Document made available under
`Patent Cooperation Treaty (PCT)
`
`the
`
`International application number: PCT/CA2009/000496
`
`International filing date:
`
`15 April 2009 (15.04.2009)
`
`Document type:
`
`Certified copy of priority document
`
`Document details:
`
`Country/Office: US
`61/045,228
`Number:
`15 April 2008 (15.04.2008)
`Filing date:
`
`Date of receipt at the International Bureau: 10 June 2009 (10.06.2009)
`
`Remark: Priority document submitted or transmitted to the International Bureau in
`compliance with Rule 17.1(a) or (b)
`
`HIPO
`RMM
`
`World Intellectual Property Organization (WIPO) - Geneva, Switzerland
`Organisation Mondiale de la Propriete Intellectuelle (OMPI) - Geneve, Suisse
`
`PROTIVA - EXHIBIT 2041
`Moderna Therapeutics, Inc. v. Protiva Biotherapeautics, Inc. - IPR2018-00739
`
`

`

`PCT/CA2009/000496
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`UNITED STATES DEPARTMENT OF COMMERCE
`
`United States Patent and Trademark Office
`
`April 28, 2009
`
`THIS IS TO CERTIFY THAT ANNEXED HERETO IS A TRUE COPY FROM
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`APPLICATION THAT MET THE REQUIREMENTS TO BE GRANTED A
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`APPLICATION NUMBER: 61/045,228
`I
`FILING DATE: April 15, 2008
`|
`THE COUNTRY CODE AND NUMBER OF YOUR PRIORITY
`APPLICATION, TO BE USED FOR FILING ABROAD UNDER THE PARIS
`CONVENTION, IS US61/045,228
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`By Authority of the
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`and Director of the United States Patent and Trademark Office
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`PCT/CA2009/000496
`
`Electronic Acknowledgement Receipt
`
`EFS ID:
`
`Application Number:
`
`3158382
`
`61045228
`
`International Application Number:
`
`Confirmation Number:
`
`2310
`
`Title of Invention:
`
`NOVEL LIPID FORMULATIONS FOR NUCLEIC ACID DELIVERY
`
`First Named Inventor/Applicant Name:
`
`Ian MacLachlan
`
`Customer Number:
`
`20350
`
`Filer:
`
`Eugenia Garrett-Wackowski/Linda Shaffer
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`Filer Authorized By:
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`Eugenia Garrett-Wackowski
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`Attorney Docket Number:
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`020801-007700US
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`PCT/CA2009/000496
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`PCT/CA2009/000496
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`This Acknowledgement Receipt evidences receipt on the noted date by the USPTO of the indicated documents,
`characterized by the applicant, and including page counts, where applicable. It serves as evidence of receipt
`similar to a Post Card, as described in MPEP 503.
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`New Applications Under 35 U.S.C. 111
`If a new application is being filed and the application includes the necessary components for a filing date (see
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`If a timely submission to enter the national stage of an international application is compliant with the conditions
`of 35 U.S.C. 371 and other applicable requirements a Form PCT/DO/EO/903 indicating acceptance of the
`application as a national stage submission under 35 U.S.C. 371 will be issued in addition to the Filing Receipt,
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`If a new international application is being filed and the international application includes the necessary
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`

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`PCT/CA2009/000496
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`Attorney Docket No.: 020801-007700US
`
`PROVISIONAL
`
`PATENT APPLICATION
`
`NOVEL LIPID FORMULATIONS FOR NUCLEIC ACID DELIVERY
`
`Inventor:
`
`Ian MacLachlan, a citizen of Canada and the United Kingdom, residing at
`8040 Aves Terrace
`Mission, B.C., Canada Y4S 1E5
`
`Edward Yaworski, a citizen of Canada, residing at
`Maple Ridge, B.C., Canada
`
`Kieu Lam, a citizen of Canada, residing at
`Surrey, B.C., Canada
`
`Assignee:
`
`Protiva Biotherapeutics, Inc.
