USOO8236943B2
`
`(12) United States Patent
`Lee et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 8.236,943 B2
`Aug. 7, 2012
`
`(54) COMPOSITIONS AND METHODS FOR
`SLENCINGAPOLIPOPROTEIN B
`
`2011/0076335 A1
`2011/O262527 A1
`2012/0058188 A1
`
`3/2011 Yaworski et al.
`10/2011 Heyes et al.
`3/2012 Maclachlan et al.
`
`(75) Inventors: Amy C. H. Lee, Burnaby (CA); Adam
`Judge, Vancouver (CA); Marjorie
`Robbins, Vancouver (CA); Ed
`Yaworski, Maple Ridge (CA); Ian
`MacLachlan, Mission (CA)
`(73) Assignee: Protiva Biotherapeutics, Inc., Burnaby
`(CA)
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(*) Notice:
`
`(21) Appl. No.: 12/828,202
`
`(22) Filed:
`
`Jun. 30, 2010
`
`(65)
`
`Prior Publication Data
`US 2011 FO1951.27 A1
`Aug. 11, 2011
`
`Related U.S. Application Data
`(60) Provisional application No. 61/351.275, filed on Jun.
`3, 2010, provisional application No. 61/222,464, filed
`on Jul. 1, 2009.
`
`(51) Int. Cl.
`(2006.01)
`C7H 2L/02
`(2006.01)
`CI2N IS/II
`(2006.01)
`CI2N 15/02
`(2006.01)
`CI2N IS/00
`(52) U.S. Cl. ......... 536/24.5; 435/450; 435/455; 514/44;
`977/800; 977/816
`(58) Field of Classification Search ........................ None
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
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`6,417,326 B1
`7/2002 Cullis et al.
`6,680,068 B2
`1/2004 Jain et al.
`7,745,651 B2
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`7,799,565 B2
`9/2010 MacLachlan et al.
`7,807,815 B2 10/2010 MacLachlan et al.
`7,838,658 B2 11/2010 MacLachlan et al.
`7,982,027 B2
`7/2011 Maclachlan et al.
`8,058,069 B2 11/2011 Yaworski et al.
`8, 101,741 B2
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`5/2005 Manoharan et al.
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`12/2005 Haeberli et al.
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`5, 2006 Soutschek et al.
`2006/01341.89 A1
`6/2006 MacLachlan et al.
`6/2006 Quay et al.
`2006, O142230 A1
`9/2006 McSwiggen et al.
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`2006/0217330 A1
`9/2006 Hartmann et al.
`2007/0042983 A1
`2/2007 Haeberli et al.
`2007/O135372 A1*
`6/2007 MacLachlan et al. .......... 514,44
`2007/0218122 A1
`9, 2007 MacLachlan et al.
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`1/2009 Sood et al.
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`FOREIGN PATENT DOCUMENTS
`WOO1,053.74 A1
`1, 2001
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`5, 2002
`WO
`WO WO 2004/091515 A2 10/2004
`WO WO 2005/007 196 A2
`1, 2005
`WO WO 2009/086558 A1 * T 2009
`WO WO 2009.127060 A1 10/2009
`WO WO 2009,132131 A1 10/2009
`WO WO 2010/042877 A1
`4/2010
`WO WO 2010/054401 A1
`5, 2010
`OTHER PUBLICATIONS
`Arpicco, S., et al., “Synthesis, characterization and transfection activ
`ity of new Saturated and unsaturated cationic lipids.” IL Farmaco,
`2004, vol. 59, pp. 869-878.
`Heyes, J., et al., "Cationic lipid Saturation influences intracellular
`delivery of encapsulated nucleic acids,” Journal of Controlled
`Release, 2005, vol. 107, pp. 276-287.
`Jaeger et al. "Preparation and characterization of glycerol-based
`cleavable surfactants and derived vesicles,” Journal of the American
`Chemical Society, 1989, vol. 111, pp. 3001-3006.
`Kiefer et al., Transfection efficiency and cytotoxicity of nonviral gene
`transfer reagents in human Smooth muscle and endothelial cells, Jun.
`2004, Pharmaceutical Research, vol. 21, pp. 1009-1017.
`Leifer, C., et al., “Heterogeneity in the Human Response to
`Immunostimulatory CpG Oligodeoxynucleotides,” Journal of
`Immunotherapy, Jul. Aug. 2003, vol. 26, pp. 313-319.
