(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`5 August 2010 (05.08.2010)
`
`(10) International Publication Number
`WO 2010/088537 A2
`
`(51) International Patent Classification:
`A61K 9/127 (2006.01)
`
`(21) International Application Number:
`PCT/US20 10/0226 14
`
`(22) International Filing Date:
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`29 January 2010 (29.01 .2010)
`
`MANOHARAN, Muthiah [US/US]; Alnylam Pharma
`ceuticals, Inc., 300 Third Street, Cambridge, MA 02142
`(US). JAYARAMAN, Muthusamy [-/US]; Alnylam
`Pharmaceuticals,
`Inc., 300 Third Street, Cambridge, MA
`02142 (US). RAJEEV, Kallanthottathil,
`G . [IN/US];
`Alnylam Pharmaceuticals,
`Inc., 300 Third Street, Cam
`bridge, MA 02142 (US).
`
`English
`
`English
`
`(74) Agent: MCCARTY, Catherine, M.; Lowrie, Lando &
`Anastasi LLP, One Main Street, Eleventh Floor, Cam
`bridge, MA 02142 (US).
`
`(30) Priority Data:
`61/148,366
`61/156,851
`61/185,7 12
`61/228,373
`61/239,686
`
`29 January 2009 (29.01 .2009)
`2 March 2009 (02.03.2009)
`10 June 2009 (10.06.2009)
`24 July 2009 (24.07.2009)
`3 September 2009 (03.09.2009)
`
`US
`us
`us
`us
`us
`(71) Applicant (for all designated States except US): ALNY-
`LAM PHARMACEUTICALS,
`INC.
`[US/US]; 300
`Third Street, Cambridge, MA 02142 (US).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO,
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI,
`NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD,
`SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR,
`TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(72) Inventors; and
`(75) Inventors/ Applicants (for US only): AKINC, Akin [US/
`(84) Designated States (unless otherwise indicated, for every
`US]; Alnylam Pharmaceuticals,
`Inc., 300 Third Street,
`kind of regional protection available): ARIPO (BW, GH,
`Cambridge, MA 02142 (US). QUERBES, William
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`[—/US]; Alnylam Pharmaceuticals, Inc., 300 Third Street,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ,
`Cambridge, MA 02142 (US). WONG, Frances [-/US];
`TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
`Alnylam Pharmaceuticals,
`Inc., 300 Third Street, Cam
`ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
`Joseph, Robert [—/
`bridge, MA 02142 (US). DORKIN,
`MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM,
`US]; Alnylam Pharmaceuticals,
`Inc., 300 Third Street,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`Cambridge, MA 02142 (US). QIN, Xiaojun; Alnylam
`ML, MR, NE, SN, TD, TG).
`Pharmaceuticals,
`Inc., 300 Third Street, Cambridge, MA
`02142 (US). CANTLEY, William [-/US]; Alnylam Published:
`Pharmaceuticals,
`Inc., 300 Third Street, Cambridge, MA
`02142 (US). BORODOVSKY, Anna [-/US]; Alnylam — without international search report and to be republished
`upon receipt of that report (Rule 48.2(g))
`Pharmaceuticals,
`Inc., 300 Third Street, Cambridge, MA
`02142 (US). DE, Soma [-/US]; Alnylam Pharmaceuti
`cals, Inc., 300 Third Street, Cambridge, MA 02142 (US).
`
`(54) Title: IMPROVED LIPID FORMULATION
`
`(57) Abstract: The invention features an improved lipid formulation comprising a cationic lipid of formula (A), a neutral lipid, a
`sterol and a PEG or PEG-modified lipid, where R i and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally sub
`stituted, and R 3 and R4 are independently lower alkyl or R 3 and R4 can be taken together to form an optionally subsituted hetero
`cyclic ring. In one embodiment, Ri and R 2 are independently selected from oleoyl, pamitoyl, steroyl, linoleyl and R 3 and R 4 are
`methyl. Also disclosed are targeting lipids, and specific lipid formulations comprising such targeting lipids.
`
`ARBUTUS - EXHIBIT 2014
`Moderna Therapeutics, Inc. v. Arbutus Biopharma Corporation
`IPR2019-00554
`
`

