(12) United States Patent
`YaWorski et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 9.404,127 B2
`* Aug. 2, 2016
`
`USOO9404127B2
`
`(54) NON-LIPOSOMAL SYSTEMS FOR NUCLEIC
`ACD DELVERY
`
`(56)
`
`(71) Applicant: PROTIVA BIOTHERAPEUTICS,
`INC., Burnaby (CA)
`
`(72) Inventors: Ed Yaworski, Maple Ridge (CA); Lloyd
`B. Jeffs, Delta (CA); Lorne R. Palmer,
`Vancouver (CA)
`
`(73) Assignee: PROTIVA BIOTHERAPEUTICS,
`INC., Burnaby, BC (CA)
`
`(*) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`This patent is Subject to a terminal dis
`claimer.
`
`(21) Appl. No.: 14/642,452
`
`(22) Filed:
`
`Mar. 9, 2015
`
`(65)
`
`Prior Publication Data
`US 2016/003232O A1
`Feb. 4, 2016
`
`Related U.S. Application Data
`(63) Continuation of application No. 13/807,288, filed as
`application No. PCT/CA2011/000778 on Jun. 30,
`2011, now Pat. No. 9,006,417.
`(60) Provisional application No. 61/360,480, filed on Jun.
`30, 2010.
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2010.01)
`(2006.01)
`
`(51) Int. Cl.
`C7H 2L/04
`CI2N 5/88
`A 6LX 9/07
`A 6LX 9/5
`A6 IK3I/7088
`A 6LX3L/705
`A6 IK3I/72
`A6 IK3I/713
`A6 IK 47/4
`CI2N IS/II3
`A61 K9/127
`(52) U.S. Cl.
`CPC .............. CI2N 15/88 (2013.01); A61K 9/1075
`(2013.01); A61 K9/5 123 (2013.01); A61 K
`3 1/7088 (2013.01); A61 K3I/712 (2013.01):
`A61 K3I/713 (2013.01); A61 K3I/7105
`(2013.01); A61 K47/14 (2013.01): CI2N
`15/113 (2013.01); A61 K9/1272 (2013.01);
`A61 K9/1274 (2013.01); C12N 23 10/14
`(2013.01); C12N 23.10/321 (2013.01)
`(58) Field of Classification Search
`None
`See application file for complete search history.
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`7/1983 Szoka, Jr. et al.
`4,394,448 A
`3, 1984 Weder et al.
`4,438,052 A
`5, 1985 Deamer
`4,515,736 A
`7, 1986 Papahadjopoulos et al.
`4,598,051 A
`1/1990 Eppstein et al.
`4,897,355 A
`5/1991 Woodle et al.
`5,013,556 A
`5,171,678 A 12/1992 Behr et al.
`5,208,036 A
`5/1993 Eppstein et al.
`5,225,212 A
`7/1993 Martin et al.
`5,264,618 A 1 1/1993 Felgner et al.
`5,279,833 A
`1/1994 Rose
`5,283,185 A
`2/1994 Epand et al.
`5,320,906 A
`6/1994 Eley et al.
`5,334,761 A
`8/1994 Gebeyehu et al.
`5,545,412 A
`8/1996 Eppstein et al.
`5,578.475 A 11/1996 Jessee et al.
`5,627,159 A
`5, 1997 Shih et al.
`5,641,662 A
`6/1997 Debs et al.
`5,656,743 A
`8, 1997 Busch et al.
`5,674,908 A 10, 1997 Haces et al.
`5,703,055 A 12/1997 Felgner et al.
`5,705.385 A
`1/1998 Bally et al.
`5,736,392 A
`4/1998 Hawley-Nelson et al.
`5,820,873. A 10, 1998 Choi et al.
`5,877,220 A
`3, 1999 Schwartz et al.
`5,885,613 A
`3, 1999 Holland et al.
`5,958,901 A
`9/1999 Dwyer et al.
`5,976,567 A 11/1999 Wheeler et al.
`5,981,501 A 11/1999 Wheeler et al.
`(Continued)
`
`FOREIGN PATENT DOCUMENTS
`
`CA
`CA
`
`4f1999
`2309.727 A1
`2271582 A1 11, 1999
`(Continued)
`
`OTHER PUBLICATIONS
`
`Arpicco, S., et al., “Preparation and Characterization of Novel
`Cationic Lipids Developed for Gene Transfection.” Proceed. Int’l
`Symp. Control. Rel. Bioact. Mater. (Controlled Release Society,
`Inc.), 1999, vol. 26, pp. 759-760.
`(Continued)
`
`Primary Examiner — Kimberly Chong
`(74) Attorney, Agent, or Firm — Kilpatrick Townsend &
`Stockton LLP
`
`ABSTRACT
`(57)
`The present invention provides novel, stable lipid particles
`having a non-lamellar structure and comprising one or more
`active agents or therapeutic agents, methods of making Such
`lipid particles, and methods of delivering and/or administer
`ing Such lipid particles. More particularly, the present inven
`tion provides stable nucleic acid-lipid particles (SNALP) that
`have a non-lamellar structure and that comprise a nucleic acid
`(such as one or more interfering RNA), methods of making
`the SNALP and methods of delivering and/or administering
`the SNALP.
`
`22 Claims, 24 Drawing Sheets
`
`PROTIVA - EXHIBIT 2029
`Moderna Therapeutics, Inc. v. Protiva Biotherapeautics, Inc. - IPR2018-00739
`
`