`Lu et al. In Vivo application of RNA interference: From functional
`genomics to therapeutics, 2005, Advances in Genetics, vol. 54, pp.
`117-142.
`Madry et al., Efficient lipid-mediated gene transfer to articular
`chondrocytes, 2000, Gene Therapy, vol. 7, pp. 286-291.
`Mashek et al., Short Communication: Net uptake of nonesterified
`long chain fatty acids by the perfused caudate lobe of the caprine
`liver, 2003, Journal of Dairy Science, vol. 86, pp. 1218-1220.
`Morrissey, D. V., et al., “Potent and persistent in vivo anti-HBV
`activity of chemically modified siRNAs.” Nature Biotechnology,
`2005, vol. 23, No. 8, pp. 1002-1007.
`Prakash, T. P. et al., “Position effects of chemical modification on
`siRNA activity MEDI 175.” General Oral Session, Division of
`Medicinal Chemistry, The 227th ACS National Meeting, 2004. 1
`page.
`Reynolds et al., “Rational siRNA design for RNA interference.”
`Nature Biotechnology, 2004, vol. 22, pp. 326-330.
`(Continued)
`Primary Examiner — Richard Schnizer
`(74) Attorney, Agent, or Firm — Kilpatrick Townsend &
`Stockton LLP
`
`ABSTRACT
`(57)
`The present invention provides compositions and methods for
`the delivery of interfering RNAs that silence APOB expres
`sion to liver cells. In particular, the nucleic acid-lipid particles
`provide efficient encapsulation of nucleic acids and efficient
`delivery of the encapsulated nucleic acid to cells in vivo. The
`compositions of the present invention are highly potent,
`thereby allowing effective knock-down of APOB at relatively
`low doses. In addition, the compositions and methods of the
`present invention are less toxic and provide a greater thera
`peutic index compared to compositions and methods previ
`ously known in the art.
`26 Claims, 17 Drawing Sheets
`
`PROTIVA - EXHIBIT 2017
`Moderna Therapeutics, Inc. v. Protiva Biotherapeautics, Inc. - IPR2018-00739
`
`

`

`US 8.236,943 B2
`Page 2
`
`OTHER PUBLICATIONS
`Semple et al. “Rational design of cationic lipids for siRNA delivery.”
`Nature Biotechnology, 2010, vol. 28, pp. 172-176.
`Sioud, siRNA delivery in vivo, 2005, Methods in Molecular Biology,
`vol. 309, pp. 237-249.
`Soutschek, J., et al., “Therapeutic silencing of an endogenous gene by
`systemic administration of modified siRNAs.” Nature, 2004, vol.
`432, pp. 173-178.
`
`Vigh et al., Does the membrane's physical state control the expres
`sion of heat shock and other genes? 1998, Trends in Biochemical
`Sciences, vol. 23, pp. 369-374.
`Wang et al. "Preparation, properties and applications of vesicle
`forming cleavable Surfactants with a 1,3-dioxane ring.” Journal of
`Colloidal and Interface Science, 1995, vol. 173, pp. 49-59.
`* cited by examiner
`
`

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`US 8,236,943 B2
`
`1.
`COMPOSITIONS AND METHODS FOR
`SLENCINGAPOLIPOPROTEIN B
`
`CROSS-REFERENCES TO RELATED
`APPLICATIONS
`
`The present application claims priority to U.S. Provisional
`Application No. 61/222,464, filed Jul. 1, 2009, and U.S. Pro
`visional Application No. 61/351,275, filed Jun. 3, 2010, the
`disclosures of which are hereby incorporated by reference in
`their entirety for all purposes.
`
`BACKGROUND OF THE INVENTION
`
`Apollipoprotein B (also known as ApoB, apolipoprotein
`B-100: ApoB-100, apolipoprotein B-48: ApoB-48 and Ag(x)
`antigen), is a large glycoprotein that serves an indispensable
`role in the assembly and secretion of lipids and in the trans
`port and receptor-mediated uptake and delivery of distinct
`classes of lipoproteins. Apollipoprotein B was cloned (Law et
`al., PNAS USA 82:8340-8344 (1985)) and mapped to chro
`mosome 2p23-2p24 in 1986 (Deeb et al., PNAS USA 83,
`419-422 (1986)). ApoB has a variety of functions, from the
`absorption and processing of dietary lipids to the regulation of
`circulating lipoprotein levels (Davidson and Shelness, Annu.