`

`IMPROVED LIPID FORMULATION
`
`Claim of Priority
`This application claims priority from U .S .S.N. 61/148,366, filed January 29,
`
`2009; U.S.S.N. 61/156,851, filed March 2, 2009; U.S.S.N. 61/185,712, filed June 10,
`
`2009; U.S.S.N. 61/228,373, filed July 24, 2009; and U.S.S.N. 61/239,686, filed
`
`September 3, 2009, each of which is incorporated by reference in its entirety.
`
`Technical Field
`The invention relates to the field of therapeutic agent delivery using lipid
`
`particles. In particular, the invention provides cationic lipids and lipid particles
`
`comprising these lipids, which are advantageous for the in vivo delivery of nucleic
`
`acids, as well as nucleic acid-lipid particle compositions suitable for in vivo
`
`therapeutic use. Additionally, the invention provides methods of preparing these
`
`compositions, as well as methods of introducing nucleic acids into cells using these
`
`compositions, e.g., for the treatment of various disease conditions.
`
`Description of the Related Art
`
`Therapeutic nucleic acids include, e.g., small interfering RNA (siRNA), micro
`
`RNA (miRNA), antisense oligonucleotides, ribozymes, plasmids, and immune
`
`stimulating nucleic acids. These nucleic acids act via a variety of mechanisms. In the
`
`case of siRNA or miRNA, these nucleic acids can down-regulate intracellular levels
`
`of specific proteins through a process termed RNA interference (RNAi). Following
`
`introduction of siRNA or miRNA into the cell cytoplasm, these double-stranded RNA
`
`constructs can bind to a protein termed RISC. The sense strand of the siRNA or
`
`miRNA 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
`
`siRNA or miRNA. Having bound the complementary mRNA the RISC complex
`
`cleaves the mRNA and releases the cleaved strands. RNAi can provide down-
`
`regulation of specific proteins by targeting specific destruction of the corresponding
`
`mRNA that encodes for protein synthesis.
`
`

`

`The therapeutic applications of RNAi are extremely broad, since siRNA and
`
`miRNA constructs can be synthesized with any nucleotide sequence directed against a
`
`target protein. To date, siRNA constructs have shown the ability to specifically
`
`down-regulate target proteins in both in vitro and in vivo models. In addition, siRNA
`
`constructs are currently being evaluated in clinical studies.
`
`However, two problems currently faced by siRNA or miRNA constructs are,
`
`first, their susceptibility to nuclease digestion in plasma and, second, their limited
`
`ability to gain access to the intracellular compartment where they can bind RISC
`
`when administered systemically as the free siRNA or miRNA. These double-stranded
`
`constructs can be stabilized by incorporation of chemically modified nucleotide
`
`linkers within the molecule, for example, phosphothioate groups. However, these
`
`chemical modifications provide only limited protection from nuclease digestion and
`
`may decrease the activity of the construct. Intracellular delivery of siRNA or miRNA
`
`can be facilitated by use of carrier systems such as polymers, cationic liposomes or by
`
`chemical modification of the construct, for example by the covalent attachment of
`
`cholesterol molecules. However, improved delivery systems are required to increase
`
`the potency of siRNA and miRNA molecules and reduce or eliminate the requirement
`
`for chemical modification.
`
`Antisense oligonucleotides and ribozymes can also inhibit mRNA translation
`
`into protein. In the case of antisense constructs, these single stranded deoxynucleic
`
`acids have a complementary sequence to that of the target protein mRNA and can
`
`bind to the mRNA by Watson-Crick base pairing. This binding either prevents
`
`translation of the target mRNA and/or triggers RNase H degradation of the mRNA
`
`transcripts. Consequently, antisense oligonucleotides have tremendous potential for
`
`specificity of action {i.e., down-regulation of a specific disease-related protein). To
`
`date, these compounds have shown promise in several in vitro and in vivo models,
`
`including models of inflammatory disease, cancer, and HIV (reviewed in Agrawal,
`
`Trends in Biotech. 14:376-387 (1996)). Antisense can also affect cellular activity by
`
`hybridizing specifically with chromosomal DNA. Advanced human clinical
`
`assessments of several antisense drugs are currently underway. Targets for these
`
`drugs include the bcl2 and apolipoprotein B genes and mRNA products.
`
`

