphatidyl-glycerol compounds U.S. Patent
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • compositions are preferably administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally.
  • the lipid particle formulations are formulated with a suitable pharmaceutical carrier.
  • a suitable pharmaceutical carrier may be employed in the compositions and methods of the present invention. Suitable formulations for use in the present invention are found, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985).
  • a variety of aqueous carriers may be used, for example, water, buffered water, 0.4% saline, 0.3% glycine, and the like, and may include glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc.
  • compositions can be sterilized by conventional liposomal sterilization techniques, such as filtration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • These compositions can be sterilized using the techniques referred to above or, alternatively, they can be produced under sterile conditions.
  • the resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • the lipid particles disclosed herein may be delivered via oral administration to the individual.
  • the particles may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, pills, lozenges, elixirs, mouthwash, suspensions, oral sprays, syrups, wafers, and the like ⁇ see, e.g., U.S. Patent Nos. 5,641,515, 5,580,579, and 5,792,451 , the disclosures of which are herein incorporated by reference in their entirety for all purposes).
  • These oral dosage forms may also contain the following: binders, gelatin; excipients, lubricants, and/or flavoring agents.
  • the unit dosage form When the unit dosage form is a capsule, it may contain, in addition to the materials described above, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. Of course, any material used in preparing any unit dosage form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • these oral formulations may contain at least about 0.1% of the lipid particles or more, although the percentage of the particles may, of course, be varied and may
  • each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound.
  • Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • Formulations suitable for oral administration can consist of: (a) liquid solutions, such as an effective amount of a packaged siR A molecule (e.g. , a siRNA molecule described in Tables A and B) suspended in diluents such as water, saline, or PEG 400; (b) capsules, sachets, or tablets, each containing a predetermined amount of a siRNA molecule, as liquids, solids, granules, or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
  • a packaged siR A molecule e.g. , a siRNA molecule described in Tables A and B
  • diluents such as water, saline, or PEG 400
  • capsules, sachets, or tablets each containing a predetermined amount of a siRNA molecule, as liquids, solids, granules, or gelatin
  • suspensions in an appropriate liquid and (d) suitable emul
  • Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers.
  • Lozenge forms can comprise a siRNA molecule in a flavor, e.g., sucrose, as well as pastilles comprising the therapeutic nucleic acid in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the siRNA molecule, carriers known in the art.
  • a flavor e.g., sucrose
  • pastilles comprising the therapeutic nucleic acid in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the siRNA molecule, carriers known in the art.
  • lipid particles can be incorporated into a broad range of topical dosage forms.
  • a suspension containing nucleic acid-lipid particles can be formulated and administered as gels, oils, emulsions, topical creams, pastes, ointments, lotions, foams, mousses, and the like.
  • lipid particles of the invention When preparing pharmaceutical preparations of the lipid particles of the invention, it is preferable to use quantities of the particles which have been purified to reduce or eliminate empty particles or particles with therapeutic agents such as siRNA associated with the external surface.
  • hosts include mammalian species, such as primates (e.g. , humans and chimpanzees as well as other nonhuman primates), canines, felines, equines, bovines, ovines, caprines, rodents (e.g., rats and mice), lagomorphs, and swine.
  • primates e.g. , humans and chimpanzees as well as other nonhuman primates
  • canines felines, equines, bovines, ovines, caprines
  • rodents e.g., rats and mice
  • lagomorphs e.g., swine.
  • the amount of particles administered will depend upon the ratio of siRNA molecules to lipid, the particular siRNA used, the strain of EBOV being treated, the age, weight, and condition of the patient, and the judgment of the clinician, but will generally be between about 0.01 and about 50 mg per kilogram of body weight, preferably between about 0.1 and about 5 mg/kg of body weight, or about 10 8 -10 10 particles per administration (e.g., injection).
  • siRNA molecules to lipid
  • the amount of particles administered will depend upon the ratio of siRNA molecules to lipid, the particular siRNA used, the strain of EBOV being treated, the age, weight, and condition of the patient, and the judgment of the clinician, but will generally be between about 0.01 and about 50 mg per kilogram of body weight, preferably between about 0.1 and about 5 mg/kg of body weight, or about 10 8 -10 10 particles per administration (e.g., injection).
  • the delivery of siRNA molecules can be to any cell grown in culture.
