(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`11111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111
`
`( 43) International Publication Date
`27 January 2005 (27.01.2005)
`
`PCT
`
`(10) International Publication Number
`WO 2005/007196 A2
`
`(51) International Patent Classification7:
`
`A61K 47/48
`
`(21) International Application Number:
`PCT /CA2004/00 1051
`
`(22) International Filing Date:
`
`16 July 2004 (16.07.2004)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/488,144
`60/503,279
`60/529,406
`
`16 July 2003 (16.07.2003) US
`15 September 2003 (15.09.2003) US
`11 December 2003 (11.12.2003) US
`
`(71) Applicant (for all designated States except US): PRO(cid:173)
`TIVABIOTHERAPEUTICS,INC. [CA/CA]; 100-3480
`Gilmore Way, Burnaby, British Columbia V5G 4Y1 (CA).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): MACLACHLAN,
`Ian [CA/CA]; 1812 W. 7th Avenue, Suite 302, Vancouver,
`British Columbia V6J 1S8 (CA). AMBEGIA, Ellen,
`Grace [CA/CA]; 3150 W. 4th Avenue, #302, Vancou(cid:173)
`ver, British Columbia V6K 1R7 (CA). HEYES, James
`[GB/CA]; 315 - 651 Moberly Road, Vancouver, British
`Columbia V5Z 4B2 (CA).
`
`(74) Agents: FETHERSTONHAUGH & CO. et a!.; Suite
`2200, 650 West Georgia Street, Box 11560, Vancouver
`Centre, Vancouver, British Columbia V6B 4N8 (CA).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, Fl,
`GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE,
`KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD,
`MG, MK, MN, MW, MX, MZ, NA, NI, NO, NZ, OM, PG,
`PH, PL, PT, RO, RU, SC, SD, SE, SG, SK, SL, SY, TJ, TM,
`TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, YU, ZA, ZM,
`zw.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, Fl,
`FR, GB, GR, HU, IE, IT, LU, MC, NL, PL, PT, RO, SE, SI,
`SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`without international search report and to be republished
`upon receipt of that report
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`iiiiiiii
`iiiiiiii
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`---iiiiiiii
`!!!!!!!! ---
`------!!!!!!!!
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`iiiiiiii ------
`--- --------------------------------------------------------------------------------------
`
`(54) Title: LIPID ENCAPSULATED INTERFERING RNA
`
`!!!!!!!!
`iiiiiiii
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`iiiiiiii ------------
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`8-gal expression in stably transfected CT26.CL25 cells is down regulated by anti-B-gal
`siRNA
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`120
`110
`100
`90
`80
`%Control 70
`60
`50
`40
`30
`20
`10
`0
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`1:2 cells only
`!E!1.5 ug SsiRNALP with PEG-DMG
`o 4.0 ug SsiRNALP with PEG-DMG
`· ······ i!ll1.5 ug L055 SPLP with PEG·DMG
`: ::: ll!l4.0 ug L055 SPLP with PEG-DMG
`.... ISJ 0.2 ug siRNA with 5 ul Oligofectamine
`.. ~ 0.2 ug siRNA with 10 ul Oligofectamine
`.. 1210.2 ug siRNA with 15 ul Oligofectamine
`
`24
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`48
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`72
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`96
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`~ (57) Abstract: The present invention provides compositions and methods for silencing gene expression by delivering nucleic acid(cid:173)
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`~ lipid particles comprising a siRNA molecule to a cell.
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`LIPID ENCAPSULATED INTERFERING RNA
`
`CROSS-REFERENCES TO RELATED APPLICATIONS
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`5
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`[0001] This application claims the benefit of U.S. Provisional Patent Application Nos.
`
`60/529,406, filed December 11, 2003; 60/503,279, filed September 15,2003, and 60/488,144,
`
`filed July 16, 2003, the disclosures of each of which are hereby incorporated by reference in
`
`their entirety for all purposes.