`100-3480 Gilmore Way
`Bumaby, BCC V5G 4Y1
`
`Entity:
`
`Small
`
`TOWNSEND and TOWNSEND and CREW LLP
`Two Embarcadero Center, Eighth Floor
`San Francisco, California 94111-3834
`Tel: 925-472-5000
`
`

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`PCT/CA2009/000496
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`PATENT
`Attorney Docket No.: 020801-007700US
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`NOVEL LIPID FORMULATIONS FOR NUCLEIC ACID DELIVERY
`BACKGROUND OF THE INVENTION
`[0001] RNA interference (RNAi) is an evolutionarily conserved process in which
`recognition of double-stranded RNA (dsRNA) ultimately leads to posttranscriptional
`suppression of gene expression. This suppression is mediated by short dsRNA, also called
`small interfering RNA (siRNA), which induces specific degradation of mRNA through
`complementary base pairing. In several model systems, this natural response has been
`developed into a powerful tool for the investigation of gene function (see, e.g., Elbashir et al.,
`Genes Dev., 15:188-200 (2001); Hammond et al, Nat. Rev. Genet., 2:110-119 (2001)). More
`recently, it was discovered that introducing synthetic 21-nucleotide dsRNA duplexes into
`mammalian cells could efficiently silence gene expression.
`[0002] Although the precise mechanism is still unclear, RNAi provides a potential new
`approach to downregulate or silence the transcription and translation of a gene of interest.
`For example, it is desirable to modulate (e.g., reduce) the expression of certain genes for the
`treatment of neoplastic disorders such as cancer. It is also desirable to silence the expression
`of genes associated with liver diseases and disorders such as hepatitis. It is further desirable
`to reduce the expression of certain genes for the treatment of atherosclerosis and its
`manifestations, e.g., hypercholesterolemia, myocardial infarction, and thrombosis.
`[0003] A safe and effective nucleic acid delivery system is required for RNAi to be
`therapeutically useful. Viral vectors are relatively efficient gene delivery systems, but suffer
`from a variety of limitations, such as the potential for reversion to the wild-type as well as
`immune response concerns. 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..
`25 Molecular Membrane Biology, 15:1 (1998)). 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 compromise delivery with
`subsequent injections.
`[0004] Plasmid DNA-cationic liposome complexes are currently the most commonly
`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
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`transfecting insect cells are disclosed in U.S. Patent No. 6,458,382. Cationic liposome
`complexes are also disclosed in U.S. Patent Publication No. 20030073640.
`[0005] Cationic liposome complexes are large, poorly defined systems that are not suited
`for systemic applications and can elicit considerable toxic side effects (Harrison et al.,
`5 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
`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)).
`[0006] Other liposomal delivery systems include, for example, the use of reverse micelles,
`anionic liposomes, and polymer liposomes. Reverse micelles are disclosed in U.S. Patent No.
`6,429,200. Anionic liposomes are disclosed in U.S. Patent Publication No. 20030026831.
`Polymer liposomes that incorporate dextrin or glycerol-phosphocholine polymers are
`disclosed in U.S. Patent Publication Nos. 20020081736 and 20030082103, respectively.
`[0007] A gene delivery system containing an encapsulated nucleic acid for systemic
`delivery should be small {i.e., less than about 100 nm diameter) and should remain intact in
`the circulation for an extended period of time in order to achieve delivery to affected tissues.
`This requires a highly stable, serum-resistant nucleic acid-containing particle that does not
`interact with cells and other components of the vascular compartment. The particle should
`also readily interact with target cells at a disease site in order to facilitate intracellular
`delivery of a desired nucleic acid.
`[0008] Recent work has shown that nucleic acids can be encapsulated in small {e.g., 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
`(DOPE), low levels of cationic lipid, and are stabilized in aqueous media by the presence of a
`poly(ethylene glycol) (PEG) coating. SPLPs 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, and can
`30 mediate transgene expression at these tumor sites. The levels of transgene expression
`observed at the tumor site following i.v. injection of SPLPs 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] Thus, 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. In addition, there is
`a need in the art for methods of downregulating the expression of genes of interest to treat or
`prevent diseases and disorders such as cancer and atherosclerosis. The present invention
`addresses these and other needs.