`

`One well known problem with the use of therapeutic nucleic acids relates to
`
`the stability of the phosphodiester internucleotide linkage and the susceptibility of this
`
`linker to nucleases. The presence of exonucleases and endonucleases in serum results
`
`in the rapid digestion of nucleic acids possessing phosphodiester linkers and, hence,
`
`therapeutic nucleic acids can have very short half-lives in the presence of serum or
`
`within cells. (Zelphati, O., et al., Antisense. Res. Dev. 3:323-338 (1993); and Thierry,
`
`A.R., et al, ppl47-161 in Gene Regulation: Biology of Antisense RNA and DNA
`
`(Eds. Erickson, RP and Izant, JG; Raven Press, NY (1992)). Therapeutic nucleic acid
`
`being currently being developed do not employ the basic phosphodiester chemistry
`
`found in natural nucleic acids, because of these and other known problems.
`
`This problem has been partially overcome by chemical modifications that
`
`reduce serum or intracellular degradation. Modifications have been tested at the
`
`internucleotide phosphodiester bridge (e.g., using phosphorothioate,
`
`methylphosphonate or phosphoramidate linkages), at the nucleotide base (e.g., 5-
`
`propynyl-pyrimidines), or at the sugar (e.g., 2' -modified sugars) (Uhlmann E., et al
`
`Antisense: Chemical Modifications. Encyclopedia of Cancer, Vol. X., pp 64-81
`
`Academic Press Inc. (1997)). Others have attempted to improve stability using 2'-5'
`
`sugar linkages (see, e.g., US Pat. No. 5,532,130). Other changes have been
`
`attempted. However, none of these solutions have proven entirely satisfactory, and in
`
`vivo free therapeutic nucleic acids still have only limited efficacy.
`
`In addition, as noted above relating to siRNA and miRNA, problems remain
`
`with the limited ability of therapeutic nucleic acids to cross cellular membranes (see,
`
`Vlassov, et al, Biochim. Biophys. Acta 1197:95-1082 (1994)) and in the problems
`
`associated with systemic toxicity, such as complement-mediated anaphylaxis, altered
`
`coagulatory properties, and cytopenia (Galbraith, et al, Antisense Nucl Acid Drug
`
`Des. 4:201-206 (1994)).
`
`In spite of recent progress, there remains a need in the art for improved lipid-
`
`therapeutic nucleic acid compositions that are suitable for general therapeutic use.
`
`Preferably, 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 intracellular
`
`

`

`delivery of the encapsulated nucleic acid. In addition, these lipid-nucleic acid
`
`particles should be well-tolerated and provide an adequate therapeutic index, such that
`
`patient treatment at an effective dose of the nucleic acid is not associated with
`
`significant toxicity and/or risk to the patient. The invention provides such
`
`compositions, methods of making the compositions, and methods of using the
`
`compositions to introduce nucleic acids into cells, including for the treatment of
`
`diseases.
`
`Summary of Invention
`
`In one aspect, the invention provides improved lipid formulations comprising
`
`a cationic lipid of formula A, a neutral lipid, a sterol and a PEG or PEG-modified
`
`lipid, wherein formula A is
`
`, where Ri and R2 are independently
`alkyl, alkenyl or alkynyl, each can be optionally substituted, and R and R are
`
`independently lower alkyl or R and R can be taken together to form an optionally
`subsituted heterocyclic ring. In one embodiment, Ri and R2 are independently
`selected from oleoyl, pamitoyl, steroyl, linoleyl and R3 and R4 are methyl.
`In one aspect, the improved lipid formulation also includes a targeting lipid
`
`(e.g., a GaINAc and/or folate containing lipid).
`
`In one aspect, the invention provides preparation for the improved lipid
`
`formulations via an extrusion or an in-line mixing method.
`
`In one aspect, the invention further provides a method of administering the
`
`improved lipid formulations containing RNA-based construct to an animal, and
`
`evaluating the expression of the target gene.
`
`In one aspect, a lipid formulation featured in the invention, such as a lipid
`
`formulation complexed with an oligonucleotide, such as a double stranded RNA
`
`(dsRNA), can be used to modify (e.g., decrease) target gene expression in a tumor cell
`
`

`

`in vivo or in vitro. In some embodiments, a lipid formulation featured in the
`
`invention can be used to modify target gene expression in a tumor cell line, including
`
`but not limited to HeLa, HCTl 16, A375, MCF7, B16F10, Hep3b, HUH7, HepG2,
`
`Skov3, U87, and PC3 cell lines.
`
`Brief Description of the Figures
`
`FIG. 1 is a flow chart of the extrusion method.
`
`FIG. 2 is a flow chart of the in-line mixing method.
`
`FIG. 3 is a schematic of a pump set-up.
`
`FIG. 4 is a graph showing the relative FVII protein with various lipid ratios.
`
`FIG. 5 is a graph showing the effect on body weight change with various lipid
`
`ratios.
`
`FIG. 6 is a graph illustrating the relative FVII protein with different amount of
`
`cationic lipid A and low PEG lipid.
`
`FIG. 7 is a graph showing the effect on body weight change with different
`
`amount of cationic lipid A and low PEG lipid.
`
`FIG. 8 is a graph illustrating the relative FVII protein with different types of
`
`phosphatidylcholine.
`
`FIG. 9 is a graph illustrating the relative FVII protein with high mol% of
`
`cationic lipid A .
`
`FIG. 10 is a graph illustrating the relative FVII protein with different
`
`cholesterol:PEG ratios.
`
`FIG. 11 is a graph illustrating the relative FVII protein at different pH levels.
`
`FIG. 12 is a graph showing the relative FVII protein with various lipid ratios
`
`prepared via an in-line mixing method.
`
`FIG. 13 is a graph showing the relative FVII protein at different charge ratios.
`
`FIG. 14 is a graph showing the efficacy of various formulations in mouse.
`
`FIGs. 15a and 15b are graphs showing the efficacy of various formulations in
`
`rat; (a) formulations preprared via an extrusion process; (b) formulations prepared via
`
`an in-line mixing process.
`
`

