`
`Exhibit D
`
`
`
`
`
`
`
`
`
`
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 2 of 181 PageID #: 18272
`
`US009364435B2
`
`(12) United States Patent
`YaWorski et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 9,364.435 B2
`*Jun. 14, 2016
`
`(54) LIPID FORMULATIONS FOR NUCLEICACID
`DELIVERY
`
`(71) Applicant: Protiva Biotherapeutics, Inc., Burnaby
`(CA)
`(72) Inventors: Edward Yaworski, Maple Ridge (CA);
`Kieu Lam, Surrey (CA); Lloyd Jeffs,
`Delta (CA); Lorne Palmer, Vancouver
`(CA); Ian MacLachlan, Mission (CA)
`
`(*) Notice:
`
`(73) Assignee: PROTIVA BIOTHERAPEUTICS,
`INC., Burnaby, BC (CA)
`s
`s
`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.: 14/462,441
`9
`Aug. 18, 2014
`
`(22) Filed:
`
`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 VE 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.
`3:32: A SE: E. et A. tal
`- - I
`epeyenu et al.
`5,545,412 A
`8/1996 Eppstein et al.
`5,578.475 A 1 1/1996 Jessee
`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 Fe1
`tal.
`5,705.385 A
`1, 1998 ES"
`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.
`6,020,202 A
`2/2000 Jessee
`(Continued)
`
`(65)
`
`Prior Publication Data
`
`FOREIGN PATENT DOCUMENTS
`
`US 2015/O164799 A1
`
`Jun. 18, 2015
`
`Related U.S. Application Data
`(63) Continuation of application No. 13/928.309, filed on
`Jun. 26, 2013, OW Pat. No. 8,822,668, which is a
`continuation of application No. 13/253,917, filed O
`Oct. 5, 2011, now Pat. No. 8,492,359, which is a
`continuation of application No. 12/424,367, filed on
`Apr. 15, 2009, now Pat. No. 8,058,069.
`(60) Provisional application No. 61/045.228, filed on Apr.
`15, 2008.
`(51) Int. Cl.
`CI2N IS/II
`A 6LX 9/27
`A6 IK 4.8/00
`C7H 2L/00
`C07J 9/00
`CI2N IS/II3
`(52) U.S. Cl.
`CPC ............. A61K 9/1272 (2013.01); A61 K9/1271
`(2013.01); A61K 48/0025 (2013.01); C07H
`21/00 (2013.01); C07J 9/00 (2013.01); C12N
`15/III (2013.01); C12N 15/113 (2013.01);
`CI2N 2310/14 (2013.01); C12N 2320/32
`(2013.01)
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2010.01)
`
`(58) Field of Classification Search
`S. E. AR No. in N. 9/1271
`ee application file for complete search history.
`References Cited
`
`(56)
`
`U.S. PATENT DOCUMENTS
`
`4,394,448 A
`4,438,052 A
`4,515,736 A
`4,598,051 A
`
`7/1983 Szoka, Jr. et al.
`3, 1984 Weder et al.
`5, 1985 Deamer
`7, 1986 Papahadjopoulos et al.
`
`CA
`CA
`
`4, 1999
`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.
`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,
`E. 8-18. M.. “Step Taken Toward I
`dVectors for GeneT
`arinaga,
`ep
`en Ioward Improve COSOC aS
`fer.” Science, 1994, vol. 266, p. 1326.
`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.
`
`(Continued)
`Primary Examiner — Brian Whiteman
`(74) Attorney, Agent, or Firm — Kilpatrick Townsend &
`Stockton LLP
`ABSTRACT
`(57)
`The present invention provides novel, stable lipid particles
`comprising one or more active agents or therapeutic agents,
`methods of making the lipid particles, and methods of deliv
`9.
`p1d p
`ering and/or administering the lipid particles. More particu
`larly, the present invention provides stable nucleic acid-lipid
`particles (SNALP) comprising a nucleic acid (such as one or
`more interfering RNA), methods of making the SNALP, and
`methods of delivering and/or administering the SNALP.
`20 Claims, 24 Drawing Sheets
`
`
`
`US 9,364.435 B2
`Page 2
`
`(56)
`
`References Cited
`
`FOREIGN PATENT DOCUMENTS
`
`U.S. PATENT DOCUMENTS
`
`CA
`CA
`CA
`2/2000 Schwartz et al.