`Rev. Nutri, 20:169-193 (2000)). Two forms of ApoB have
`been characterized: ApoB-100 and ApoB-48. ApoB-100 is
`the major protein component of LDL, contains the domain
`required for interaction of this lipoprotein species with the
`LDL receptor, and participates in the transport and delivery of
`endogenous plasma cholesterol (Davidson and Shelness,
`2000, Supra). ApoB-48 circulates in association with chylo
`microns and chylomicron remnants which are cleared by the
`LDL-receptor-related protein (Davidson and Shelness, 2000,
`supra). ApoB-48 plays a role in the delivery of dietary lipid
`from the small intestine to the liver.
`Susceptibility to atherosclerosis is highly correlated with
`the ambient concentration of apolipoprotein B-containing
`lipoproteins (Davidson and Shelness, 2000, supra). Elevated
`plasma levels of the ApoB-100-containing lipoprotein Lp(a)
`are associated with increased risk for atherosclerosis and its
`manifestations, which may include hypercholesterolemia
`(Seed et al., N. Engl.J. Med. 322:1494-1499 (1990), myocar
`dial infarction (Sandkamp et al., Clin. Chem. 36:20-23
`(1990), and thrombosis (Nowak-Gottl et al., Pediatrics,
`99:E11 (1997)).
`Apollipoprotein B knockout mice (bearing disruptions of
`both ApoB-100 and ApoB-48) have been generated which are
`protected from developing hypercholesterolemia when fed a
`high-fat diet (Farese et al., PNAS USA. 92:1774-1778 (1995)
`and Kim and Young, J. Lipid Res., 39:703-723 (1998)). The
`incidence of atherosclerosis has been investigated in mice
`expressing exclusively ApoB-100 or ApoB-48 and suscepti
`bility to atherosclerosis was found to be dependent on total
`cholesterol levels.
`In view of Such findings, significant efforts have been made
`to modulate serum cholesterol levels by modulating ApoB
`expression using therapeutic nucleic acids, e.g., antisense
`oligonucleotides, ribozymes, etc. (see, e.g., U.S. Pat. No.
`7,407,943, which is directed to modulation of ApoB using
`antisense oligonucleotides). More recent efforts have focused
`on the use of interfering RNA molecules, such as siRNA and
`miRNA, to modulate ApoB (see, Zimmermann et al., Nature,
`441: 111-114 (2006), U.S. Patent Publication Nos.
`2006O1341.89 and 20060105976, and PCT Publication No.
`WO 04/091515). Interfering RNA molecules can down-regu
`late intracellular levels of specific proteins, such as ApoB,
`
`2
`through a process termed RNA interference (RNAi). Follow
`ing introduction of interfering RNA into the cell cytoplasm,
`these double-stranded RNA constructs can bind to a protein
`termed RISC. The sense strand of the interfering RNA is
`displaced from the RISC complex, providing a template
`within RISC that can recognize and bind mRNA with a
`complementary sequence to that of the bound interfering
`RNA. Having bound the complementary mRNA, the RISC
`complex cleaves the mRNA and releases the cleaved strands.
`RNAi can provide down-regulation of specific proteins, such
`as ApoB, by targeting specific destruction of the correspond
`ing mRNA that encodes for protein synthesis.
`Despite the high therapeutic potential of RNAi, two prob
`lems currently faced by interfering RNA constructs are, first,
`their susceptibility to nuclease digestion in plasma and, sec
`ond, their limited ability to gain access to the intracellular
`compartment where they can bind RISC when administered
`systemically as free interfering RNA molecules. These
`double-stranded constructs can be stabilized by the incorpo
`ration of chemically modified nucleotide linkers within the
`molecule, e.g., phosphothioate groups. However, Such
`chemically modified linkers provide only limited protection
`from nuclease digestion and may decrease the activity of the
`COnStruct.
`In an attempt to improve efficacy, investigators have
`employed various lipid-based carrier systems to deliver
`chemically modified or unmodified therapeutic nucleic acids,
`including anionic (conventional) liposomes, pH sensitive
`liposomes, immunoliposomes, fusogenic liposomes, and cat
`ionic lipid/nucleic acid aggregates. In particular, one lipid
`based carrier System, i.e., the stable nucleic-acid lipid particle
`(SNALP) system, has been found to be particularly effective
`for delivering interfering RNA (see, U.S. Patent Publication
`No. 2005OO64595 and U.S. Patent Publication No.