`

`FIGs. 16a-16c compare the effect of ApoE pre-association on (a) LNPOl, (b)
`
`SNALP, (c) LNP05.
`
`FIG. 17 depicts graphs that show the ApoE dependence of efficacy of
`
`formulations comprising LNP08. Wildtype but not ApoE knockout mice showed
`
`dose-dependent reduction in FVII protein levels.
`
`FIG. 18 depicts a graph that demonstrates that ApoE dependence of the
`
`LNP09 liposomal formulation and the lack of silencing in ApoE KO mice using
`
`LNP09 can be effectively rescued by premixing with ApoE.
`
`FIGs. 19a and 19b depict graphs that demonstrate in vivo results of a mouse
`
`FVII silencing model, wherein LNP08 formulations also containing varying amounts
`
`GalNAc3-DSG or GalNAc3-PEG-DSG are administered to ApoE deficient (KO)
`
`mice.
`
`FIG. 20 is a graph showing the efficacy of Lipid A liposomal formulations
`containing (GaINAc) 3-PEG-LCO in ApoE KO mice.
`FIG. 2 1 is a graph showing the efficacy of Lipid A liposomal formulations
`containing (GaINAc) 3-PEG-DSG in ApoE KO mice.
`FIG. 22 is a graph showing the effect of precomplexing with varying amounts
`
`of ApoE on the uptake of LNPOl and LNP08 formulations in Hep3B cells (4 hours
`
`incubation).
`
`FIG. 23 depicts increased uptake of siRNA as well as lipid carrier in the
`
`presence of ApoE in Hep3B cells as demonstrated by BODIPY labeling of lipid A .
`
`FIG. 24 depicts the effect of ApoE on silencing in HeLa-GFP cells (2OnM
`
`with serum). ApoE was pre-complexed with liposomes for 10 minutes at 37°C.
`
`FIG. 25 depicts a graph that demonstrates the effect of ApoE on silencing in
`
`HeLa cells (2OnM serum-free DMEM). ApoE was pre-complexed with liposomes for
`
`1 hour at 37°C.
`
`FIG. 26 is a graph showing that other ApoE isoforms, ApoE2 or ApoE4,
`
`enhance LNP08 silencing comparably to ApoE3 in HeLa cells.
`
`FIG. 27 is a graph showing the uptake of folate liposome in KB cells as
`
`demonstrated by FACS.
`
`

`

`FIG. 28 is a graph showing the uptake of liposomes containing folate
`
`conjugated lipids in KB cells as demonstrated by microscopy.
`
`FIGs. 29a and 29b show silencing of GFP mediated by liposomal formulations
`
`containing folate conjugated lipids (a) in the presence of serum or (b) in the absence
`
`of serum.
`
`FIG. 30 is a bar graph illustrating the levels of relative serum FVII protein in a
`
`dose response study.
`
`FIG. 3 1 is a bar graph showing the efficacy of Lipid A liposomal formulations
`
`containing GalNAc3 in ApoE wildtype mice.
`
`FIG. 32 is a graph showing the time-dependent degradation of Lipid A
`
`liposomal formulation in 100 mM NaOAc buffer (pH=5).
`
`FIG. 33 a graph showing the effect of BHT on inhibition of the degration of
`
`Lipid A liposomal formulation.
`
`FIG. 34 is a graph showing the effect of vitamine E on inhibition of the
`
`degration of Lipid A liposomal formulation.
`
`FIG. 35 is a graph showing the effect of LNP09 on Serum FVII protein levels
`
`in wildtype and LDLR KO mice.
`
`FIG. 36 is a graph showing the effect of LNP09 in which 0.5 mol% of the
`
`PEG-DMG was replaced with GALNac3-PEG-lipid on Serum FVII protein levels in
`
`wildtype and LDLR KO mice.
`
`Detailed Description
`
`Described herein is an improved lipid formulation, which can be used, for
`
`example, as a delivering an agent, e.g., a nucleic acid-based agent, such as an RNA-
`
`based construct, to a cell or subject. Also described herein are methods of
`
`administering the improved lipid formulations containing an RNA-based construct to
`
`an animal, and in some embodiments, evaluating the expression of the target gene. In
`
`some embodiments the improved lipid formulation includes a targeting lipid (e.g., a
`
`targeting lipid described herein such as a GaINAc or folate containing lipid).
`
`The invention provides improved lipid formulations comprising a cationic
`
`lipid of formula A, a neutral lipid, a sterol and a PEG or PEG-modified lipid, wherein
`
`