`
`FIELD OF THE INVENTION
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`10
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`[0002] The present invention relates to compositions and methods for the therapeutic
`delivery of a nucleic acid by delivering a serum-stable lipid delivery vehicle encapsulating
`the nucleic acid to provide efficient RNA interference (RNAi) in a cell or mammal. More
`particularly, the present invention is directed to using a small interfering RNA (siRNA)
`encapsulated in a serum-stable lipid particle having a small diameter suitable for systemic
`
`15
`
`delivery.
`
`BACKGROUND OF THE INVENTION
`
`[0003] RNA interference (RNAi) is an evolutionarily conserved, sequence specific
`
`mechanism triggered by double stranded RNA (dsRNA) that induces degradation of
`complementary target single stranded mRNA and "silencing" of the corresponding translated
`
`20
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`sequences (McManus and Sharp, Nature Rev. Genet. 3:737 (2002)). RNAi functions by
`enzymatic cleavage oflonger dsRNA strands into biologically active "short-interfering RNA"
`(siRNA) sequences of about 21-23 nucleotides in length (Elbashir, et al., Genes Dev. 15:188
`(2001 )). siRNA can be used downregulate or silence the translation of a gene product of
`interest. For example, it is desirable to downregulate genes associated with various diseases
`
`25
`
`and disorders.
`
`[0004] Delivery of siRNA remains problematic (see, e.g., Novina and Sharp, Nature
`430::161-163 (2004); and Garber, J. Nat!. Cancer lnst. 95(7):500-2 (2003)). An effective
`and safe nucleic acid delivery system is required for siRNA to be therapeutically useful.
`
`Naked dsRNA administered to most subjects will: (1) be degraded by endogenous nucleases;
`and (2) will not be able to cross cell membranes to contact and silence their target gene
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`30
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`sequences.
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`[0005] Viral vectors are relatively efficient gene delivery systems, but suffer from a variety
`
`of safety concerns, such as potential for undesired immune responses. Furthermore, viral
`
`systems are rapidly cleared from the circulation, limiting transfection to "first-pass" organs
`
`such as the lungs, liver, and spleen. In addition, these systems induce immune responses that
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`5
`
`compromise delivery with subsequent injections. As a result, nonviral gene delivery systems
`
`are receiving increasing attention (Worgall, et al., Human Gene Therapy 8:37 (1997);
`
`Peeters, et al., Human Gene Therapy 7:1693 (1996); Yei, et al., Gene Therapy 1: 192 (1994);
`
`Hope, et al., Molecular Membrane Biology 15:1 (1998)).
`
`[0006] Plasmid DNA-cationic liposome complexes are currently the most commonly
`
`10
`
`employed nonviral gene delivery vehicles (Feigner, Scientific American 276:102 (1997);
`
`Chonn, et al., Current Opinion in Biotechnology 6:698 (1995)). For instance, cationic
`
`liposome complexes made of an amphipathic compound, a neutral lipid, and a detergent for
`
`transfecting insect cells are disclosed in U.S. Patent No. 6,458,382. Cationic liposome
`
`complexes are also disclosed in U.S. Patent Application Publication No. 2003/0073640.
`
`15
`
`Cationic liposome complexes, however, are large, poorly defined systems that are not suited
`
`for systemic applications and can elicit considerable toxic side effects (Harrison, et al.,
`
`Biotechniques 19:816 (1995); Li, et al., The Gene 4:891 (1997); Tam, et al, Gene Ther.
`
`7:1867 (2000)). As large, positively charged aggregates, lipoplexes are rapidly cleared when
`
`administered in vivo, with highest expression levels observed in first-pass organs, particularly
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`20
`
`the lungs (Huang, et al., Nature Biotechnology 15:620 (1997); Templeton, et al., Nature
`
`Biotechnology 15:647 (1997); Hofland, et al., Pharmaceutical Research 14:742 (1997)).