`

`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`6,020,202
`6,020,526
`6,034,135
`6,051.429
`6,075,012
`6,165,501
`6,172,049
`6,251,939
`6,284.267
`6,287,591
`6,339,173
`6,376,248
`6,534,484
`6,586.410
`6,638,529
`6,649,780
`6,671,393
`6,696,424
`6,815,432
`6,858,224
`7,166,745
`7,422,902
`7,479,573
`7,601,872
`7,687,070
`7,745,651
`7,799,565
`7,803,397
`7,807,815
`7,838,658
`7,901,708
`7,915,450
`7,982,027
`8,058,068
`8,058,069
`8, 101,741
`8, 158,827
`8, 188,263
`8,227.443
`8,236,943
`8,283,333
`8,455.455
`8.492.359
`8,513,403
`8,569.256
`8,598.333
`8,822,668
`9,006,417
`2001/0048940
`2003, OO69.173
`2003/OO72794
`2003/OO77829
`2003.0143732
`2004, OO63654
`2004/O142892
`2004/O253723
`2004/O259247
`2005, OO64595
`2005, 0118253
`2005/026O757
`2006, OOO8910
`2006/O147514
`2006/02284.06
`2006/0240093
`2007/OO42O31
`2007/0202598
`2007/0202600
`2009.0143583
`2009,0291131
`2010/O130588
`2010. O159593
`2012/0058.188
`2012/0136O73
`2012/014.8663
`2012/0183581
`
`2, 2000
`2, 2000
`3, 2000
`4, 2000
`6, 2000
`12, 2000
`1, 2001
`6, 2001
`9, 2001
`9, 2001
`1, 2002
`4, 2002
`3, 2003
`T/2003
`10, 2003
`11, 2003
`12, 2003
`2, 2004
`11, 2004
`2, 2005
`1/2007
`9, 2008
`1/2009
`10, 2009
`3, 2010
`6, 2010
`9, 2010
`9, 2010
`10, 2010
`11, 2010
`3, 2011
`3, 2011
`7/2011
`11, 2011
`11, 2011
`1, 2012
`4, 2012
`5, 2012
`T/2012
`8, 2012
`10, 2012
`6, 2013
`T/2013
`8, 2013
`10, 2013
`12, 2013
`9, 2014
`4, 2015
`12, 2001
`4, 2003
`4, 2003
`4, 2003
`T/2003
`4, 2004
`T/2004
`12, 2004
`12, 2004
`3, 2005
`6, 2005
`11/2005
`1, 2006
`T/2006
`10, 2006
`10, 2006
`2, 2007
`8, 2007
`8, 2007
`6, 2009
`11/2009
`5, 2010
`6, 2010
`3/2012
`5, 2012
`6, 2012
`T/2012
`
`Jessee
`Schwartz et al.
`Schwartz et al.
`Hawley-Nelson et al.
`Gebeyehu et al.
`Tirosh et al.
`Dwyer et al.
`Schwartz et al.
`Aneja
`Semple et al.
`Schwartz et al.
`Hawley-Nelson et al.
`Wheeler et al.
`Wheeler et al.
`Schwartz et al.
`Eiblet al.
`Hays et al.
`Wheeler
`Wheeler et al.
`Wheeler et al.
`Chu et al.
`Wheeler et al.
`Chu et al.
`Chu et al.
`Gebeyehu et al.
`Heyes et al.
`MacLachlan et al.
`Heyes et al.
`MacLachlan et al.
`MacLachlan et al.
`MacLachlan et al.
`Chu et al.
`MacLachlan et al
`Hawley-Nelson et al.
`Yaworski
`MacLachlan et al.
`Chu et al.
`MacLachlan et al.
`MacLachlan et al.
`Lee et al.
`Yaworski et al.
`Robbins et al.
`Yaworski et al.
`MacLachlan et al.
`Heyes et al.
`MacLachlan et al.
`Yaworski et al.
`Yaworski et al.
`Tousignant et al.
`Hawley-Nelson et al.
`Boulikas
`MacLachlan
`Fosnaugh et al.
`Davis et al.
`Finn et al.
`Tachas et al.