`

`fop nula A is
`where Ri and R2 are independently alkyl, alkenyl
`or alkynyl, each can be optionally substituted, and R and R are independently lower
`
`alkyl or R and R can be taken together to form an optionally subsituted heterocyclic
`ring. In one embodiment, Ri and R2 are independently selected from oleoyl,
`pamitoyl, steroyl, linoleyl and R3 and R4 are methyl. In one embodiment, Ri and R2
`are linoleyl. In one embodiments, Ri and R2 are linoleyl and R3 and R4 are methyl.
`In one embodiment, the formulation include from about 25% to about 75% on
`
`a molar basis of cationic lipid of formula A e.g., from about 35 to about 65%, from
`
`about 45 to about 65%, about 60%, about 57.5%, about 57.1%, about 50% or about
`
`40% on a molar basis. In one embodiment, the cationic lipid of formula A is 2,2-
`
`Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (Lipid A).
`
`In one embodiment, the formuation includes from about 0% to about 15% on a
`
`molar basis of the neutral lipid e.g., from about 3 to about 12%, from about 5 to about
`
`10%, about 15%, about 10%, about 7.5%, about 7.1% or about 0% on a molar basis.
`
`In one embodiment, the neutral lipid is DPPC. In one embodiment, the neutral lipid is
`
`DSPC
`
`In one embodiment, the formulation includes from about 5% to about 50% on
`
`a molar basis of the sterol (e.g., about 15 to about 45%, about 20 to about 40%, about
`
`48%, about 40%, about 38.5%, about 35%, about 34.4%, about 31.5% or about 31%
`
`on a molar basis. In one embodiment, the sterol is cholesterol.
`
`In one embodiment, the formulation includes from about 0.1% to about 20%
`
`on a molar basis of the PEG or PEG-modified lipid (e.g., about 0.5 to about 10%,
`
`about 0.5 to about 5%, about 10%, about 5%, about 3.5%, about 1.5%, about 0.5%,
`
`or about 0.3% on a molar basis. In one embodiment, the PEG-modified lipid is PEG-
`
`DMG. In one embodiment, the PEG-modified lipid is PEG-cDMA.
`
`

`

`In one embodiment, the formulations of the inventions include 25-75% of
`
`cationic lipid of formula A, 0.5-15% of the neutral lipid, 5-50% of the sterol, and 0.5-
`
`20% of the PEG or PEG-modified lipid on a molar basis.
`
`In one embodiment, the formulations of the inventions include 35-65% of
`
`cationic lipid of formula A, 3-12% of the neutral lipid, 15-45% of the sterol, and 0.5-
`
`10% of the PEG or PEG-modified lipid on a molar basis.
`
`In one embodiment, the formulations of the inventions include 45-65% of
`
`cationic lipid of formula A, 5-10% of the neutral lipid, 25-40% of the sterol, and 0.5-
`
`5% of the PEG or PEG-modified lipid on a molar basis.
`
`In one embodiment, the formulations of the inventions include about 60% of
`
`cationic lipid of formula A, about 7.5% of the neutral lipid, about 3 1 % of the sterol,
`
`and about 1.5% of the PEG or PEG-modified lipid on a molar basis. In one preferred
`
`embodiment, the cationic lipid of formula A is 2,2-Dilinoleyl-4-dimethylaminoethyl-
`
`[1,3]-dioxolane, the neutral lipid is DSPC, the sterol is cholesterol and the PEG lipid
`
`is PEG-DMG. In one embodiment, the PEG or PEG modified lipid comprises a PEG
`
`molecule of an average molecular weight of 2,000 Da. In one embodiment, the PEG
`
`or PEG modified lipid is a compound of the following formula 1:
`
`In one embodiment,
`
`the PEG or PEG modified lipid is PEG-distyryl glycerol (PEG-DSG).
`
`In one embodiment, the PEG or PEG modified lipid is a compound of the
`
`formula 1 or PEG-DSG, wherein the PEG molecule has an average molecular weight
`
`of 2,000 Da.
`
`In one embodiment, the formulations of the inventions include about 57.5% of
`
`cationic lipid of formula A, about 7.5% of the neutral lipid, about 31.5 % of the sterol,
`
`and about 3.5% of the PEG or PEG-modified lipid on a molar basis. In one preferred
`
`embodiment, the cationic lipid of formula A is 2,2-Dilinoleyl-4-dimethylaminoethyl-
`
`[1,3]-dioxolane (Lipid A), the neutral lipid is DSPC, the sterol is cholesterol and the
`
`PEG lipid is PEG-DMG (also known as PEG-dimyristoyl glycerol (C14-PEG, or
`
`PEG-C14) (PEG with an average mol. Weight of 2000)).
`
`