`
`[0007] Other liposomal delivery systems include, for example, the use of reverse micelles,
`
`anionic and polymer liposomes as disclosed in, e.g., U.S. Patent No. 6,429,200; U.S. Patent
`
`Application No. 2003/0026831; and U.S. Patent Application Nos. 2002/0081736 and
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`25
`
`2003/0082103, respectively.
`
`[0008] Recent work has shown that nucleic acids can be encapsulated in small (about 70
`
`nm diameter) "stabilized plasmid-lipid particles" (SPLP) that consist of a single plasmid
`
`encapsulated within a bilayer lipid vesicle (Wheeler, et al., Gene Therapy 6:271 (1999)).
`
`These SPLPs typically contain the "fusogenic" lipid dioleoylphosphatidylethanolamine
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`30
`
`(DOPE), low levels of cationic lipid (i.e., 10% or less), and are stabilized in aqueous media
`
`by the presence of a poly( ethylene glycol) (PEG) coating. SPLP have systemic application as
`
`they exhibit extended circulation lifetimes following intravenous (i.v.) injection, accumulate
`
`preferentially at distal tumor sites due to the enhanced vascular permeability in such regions,
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`and can mediate trans gene expression at these tumor sites. The levels of trans gene
`
`expression observed at the tumor site following i.v. injection of SPLP containing the
`
`luciferase marker gene are superior to the levels that can be achieved employing plasmid
`
`DNA-cationic liposome complexes (lipoplexes) or naked DNA.
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`[0009] However, there remains a strong need in the art for novel and more efficient
`
`methods and compositions for introducing nucleic acids, such as siRNA, into cells. The
`
`present invention addresses this and other needs.
`
`BRIEF SUMMARY OF THE INVENTION
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`10
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`[0010] The present invention provides stable nucleic acid-lipid particles (SNALP) useful
`
`for encapsulating one or more siRNA molecules, methods of making SNALPs comprising
`
`siRNA, SNALPs comprising siRNA and methods of delivering and/or administering the
`
`SNALPs to a subject to silence expression of a target gene sequence.
`
`[0011]
`
`In one embodiment, the invention provide nucleic acid-lipid particles comprising: a
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`15
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`cationic lipid, a non-cationic lipid, a conjugated lipid that inhibits aggregation of particles and
`
`a siRNA. In some embodiments, the siRNA molecule is fully encapsulated within the lipid
`
`bilayer of the nucleic acid-lipid particle such that the nucleic acid in the nucleic acid-lipid
`
`particle is resistant in aqueous solution to degradation by a nuclease. The nucleic acid
`particle are substantially non-toxic to mammals. The siRNA molecule may comprise about
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`20
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`15 to about 60 nucleotides. The siRNA molecule may be derived from a double-stranded
`
`RNA greater than about 25 nucleotides in length. In some embodiments the siRNA is
`
`transcribed from a plasmid, in particular a plasmid comprising a DNA template of a target
`
`sequence.
`
`[0012] The cationic lipid may be one or more ofN,N-dioleyl-N,N-dimethylammonium
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`25
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`chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-
`
`dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-
`
`dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), and N,N-dimethyl-2,3-
`
`dioleyloxy)propylamine (DODMA), and a mixture thereof. The non-cationic lipid may be
`
`one or more of dioleoylphosphatidylethanolamine (DOPE),
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`30
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`palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC),
`
`distearoylphosphatidylcholine (DSPC), cholesterol, and combinations thereof.