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`SUMMARY OF THE INVENTION
`[0010] The present invention provides novel, stable nucleic acid-lipid particles (SNALP)
`encapsulating a nucleic acid (such as one or more interfering RNA molecules), methods of
`making the SNALPs, and methods of delivering and/or administering the SNALPs. More
`particularly, the present invention provides nucleic acid-lipid particle comprising: (a) a
`nucleic acid; (b) a cationic lipid comprising from about 50 mol % to about 85 mol % of the
`total lipid present in the particle; (c) a non-cationic lipid comprising from about 13 mol % to
`about 49.5 mol % of the total lipid present in the particle; and (d) a conjugated lipid that
`inhibits aggregation of particles comprising from about 0.5 mol % to about 2 mol % of the
`total lipid present in the particle. In a preferred embodiment, the nucleic acid (e.g., the
`siRNA molecule) is fully encapsulated within the lipid 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. In a preferred embodiment, the nucleic acid particle is
`substantially non-toxic to mammals.
`[0011]
`In one aspect, the nucleic acid is an interfering RNA molecule, such as an small
`interfering RNA molecule (siRNA). In one embodiment, the siRNA comprises a double-
`stranded region of about 15 to about 60 nucleotides in length (e.g., about 15-60, 15-50, 15-40,
`15-30, 15-25, or 19-25 nucleotides in length, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25
`nucleotides in length. In another embodiment, the siRNA molecule comprises at least one
`25 modified nucleotide. In a preferred embodiment, the modified siRNA molecule comprises
`one, two, three, four, five, six, seven, eight, nine, ten, or more modified nucleotides in the
`double-stranded region. In some embodiments, the modified siRNA comprises from about
`1% to about 100% (e.g., about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
`55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) modified nucleotides in the
`double-stranded region. In preferred embodiments, less than about 25% (e.g., less than about
`25%, 20%, 15%, 10%, or 5%) or from about 1% to about 25% (e.g., from about l%-25%,
`5%-25%, 10%-25%, 15%-25%, 20%-25%, or 10%-20%) of the nucleotides in the double-
`stranded region comprise modified nucleotides.
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`[0012]
`In some embodiments, the modified siRNA comprises modified nucleotides
`including, but not limited to, 2'-0-methyl (2'OMe) nucleotides, 2'-deoxy-2'-fluoro (2'F)
`nucleotides, 2'-deoxy nucleotides, 2'-0-(2-methoxyethyl) (MOE) nucleotides, locked nucleic
`acid (LNA) nucleotides, and mixtures thereof. In preferred embodiments, the modified
`siRNA comprises 2'OMe nucleotides (e.g., 2'OMe purine and/or pyrimidine nucleotides) such
`as, for example, 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine
`nucleotides, 2'OMe-cytosine nucleotides, and mixtures thereof. In certain instances, the
`modified siRNA does not comprise 2'OMe-cytosine nucleotides. In other embodiments, the
`modified siRNA comprises a hairpin loop structure.
`[0013] The modified siRNA can comprise modified nucleotides in one strand (i.e., sense or
`antisense) or both strands of the double-stranded region of the siRNA molecule. Preferably,
`uridine and/or guanosine nucleotides are modified at selective positions in the double-
`stranded region of the siRNA duplex. With regard to uridine nucleotide modifications, at
`least one, two, three, four, five, six, or more of the uridine nucleotides in the sense and/or
`antisense strand can be a modified uridine nucleotide such as a 2'OMe-uridine nucleotide. In
`some embodiments, every uridine nucleotide in the sense and/or antisense strand is a 2'OMe-
`uridine nucleotide. With regard to guanosine nucleotide modifications, at least one, two,
`three, four, five, six, or more of the guanosine nucleotides in the sense and/or antisense strand
`can be a modified guanosine nucleotide such as a 2lOMe-guanosine nucleotide. In some
`embodiments, every guanosine nucleotide in the sense and/or antisense strand is a 2'OMe-
`guanosine nucleotide.