`

`In one embodiment, the formulation of the inventions include about 57.1% of
`
`the cationic lipid of formula A, about 7.1% of the neutral lipid, about 34.4% of the
`
`sterol and about 1.4% of the PEG or PEG-modified lipid on a molar basis. In one
`
`preferred embodiment, the cationic lipid of formula A is 2,2-Dilinoleyl-4-
`
`dimethylaminoethyl-[1,3]-dioxolane (Lipid A), the neutral lipid is DPPC, the sterol is
`
`cholesterol and the PEG lipid is PEG-cDMA (also known as PEG-carbamoyl-1,2-
`
`dimyristyloxypropylamine (PEG with an average mol. weight of 2000)).
`
`In one embodiment, the formulation of the inventions include about 60% of
`
`the cationic lipid of formula A, about 7.5% of the neutral lipid, about 31% of the
`
`sterol and about 1.5% of the PEG or PEG-modified lipid on a molar basis. In one
`
`preferred embodiment, the cationic lipid of formula A is 2,2-Dilinoleyl-4-
`
`dimethylaminoethyl-[1,3]-dioxolane (Lipid A), the neutral lipid is DSPC, the sterol is
`
`cholesterol and the PEG lipid is PEG-DMG (also known as PEG-dimyristoyl glycerol
`
`(C14-PEG, or PEG-C14) (PEG with an average mol. Weight of 2000)).
`
`In one embodiment, the formulation of the inventions include about 50% of
`
`the cationic lipid of formula A, about 10% of the neutral lipid, about 38.5% of the
`
`sterol and about 1.5% of the PEG or PEG-modified lipid on a molar basis. In one
`
`preferred embodiment, the cationic lipid of formula A is 2,2-Dilinoleyl-4-
`
`dimethylaminoethyl-[1,3]-dioxolane (Lipid A), the neutral lipid is DSPC, the sterol is
`
`cholesterol and the PEG lipid is PEG-DMG (also known as PEG-dimyristoyl glycerol
`
`(C14-PEG, or PEG-C14) (PEG with an average mol. Weight of 2000)).
`
`In one embodiment, the ratio of lipid:siRNA is at least about 0.5:1, at least
`
`about 1:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at
`
`least about 6:1, at least about 7:1, at least about 11:1 or at least about 33:1. In one
`
`embodiment, the ratio of lipid: siRNA ratio is between about 1:1 to about 35:1, about
`
`3:1 to about 15:1, about 4:1 to about 15:1, about 5:1 to about 13:1. In one
`
`embodiment, the ratio of lipid:siRNA ratio is between about 0.5:1 to about 12:1.
`
`In one aspect, the improved lipid formulation also includes a targeting lipid.
`
`In some embodiments, the targeting lipid includes a GaINAc moiety (i.e., an N-
`
`galactosamine moiety). For example, a targeting lipid including a GaINAc moiety can
`
`include those disclosed in USSN 12/328,669, filed 12/4/2008, which is incorporated
`
`

`

`herein by reference in its entirety. A targeting lipid can also include any other lipid
`
`(e.g., targeting lipid) known in the art, for example, as described in USSN 12/328,669
`
`or International Publication No. WO 2008/042973, the contents of each of which are
`
`incorporated herein by reference in their entirety. In some embodiments, the targeting
`
`lipid includes a plurality of GaINAc moieties, e.g., two or three GaINAc moieties. In
`
`some embodiments, the targeting lipid contains a plurality, e.g., two or three N-
`
`acetylgalactosamine (GaINAc) moieties. In some embodiments, the lipid in the
`
`targeting lipid is l^-Di- O-hexadecyl-sra-glyceride (i.e., DSG). In some embodiments,
`
`the targeting lipid includes a PEG moiety (e.g., a PEG moiety having a molecular
`
`weight of at least about 500 Da, such as about 1000 Da, 1500 Da, 2000 Da or greater),
`
`for example, the targeting moiety is connected to the lipid via a PEG moiety.
`
`In some embodiments, the targeting lipid includes a folate moiety. For
`
`example, a targeting lipid including a folate moiety can include those disclosed in
`
`USSN 12/328,669, filed 12/4/2008, which is incorporated herein by reference in its
`
`entirety. In another embodiment, a targeting lipid including a folate moiety can
`
`include the compound of formula 5 .
`
`Exemplary targeting lipids are represented by formula L below:
`(Targeting group)n-L-Lipid
`formula L
`
`wherein:
`
`Targeting group is any targeting group that known by one skilled in the art
`
`and/or described herein (e.g., a cell surface receptor);
`
`n is an integer from 1 to 5, (e.g., 3)
`
`L is a linking group; and
`
`Lipid is a lipid such as a lipid described herein (e.g., a neutral lipid such as
`
`DSG).
`
`In some embodiments, the linking group includes a PEG moiety. In another
`
`embodiment, the PEG moiety can vary in size from a molecular weight of about 1,000
`
`to about 20,000 daltons (e.g., from about 1,500 to about 5,000 daltons, e.g., about
`
`1000 daltons, about 2000 daltons, about 3400 daltons, or about 5000 daltons.
`
`