  • the cells are animal cells, more preferably mammalian cells, and most preferably human cells.
  • the concentration of particles varies widely depending on the particular application, but is generally between about 1 ⁇ and about 10 mmol.
  • Treatment of the cells with the lipid particles is generally carried out at physiological temperatures (about 37°C) for periods of time of from about 1 to 48 hours, preferably of from about 2 to 4 hours.
  • a lipid particle suspension is added to 60-80% confluent plated cells having a cell density of from about 10 3 to about 10 5 cells/ml, more preferably about 2 x 10 4 cells/ml.
  • the concentration of the suspension added to the cells is preferably of from about 0.01 to 0.2 ⁇ g/ml, more preferably about 0.1 ⁇ g/ml.
  • tissue culture of cells may be required, it is well-known in the art.
  • Freshney Culture of Animal Cells, a Manual of Basic Technique, 3rd Ed., Wiley- Liss, New York (1994), Kuchler et al, Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc. (1977), and the references cited therein provide a general guide to the culture of cells.
  • Cultured cell systems often will be in the form of monolayers of cells, although cell suspensions are also used.
  • ERP Endosomal Release Parameter
  • an ERP assay is to distinguish the effect of various cationic lipids and helper lipid components of the lipid particle based on their relative effect on binding/uptake or fusion with/destabilization of the endosomal membrane. This assay allows one to determine quantitatively how each component of the lipid particle affects delivery efficiency, thereby optimizing the lipid particle.
  • an ERP assay measures expression of a reporter protein (e.g., luciferase, ⁇ - galactosidase, green fluorescent protein (GFP), etc.), and in some instances, a lipid particle formulation optimized for an expression plasmid will also be appropriate for encapsulating a siRNA.
  • a reporter protein e.g., luciferase, ⁇ - galactosidase, green fluorescent protein (GFP), etc.
  • an ERP assay can be adapted to measure downregulation of transcription or translation of a target sequence in the presence or absence of a siRNA. By comparing the ERPs for each of the various lipid particles, one can readily determine the optimized system, e.g., the lipid particle that has the greatest uptake in the cell. Detection of Lipid Particles
  • the lipid particles of the present invention are detectable in the subject at about 1, 2, 3, 4, 5, 6, 7, 8 or more hours. In other embodiments, the lipid particles of the present invention are detectable in the subject at about 8, 12, 24, 48, 60, 72, or 96 hours, or about 6, 8, 10, 12, 14, 16, 18, 19, 22, 24, 25, or 28 days after administration of the particles.
  • the presence of the particles can be detected in the cells, tissues, or other biological samples from the subject.
  • the particles may be detected, e.g. , by direct detection of the particles, detection of a siRNA sequence, detection of the target sequence of interest (i. e. , by detecting expression or reduced expression of the sequence of interest), detection of a compound modulated by an EBOV protein (e.g., interferon), detection of viral load in the subject, or a combination thereof.
  • Lipid particles of the invention can be detected using any method known in the art.
  • a label can be coupled directly or indirectly to a component of the lipid particle using methods well-known in the art.
  • a wide variety of labels can be used, with the choice of label depending on sensitivity required, ease of conjugation with the lipid particle component, stability requirements, and available instrumentation and disposal provisions.
  • Suitable labels include, but are not limited to, spectral labels such as fluorescent dyes (e.g., fluorescein and derivatives, such as fluorescein isothiocyanate (FITC) and Oregon GreenTM; rhodamine and derivatives such Texas red, tetrarhodimine isothiocynate (TRITC), etc., digoxigenin, biotin, phycoerythrin, AMCA, CyDyesTM, and the like; radiolabels such as 3 H, 125 1, 35 S, 14 C, 32 P, 33 P, etc.; enzymes such as horse radish peroxidase, alkaline phosphatase, etc.; spectral colorimetric labels such as colloidal gold or colored glass or plastic beads such as polystyrene,
  • fluorescent dyes e.g., fluorescein and derivatives, such as fluorescein isothiocyanate (FITC) and Oregon GreenTM
  • rhodamine and derivatives such Texas
  • the label can be detected using any means known in the art.
  • Nucleic acids e.g., siRNA molecules
  • the detection of nucleic acids may proceed by well-known methods such as Southern analysis. Northern analysis, gel
  • electrophoresis capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography may also be employed.