`Tuschl et al.
`MacLachlan et al.
`MacLachlan et al.
`Gebeyehu et al.
`MacLachlan et al.
`Gebeyehu et al.
`Chiou et al.
`MacLachlan et al.
`MacLachlan et al.
`Chu et al.
`Chu et al.
`Chu et al.
`MacLachlan et al.
`Yaworski et al.
`Chu et al.
`MacLachlan et al.
`Yang et al.
`Nilssen et al.
`Yaworski et al.
`
`US 9.404,127 B2
`Page 2
`
`9/2012 Chu et al.
`2012fO238747 A1
`2012fO276209 A1 11, 2012 Cullis et al.
`2013/0303587 A1 11/2013 Yaworski et al.
`2014f0065228 A1
`3/2014 Yaworski et al.
`2014/0294937 A1 10/2014 MacLachlan et al.
`2015,0164799 A1
`6, 2015 Yaworski et al.
`
`FOREIGN PATENT DOCUMENTS
`
`CA
`CA
`CA
`JP
`JP
`JP
`JP
`JP
`JP
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`
`2330741 A1 11, 1999
`2397O16 A1
`T 2001
`2513623
`8, 2004
`O3-126211
`5, 1991
`05-202085
`8, 1993
`06-080560
`3, 1994
`2002-52.5063
`8, 2002
`2003-505401
`2, 2003
`2007-524349
`8, 2007
`91 (16024 A1 10, 1991
`93,05162 A1
`3, 1993
`93.1224.0 A1
`6, 1993
`93.12756 A2
`7, 1993
`93.24640 A2 12, 1993
`93.25673 A1 12, 1993
`95,02698 A1
`1, 1995
`95, 18863 A1
`7, 1995
`95.35301 A1 12/1995
`96.O2655 A1
`2, 1996
`96,10390 A1
`4f1996
`96.40964 A2 12, 1996
`96.41873 A1 12, 1996
`98.51285 A2 11/1998
`OO?O3683 A2
`1, 2000
`00.15820 A1
`3, 2000
`OO62813 A2 10, 2000
`O1/O53.74 A1
`1, 2001
`O1/O5873 A1
`1/2001
`01,75164 A2 10, 2001
`01.93836
`12/2001
`02A34236 A2
`5, 2002
`02/087541 A1
`11, 2002
`O3,O97805 A2 11/2003
`2004/065546 A2
`8, 2004
`2004/110499 A1 12/2004
`2005/007 196 A2
`1, 2005
`2005/026372 A1
`3, 2005
`2005/035764 A1
`4/2005
`2005/120152 A2 12/2005
`2006/002538 A1
`1, 2006
`2006/053430 A1
`5, 2006
`2007/O12191
`2, 2007
`2007/056861 A1
`5/2007
`2009/086558 A1
`T 2009
`2009/11 1658 A2
`9, 2009
`2009/127O60
`10/2009
`2010/042877 A1
`4/2010
`2010/048.228 A2
`4/2010
`2010/088537 A2
`8, 2010
`2010, 105209 A1
`9, 2010
`2010, 14474.0 A1 12/2010
`2011/000107 A1
`1, 2011
`2011, 140627
`11, 2011
`2012/O19168 A2
`2, 2012
`2012/045075 A1
`4/2012
`2012/13.5805 A2 10/2012
`2012, 158736 A1 11, 2012
`2015, 011633 A1
`1, 2015
`OTHER PUBLICATIONS
`
`Arpicco, S., et al., “Synthesis, characterization and transfection activ
`ity of new Saturated and unsaturated cationic lipids.” IL Farmaco,
`2004, vol. 59, pp. 869-878.
`Ballas, N., et al., "Liposomes bearing a quaternary ammonium deter
`gent as an efficient vehicle for functional transfer of TMV-RNA into
`plant protoplasts.” Biochimica et Biophysica Acta, 1988, vol. 939,
`pp. 8-18.
`Barinaga, M., “Step Taken Toward Improved Vectors for Gene Trans
`fer.” Science, 1994, vol. 266, p. 1326.
`
`