`

`In some embodiments, the targeting lipid is a compound of formula 2, 3, 4, 5,
`
`or 7 as provided below:
`
`f
`
`GalNAc3-PEG-DSG
`
`GalNAc3-PEG-DSG
`
`

`

`Folate-PEG-DSPE
`
`1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene
`
`glycol)-2000] (ammonium salt)
`fop nula 5
`
`MoI Wt - 3028
`
`Folate-PEG2000-DSG
`fop nula 6
`
`MW ~ 4761
`Folate-PEG3400-DSG
`Fop nula 7
`In some embodiments, the targeting lipid is present in the formulation in an
`
`amount of from about 0.001% to about 5% (e.g., about 0.005%, 0.15%, 0.3%, 0.5%,
`
`1.5%, 2%, 2.5%, 3%, 4%, or 5%) on a molar basis. In some embodiments, the
`
`targeting lipid is included in a formulation described herein such as LNP05 or LNP08.
`
`In some embodiments, the lipid formulation also included an antioxidant (e.g.,
`
`a radical scavenger). The antioxidant can be present in the formulation, for example,
`
`at an amound from about 0.01% to about 5%. The antioxidant can be hydrophobic or
`
`hydrophilic (e.g., soluble in lipids or soluble in water). In some embodiments, the
`
`antioxidant is a phenolic compound, for example, butylhydroxytoluene, resveratrol,
`
`coenzyme QlO, or other flavinoids, or a vitamin, for example, vitamin E or vitamin C .
`
`Other exemplary antioxidants include lipoic acid, uric acid, a carotene such as beta-
`
`carotene or retinol (vitamin A), glutathione, melatonin, selenium, and ubiquinol.
`
`In some embodiments, the receptor for the targeting lipid (e.g., a GaINAc
`
`containing lipid) is the asialoglycoprotein receptor (i.e., ASGPR).
`
`

`

`In one embodiment, the formulations of the invention are produced via an
`
`extrusion method or an in-line mixing method.
`
`The extrusion method (also refer to as preformed method or batch process) is a
`
`method where the empty liposomes (i.e. no nucleic acid) are prepared first, followed
`
`by the the addition of nucleic acid to the empty liposome. Extrusion of liposome
`
`compositions through a small-pore polycarbonate membrane or an asymmetric
`
`ceramic membrane results in a relatively well-defined size distribution. Typically, the
`
`suspension is cycled through the membrane one or more times until the desired
`
`liposome complex size distribution is achieved. The liposomes may be extruded
`
`through successively smaller-pore membranes, to achieve a gradual reduction in
`
`liposome size. In some instances, the lipid-nucleic acid compositions which are
`
`formed can be used without any sizing. These methods are disclosed in the US
`
`5,008,050; US 4,927,637; US 4,737,323; Biochim Biophys Acta. 1979 Oct
`
`19;557(l):9-23; Biochim Biophys Acta. 1980 Oct 2;601(3):559-7; Biochim Biophys
`
`Acta. 1986 Jun 13;858(l):161-8; and Biochim. Biophys. Acta 1985 812, 55-65, which
`
`are hereby incorporated by reference in their entirety.
`
`The in-line mixing method is a method wherein both the lipids and the nucleic
`
`acid are added in parallel into a mixing chamber. The mixing chamber can be a
`
`simple T-connector or any other mixing chamber that is known to one skill in the art.
`
`These methods are disclosed in US patent nos. 6,534,018 and US 6,855,277; US
`
`publication 2007/0042031 and Pharmaceuticals Research, Vol. 22, No. 3, Mar. 2005,
`
`p . 362-372, which are hereby incorporated by reference in their entirety.
`
`It is further understood that the formulations of the invention can be prepared
`
`by any methods known to one of ordinary skill in the art.
`
`In a further embodiment, representative formulations prepared via the
`
`extrusion method are delineated in Table 1, wherein Lipid A is a compound of
`
`formula A, where Ri and R are linoleyl and R and R are methyl, i.e., 2,2-
`
`Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane. References to "Lipid A"
`
`throughout the application, for example, in other tables and in the Examples, refer to
`this same lipid of formula A, where Ri and R2 are linoleyl and R3 and R4 are methyl,
`unless explicitly otherwise defined.
`
`