`6,020,526 A
`JP
`3, 2000 Schwartz et al.
`6,034,135 A
`JP
`4/2000 Hawley-Nelson et al.
`6,051,429 A
`JP
`6/2000 Gebeyehu et al.
`6,075,012 A
`JP
`6,165,501 A 12/2000 Tirosh et al.
`JP
`6,172,049 B1
`1/2001 Dwyer et al.
`JP
`6,251,939 B1
`6, 2001 Schwartz et al.
`WO
`6,284,267 B1
`9/2001 Aneja
`WO
`6,287,591 B1
`9/2001 Semple et al.
`WO
`6,339,173 B1
`1/2002 Schwartz et al.
`WO
`6,376,248 B1
`4/2002 Hawley-Nelson et al.
`WO
`6,534,484 B1
`3/2003 Wheeler et al.
`WO
`6,586.410 B1
`7/2003 Wheeler et al.
`WO
`6,638,529 B2 10/2003 Schwartz et al.
`WO
`6,649,780 B1 1 1/2003 Eiblet al.
`WO
`6,671,393 B2 12/2003 Hays et al.
`WO
`6,696,424 B1
`2/2004. Wheeler et al.
`WO
`6,815,432 B2 11/2004 Wheeler et al.
`WO
`6,858,224 B2
`2/2005 Wheeler et al.
`WO
`7,166,745 B1
`1/2007 Chu et al.
`WO
`7,422,902 B1
`9, 2008 Wheeler et al.
`WO
`7,479,573 B2
`1/2009 Chu et al.
`WO
`7,601,872 B2 10/2009 Chu et al.
`WO
`7,687,070 B2
`3/2010 Gebeyehu et al.
`WO
`7,745,651 B2
`6/2010 Heyes et al.
`WO
`7,799,565 B2
`9/2010 MacLachlan et al.
`WO
`7,803,397 B2
`9/2010 Heyes et al.
`WO
`7,807,815 B2 10/2010 MacLachlan et al.
`WO
`7,838,658 B2 11/2010 MacLachlan et al.
`WO
`7,901,708 B2
`3/2011 MacLachlan et al.
`WO
`7,915.450 B2
`3/2011 Chu et al.
`WO
`7,982,027 B2
`7/2011 MacLachlan et al.
`WO
`8,058,068 B2 11/2011 Hawley-Nelson et al.
`WO
`8,058,069 B2 11/2011 Yaworski et al.
`WO
`8, 101,741 B2
`1/2012 MacLachlan et al.
`WO
`8, 158,827 B2
`4/2012 Chu et al.
`WO
`8, 188,263 B2
`5/2012 MacLachlan et al.
`WO
`8,227,443 B2
`7/2012 MacLachlan et al.
`WO
`8,236,943 B2
`8/2012 Lee et al. ..................... 536, 24.5
`WO
`8,283,333 B2 10/2012 Yaworski et al.
`WO
`8,455.455 B1
`6, 2013 Robbins et al.
`8.492.359 B2
`7/2013 Yaworski et al. ........... 514,44 A WO
`8,513.403 B2
`8/2013 MacLachlan et al.
`WO
`8,569.256 B2 10/2013 Heyes et al.
`WO
`8,598,333 B2 12/2013 MacLachlan et al.
`WO
`8,822,668 B2
`9/2014 Yaworski et al. ............ 536,245
`WO
`9,006,417 B2
`4/2015 Yaworski et al. ............ 536,245
`2001/0048940 Al 12/2001 Tousignant et al.
`2003, OO69173 A1
`4/2003 Hawley-Nelson et al.
`2003/OO72794 A1
`4/2003 Boulikas
`2003/OO77829 A1
`4/2003 MacLachlan
`2003.0143732 A1
`7/2003 Fosnaugh et al.
`2004/0063654 A1
`4/2004 Davis et al.
`2004/O142892 A1
`7/2004 Finn et al.
`2004/0253723 A1 12/2004 Tachaset al.
`2004/0259247 A1 12/2004 Tuschi et al.
`2005, OO64595 A1
`3/2005 MacLachlan et al.
`2005, 0118253 A1
`6/2005 MacLachlan et al.
`2005/0260757 A1 1 1/2005 Gebeyehu et al.
`2006, OOO891.0 A1
`1/2006 MacLachlan et al.
`2006/0147514 A1
`7/2006 Gebeyehu et al.