`

`US 9.404,127 B2
`Page 3
`
`(56)
`
`References Cited
`
`OTHER PUBLICATIONS
`
`Bass, “The Short Answer,” Nature, 2001, 411: 428-9.
`Beale, G. et al., “Gene Silencing Nucleic Acids Designed by Scan
`ning Arrays: Anti-EGFR Activity of siRNA, Ribozyme and DNA
`Enzymes Targeting a Single Hybridization-accessible Region using
`the Same Delivery System.” Journal of Drug Targeting, 2003, vol. 11,
`No. 7, pp. 449-456.
`Behr, J.-P. “Synthetic Gene-Transfer Vectors.” Acc. Chem. Res.,
`1993, vol. 26, pp. 274–278.
`Brigham, K., et al., “Rapid Communication: In vivo Transfection of
`Murine Lungs with a Functioning Prokaryotic Gene Using a Lipo
`some Vehicle.” The American Journal of the Medical Sciences, vol.
`298, No. 4, pp. 278-281.
`Brummelkamp, et al., “A System for Stable Expression of Short
`Interfering RNAs in Mammalian Cells.” Science, 2002, V. 296. pp.
`550-553.
`Cevic, G., “How Membrane Chain-Melting Phase-Transition Tem
`perature is Affected by the Lipid Chain Asymmetry and Degree of
`Unsaturation: An Effective Chain-Length Model.” Biochemistry,
`1991, vol. 30, pp. 7186-7193.
`Choi. S. et al., “Novel cationic solid lipid nanoparticles enhanced p53
`gene transfer to lung cancer cells.' European J. of Pharmaceutics and
`Biopharmaceutics, 68:545-554, 2008.
`Chonnet al., “Recent advances in liposomal drug-delivery systems.”
`Current Opinion in Biotechnology, 1995, vol. 6, pp. 698-708.
`Cortesi, R., et al., “Effect of cationic liposome composition on in
`vitro cytotoxicity and protective effect on carried DNA.” Interna
`tional Journal of Pharmaceutics, 1996, vol. 139, pp. 69-78.
`Crystal, R. “Transfer of Genes to Humans: Early Lessons and
`Obstacles to Success.” Science, 1995, vol. 270, pp. 404-410.
`Culver K. “The First Human Gene Therapy Experiment.” Gene
`Therapy: A Handbook for Physicians, 1994, pp. 33-40.
`Duzgunes, N., “Membrane Fusion.” Subcellular Biochemistry, 1985,
`vol. 11, pp. 195-286.
`Dwarki, V.J., et al., “Cationic Liposome-Mediated RNA Transfec
`tion.” Methods in Enzymology, 1993, vol. 217, pp. 644-654.
`Elbashir, et a..., "Duplexes of 21-nucleotide RNAs mediate RNA
`interference in cultured mammalian cells.” Nature, May 2001, pp.
`494-498, vol. 411.
`Enoch, H., et al., “Formation and properties of 1000-A-diameter,
`single-bilayer phospholipid vesicles.” Proc. Natl. Acad. Sci. USA,
`1979, vol. 76, No. 1, pp. 145-149.
`Epand, Richard M. et al., “Phosphatidylcholine structure determines
`cholesterol solubility and lipid polymorphism.” Chemistry and Phys
`ics of Lipids, 2005, vol. 135, pp. 39-53.
`Felgner, J., et al., "Cationic Lipid-Mediated Transfection in Mam
`malian Cells: Lipofection.” J. Tiss. Cult. Meth., 1993, vol. 15, pp.
`63-68.
`Felgner, J., et al., “Enhanced Gene Delivery and Mechanism Studies
`with a Novel Series of Cationic Lipid Formulations.” The Journal of
`Biological Chemistry, 1994, vol. 269, No. 4, pp. 2550-2561.
`Felgner, P. et al., “Lipofection: A highly efficient, lipid-mediated
`DNA-transfection procedure.” Proc. Natl. Acad. Sci. USA, 1987, vol.
`84, pp. 7413-7417.
`Felgner, P.L., et al., "Cationic Liposome Mediated Transfection.”
`Proc. West. Pharmacol. Soc., 1989, vol. 32, pp. 115-121.
`Gao, X., et al., “A Novel Cationic Liposome Reagent for Efficient
`Transfection of Mammalian Cells.” Biochem. Biophys.Res. Comm.
`1991, vol. 179, No. 1, pp. 280-285.
`Gershon, H., et al., “Mode of Formation and Structural Feature of
`DNA-Cationic Liposome Complexes Used for Transfection.” Bio
`chemistry, 1993, vol. 32, pp. 7143-7151.
`Global Newswire, retrieved from http://globalnewswire.com on Feb.
`27, 2013, Tekmira Sues Alnylam Pharmaceuticals for repeated mis
`use of trade secrets and confidential information, Mar. 16, 2011, pp.
`1-3.
`Guy-Caffey, J., et al., “Novel Polyaminolipids Enhance the Cellular
`Uptake of Oligonucleotides.” The Journal of Biological Chemistry,
`1995, vol. 270, No. 52, pp. 31391-31396.
`
`Hawley-Nelson, P. et al., "Lipofect AmineTM Reagent: A New,