`

`Composition (mole %)
`
`Lipid
`A
`
`DSPC
`
`Choi
`
`PEG
`
`siRNA
`
`20
`20
`20
`20
`20
`
`30
`30
`30
`30
`30
`
`40
`40
`40
`40
`40
`
`30
`30
`30
`30
`30
`
`20
`20
`20
`20
`20
`
`10
`10
`10
`10
`10
`
`40
`40
`40
`40
`40
`
`40
`40
`40
`40
`40
`
`40
`40
`40
`40
`40
`
`1955
`1955
`1955
`1955
`1955
`
`1955
`1955
`1955
`1955
`1955
`
`1955
`1955
`1955
`1955
`1955
`
`10
`10
`10
`10
`10
`
`10
`10
`10
`10
`10
`
`10
`10
`10
`10
`10
`
`10
`10
`10
`10
`10
`
`Lipid
`A/
`siRN
`A
`2.13
`2.35
`2.37
`3.23
`3.91
`
`2.89
`3.34
`3.34
`4.10
`5.64
`
`3.02
`3.35
`3.74
`5.80
`8.00
`
`Table 1
`
`Charge
`ratio
`
`Total
`Lipid/
`siRNA
`
`Entrapment
`(%)
`
`Zeta
`potential
`
`Particle
`size
`(nm)
`
`39
`53
`70
`77
`85
`
`44
`57
`76
`93
`90
`
`57
`77
`92
`89
`86
`
`12.82
`14. 15
`14.29
`19.48
`23.53
`
`11.36
`13. 16
`13. 16
`
`16. 13
`22.22
`
`8.77
`9.74
`10.87
`16.85
`23.26
`
`1.12
`1.23
`1.25
`1.70
`2.05
`
`1.52
`1.76
`1.76
`2.15
`2.97
`
`1.59
`1.76
`1.97
`3.05
`4.20
`
`1.72
`1.74
`2.34
`3.68
`5.15
`
`-0.265
`-0.951
`0.374
`5.89
`10.7
`
`-9.24
`-4.32
`-1.75
`3.6
`4.89
`
`-12.3
`7.73
`13.2
`13.8
`14.7
`
`-10.7
`12.6
`12.4
`13.2
`13.9
`
`85.3
`86.8
`79. 1
`81.4
`80.3
`
`82.7
`76.3
`74.8
`72.8
`70.8
`
`63.3
`57
`56.9
`64
`65.2
`
`56.4
`40.8
`51.4
`78. 1
`64.2
`
`PDI
`
`0 .109
`0.081
`0.201
`0.099
`0 .105
`
`0 .142
`0.083
`0.067
`0.082
`0.202
`
`0 .146
`0 .192
`0.203
`0 .109
`0 .132
`
`0.219
`0.238
`0.099
`0.055
`0 .106
`
`45
`45
`45
`45
`45
`
`50
`
`20
`20
`20
`20
`20
`
`30
`30
`30
`30
`30
`
`40
`40
`40
`
`5
`5
`5
`5
`5
`
`0
`
`35
`35
`35
`35
`35
`
`25
`25
`25
`25
`25
`
`15
`
`15
`
`15
`
`40
`40
`40
`40
`40
`
`40
`
`40
`40
`40
`40
`40
`
`40
`40
`40
`40
`40
`
`40
`40
`40
`
`1955
`1955
`1955
`1955
`1955
`
`1955
`
`1955
`1955
`1955
`1955
`1955
`
`1955
`1955
`1955
`1955
`1955
`
`1955
`1955
`1955
`
`3.27
`3.30
`4.45
`7.00
`9.80
`27.0
`3
`3.00
`3.32
`3.05
`3.67
`4.71
`
`2.47
`2.98
`3.29
`4.99
`7.15
`
`2.79
`3.29
`4.33
`
`8.33
`8.43
`11.36
`17.86
`25.00
`
`14.21
`
`68.97
`
`1.58
`1.75
`1.60
`1.93
`2.48
`
`1.30
`1.57
`1.73
`2.62
`3.76
`
`1.46
`1.73
`2.28
`
`16. 13
`17.86
`16.39
`19.74
`25.32
`
`8.62
`10.42
`11.49
`17.44
`25.00
`
`7.14
`8.43
`11. 11
`
`10
`
`5
`5
`5
`5
`5
`
`5
`5
`5
`5
`5
`
`5
`5
`5
`
`60
`89
`88
`84
`80
`
`29
`
`31
`42
`61
`76
`79
`
`58
`72
`87
`86
`80
`
`70
`89
`90
`
`42.0
`
`76.8
`79.3
`64.4
`72.9
`76.6
`
`79. 1
`74. 1
`72.5
`72.3
`75.8
`
`65.4
`58.8
`62.3
`
`0 .155
`
`0.068
`0.093
`0.12
`0 .161
`0.067
`
`0 .153
`0.046
`0.079
`0.057
`0.069
`
`0.068
`0.078
`0.093
`
`-8. 14
`-4.88
`-4.48
`3.89
`10.7
`
`-2.8
`-2.73
`13.6
`14.6
`13.8
`
`-3.52
`13.3
`14.9
`
`

`

`WO 2010/