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`[0013] The conjugated lipid that inhibits aggregation of particles may be one or more of a
`
`polyethyleneglycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, and
`
`combinations thereof. The PEG-lipid conjugate may be one or more of a PEG(cid:173)
`
`dialkyloxypropyl (DAA), a PEG-diacylglycerol (DAG), a PEG-phospholipid, a PEG-
`
`5
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`ceramide, and combinations thereof. The PEG-DAG conjugate may be one or more of a
`PEG-dilauroylglycerol (CI2), a PEG-dimyristoylglycerol (CI 4), a PEG-dipalmitoylglycerol
`(CI6), and a PEG-distearoylglycerol (Cis), and combinations thereof. The PEG-DAA
`
`conjugate may be one or more of a PEG-dilauryloxypropyl (CI 2), a PEG-dimyristyloxypropyl
`(CI4), a PEG-dipalmityloxypropyl (CI6), and a PEG-distearyloxypropyl (Cis), and
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`10
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`combinations thereof. The nucleic acid-lipid particle may further comprise a cationic
`
`polymer lipid.
`
`In some embodiments, the particles are made by providing an aqueous solution in a
`[0014]
`first reservoir and an organic lipid solution in a second reservoir and mixing the aqueous
`
`solution with the organic lipid solution so as to substantially instantaneously produce a
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`15
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`liposome encapsulating an interfering RNA. In some embodiments, the particles are made by
`
`formation ofhydrophobic intermediate complexes in either detergent-based or organic
`
`solvent-based systems, followed by removal of the detergent or organic solvent. Preferred
`
`embodiments are charge-neutralized.
`
`[0015]
`
`In one embodiment, the interfering RNA is transcribed from a plasmid and the
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`plasmid is combined with cationic lipids in a detergent solution to provide a coated nucleic
`
`acid-lipid complex. The complex is then contacted with non-cationic lipids to provide a
`
`solution of detergent, a nucleic acid-lipid complex and non-cationic lipids, and the detergent
`
`is then removed to provide a solution of serum-stable nucleic acid-lipid particles, in which
`
`the plasmid comprising an interfering RNA template is encapsulated in a lipid bilayer. The
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`particles thus formed have a size of about 50-150 nm.
`
`[0016]
`
`In another embodiment, serum-stable nucleic acid-lipid particles are formed by
`
`preparing a mixture of cationic lipids and non-cationic lipids in an organic solvent; contacting
`
`an aqueous solution of nucleic acids comprising interfering RNA with the mixture of cationic
`
`and non-cationic lipids to provide a clear single phase; and removing the organic solvent to
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`30
`
`provide a suspension of nucleic acid-lipid particles, in which the nucleic acid is encapsulated
`
`in a lipid bilayer, and the particles are stable in serum and have a size of about 50-150 nm.
`
`[0017] The nucleic acid-lipid particles of the present invention are useful for the
`
`therapeutic delivery of nucleic acids comprising a siRNA sequence. In particular, it is an
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`object of this invention to provide in vitro and in vivo methods for treatment of a disease in a
`mammal by downregulating or silencing the translation of a target nucleic acid sequence. In
`these methods, a siRNA molecule is formulated into a nucleic acid-lipid particle, and the
`particles are administered to patients requiring such treatment (e.g., a patient diagnosed with
`a disease or disorder associated with the expression or overexpression of a gene comprising
`the target nucleic acid sequence). Alternatively, cells are removed from a patient, the siRNA
`is delivered in vitro, and the cells are reinjected into the patient. In one embodiment, the
`present invention provides for a method of introducing a siRNA molecule into a cell by
`contacting a cell with a nucleic acid-lipid particle comprising of a cationic lipid, a non-
`cationic lipid, a conjugated lipid that inhibits aggregation, and a siRNA.