`2006/02284.06 A1 10, 2006 Chiou et al.
`2006/0240093 A1 10, 2006 MacLachlan et al.
`2007/0042031 A1
`2/2007 MacLachlan et al.
`2007/0202598 A1
`8, 2007 Chu et al.
`2007/0202600 A1
`8, 2007 Chu et al.
`299;
`A1
`6, 2009 Chu et al.
`291131 A1 1 1/2009 MacLachlan et al.
`2010/013 0588 A1
`5, 2010 Yaworski et al.
`2010. O159593 A1
`6, 2010 Chu et al.
`2012/0058188 A1
`3/2012 MacLachlan et al.
`2012/0136073 A1
`5/2012 Yang et al.
`2012/0183581 A1
`7, 2012 Yaworski et al.
`2012fO238747 A1
`9, 2012 Chu et al.
`2014f0065228 A1
`3/2014 Yaworski et al.
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 3 of 181 PageID #: 18273
`
`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
`2007/056861 A1
`5/2007
`2009/086558 A1
`T 2009
`2009/11 1658 A2
`9, 2009
`2010/042877 A1
`4/2010
`2010/048.228 A2
`4/2010
`2010/088537 A2
`8, 2010
`2010, 105209 A1
`9, 2010
`
`OTHER PUBLICATIONS
`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 L.
`ith a Functioning Prokarvotic Gene Usi
`Li
`urine tungswan a functioning Tokaryote ene sing 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
`ture is Affected by the Lipid Chain A
`d D
`f
`perature is Affected by the Lipid Chain Asymmetry and Degree o
`Unsaturation: An Effective Chain-Length Model.” Biochemistry,
`1991, vol. 30, pp. 7186-7193.
`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.
`
`
`
`US 9,364.435 B2
`Page 3
`
`(56)
`
`References Cited
`
`OTHER PUBLICATIONS
`
`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.
`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 tradesecrets 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.
`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.
`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.
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 4 of 181 PageID #: 18274
`
`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.
`Murahashi et al., “Synthesis and evaluation of neoglycolipid for
`liposome modification.” Biol. Pharm. Bull., 1997, 2006):704-707.
`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.
`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.
`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.
`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.
`Zhu, N., et al., “Systemic Gene Expression. After Intravenous DNA
`Delivery into Adult Mice.” Science, 1993, vol. 261, pp. 209-211.
`* cited by examiner
`
`
`
`U.S. Patent
`
`US 9,364.435 B2
`
`
`
`
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 5 of 181 PageID #: 18275
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 2 of 24
`
`US 9,364.435 B2
`
`
`
`6 ?IduueS
`
`k k
`
`+
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 6 of 181 PageID #: 18276
`
`(seo peee Jun%) AqelA
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 3 of 24
`
`US 9,364.435 B2
`
`Activity of SNALP Upon intravenous Administration in Mice
`group mean + SD (n=4)
`
`2. O
`
`O. 5 w
`
`0. O
`«? SS s
`cS c. c.
`
`X
`SS s
`c.
`
`Nb N^
`NS NN N
`o
`o A Q
`o
`S S S S S S S S S
`c.c. c. c9; c.9 c. c9cs
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 7 of 181 PageID #: 18277
`
`FIG. 2
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 4 of 24
`
`US 9,364.435 B2
`
`
`
`3.0
`
`2. 5
`
`2. O
`
`O15
`
`O. 5
`
`0. O
`
`-47%. VS PBS Control
`
`-77% WS PBS Control
`
`PBS
`
`2:30 SNALP 5x1 mg/kg 1:57 SNALP 5x0.1 mg/kg
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 8 of 181 PageID #: 18278
`
`FIG. 3
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 5 of 24
`
`US 9,364.435 B2
`
`Activity of SNALP Upon Intravenous Administration in Mice
`group mean + SD (n=4)
`
`Int
`
`PBS Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8
`
`112 O-5O
`
`O 5 O.O
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 9 of 181 PageID #: 18279
`
`FIG. 4
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 6 of 24
`
`US 9,364.435 B2
`
`Activity of SNALP Upon intravenous Administration in Mice
`group meant SD (n=4)
`
`112 O-5-O-
`
`O 5 OO
`
`Nix No
`NS NN N. N.
`Q to
`so o 1,
`b
`se in
`<ssssssss Sess’ssess.