`20060008910 (collectively referred to as “MacLachlan et
`al.)). MacLachlan et al. have demonstrated that interfering
`RNA, such as siRNA, can be effectively systemically admin
`istered using nucleic acid-lipid particles containing a cationic
`lipid, and that these nucleic acid-lipid particles provide
`improved down-regulation of target proteins in mammals
`including non-human primates (see, Zimmermann et al.,
`Nature, 441: 111-114 (2006)).
`Eveninspite of this progress, there remains a need in the art
`for improved SNALPs that are useful for delivering therapeu
`tic nucleic acids, such as siRNA and miRNA, to the liver of a
`mammal (e.g., a human), and that result in increased silencing
`of target genes of interest in the liver, such as ApoB. Prefer
`ably, these compositions would encapsulate nucleic acids
`with high-efficiency, have high drug:lipid ratios, protect the
`encapsulated nucleic acid from degradation and clearance in
`serum, be suitable for systemic delivery, and provide intrac
`ellular delivery of the encapsulated nucleic acid. In addition,
`these nucleic acid-lipid particles should be well-tolerated and
`provide an adequate therapeutic index. Such that patient treat
`ment at an effective dose of the nucleic acid is not associated
`with significant toxicity and/or risk to the patient. The present
`invention provides such compositions, methods of making
`the compositions, and methods of using the compositions to
`introduce nucleic acids, such as siRNA and miRNA, into the
`liver, including for the treatment of diseases, such as hyper
`cholesterolemia (e.g., atherosclerosis, angina pectoris or high
`blood pressure).
`
`BRIEF SUMMARY OF THE INVENTION
`
`The present invention is based, in part, on the discovery
`that the use of certain cationic (amino) lipids in nucleic acid
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`lipid particles provides advantages when the particles are
`used for the in vivo delivery of therapeutic nucleic acids, such
`as siRNA, into the liver of a mammal. In particular, it has been
`unexpectedly found that the nucleic acid-lipid particles of the
`present invention comprising at least one cationic lipid of
`Formula I-XIV and at least one interfering RNA as described
`herein demonstrate increased potency (i.e., increased silenc
`ing activity) and/or increased tolerability (e.g., a more favor
`able toxicity profile) when targeting a gene of interest in the
`liver such as APOB, APOC3, PCSK9, DGAT1, and/or
`DGAT2 when compared to other nucleic acid-lipid particle
`compositions previously described. In preferred embodi
`ments, the present invention provides nucleic acid-lipid par
`ticles (e.g., SNALP) comprising APOB siRNA 3/5 and the
`cationic lipid DLin-K-C2-DMA and methods of use thereof,
`which nucleic acid-lipid particles unexpectedly possess
`increased potency and increased tolerability when silencing
`APOB expression in vivo compared to other nucleic acid
`lipid particle compositions previously described.
`In particular embodiments, the present invention provides
`cationic lipids that enable the formulation of compositions for
`the in vitro and in vivo delivery of interfering RNA, such as
`siRNA, to the liver that result in increased silencing of the
`target gene of interest, such as APOB. It is shown herein that
`these improved lipid particle compositions are particularly
`effective in down-regulating (e.g., silencing) the protein lev
`els and/or mRNA levels of target genes in the liver, such as
`APOB. Furthermore, it is shown herein that the activity of
`these improved lipid particle compositions is dependent on
`30
`the presence of the cationic lipids of Formula I-XIV of the
`invention.
`In one aspect, the present invention provides a nucleic
`acid-lipid particle (e.g., SNALP) comprising:
`(a) an interfering RNA that silences Apollipoprotein B
`(APOB) expression and/or the expression of another liver
`target gene such as APOC3, PCSK9, DGAT1, and/or
`DGAT2:
`(b) a cationic lipid of Formula I having the following struc
`ture:
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`4
`In another aspect, the present invention provides a nucleic
`acid-lipid particle (e.g., SNALP) comprising:
`(a) an interfering RNA that silences Apolipoprotein B
`(APOB) expression and/or the expression of another liver
`target gene such as APOC3, PCSK9, DGAT1, and/or
`DGAT2:
`(b) a cationic lipid of Formula II having the following
`Structure:
`
`II
`
`R ,
`N
`
`R3
`
`R2
`RI
`
`N,
`
`iii.