`[0018] The nucleic acid-lipid particle may be administered, e.g., intravenously, parenterally
`or intraperitoneally. In one embodiment, at least about 10% of the total administered dose of
`the nucleic acid-lipid particles is present in plasma about 24, 36, 48, 60, 72, 84, or 96 hours
`after injection. In other embodiments, more than 20%, 30%, 40% and as much as 60%, 70%
`or 80% of the total injected dose of the nucleic acid-lipid particles is present in plasma 24, 36,
`48, 60, 72, 84, or 96 hours after injection. In one embodiment, the presence of a siRNA in
`cells in a target tissue (i.e., lung, liver, tumor or at a site of inflammation) is detectable at 24,
`48, 72 and 96 hours after administration. In one embodiment, downregulation of expression
`of the target sequence is detectable at 24, 48, 72 and 96 hours after administration. In one
`embodiment, downregulation of expression of the target sequence occurs preferentially in
`tumor cells or in cells at a site of inflammation. In one embodiment, the presence of a siRNA
`in cells at a site distal to the site of administration is detectable at least four days after
`intravenous injection of the nucleic acid-lipid particle. In another embodiment, the presence
`of a siRNA in of cells in t a target tissue (i.e., lung, liver, tumor or at a site of inflammation)
`is detectable at least four days after injection of the nucleic acid-lipid particle.
`[0019] The particles are suitable for use in intravenous nucleic acid transfer as they are
`stable in circulation, of a size required for pharmacodynamic behavior resulting in access to
`extravascular sites and target cell populations. The invention also provides for
`pharmaceutically acceptable compositions comprising a nucleic acid-lipid particle.
`[0020] Another embodiment of the present invention provides methods for in vivo delivery
`ofsiRNA. A nucleic acid-lipid particle comprising a cationic lipid, a non-cationic lipid, a
`conjugated lipid that inhibits aggregation of particles, and siRNA is administered (e.g.,
`intravenously, subcutaneously, intraperitoneally, or subdermally) to a subject (e.g., a mammal
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`such as a human). In some embodiments, the invention provides methods for in vivo delivery
`
`of interfering RNA to the liver of a mammalian subject.
`
`[0021] A further embodiment of the present invention provides a method of treating a
`
`disease or disorder in a mammalian subject. A therapeutically effective amount of a nucleic
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`acid-lipid particle comprising a cationic lipid, a non-cationic lipid, a conjugated lipid that
`
`inhibits aggregation of particles, and siRNA is administered to the mammalian subject (e.g., a
`
`rodent such as a mouse, a primate such as a human or a monkey) with the disease or disorder.
`
`In some embodiments, the disease or disorder is associated with expression and/or
`
`overexpression of a gene and expression or overexpression of the gene is silenced by the
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`siRNA. In some embodiments, the disease is a viral disease such as, for example, hepatitis
`
`(e.g., Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Hepatitis G, or a
`
`combination thereof). In some embodiment, the disease or disorder is a liver disease or
`
`disorder, such as, for example, dyslipidemia.
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`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0022] Figure 1 illustrates downregulating 13-galactosidase expression in CT26.CL25 cells
`
`via in vitro delivery of encapsulated anti-13-galactosidase siRNA in
`
`DSPC:Cholesterol:DODMA:PEG-DMG liposomes.
`[0023] Figure 2 illustrates the structures ofPEG-Diacylglycerols and PEG-Ceramide C2o.
`[0024] Figure 3 illustrates that clearance studies with LUVs showed that SNALPs
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`20
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`containing PEG-DAGs were comparable to SNALPs containing PEG-CeramideC20.
`
`[0025] Figure 4 illustrates that SNALPs containing PEG-DAGs can be formulated via a
`
`detergent dialysis method.
`
`[0026] Figure 5 illustrates the pharmacokinetic properties of SNALPs containing PEG-
`
`25
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`DAGs.
`
`[0027] Figure 6 illustrates the biodistribution properties ofSNALPs containing PEG(cid:173)
`
`DAGs.
`
`[0028] Figure 7 illustrates the luciferase gene expression 24 hrs post IV administration of
`SPLPs containing PEG-CeramideC20 versus PEG-DAGs in Neuro-2a Tumor Bearing Male
`AIJ Mice.
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`[0029] Figure 8 illustrates the luciferase gene expression 48 hrs post IV administration of
`SPLPs containing PEG-CeramideC20 versus PEG-DAGs in Neuro-2a Tumor Bearing Male
`AIJ Mice.