`c. c c c c
`s c c c c c c c CS
`
`I
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 10 of 181 PageID #: 18280
`
`FIG. 5
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 7 of 24
`
`US 9,364.435 B2
`
`Tolerability of IV 1:57 SNALP in Female BALB/c Mice, n=4, SD error
`
`ALT levels indicate hepatocyte damag
`96
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Tolerability of
`IV 1:57 SNALP in Female BALB?c Mice,
`4, SD error
`n
`Alanine Aminotransferase
`Aspartate Aminotransferase
`Sorbital Dehydrogenase
`is considered clinically
`2-fold increase ("3XULN")
`significant
`
`çN? P |(-)-(');
`
`**********************j CN ·
`-XXXXXXXXXXXXXXXXXX?
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 11 of 181 PageID #: 18281
`
`Lipid dose
`siRNA dose
`
`F.G. 6B
`
`9 mg/kg
`
`102
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 8 of 24
`
`US 9,364.435 B2
`
`S. 2.0
`FIG. 7A is -
`CC
`É 1.5
`8
`1.0
`
`CC
`(D
`g 0.5
`C.
`CC
`2
`
`0.0
`
`SNALP Activity From Different Manufacturing Processes
`IV, 48 h, ApoB-10048 U2/2 G1/2 (Dow),
`female BALB/C mice, n=4, SD error
`
`1:57
`1:57
`1:57
`PBS 1:57
`Syringe Gear Syringe Gear
`0.05mg 0.05mg 0.1mg/ 0.1mg/
`/kg
`/kg
`kg
`kg
`
`0.6 SNALP Re-Formulation - Activity Assessment in BALB/c Mice
`48 h time point, n=4, SD error bars
`
`FIG. 7B as
`O 0.5
`ch
`8. 0.4
`sC.
`0.3
`& 02
`n
`$ 0.1
`c
`2 -0.1
`
`Syringe Gear Syringe Gear
`Press Pump Press Pump
`0.05 mg/kg
`0.1 mg/kg
`
`FIG. 7C
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 12 of 181 PageID #: 18282
`
`Efficacy of SNALP. Formulations
`1. Fresh Terminal Plasma, n=4 female Balb/c mice, SD Error Bars
`g
`70
`co)
`5 60
`S
`50
`92 8
`40
`30
`20
`10
`O
`
`
`
`st
`o
`
`Syringe Gear 'Syringe Gear
`Press Pump Press Pump
`0.05 mg/kg
`0.1 mg/kg
`
`r
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 9 of 24
`
`US 9,364.435 B2
`
`Tolerability of 157 SNALP IV in BALB/c Mice, n=4 (Grp1-3 n=3), SD error
`
`6%
`
`d g
`G 4%
`92
`
`W
`
`3t 2% . . .
`
`.
`
`. .
`
`.
`
`.
`
`. . . .
`
`.
`
`. . . . .
`
`. . .
`
`. . . . . .
`
`. . . . . . .T. . . . . .
`
`. . . . .
`
`.
`
`. . . . . . . . . . . . . .
`
`. . . . . . . . .
`
`. . . . .
`
`. . . . . . . 3% H
`
`km
`
`7
`
`TT 4
`
`W. %
`N
`3% H
`: o, Illir. 2. H%.1% 3% E
`91 on 61 sh 6:1
`6:1
`9:1
`W2
`|
`9mg/ 11 mg 11 mg 13mg/15mg/ 17mg/11mg/
`
`:
`E
`
`- -2%
`
`-
`
`-
`
`- - -
`
`- -
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`. - - -
`
`-
`
`-
`
`- -
`
`-
`
`-
`
`- - - -
`
`- - - -
`
`-
`
`-
`
`-
`
`- kg
`
`/kg
`
`/kg
`
`kg
`
`kg
`
`kg
`
`kg
`
`PBS 91
`7mg/kg
`-40A . . . . . . . . . . . . . . . . . . . . . . . .
`. . . . . . . . .
`9.1 PBS
`11 mg/kg
`
`. . . . . .
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`. .
`
`. . . .
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`. . . . . . . . . . . . . . .
`
`is 4%
`S
`f
`
`-6%
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 13 of 181 PageID #: 18283
`
`FIG. 8
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 10 of 24
`
`US 9,364.435 B2
`
`Tolerability of IV 157 SNALP Prepared at 9:1 Lipid:Drug Ratio
`
`813
`
`828
`
`No.