  • HPLC high performance liquid chromatography
  • TLC thin layer chromatography
  • hyperdiffusion chromatography may also be employed.
  • nucleic acid hybridization format is not critical.
  • a variety of nucleic acid hybridization formats are known to those skilled in the art.
  • common formats include sandwich assays and competition or displacement assays.
  • Hybridization techniques are generally described in, e.g., "Nucleic Acid Hybridization, A Practical Approach,” Eds. Hames and Higgins, IRL Press (1985).
  • the sensitivity of the hybridization assays may be enhanced through the use of a nucleic acid amplification system which multiplies the target nucleic acid being detected.
  • a nucleic acid amplification system which multiplies the target nucleic acid being detected.
  • In vitro amplification techniques suitable for amplifying sequences for use as molecular probes or for generating nucleic acid fragments for subsequent subcloning are known. Examples of techniques sufficient to direct persons of skill through such in vitro amplification methods, including the polymerase chain reaction (PCR), the ligase chain reaction (LCR), QP-replicase amplification, and other RNA polymerase mediated techniques (e.g. , NASBATM) are found in Sambrook et al.
  • the select sequences can be generally amplified using, for example, nonspecific PCR primers and the amplified target region later probed for a specific sequence indicative of a mutation.
  • Nucleic acids for use as probes e.g., in in vitro amplification methods, for use as gene probes, or as inhibitor components are typically synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage et al., Tetrahedron Letts., 22: 1859 1862 (1981), e.g. , using an automated synthesizer, as described in Needham
  • In situ hybridization assays are well-known and are generally described in Angerer et al. , Methods Enzymol., 152:649 (1987).
  • in situ hybridization assay cells are fixed to a solid support, typically a glass slide. If DNA is to be probed, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of specific probes that are labeled.
  • the probes are preferably labeled with radioisotopes or fluorescent reporters.
  • This Example describes the selection of the 2 Guinea/Sierra Leone (SL) variant of Ebola virus siRNA molecules set forth in Table A.
  • the 2 siRNAs were originally designed against a Mayinga variant, perfectly match the 1995 Kikwit variant, and were revised to address target site mutations in the current outbreak Ebola virus variant.
  • the full genome sequence identity generally remains very high (at least 97%).
  • All publically available viral sequences (3 Guinea, 99 Sierra Leone) from the current outbreak were collected from sources such as GenBank - the NIH genetic sequence database. Table A.
  • This Example describes the biological assay used to test the activity of the 2 siRNA sequences set forth in Table B.
  • the Ebola virus genome is a ⁇ 19-kb single-stranded RNA.
  • the L and VP35 proteins together comprise the polymerase complex that is responsible for transcribing and replicating the Ebola virus genome. These genes and their products represent targets for the development of therapeutic agents and vaccines.
  • An Ebola virus Guinea variant genomic sequence (accession number KJ660347) was applied to perfectly match the candidate siRNAs. Specifically, the following two sequence regions, containing the target sites for candidate siRNAs, were joined in silico: 3817 to 4016, and 17287 to 17488 to ensure that the candidate siRNAs were perfectly complementary to this synthetic consensus Ebola virus Guinea variant target fragment.
  • the synthetic consensus target fragment was synthesized with restriction enzyme sites Xhol and Notl added to the 5' and 3' end, respectively, to facilitate cloning into the psiCHECK-2 Dual Luciferase vector.
  • the Xhol/Notl cloning site is between the stop codon and polyadenylation signal of Renilla luciferase on the psiCHECK-2 Dual Luciferase vector.
  • siRNAs against Ebola virus Guinea variant VP35 and Lpol mRNA were confirmed in vitro using a dual luciferase reporter assay assessing reduction in the expression of the chimeric viral transgenes cloned into the psiCHECK2 vector (Promega) in both Cos-7 cells (ATCC No. CRL-1651) grown in DMEM (Gibco) supplemented media, and HepG2 cells (ATCC No. HB-8065) grown in MEM (Gibco) supplemented media.
  • Cos-7 cells were seeded at a density of 25,000 cells per well in 96-well plates, and reverse-transfected with Lipofectamine 2000 (Invitrogen) complexes containing 40 ng of reporter plasmid and siRNA (0.01 -18.1 nM) per well, with technical triplicates.