`Higher Efficiency Polycationic Liposome Transfection Reagent.”
`Focus, 1993, vol. 15, No. 3, pp. 73-80.
`Heyes et al., “Cationic lipid saturation influences intracellular deliv
`ery of encapsulated nucleic acids.” Journal of Controlled Release,
`2005, vol. 107, pp. 276-287.
`Heyes et al., “Synthesis of novel cationic lipids: effect of structural
`modification on the efficiency of genetransfer.” J. Med. Chem., 2002,
`vol. 45, pp. 99-114.
`Hyde, S., et al., “Correction of the ion transport defect in cystic
`fibrosis transgenic mice by genetherapy.” Nature, 1993, vol. 362, pp.
`250-255.
`Jeffs, L. et al., “A Scalable, extrusion-free method for efficient
`liposomal encapsulation of plasmid DNA.” Pharmaceutical
`Research, 22(3):362-372, 2005.
`Jiang, L., et al., “Comparison of protein precipitation methods for
`sample preparation prior to proteomic analysis,” Journal of Chroma
`tography A. 2004, vol. 1023, pp. 317-320.
`Juliano, R., et al., “The Effect of Particle Size and Charge on the
`Clearance Rates of Liposomes and Liposome Encapsulated Drugs.”
`Biochem. Biophys.Res. Commun., 1975, vol. 63, No.3, pp. 651-658.
`Keough, K., “Influence of chain unsaturation and chain position on
`thermotropism and intermolecular interactions in membranes.”
`Biochem. Soc. Transactions, 1990, vol. 18, No. 5, pp. 835-837.
`Kim, H. et al., “Cationic solid lipid nanoparticles reconstituted from
`low density lipoprotein components for delivery of siRNA.” Molecu
`lar Pharmaceutics, 5(4):622-631, 2008.
`Krichevsky, A. et al., “RNAi functions in cultured mammalian neu
`rons.” PNAS, 99(18): 11926-29, 2002.
`Lawrence et al. “The formation, characterization and stability of
`non-ionic surfactant vesicles.” S.T.P. Pharma Sciences, 1996, vol. 6,
`No. 1, pp. 49-60.
`Lawrence et al., "Synthesis and aggregation properties of dialkyl
`polyoxyethylene glycerol ethers.” Chemistry and Physics of Lipids,
`1996, 82(2):89-100.
`Legendre, J.-Y. et al., “Delivery of Plasmid DNA into Mammalian
`Cell Lines Using pH-Sensitive Liposomes: Comparison with
`Cationic Liposomes.” Pharm. Res., 1992, vol. 9, No. 10, pp. 1235
`1242.
`Leventis, R., et al., “Interactions of mammalian cells with lipid dis
`persions containing novel metabolizable cationic amphiphiles.”
`Biochem. Biophys. Acta, 1990, vol. 1023, pp. 124-132.
`Liu, et al., "Cationic Liposome-mediated Intravenous Gene Deliv
`ery”. J. Biol. Chem., 1995, V. 270, pp. 24864-24870.
`Marshall, E., “Gene Therapy's Growing Pains.” Science, 1995, vol.
`269, pp. 1050-1055.
`Montana, G. et al., “Employment of cationic Solid-lipid nanoparticles
`as RNA carriers.” Bioconjugate Chemistry, 18:302-308, 2007.
`Murahashi et al., “Synthesis and evaluation of neoglycolipid for
`liposome modification.” Biol. Pharm. Bull., 1997, 2006):704-707.
`Olbrich, C. et al., “Cationic solid-lipid nanoparticles can efficiently
`bind and transfect plasmid DNA. J. Controlled Release, 77:345-355.
`2007.
`Orkin, S., et al., NIH Report, Report and Recommendations of the
`Panel to Assess the NIH Investment in Research on Gene Therapy,
`1995.
`Parret al., Factors influencing the retention and chemical stability of
`poly(ethylene glycol)-lipid conjugates incorporated into large
`unilamellar vesicles, Biochimica et Biophysica Acta, 1994, 1195:21
`30.
`Paul, C., et al., “Effective expression of Small interfering RNA in
`human cells.” Nature Biotech., 2002, vol. 20, pp. 505-508.
`Puyal, C., et al., “A new cationic liposome encapsulating genetic
`material: A potential delivery system for polynucleotides.” Eur, J.
`Biochem., 1995, vol. 228, pp. 697-703.
`Rotenberg, M. et al., “Physico-chemical characterization of
`Intralipid TM emulsions.” Biochemica et Biophysica Acta, 1086:265
`272, 1991.
`Rudolph, C. et al., "Application of novel solid lipid nanoparticle
`(SLN)-gene vector formulations based on a dimeric HIV-1 TAT
`peptide in vitro and in vivo..” Pharmaceutical Research, 21 (9): 1662
`1669, 2004.
`
`