`[0014]
`In the SNALPs of the present invention, the cationic lipid may be, e.g., 1,2-
`Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-
`dimethylaminopropane (DLenDMA), N,N-dioleyl-N,N-dimethylammonium chloride
`(DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-
`dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),
`distearyldimethylammonium (DSDMA), N-(l-(2,3-dioleyloxy)propyl)-N,N,N-
`trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxypropylamine
`(DODMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Choi), N-(l,2-
`dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 2,3-
`dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-
`propanaminiumtrifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 3-
`dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1 -(cis,cis-9,12-
`octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-
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`dimethy-l-(cis,cis-9',l-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-
`dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane
`(DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-
`Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-
`dimethylaminopropane (DLinCDAP), and a mixture thereof. In a preferred embodiment, the
`cationic lipid is DLinDMA. The cationic lipid typically comprises from about 50 mol % to
`about 85 mol %, about 50 mol % to about 80 mol %, about 50 mol % to about 75 mol %,
`about 50 mol % to about 65 mol %, or about 55 mol % to about 65 mol % of the total lipid
`present in the particle.
`[0015] The non-cationic lipid in the SNALPs of the present invention may be an anionic
`lipid or a neutral lipid. In one embodiment, the non-cationic lipid comprises cholesterol or a
`derivative thereof. Examples of suitable cholesterol derivatives include, but are not limited
`to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether,
`and cholesteryl-4'-hydroxybutyl ether. In this embodiment, the cholesterol or cholesterol
`derivative comprises from about 30 mol % to about 40 mol % of the total lipid present in the
`particle. In another embodiment, the non-cationic lipid comprises a phospholipid. In yet
`another embodiment, the non-cationic lipid comprises a mixture of a phospholipid and
`cholesterol or a cholesterol derivative.
`[0016] Phospholipids suitable for use in either of these embodiments include, but are not
`limited to, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC),
`dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC),
`palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleyolphosphatidylglycerol
`(POPG), dipalmitoylphosphatidylethanolamine (DPPE),
`dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE),
`25 monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine,
`dielaidoylphosphatidylethanolamine (DEPE), stearoylolcoylphosphatidylethanolamine
`(SOPE), egg phosphatidylcholine (EPC), and a mixture thereof. When the non-cationic lipid
`is a mixture of a phospholipid and cholesterol or a cholesterol derivative, the phospholipid
`comprise from about 4 mol % to about 10 mol % of the total lipid present in the particle, and
`the cholesterol or cholesterol derivative comprises from about 30 mol % to about 40 mol %
`of the total lipid present in the particle. If a cholesterol derivative is used, the cholesterol
`derivative includes, but is not limited to, cholestanol, cholestanone, cholestenone,
`coprostanol, cholesteryl-2'-hydroxyethyl ether, and cholesteryl-4'-hydroxybutyl ether. In a
`preferred embodiment, the phospholipid comprises DPPC.
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`[0017] The SNAPs of the present invention also comprise a conjugated lipid that inhibits
`aggregation of the particles. Examples of suitable conjugated lipids include, but are not
`limited to, a polyethyleneglycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate,
`a cationic-polymer-lipid conjugates (CPLs), or mixtures thereof. In one preferred
`embodiment, the nucleic acid-lipid particles comprise either a PEG-lipid conjugate or an
`ATTA-lipid conjugate. In certain embodiments, the PEG-lipid conjugate or ATTA-lipid
`conjugate is used together with a CPL. In a preferred embodiment, the conjugated lipid is a
`PEG-lipid.
`[0018] Examples of suitable PEG-lipids include, but are not limited to, a PEG-
`diacylglycerol (DAG), a PEG dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-
`ceramide (Cer), or mixtures thereof. In a preferred embodiment, the PEG-lipid is a PEG-
`DAA conjugate. Examples of suitable PEG-DAA conjugates include, but are not limited to,
`PEG-dilauryloxypropyl (CI2), a PEG-dimyristyloxypropyl (CI4), a PEG-
`dipalmityloxypropyl (CI6), and a PEG-distearyloxypropyl (CI8). In a preferred
`embodiment, the PEG-DAA is PEG-dimyristyloxypropyl (C14). In another preferred
`embodiment, the PEG-DAA is PEG-distearyloxypropyl (CI8). The conjugated lipid
`typically comprises about 0.5 mol % to about 2 mol % of the total lipid present in the particle.