  • Lipofectamine 2000 Invitrogen
  • MARV NP were used as positive and non-specific targeting negative controls, respectively.
  • HepG2 cells were seeded at a density of 80,000 cells per well in 96-well plates, and reverse-transfected with Lipofectamine 2000 (Invitrogen) complexes containing 80 ng of reporter plasmid and nucleic acid-lipid particles (0.4-54.3 nM) per well, with technical triplicates.
  • the nucleic acid-lipid particles tested in HepG2 cells contain individual siRNA and/or combined siRNA. Nucleic acid-lipid particles containing siRNA targeting Renilla luciferase (Rluc) or a non-specific target (MARV NP) were used as positive and negative controls respectively.
  • Renilla luciferase (fused to the Ebola virus target transgene) and firefly luciferase signals were detected using the Dual Luciferase Reporter Assay kit according to manufacturer's protocol (Promega) using a Berthold luminometer (Berthold Detection Systems).
  • the Renilla luciferase signal (reflecting target transgene expression) was normalized to the Firefly luciferase signal and expressed as percentage gene expression relative to a plasmid-only control assigned a value of 100%.
  • EBOV titration was performed by conventional plaque assay on Vero E6 cells from plasma collected from nonhuman primates as described elsewhere (Jones et al., Nat Med; 1 1, 786-790 (2005)). Samples were collected from rhesus macaques at day 0, 3, 6, 10, 14, 22 and 28 or at euthanasia. In brief, increasing 10-fold dilutions of the samples were adsorbed to Vero E6 monolayers in duplicate wells (200 uL). The limit of detection was 5 pfu/mL.
  • R A was isolated from whole blood utilizing the Viral RNA mini-kit (Qiagen). Primers and a FAM-labelled probe targeting the VP30 gene of EBOV were used for qRT-PCR.
  • EBOV RNA was detected using One-step probe qRT-PCR kits with the following cycle conditions: 50°C for 10 minutes, 95°C for 10 seconds, and 40 cycles of 95°C for 10 seconds and 59°C for 30 seconds.
  • Threshold cycle (CT) values representing EBOV genomes were used to calculate genomic equivalents based on CT values obtained using a genomic equivalent standard obtained using RNA extracted from EBOV viral stocks.
  • CT Threshold cycle
  • siEbola-3 lipid particle to confer survival benefit in lethally challenged nonhuman primates, a model that more closely recapitulates human disease, was assessed.
  • Rhesus macaques were challenged with EBOV Makona, and treatment with siEbola-3 lipid particle was initiated at three days post-infection.
  • a delay in treatment initiation after viral infection in this nonhuman primate model experimentally mimics the clinical patient situation, where the timing of infection is generally unknown and patients at the point of treatment may be seen at various stages of the disease.
  • Animals that were untreated succumbed either 8-9 days after infection (untreated, 0% survival, 0/3) and displayed clinical signs of disease (Table 1).
  • Animals that were treated with siEbola-3 lipid particle also had ameliorated clinical disease signs as assessed by lower clinical scores in a scoring system that measured changes in activity, behavior, food and water intake, weight, respiration, and disease manifestations such as visible rash, hemorrhage, ecchymosis, or flushed skin (Table 4).
  • siRNA nucleic acid-lipid particles
  • EBOV Makona West African Ebola virus variant
  • the data demonstrate that siEbola-3 siRNA in lipid particles provided 100% protection against a high infectious dose of EBOV Makona in the rhesus macaque nonhuman primate model, which represents a potential patient worst case scenario of high viral loads and shortened infection time course with a fatal outcome.
  • Treatment with siEbola-3 lipid particle was able to confer survival benefit and significantly reduce viral load in infected animals (Thi et al., Nature, 521, 362-365 (2015); which publication is specifically incorporated by reference).

Abstract

L'invention se rapporte à des compositions qui contiennent des acides nucléiques thérapeutiques, tels qu'un ARN interférent court qui ciblent l'expression du gène du virus Ebola, des particules lipidiques comprenant un ou plusieurs acides nucléiques thérapeutiques (par ex., une combinaison), et des méthodes de livraison et/ou d'administration des particules lipidiques (par ex., pour traiter l'infection par le virus Ebola chez l'homme).
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