`

`US 9.404,127 B2
`Page 4
`
`(56)
`
`References Cited
`
`OTHER PUBLICATIONS
`
`Sawada et al., “Microemulsions in Supercritical CO utilizing the
`polyethyleneglycol dialkylglycerol and their use for the solubiliza
`tion of hydrophiles.” Dyes and Pigments, 2005, pp. 64-74, vol. 65.
`Shin, et al. "Acid-triggered release via dePEGylation of DOPE
`liposomes containing acid-labile vinyl ether PEG-lipids,” Journal of
`Controlled Release, 2003, vol. 91, pp. 187-200.
`Smisterova, J. et al., “Molecular shape of the cationic lipid controls
`the structure of cationic lipid? dioleylphosphatidylethanolamine
`DNA complexes and the efficiency of gene delivery,” J. Biol. Chem.
`276(50):47615-47622, 2001.
`Song et al., “Characterization of the inhibitory effect of PEG-lipid
`conjugates on the intracellular delivery of plasmid and antisense
`DNA mediated by cationic lipid liposomes.” Biochimica et
`Biophysica Acta, 2002, 1558:1-13.
`Sorensen, et al., “Gene Silencing by Systemic Delivery of Synthetic
`siRNAs in Adult Mice”. J. Biol. Chem., 2003, V. 327, pp. 761-766.
`Spagnou, S., et al., “Lipidic Carriers of siRNA: Differences in the
`Formulation, Cellular Uptake, and Delivery with Plasmid DNA.”
`Biochemistry, 2004, vol.43, pp. 13348-13356.
`Stamatatos, L., et al., “Interactions of Cationic Lipid Vesicles with
`Negatively Charged Phospholipid Vesicles and Biological Mem
`branes.” Biochemistry, 1988, vol. 27, pp. 3917-3925.
`Szoka, F., et al., “Comparative Properties and Methods of Preparation
`of Lipid Vesicles (Liposomes).” Ann. Rev. Biophys. Bioeng. 1980,
`vol. 9, pp. 467-508.
`Szoka, F., et al., “Procedure for preparation of liposomes with large
`internal aqueous space and high capture by reverse-phase evapora
`
`tion.” Proc. Natl. Acad. Sci. USA, 1978, vol. 75, No. 9, pp. 4194
`4.198.
`Tabatt, K. et al., “Effect of cationic lipid and matrix lipid composition
`on Solid lipid nanoparticle-mediated gene transfer, European J. of
`Pharmaceutics and Biopharmaceutics, 57:155-162, 2004.
`Teixeira, H. et al., "Characterization of oligonucleotide? lipid interac
`tions in Submicron cationic emulsions: influence of the cationic lipid
`structure and the presence of PEG-lipids.” Biophysical Chemistry,
`92: 169-181, 2001.
`Tekmira Pharmaceuticals and Protiva Biotherapeutics Inc., Poster
`entitled “Manufacturing, Safety and Efficacy of SNALP Formulated
`siRNA.” Presented at CHI Discovery on Target—Oct. 23, 2008,
`Boston, MA, by Ian MacLachlan.
`Templeton, “Cationic Liposome-mediated Gene Delivery in vivo”.
`Bioscience Reports, 2002, vol. 22, No. 2, pp. 283-295.
`VanDerWoude, I., et al., “Parameters influencing the introduction of
`plasmid DNA into cells by the use of synthetic amphiphiles as a
`carrier system.” Biochimica et Biophysica Acta, 1995, vol. 1240, pp.
`34-40.
`Wheeler, et al., "Stabilized Plasmid-lipid Particles: Constructions
`and Characterization.” Gene Therapy, V. 6, pp. 271-281.
`Wilson, R., et al., "Counterion-Induced Condensation of
`Deoxyribonucleic Acid.” A Light-Scattering Study, Biochemistry,
`1979, vol. 18, No. 11, pp. 2192-2196.
`Woodle, M.C., et al., “Versatility in lipid compositions showing
`prolonged circulation with sterically stabilized liposomes.”
`Biochimica et Biophysica Acta, 1992, vol. 1105, pp. 193-200.
`Xu, Y. et al., “Physicochemical characterization and purification of
`cationic lipoplexes.” Biophysical Journal, 77:341-353, 1999.
`Zhu, N., et al., “Systemic Gene Expression. After Intravenous DNA
`Delivery into Adult Mice.” Science, 1993, vol. 261, pp. 209-211.
`
`

`

`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 1 of 24
`
`US 9.404,127 B2
`
`Step One: Blending
`Lipids Stock in 90% Ethanol
`
`Impingement
`
`siRNA stock in EDTA
`
`Stabilized
`Peristaltic
`Pump NALP in 45%
`Ethano
`
`Step Two: Diluting
`
`Stabilized NALP
`in 45%. Ethano
`
`
`
`Dilution Zone
`
`Peristatic
`Pump
`
`Warm
`citrate/NaC
`buffer
`
`Stabilized
`NAP in 22.5%
`Ethanol
`
`FIG. 1A
`
`

`

`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 2 of 24
`
`US 9.404,127 B2
`
`
`
`
`
`Impingement
`Zone
`
`
`
`Stabilized NALP in 17%. Ethanol
`
`FIG. 1B
`
`

`

`U.S. Patent
`U.S. Patent
`
`Aug. 2, 2016
`Aug. 2, 2016
`
`Sheet 3 of 24
`Sheet 3 of 24
`
`US 9.404,127 B2
`US 9,404,127 B2
`
`
`
`
`
`FIG. 2A
`FIG. 2A
`
`

`

`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 4 of 24
`
`US 9.404,127 B2
`
`
`
`The Cryo vitrification technique
`
`
`
`- CD
`
`Bare HPF Sample drop placed ... and thinned Sample spanning
`on the grid
`by blotting
`holes in film
`
`... and vitrified in
`liquid ethane
`
`m 5um
`
`
`
`Grid with holey polymer film
`(HPF)
`
`FIG. 2B
`
`