`In the SNALPs of the present invention, the nucleic acid is fully encapsulated in the
`[0019]
`lipid, thereby protecting the nucleic acid from nuclease degradation. In one embodiment, the
`nucleic acid in the nucleic acid-lipid particle is not substantially degraded after exposure of
`the particle to a nuclease at 370C for 20 minutes. In another embodiment, the nucleic acid in
`the nucleic acid-lipid particle is not substantially degraded after exposure of the particle to a
`nuclease at 370C for 30 minutes. Again, the nucleic acid is encapsulated in the nucleic acid-
`lipid particle.
`[0020] Typically, the SNALPs of the present invention have a lipidmuclcic acid ratio of
`about 1 to about 100. In a preferred embodiment, the SNALPs of the present invention have
`a lipid:nucleic acid ratio of about 5 to about 15. In another preferred embodiment, the
`SNALPs of the present invention have a lipid:nucleic acid ratio of about 6. Typically, the
`SNALPs of the present invention have a mean diameter of from about 50 nm to about 150
`nm. In a preferred embodiment, the SNALPs of the present invention have a mean diameter
`of from about 70 nm to about 90 nm.
`In another aspect, the present invention provides a nucleic acid-lipid particle
`[0021]
`comprising: (a) an siRNA; (b) a cationic lipid comprising from about 56.5 mol % to about
`66.5 mol % of the total lipid present in the particle; (c) a non-cationic lipid comprising from
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`about 31.5 mol % to about 42.5 mol % of the total lipid present in the particle; and (d) a
`conjugated lipid that inhibits aggregation of particles comprising from about 1 mol % to
`about 2 mol % of the total lipid present in the particle. This embodiment of SNALP is
`generally referred to herein as the "1:62" formulation. In a preferred embodiment, the nucleic
`acid is a small interfering RNA (siRNA). In another preferred embodiment, the cationic lipid
`is DLinDMA, the non-cationic lipid is cholesterol and the conjugated lipid is a PEG-DAA
`conjugate. Although these are preferred embodiments of the 1:62 formulation, those of skill
`in the art will appreciate that other cationic lipids, non-cationic lipids, including other
`cholesterol derivatives, and conjugated lipids can be used in the 1:62 formulation as
`described herein.
`In another aspect, the present invention provides a nucleic acid-lipid particle
`[0022]
`comprising: (a) an siRNA; (b) a cationic lipid comprising from about 52 mol % to about 62
`mol % of the total lipid present in the particle; (c) a non-cationic lipid comprising from about
`36 mol % to about 47 mol % of the total lipid present in the particle; and (d) a conjugated
`lipid that inhibits aggregation of particles comprising from about 1 mol % to about 2 mol %
`of the total lipid present in the particle. This embodiment of SNALP is generally referred to
`herein as the " 1:57" formulation. In a preferred embodiment, the nucleic acid is a small
`interfering RNA (siRNA). In another preferred embodiment, the cationic lipid is DLinDMA,
`the non-cationic lipid is a mixture of a phospholipid (such as DPPC) and cholesterol, wherein
`the phospholipid comprises about 5 mol % to about 9 mol % of the total lipid present in the
`particle, and the cholesterol (or cholesterol derivative) comprises about 32 mol % to about 37
`mol % of the total lipid present in the particle, and the PEG-lipid is PEG-DAA. Although
`these are preferred embodiments of the 1:57 formulation, those of skill in the art will
`appreciate that other cationic lipids, non-cationic lipids (including other phospholipids and
`other cholesterol derivatives) and conjugated lipids can be used in the 1:57 formulation as
`described herein.
`In yet another aspect, the present invention provides a nucleic acid-lipid particle and
`[0023]
`a pharmaceutically acceptable carrier.
`In a further aspect, the present invention provides a method for introducing a
`[0024]
`nucleic acid into a cell, the method comprising contacting the cell with a nucleic acid-lipid
`particle of the present invention. In one embodiment, the cell is in a mammal and the
`mammal is a human. In still a further embodiment, the present invention provides a method
`for the in vivo delivery of a nucleic acid, the method comprising administering to a
`mammalian subject a nucleic acid-lipid particle fo the present invention. In a preferred
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`embodiment, the mode of administration includes, but is not limited to, oral, intranasal,
`intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal,
`subcutaneous, and intradermal. In a preferred embodiment, the mammalian subject is a
`human.
`[0025] As explained herein, the nucleic acid-lipid particles of the present invention are
`useful for the therapeutic delivery of nucleic acids comprising an interfering RNA sequence.