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`5
`
`[0030] Figure 9 illustrates the luciferase gene expression 72 hrs post IV administration of
`SPLPs containing PEG-CeramideC20 versus PEG-DAGs in Neuro-2a Tumor Bearing Male
`AIJ Mice.
`
`[0031] Figure 10 illustrates the structures of three exemplary PEG-dialkyloxypropyl
`
`derivatives suitable for use in the present invention, i.e., N-(2,3-dimyristyloxypropyl)
`carbamate PEG2000 methyl ether (i.e., PEG-C-DMA), N-(2,3-dimyristyloxypropyl) amide
`PEG2000 methyl ether (i.e., PEG-A-DMA), and N-(2,3-dimyristyloxypropyl) succinamide
`PEG2ooo methyl ether (i.e., PEG-S-DMA).
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`[0032] Figure 11 illustrates data showing luciferase gene expression in tumors 48 hours
`
`after intravenous administration ofSPLP comprising PEG-DAA conjugates and PEG-DAG
`
`conjugates.
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`[0033] Figure 12 illustrates data showing luciferase gene expression in liver, lung, spleen,
`
`heart, and tumor following intravenous administration ofSPLP comprising PEG-DAA
`
`conjugates and PEG-DAG conjugates.
`
`[0034]
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`Figure 13 illustrates data from clearance studies in Neuro-2a tumor bearing male
`
`AIJ mice after administration of SPLPs comprising a PEG-DAA conjugate and containing a
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`20
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`plasmid encoding luciferase under the control of the CMV promoter and SNALPs comprising
`
`a PEG-DAA conjugate and containing anti-luciferase siRNA.
`
`[0035] Figure 14 illustrates data from studies of the pharmacokinetic properties of SPLPs
`
`comprising a PEG-DAA conjugate and containing a plasmid encoding luciferase under the
`
`control of the CMV promoter and SNALPs comprising a PEG-DAA conjugate and
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`25
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`containing anti-luciferase siRNA in Neuro-2a tumor bearing male A/J mice.
`
`[0036] Figure 15 illustrates data from clearance studies in Neuro-2a tumor bearing male
`
`AIJ mice after administration of SPLPs comprising a PEG-DAA conjugate or a PEG-DAG
`
`conjugate and containing a plasmid encoding luciferase under the control of the CMV
`
`promoter, pSPLPs comprising a PEG-DAG conjugate and containing a plasmid encoding
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`30
`
`luciferase under the control of the CMV promoter and SNALPs comprising a PEG-DAA
`
`conjugate and containing anti-luciferase siRNA.
`
`[0037] Figure 16 illustrates data from studies of the pharmacokinetic properties of SPLPs
`
`comprising a PEG-DAA conjugate or a PEG-DAG conjugate and containing a plasmid
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`encoding luciferase under the control of the CMV promoter, pSPLPs comprising a PEG(cid:173)
`
`DAG conjugate and containing a plasmid encoding luciferase under the control of the CMV
`
`promoter and SNALPs comprising a PEG-DAA conjugate and containing anti-luciferase
`
`siRNA in Neuro-2a tumor bearing male NJ mice.
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`5
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`[0038] Figure 17 illustrates in vitro data demonstrating silencing ofluciferase expression in
`
`luciferase expressing cells treated with SPLPs comprising a PEG-lipid conjugate and
`
`containing a plasmid encoding luciferase under the control of the CMV promoter and
`
`SNALPs comprising a PEG-lipid conjugate conjugate and containing anti-luciferase siRNA.
`
`[0039] Figure 18 illustrates in vivo data demonstrating silencing of luciferase expression in
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`10 Neuro-2a tumor bearing male NJ mice treated with SPLPs comprising a PEG-DAA
`
`conjugate and containing a plasmid encoding luciferase under the control of the CMV
`
`promoter and SNALPs comprising a PEG-DAA conjugate and containing anti-luciferase
`
`siRNA.