`

`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 5 of 24
`
`US 9.404,127 B2
`
`
`
`Hole size: 1-6 pum
`
`Thickness of sample film:
`10-500 am
`
`FIG. 2C
`
`

`

`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 6 of 24
`
`US 9.404,127 B2
`
`
`
`lipid mg/mL.
`
`Particle size (nm)
`
`Final encapsulation (%)
`
`15
`
`93 (0.12)
`
`
`
`Number-averaged
`Diameter size (nm)
`46*
`
`

`

`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 7 of 24
`
`US 9.404,127 B2
`
`
`
`lipid mg/mL.
`
`Particle size
`(nm)
`
`Final encapsulation (%)
`
`Number-averaged
`Diameter size (nm)
`
`

`

`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 8 of 24
`
`US 9.404,127 B2
`
`
`
`lipid) mg/mL.
`
`15
`
`Particle size
`(nm)
`116 (0.06)
`
`Final encapsulation (%)
`
`Number-averaged
`Diameter size (nm)
`64*
`
`

`

`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 9 of 24
`
`US 9.404,127 B2
`
`
`
`lipid mg/mL
`
`15
`
`Particle Size
`(nm)
`108 (0.09)
`
`Final encapsulation (%)
`
`Number-averaged
`Diameter size (nm)
`59*
`
`

`

`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 10 of 24
`
`US 9.404,127 B2
`
`25
`
`2O
`
`3.
`.9
`E 15
`s
`C
`e
`
`10
`
`SS
`
`5 -
`
`O
`
`Presence of lamellar particles using SDM
`
`O:15
`
`2:30
`
`157
`
`13
`2
`82
`
`
`
`Number of non-
`lamellar particles
`Number Of
`lamellar particles
`
`10:15 SNAP
`
`2:30 SNAP
`
`1:57 SNALP
`
`162 SNALP
`
`594 (77%)
`
`665 (91%)
`
`325 (95%)
`
`313 (99%)
`
`173 (23%)
`
`67 (9%)
`
`16 (5%)
`
`4 (1%)
`
`FIG. 7
`
`

`

`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 11 of 24
`
`US 9.404,127 B2
`
`
`
`lipid mg/mL
`
`Particle size
`(nm)
`58 (0.07)
`
`Final encapsulation (%)
`
`Number-averaged
`Diameter size (nm)
`37
`
`

`

`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 12 of 24
`
`US 9.404,127 B2
`
`lipid mg/ml.
`
`
`
`
`
`Particle size
`(nm)
`65 (O. 11)
`
`
`
`Final encapsulation (%)
`
`Number-averaged
`Diameter size (nm)
`
`
`
`

`

`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 13 of 24
`
`US 9.404,127 B2
`
`
`
`lipid) mg/mL.
`
`15
`
`Particle size
`(nm)
`78 (0.03)
`
`Final encapsulation (%)
`
`96
`
`Number-averaged
`Diameter size (nm)
`55
`
`

`

`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 14 of 24
`
`US 9.404,127 B2
`
`
`
`lipid mg/mL
`
`Particle size
`(nm)
`
`Final encapsulation (%)
`
`Number-averaged
`Diameter size (nm)
`
`

`

`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 15 of 24
`
`US 9.404,127 B2
`
`Presence of lamellar particles using DDM
`
`
`
`Number of non-
`Iamellar particles
`Number of
`lamellar particles
`
`2:3O SNAP
`
`24O SNAP
`
`1:57 SNAP
`
`162 SNAP
`
`1386 (99%)
`
`1191 (99%)
`
`694 (>99%)
`
`707 (>99%)
`
`14 (1%)
`
`1 O (1%)
`
`2 (<1%)
`
`2 (<1%)
`
`FIG, 12
`
`

`

`U.S. Patent
`U S. Patent
`
`Aug. 2, 2016
`Aug. 2, 2016
`
`Sheet 16 of 24
`Sheet 16 of 24
`
`US 9.404,127 B2
`US 9,404,127 132
`
`
`
`
`
`i:3: SE
`
`

`

`U.S. Patent
`U S. Patent
`
`Aug. 2, 2016
`Aug. 2, 2016
`
`Sheet 17 of 24
`Sheet 17 of 24
`
`US 9.404,127 B2
`US 9,404,127 132
`
`
`
`
`
`FIG. 14
`F.G. 14
`
`

`

`U.S. Patent
`US. Patent
`
`Aug. 2, 2016
`Aug. 2, 2016
`
`Sheet 18 of 24
`Sheet 18 of 24
`
`US 9.404,127 B2
`US 9,404,127 132
`
`
`
`
`
`FIG. 15
`FIG. 15
`
`

`

`U.S. Patent
`U-S- Patent
`
`US 9.404,127 B2
`US 9,
`404,127 B2
`
`
`
`
`
`505.0.5.U5.O,
`33333333
`
`
`
`2
`
`
`
`
`
`
`
`
`
`
`