`NOEN 794
`
`C) O C O o co o
`<t. CN o co co < c\!
`O O CO O C O o CD
`
`
`
`
`
`9
`8
`7
`Gear PBS InLine at
`11 (112) mg/kg
`
`ine at
`7 (71) mg/kg
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 14 of 181 PageID #: 18284
`
`FIG. 9
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 11 of 24
`
`US 9,364.435 B2
`
`Tolerability of IV 1:57 Gear PBS in-Line SNALP in Female BALB/c
`Mice, n=4, SD error
`
`Alanine Aminotransferase
`Aspartate Aminotransferase
`E3 Sorbital Dehydrogenase
`Samples taken at 24 h time point except
`for last grp (48 h).
`
`2
`
`6 8 8
`
`6
`
`1200
`s
`
`1,000
`
`s
`
`800
`
`600
`
`400
`
`200
`
`%
`:
`::Kx.
`sia
`orza::125, 22:EX), 12:33, P7:
`6:1
`:1
`6:1
`PBS
`9:1
`9:1
`6:1
`SNALP SNALP SNALP SNALP SNALP SNALP SNALP
`9mg/ 11 mg/ 11 mg/ 13mg/ 15mg/ 17mg/ 11 mg/kg
`kg
`kg
`kg
`kg
`kg
`kg
`48h
`
`: 2
`
`al
`
`*
`
`*
`
`
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 15 of 181 PageID #: 18285
`
`FIG. 10A
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 12 of 24
`
`US 9,364.435 B2
`
`
`
`Zaaya
`X.
`-xxxx
`
`XXX SS
`X
`
`SS
`
`YYYYYYZ
`
`77,777-??
`Sassass
`22
`
`N
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 16 of 181 PageID #: 18286
`
`09
`
`07
`
`09
`
`|euONJO -ul Jeddin plo--X
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 13 of 24
`
`US 9,364.435 B2
`
`FIG. 11A
`157 Gear PBS In-Line SNALP Activity From Different input Lipid: Drug Ratios
`IV, 48 h, ApoB-10048 U2/2 G1/2 (Dow), female BALB/cmice, n=4, SD error
`30
`
`2-tailed T-test: p=0.078
`
`N
`
`N
`N
`Y N
`QNS)
`N
`Q
`'.
`N
`.N
`SS SSNS SS SS R&S SSS's
`
`SNALP Re-Formulation - Activity Assessment in BALB/c Mice
`DOWApoB lead siRNA, 48 h time point, n=4, SD error bars
`LLO =9%
`
`-53%
`excluded
`
`.9
`
`2.5
`s
`2.0
`
`?
`
`9. (D 15
`
`O gd.
`CC 10
`al
`2
`-
`
`0.5
`
`O.O
`
`FIG. 11B
`
`0.40
`
`0.35 -
`
`0.30.
`
`0. 2 5
`
`O 2 O
`
`0.10
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 17 of 181 PageID #: 18287
`
`0.05 -
`
`O.OO
`
`kg
`kg
`mg/kg
`1:57 SNALP (10:1)
`
`0.1 mg/ 0.2 mg/l 0.4 mg/
`kg
`kg
`kg
`mg/kg
`New 1:57 SNALP (7:1)
`
`
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 14 of 24
`
`US 9,364.435 B2
`
`N
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 18 of 181 PageID #: 18288
`
`FIG. 12
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 15 of 24
`
`US 9,364.435 B2
`
`O
`120%
`
`11.5%
`
`5. 110%-
`
`10.5%
`
`100%-
`
`95%-
`
`90%-
`8
`
`-0- Luc - A - PLK1424
`
`12 16 20 24 28 32 36 40 44 48 52 56 60
`Study Day
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 19 of 181 PageID #: 18289
`
`FIG. 13
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 16 of 24
`
`US 9,364.435 B2
`
`
`
`- A - Control SNALP -d- Active SNALP
`
`15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90
`Days after seeding
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 20 of 181 PageID #: 18290
`
`FIG. 14
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 17 of 24
`
`US 9,364.435 B2
`
`
`
`2. O
`
`1 6
`
`1 2
`
`0. 8
`
`0. 4
`
`CC
`Z
`Y
`E
`?