`
`or salts thereof, wherein: R' and R are either the same or
`different and are independently optionally substituted C
`C alkyl, optionally Substituted C-C alkenyl, optionally
`Substituted C-C alkynyl, or optionally Substituted C
`C. acyl: R and R are either the same or different and are
`independently optionally Substituted C-C alkyl, optionally
`Substituted C-C alkenyl, or optionally Substituted C-C,
`alkynyl or R and R may join to form an optionally substi
`tuted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2
`heteroatoms chosen from nitrogen and oxygen; R is either
`absent or is hydrogen or C-C 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; Y and Z are either the same or
`different and are independently O, S, or NH; and
`(c) a non-cationic lipid.
`In some embodiments, cationic lipids falling within the
`scope of Formulas I and/or II that are useful in the nucleic
`acid-lipid particles of the present invention include, but are
`not limited to, the following: 2,2-dilinoleyl-4-(2-dimethy
`laminoethyl) 1,3-dioxolane (DLin-K-C2-DMA; “XTC2” or
`“C2K), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-1,3-di
`oxolane (DLin-K-C3-DMA; “C3K), 2,2-dilinoleyl-4-(4-
`dimethylaminobutyl)-1,3-dioxolane (DLin-K-C4-DMA;
`“C4K), 2,2-dilinoleyl-5-dimethylaminomethyl-1,3-diox
`ane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino
`1,3-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethy
`laminomethyl-1,3-dioxolane
`(DLin-K-DMA),
`2.2-
`dioleoyl-4-dimethylaminomethyl-1,3-dioxolane (DO-K-
`DMA),
`2,2-distearoyl-4-dimethylaminomethyl-1,3-
`dioxolane (DS-K-DMA), 2,2-dilinoleyl-4-N-morpholino-1,
`3-dioxolane
`(DLin-K-MA),
`2,2-Dillinoleyl-4-
`trimethylamino-1,3-dioxolane
`chloride
`(DLin-K-
`TMAC1), 2,2-dilinoleyl-4,5-bis(dimethylaminomethyl) 1,
`3-dioxolane
`(DLin-K-DMA),
`2,2-dilinoleyl-4-
`methylpiperzine-1,3-dioxolane
`(D-Lin-K-N-
`methylpiperzine), analogs thereof, salts thereof, and mixtures
`thereof.
`In yet another aspect, the present invention provides a
`nucleic acid-lipid particle (e.g., SNALP) comprising: (a) an
`interfering RNA that silences Apollipoprotein B (APOB)
`expression and/or the expression of another liver target gene
`such as APOC3, PCSK9, DGAT1, DGAT2, etc.); (b) a cat
`ionic lipid having the structure of Formula III-XIV; and (c) a
`non-cationic lipid. Examples of cationic lipids falling within
`the scope of Formula III-XIV that are useful in the nucleic
`acid-lipid particles of the present invention include, but are
`not limited to, the following: 1,2-di-y-linolenyloxy-N,N-dim
`ethylaminopropane (Y-DLenDMA), Y-DLen-C2K-DMA,
`
`Y.
`R R5
`(),
`V /
`N-(CH), r1
`R3
`Q
`
`)
`p R2
`R1
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`or salts thereof, wherein: R' and R are either the same or
`50
`different and are independently optionally substituted C
`Calkyl, optionally Substituted C-C alkenyl, optionally
`Substituted C-C alkynyl, or optionally Substituted C
`C. acyl, with the proviso that at least one of RandR has at
`least two sites of unsaturation; RandR are either the same
`or different and are independently optionally substituted
`C-C alkyl, optionally Substituted C-C alkenyl, or option
`ally substituted C-C alkynyl or RandR 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 is either absent or hydrogen or C-C alkyl to
`provide a quaternary amine; m, n and pare either the same or
`different and are independently either 0, 1 or 2, with the
`proviso that m, n, and pare not simultaneously 0; q is 0, 1, 2,
`3, or 4: Y and Z are either the same or different and are
`independently O, S, or NH; and
`(c) a non-cationic lipid.