`

`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 20 of 24
`
`US 9.404,127 B2
`
`3.0 --m-m-my------- -----------. v.--------8
`
`-----
`
`2.5
`
`
`
`47% vs. PBS Control
`
`-77% vs. PBS Contro
`
`
`
`PBS
`
`2:30 SNALP 5 x 1 mg/kg
`
`1:57 SNALP 5 x 0.1 mg/kg
`
`FIG. 17
`
`

`

`U.S. Patent
`US, Patent
`
`Aug 2, 2016
`
`Sheet 21 of 24
`Sheet 21 of 24
`
`US 9.404,127 B2
`
`
`
`
`
`
`
`2
`
`
`
`
`
`
`
`
`
`

`

`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 22 of 24
`
`US 9.404,127 B2
`
`O)e. OdVSD Xc
`
`
`
`o
`N
`Ole J Old We : godw
`
`C
`
`wer
`
`

`

`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 23 of 24
`
`US 9.404,127 B2
`
`Anti-tumor efficacy of SNALP in subcutaneous Hep38 tumor-bearing
`mice after 6 x 3 mg/kg doses intravenously adminstered twice weekly
`for 3 Weeks
`
`18OO -
`
`
`
`16OO
`
`-- PBS
`
`-- 1:57 DLinDMA (PEG2000-C-DMA)
`
`-A-7:54 DLinDMA (PEG750-C-DMA)
`
`1400
`
`12OO
`
`1 OOO
`
`6OO -
`
`4OO -
`
`2OO
`
`O
`16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
`Study day
`
`FIG. 20
`
`

`

`U.S. Patent
`U.S. Patent
`
`Aug. 2, 2016
`Aug. 2, 2016
`
`Sheet 24 of 24
`Sheet 24 0f 24
`
`US 9.404,127 B2
`US 9,404,127 B2
`
`
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`

`

`1.
`NON-LPOSOMAL SYSTEMIS FOR NUCLEC
`ACD DELIVERY
`
`US 9,404,127 B2
`
`CROSS-REFERENCE TO RELATED
`APPLICATION
`
`This application is a continuation of U.S. application Ser.
`No. 13/807,288, filed Apr. 18, 2013, which application is a
`National Phase application under 35 U.S.C. S371 of PCT/
`CA2011/000778, filed Jun. 30, 2011, which application
`claims the benefit of U.S. Provisional Application No.
`61/360,480, filed Jun. 30, 2010, the disclosures of which are
`incorporated herein by reference for all purposes.
`
`REFERENCE TO A “SEQUENCE LISTING. A
`TABLE, ORACOMPUTER PROGRAM LISTING
`APPENDIX SUBMITTED AS ANASCII TEXT
`FILE
`
`10
`
`15
`
`The Sequence Listing written in file -100-1.TXT, created
`on May 15, 2013, 4,096 bytes, machine format IBM-PC,
`MS-Windows operating system, is hereby incorporated by
`reference in its entirety for all purposes.
`
`BACKGROUND OF THE INVENTION
`
`25
`
`30
`
`35
`
`40
`
`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 complemen
`tary 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., Elbashiret 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.
`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 expres
`sion of certain genes for the treatment of neoplastic disorders
`Such as cancer. It is also desirable to silence the expression of
`45
`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 inf
`arction, and thrombosis.
`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 increas
`ing 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)). 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 com
`promise delivery with Subsequent injections.
`Plasmid DNA-cationic liposome complexes are currently
`the most commonly employed nonviral gene delivery
`vehicles (Feigner, Scientific American, 276:102 (1997);
`
`50
`
`55
`
`60
`
`65
`
`2
`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. Pat. No. 6,458,
`382. Cationic liposome complexes are also disclosed in U.S.
`Patent Publication No. 20030073640.
`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., Biotech
`niques, 19:816 (1995); Lietal. The Gene, 4:891 (1997); Tam
`etal, 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 Biotech
`nology, 15:620 (1997); Templeton et al., Nature Biotechnol
`ogy, 15:647 (1997); Hofland et al., Pharmaceutical Research,
`14:742 (1997)).
`Other liposomal delivery systems include, for example, the
`use of reverse micelles, anionic liposomes, and polymer lipo
`somes. Reverse micelles are disclosed in U.S. Pat. No. 6,429,
`200. Anionic liposomes are disclosed in U.S. Patent Publica
`tion No. 20030026831. Polymer liposomes that incorporate
`dextrinor glycerol-phosphocholine polymers are disclosed in
`U.S.
`Patent
`Publication
`Nos. 20020081736 and
`20030082103, respectively.
`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 cir
`culation 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 com
`partment. The particle should also readily interact with target
`cells at a disease site in order to facilitate intracellular delivery
`of a desired nucleic acid.
`Recent work has shown that nucleic acids can be encapsu
`lated in small (e.g., about 70 nm diameter) “stabilized plas
`mid-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 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 (li
`poplexes) or naked DNA.
`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.
`
`BRIEF SUMMARY OF THE INVENTION
`
`The