`
`[0040] Figure 19 illustrates in vivo data demonstrating silencing ofluciferase expression in
`
`15 Neuro-2a tumor bearing male NJ mice treated with SPLPs comprising a PEG-DAA
`
`conjugate and containing a plasmid encoding luciferase under the control of the CMV
`
`promoter and SNALPs comprising a PEG-DAA conjugate and containing anti-luciferase
`
`siRNA.
`
`[0041] Figure 20 illustrates in vivo data demonstrating silencing ofluciferase expression in
`
`20 Neuro-2a tumor bearing male NJ mice treated with SPLPs comprising a PEG-DAA
`
`conjugate and containing a plasmid encoding luciferase under the control of the CMV
`
`promoter and SNALPs comprising a PEG-DAA conjugate and containing anti-luciferase
`
`siRNA.
`
`[0042] Figure 21 illustrates in vivo data demonstrating silencing ofluciferase expression in
`
`25 Neuro-2a tumor bearing male NJ mice treated with SPLPs comprising a PEG-DAA
`
`conjugate and containing a plasmid encoding luciferase under the control of the CMV
`
`promoter and SNALPs comprising a PEG-DAA conjugate and containing anti-luciferase
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`siRNA.
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`[0043] Figure 22 illustrates in vivo data demonstrating silencing of luciferase expression in
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`30 Neuro-2a tumor bearing male NJ mice treated with SPLPs comprising a PEG-DAA
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`conjugate and containing a plasmid encoding luciferase under the control of the CMV
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`promoter and SNALPs comprising a PEG-DAA conjugate and containing anti-luciferase
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`siRNA.
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`DETAILED DESCRIPTION OF THE INVENTION
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`I.
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`Introduction
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`[0044] The present invention provides stable nucleic acid-lipid particles (SNALP) useful
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`for encapsulating one or more siRNA molecules, methods of making SNALPs comprising
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`siRNA, SNALPs comprising siRNA and methods of delivering and/or administering the
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`SNALPs to a subject to silence expression of a target gene sequence.
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`[0045] The present invention is based on the unexpected success of encapsulating short
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`interfering RNA (siRNA) molecules in SNALPs. Using the methods of the present
`
`invention, siRNA molecules are encapsulated in SNALPs with an efficiency greater than
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`70%, more usually with an efficiency greater than 80 to 90%. The SNALPs described herein
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`can conveniently be used in vitro and in vivo to efficiently deliver administer siRNA
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`molecules locally or systemically to cells expressing a target gene. Once delivered, the
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`siRNA molecules in the SNALPs silence expression of the target gene.
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`[0046] The SNALPs described herein are typically < 150 nm diameter and remain intact in
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`the circulation for an extended period of time in order to achieve delivery of siRN A to target
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`tissues. The SNALPs are highly stable, serum-resistant nucleic acid-containing particles that
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`does not interact with cells and other components of the vascular compartment. Moreover,
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`the SNALPs also readily interact with target cells at a disease site in order to facilitate
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`intracellular delivery of a desired nucleic acid (e.g., a siRNA or a plasmid encoding a
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`siRNA).
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`II.
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`Definitions
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`[0047] The term "lipid" refers to a group of organic compounds that include, but are not
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`limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble
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`in many organic solvents. They are usually divided into at least three classes: (1) "simple
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`lipids' which include fats and oils as well as waxes; (2) "compound lipids" which include
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`phospholipids and glycolipids; (3) "derived lipids" such as steroids.
`
`[0048]
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`"Lipid vesicle" refers to any lipid composition that can be used to deliver a
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`compound including, but not limited to, liposomes, wherein an aqueous volume is
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`encapsulated by an amphipathic lipid bilayer; or wherein the lipids coat an interior
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`comprising a large molecular component, such as a plasmid comprising an interfering RNA
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`sequence, with a reduced aqueous interior; or lipid aggregates or micelles, wherein the
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`encapsulated component is contained within a relatively disordered lipid mixture.