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`5
`DLen-C2K-DMA, DPan-C2K-DMA, DPan-C3K-DMA,
`1,2-dilinoleyloxy-3-piperidinopropylamine (DLinPip), 1,2-
`dilinoleyloxy-3-(3'-hydroxypiperidino)-propylamine (DLin
`Pip-3OH), 1,2-dilinoleyloxy-3-(4-hydroxypiperidino)-pro
`pylamine
`(DLinPip-4OH),
`1,2-dilinoleyloxy-3-(N.N
`dimethyl)-propylamine (DLinDEA), N1-((2.3-linoleyloxy)
`propyl)-N1,N3.N3-trimethylpropane-1,3-diamine
`(2N
`DLinDMA), 1,2-Dillinoleyloxy-3-(1-imidazole)propylamine
`(DLinIm), 1,2-dilinoleyloxy-(N,N-dimethyl)-butyl-4-amine
`(C2-DLinDMA), 1,2-diphytanyloxy-(N,N-dimethyl)-butyl
`4-amine (C2-DPan MA), 1,2-dilinoleoyloxy-(N,N-dim
`ethyl)-butyl-4-amine (C2-DLinDAP), Linoley1/Oleyl DMA,
`Linoleyl/Phytanyl DMA, Linoleyl/Linolenyl DMA, Lino
`leyl/Stearyl DMA, Linoleyl/C:0 DMA, Linoleyl/C:1
`DMA, 1-(2.3-linoleyloxypropoxy)-2-(linoleyloxy)-(N.N-
`dimethyl)-propyl-3-amine (TLinDMA), C2-TLinDMA,
`DHep-C2K-DMA, DLin-C2K-Pip-3OH, 1,2-diarachidony
`loxy-(N,N-dimethyl)-propyl-3-amine (DAraDMA), 1,2-di
`docosahexaenyloxy-(N,N-dimethyl)-propyl-3-amine
`(DDocDMA), 1,2-diphytanyloxy-3-(N,N-dimethyl)-propy
`lamine (DPanDMA), 6-membered ketal lipids such as DPan
`C1K6-DMA, analogs thereof, salts thereof, and mixtures
`thereof.
`In some embodiments, the lipid particles of the invention
`preferably comprise an interfering RNA that silences APOB
`25
`and/or other liver target genes such as APOC3, PCSK9.
`DGAT1, DGAT2, or combinations thereof, a cationic lipid of
`Formula I-XIV as disclosed herein, a non-cationic lipid, and
`a conjugated lipid that inhibits aggregation of particles.
`In certain embodiments, the non-cationic lipid component
`of the lipid particle may comprise a phospholipid, cholesterol
`(or cholesterol derivative), or a mixture thereof. In one par
`ticular embodiment, the phospholipid comprises dipalmi
`toylphosphatidylcholine (DPPC), distearoylphosphatidyl
`choline (DSPC), or a mixture thereof. In some embodiments,
`35
`the conjugated lipid component of the lipid particle com
`prises a polyethyleneglycol (PEG)-lipid conjugate. In certain
`instances, the PEG-lipid conjugate comprises a PEG-diacylg
`lycerol (PEG-DAG) conjugate, a PEG-dialkyloxypropyl
`(PEG-DAA) conjugate, or a mixture thereof.
`In some embodiments, the interfering RNA is fully encap
`sulated within the lipid portion of the lipid particle such that
`the interfering RNA in the lipid particle is resistant in aqueous
`Solution to enzymatic degradation, e.g., by a nuclease or
`protease. Non-limiting examples of interfering RNA include
`siRNA, aiRNA, miRNA, Dicer-substrate dsRNA, shRNA,
`and mixtures thereof. In other embodiments, the lipid par
`ticles described herein are Substantially non-toxic to mam
`mals such as humans.
`In other embodiments, the nucleic acid-lipid particle com
`50
`prises an interfering RNA (e.g., siRNA) that targets APOB,
`wherein the interfering RNA comprises an antisense strand
`comprising the sequence 5'-UAUUCAGUGUGAUGA
`CACU-3' (SEQ ID NO:13). In still other embodiments, the
`nucleic acid-lipid particle further comprises a sense strand
`55
`comprising
`the
`Sequence
`5'-AGUGUCAUCA
`CACUGAAUA-3' (SEQID NO:14). In certain embodiments,
`the interfering RNA comprises a 3' overhang in one or both
`strands of the interfering RNA molecule. In certain embodi
`ments, the interfering RNA comprises an antisense strand
`60
`comprising a 5'-UG-3' overhang and/or a sense Strand com
`prising a 5'-CC-3' overhang.