`In particular, it is an 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 transcription and
`translation of a target nucleic acid sequence of interest. In some embodiments, an interfering
`10 RNA is formulated into a nucleic acid-lipid particle, and the particles are administered to
`patients requiring such treatment. In other embodiments, cells are removed from a patient,
`the interfering RNA delivered in vitro, and reinjected into the patient. In one embodiment,
`the present invention provides for a method of introducing a nucleic acid into a cell by
`contacting a cell with a nucleic acid-lipid particle comprised of a cationic lipid, a non-
`cationic lipid, a conjugated lipid that inhibits aggregation, and an interfering RNA.
`[0026]
`In one embodiment, at least about 5%, 10%, 15%, 20%, or 25% of the total injected
`dose of the nucleic acid-lipid particles is present in plasma about 8, 12, 24, 36, or 48 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 about
`8, 12, 24, 36, or 48 hours after injection. In one embodiment, the presence of an interfering
`RNA in cells of the lung, liver, tumor or at a site of inflammation is detectable at about 8, 12,
`24, 36, 48, 60, 72 or 96 hours after administration. In one embodiment, downregulation of
`expression of the target sequence is detectable at about 8, 12, 24, 36, 48, 60, 72 or 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 an interfering RNA 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 an interfering RNA in of cells in the
`lung, liver or a tumor is detectable at least four days after injection of the nucleic acid-lipid
`particle. In another embodiment, the nucleic acid-lipid particle is administered parenterally
`or intraperitoneally.
`[0027] The SNALPs of the present invention 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
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`also provides for pharmaceutically acceptable compositions comprising a nucleic acid-lipid
`particle and a pharmaceutically acceptable carrier.
`[0028] Another embodiment of the present invention provides methods for in vivo delivery
`of interfering RNA. A nucleic acid-lipid particle comprising a cationic lipid, a non-cationic
`lipid, a conjugated lipid that inhibits aggregation of particles, and an interfering RNA (such
`as an siRNA molecule) is administered {e.g., intravenously) to a subject (e.g., a mammal 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.
`[0029] 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
`acid-lipid particle comprising a cationic lipid, a non-cationic lipid, a conjugated lipid that
`inhibits aggregation of particles, and an interfering RNA (such as an siRNA) is administered
`to the mammalian subject {e.g., a rodent such as a mouse, a primate such as a human or a
`monkey). 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 reduced by the
`interfering RNA.
`[0030] Other objects, features, and advantages of the present invention will be apparent to
`one of skill in the art from the following detailed description and figures.
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`BRIEF DESCRIPTION OF THE DRAWINGS
`[00311 Figure 1 illustrates data demonstrating the activity of 1:57 SNALPs containing Eg5
`siRNA in a human colon cancer cell line.
`[0032] Figure 2 illustrates data demonstrating the activity of 1:57 SNALPs containing
`ApoB siRNA following intravenous administration in mice.
`[0033] Figure 3 illustrates additional data demonstrating the activity of 1:57 SNALPs
`containing ApoB siRNA following intravenous administration in mice. Each bar represents
`the group mean of five animals. Error bars indicate the standard deviation.
`[0034] Figure 4 illustrates data demonstrating the activity of 1:57 and 1:62 SNALPs
`containing ApoB siRNA following intravenous administration in mice.
`[0035] Figure 5 illustrates data demonstrating the activity of 1:62 SNALPs containing
`30 ApoB siRNA following intravenous administration in mice.
`[0036] Figure 6 illustrates data demonstrating that the tolerability of 1:57 SNALPs
`containing ApoB siRNA prepared by citrate buffer versus PBS direct dilution did not differ
`significantly in terms of blood clinical chemistry parameters.
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`[0037] Figure 7 illustrates data demonstrating that the efficacy of 1:57 SNALPs containing
`ApoB siRNA prepared by gear pump was similar to the same SNALP prepared by syringe
`press.
`[0038] Figure 8 illustrates data demonstrating that there was very little effect on body
`5 weight 24 hours after administration of 1:57 SNALPs containing ApoB siRNA.
`[0039] Figure 9 illustrates data demonstrating that there were no obvious changes i