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`[0049] As used herein, "lipid encapsulated" can refer to a lipid formulation that provides a
`
`compound with full encapsulation, partial encapsulation, or both. In a preferred embodiment,
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`the nucleic acid is fully encapsulated in the lipid formulation.
`
`[0050] As used herein, the term "SNALP" refers to a stable nucleic acid lipid particle. A
`
`SNALP represents a vesicle oflipids coating a reduced aqueous interior comprising a nucleic
`
`acid such as an interfering RNA sequence or a plasmid from which an interfering RNA is
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`transcribed.
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`[0051] The term "vesicle-forming lipid" is intended to include any amphipathic lipid
`
`having a hydrophobic moiety and a polar head group, and which by itself can form
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`spontaneously into bilayer vesicles in water, as exemplified by most phospholipids.
`
`[0052] The term "vesicle-adopting lipid" is intended to include any amphipathic lipid that is
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`stably incorporated into lipid bilayers in combination with other amphipathic lipids, with its
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`hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane,
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`and its polar head group moiety oriented toward the exterior, polar surface of the membrane.
`
`Vesicle-adopting lipids include lipids that on their own tend to adopt a nonlamellar phase, yet
`
`which are capable of assuming a bilayer structure in the presence of a bilayer-stabilizing
`
`component. A typical example is DOPE (dioleoylphosphatidylethanolamine). Bilayer
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`stabilizing components include, but are not limited to, conjugated lipids that inhibit
`
`aggregation ofthe SNALPs, polyamide oligomers (e.g., ATTA-lipid derivatives), peptides,
`
`proteins, detergents, lipid-derivatives, PEG-lipid derivatives such as PEG coupled to
`
`dialkyloxypropyls, PEG coupled to diacylglycerols, PEG coupled to phosphatidyl(cid:173)
`
`ethanolamines, and PEG conjugated to ceramides (see, U.S. Pat. No. 5,885,613, which is
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`incorporated herein by reference).
`
`[0053] The term "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. Amphipathic lipids are usually the
`
`major component of a lipid vesicle. Hydrophilic characteristics derive from the presence of
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`polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfato, amino,
`
`sulfhydryl, nitro, hydroxy 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
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`aromatic, cycloaliphatic or heterocyclic group(s). Examples of amphipathic compounds
`include, but are not limited to, phospholipids, aminolipids and sphingolipids. Representative
`examples of phospholipids include, but are not limited to, phosphatidylcholine,
`phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid,
`palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine,
`lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,
`distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine. Other compounds lacking
`in phosphorus, such as sphingolipid, glycosphingolipid families, diacylglycerols and .beta.(cid:173)
`acyloxyacids, are also within the group designated as amphipathic lipids. Additionally, the
`amphipathic lipid described above can be mixed with other lipids including triglycerides and
`sterols.
`[0054] The term "neutral lipid" refers to any of a number oflipid species that exist either in
`an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids
`include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,
`sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols.
`[0055] The term "noncationic lipid" refers to any neutral lipid as described above as well as
`
`anionic lipids.
`[0056] The term "anionic lipid" refers to any lipid that is negatively charged at
`physiological pH. These lipids include, but are not limited to, phosphatidylglycerol,
`cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl
`phosphatidylethanolamines, N -succinyl phosphatidylethanolamines, N(cid:173)
`glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,
`palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to
`
`neutral lipids.
`[0057] The term "cationic lipid" refers to any of a number oflipid species that carry a net
`positive charge at a selected pH, such as physiological pH. Such lipids include, but are not
`limited to, N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N-(2,3-
`dioleyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTMA"); N,N-distearyl-N,N(cid:173)
`dimethylammonium bromide ("DDAB"); N-(2,3-dioleoyloxy)propyl)-N,N,N-
`trimethylammonium chloride ("D