`In yet other embodiments, the nucleic acid-lipid particle
`comprises an interfering RNA (e.g., siRNA) that targets
`APOB, wherein the interfering RNA comprises at least one
`modified nucleotide. In certain embodiments, one or more of
`the nucleotides in the double-stranded region of the interfer
`
`6
`ing RNA comprise modified nucleotides. In certain other
`embodiments, one or more of the nucleotides in the 3' over
`hang in one or both strands of the interfering RNA comprise
`modified nucleotides. In particular embodiments; the modi
`fied nucleotides comprise 2'-O-methyl (2'OMe) nucleotides.
`In further embodiments, the nucleic acid-lipid particle
`comprises an interfering RNA (e.g., siRNA) that targets
`APOB, wherein the interfering RNA comprises an antisense
`strand comprising the sequence 5'-UAUUCAGUGUGA
`UGACACU-3' (SEQ ID NO:15), wherein the bolded and
`underlined nucleotides are 2'OMe nucleotides. In other
`embodiments, the particle further comprises a sense Strand
`comprising the sequence 5'-AGUGUCAUCACACUGAA
`UA-3' (SEQID NO:16), wherein the bolded and underlined
`nucleotides are 2'OMe nucleotides. In certain embodiments,
`the interfering RNA comprises a 3' overhang in one or both
`strands of the interfering RNA molecule. In some embodi
`ments, the interfering RNA comprises an antisense strand
`comprising a 5'-UG-3' overhang and/or a sense Strand com
`prising a 5'-CC-3 overhang, wherein the bolded and under
`lined nucleotides are 2"OMe nucleotides. In other embodi
`ments, the nucleic acid-lipid particle comprises an interfering
`RNA consisting of the following sequences:
`
`(SEQ ID NO:
`5'-AGUGUCAUCACACUGAAUACC-3'
`and
`
`4)
`
`11)
`
`(SEQ ID NO:
`3'-GUUCACAGUAGUGUGACUUAU-5'
`wherein the bolded and underlined nucleotides are 2'OMe
`nucleotides.
`The present invention also provides pharmaceutical com
`positions comprising a nucleic acid-lipid particle described
`herein (e.g., SNALP) and a pharmaceutically acceptable car
`1.
`In another aspect, the present invention provides methods
`for introducing one or more interfering RNA molecules (e.g.,
`siRNAs that silence APOB expression and/or the expression
`of other liver target genes such as APOC3, PCSK9, DGAT1,
`and/or DGAT2) into a cell (e.g., a liver cell), the method
`comprising contacting the cell with a nucleic acid-lipid par
`ticle described herein (e.g., SNALP). In one embodiment, the
`cell is in a mammal and the mammal is a human.
`In yet another aspect, the present invention provides meth
`ods for the in vivo delivery of one or more interfering RNA
`molecules (e.g., siRNAs) to liver cells, the method compris
`ing administering to a mammal a nucleic acid-lipid particle
`described herein (e.g., SNALP). Advantageously, the nucleic
`acid-lipid particles of the invention are particularly effective
`at silencing target gene expression in the liver and, thus, are
`well suited for targeting genes such as APOB, APOC3,
`PCSK9, DGAT1, DGAT2, and combinations thereof. In cer
`tain embodiments, the nucleic acid-lipid particles (e.g.,
`SNALP) are administered by one of the following routes of
`administration: oral, intranasal, intravenous, intraperitoneal,
`intramuscular, intra-articular, intralesional, intratracheal,
`Subcutaneous, and intradermal. In particular embodiments,
`the nucleic acid-lipid particles (e.g., SNALP) are adminis
`tered systemically, e.g., via enteral or parenteral routes of
`administration. In preferred embodiments, the mammal is a
`human.
`In certain embodiments, the present invention provides
`methods for treating a liver disease or disorder by adminis
`tering an interfering RNA (e.g., one or more siRNAS targeting
`APOB, APOC3, PCSK9, DGAT1, and/or DGAT2 expres
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`sion) in nucleic acid-lipid particles (e.g., SNALP) as
`described herein, alone or in combination with a lipid-lower
`ing agent. Examples of lipid diseases and disorders include,
`but are not limited to, dyslipidemia (e.g., hyperlipidemias
`Such as elevated triglyceride levels (hypertriglyceridemia)
`and/or elevated cholesterol levels (hypercholesterolemia)),
`atherosclerosis, coronary heart disease, coronary artery dis
`ease, atherosclerotic cardiovascular disease (CVD), fatty
`liver disease (hepatic steatosis), abnormal lipid metabolism,
`abnormal cholesterol metabolism, diabetes (including Type