`d
`CC
`CD
`C
`Na
`-
`n
`C
`C
`c
`CD
`
`D 0. O
`
`PBS
`
`Luc
`
`PLK1424
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 21 of 181 PageID #: 18291
`
`FIG. 15
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 18 of 24
`
`US 9,364.435 B2
`
`
`
`-- PLK1424 5'RACE
`product
`476bp
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 22 of 181 PageID #: 18292
`
`FIG, 16
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 19 of 24
`
`US 9,364.435 B2
`
`
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 23 of 181 PageID #: 18293
`
`FIG. 17
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 20 of 24
`
`US 9,364.435 B2
`
`6x2mg/kg Mean tumor volume
`
`--Luc-DMA
`-- PLK-DMA
`- A - PLK-DSA
`
`
`
`100
`
`800
`
`CD 60
`C
`c
`d
`
`40
`
`20
`
`O
`8
`
`T
`10
`
`12
`
`--
`14
`
`-Thor
`18
`20
`22
`24
`
`16
`Days
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 24 of 181 PageID #: 18294
`
`FIG. 18
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 21 of 24
`
`US 9,364.435 B2
`
`
`
`
`
`PLK mRNA silencing in scid/beige mice treated with 2mg/kg 1:57 SNALP against
`subcutaneous Hep3B tumors
`
`0.60
`
`100%
`
`
`
`0.30
`
`0.20
`
`0.10
`
`S s NS)
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 25 of 181 PageID #: 18295
`
`FIG. 19
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 22 of 24
`
`US 9,364.435 B2
`
`6x2mg/kg Mean tumor volume
`
`Crossover dosing of Luc
`cDMA group with 6 x 2
`mg/kg PLK cDSA SNALP
`
`2200
`2000
`1800
`1600-
`E
`1400
`O
`> is 1200
`
`
`
`H
`
`-0- LUC-DMA
`-- PLK-DMA
`- A - PLK-DSA
`
`Initial 6 x 2 mg/kg
`
`8 10 12 14 16 18 20 22 24 26 2. 30 32 34 36 38 40 42 44 46 48 50
`ays
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 26 of 181 PageID #: 18296
`
`FIG. 20
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 23 of 24
`
`US 9,364.435 B2
`
`Mean hPLK (1:4): hCGAPDH (1:40) minus "background"
`
`120
`
`
`
`1.OO
`
`O.80
`
`O.60
`
`0.40
`
`0.20
`
`O.00
`
`24h Luc 1:57 24h PLK 1:57 24h PLK 1:57 96h PLK 1:57 96h PLK 1.57
`CDMA
`CDMA
`CDSA
`CDMA
`CDSA
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 27 of 181 PageID #: 18297
`
`FIG. 21
`
`
`
`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 24 of 24
`
`US 9,364.435 B2
`
`
`
`-0 - 1:57 PEG-CDSA SNALP
`
`-A - 1:57 PEG-CDMA SNALP
`
`Case 1:22-cv-00252-MSG Document 316-5 Filed 05/13/24 Page 28 of 181 PageID #: 18298
`
`FIG. 22
`
`
`
`US 9,364,435 B2
`
`1.
`LPID FORMULATIONS FOR NUCLECACD
`DELIVERY
`
`CROSS-REFERENCES TO RELATED
`APPLICATIONS
`
`The present application is a continuation of U.S. applica
`tion Ser. No. 13/928.309, filed Jun. 26, 2013, which applica
`tion is a continuation of Ser. No. 13/253,917, filed Oct. 5,
`2011, now U.S. Pat. No. 8,492.359, which application is a
`continuation of Ser. No. 12/424,367 filed Apr. 15, 2009, now
`U.S. Pat. No. 8,058,069, which application claims priority to
`U.S. Provisional Application No. 61/045.228, filed Apr. 15,
`2008, the disclosures of which are herein incorporated by
`reference in their entirety for all purposes.
`
`STATEMENT REGARDING FEDERALLY
`SPONSORED RESEARCH ORDEVELOPMENT
`
`Not applicable.
`
`NAMES OF PARTIES TO AJOINT RESEARCH
`AGREEMENT
`
`Not applicable.
`
`REFERENCE TO A “SEQUENCE LISTING. A
`TABLE, ORACOMPUTER PROGRAM LISTING
`APPENDIX SUBMITTED AS ANASCII TEXT
`FILE
`
`5
`
`10
`
`15
`
`25
`
`30
`
`The Sequence Listing written in file-77-3.TXT, created on
`Aug. 22, 2013, 8,192 bytes, machine format IBM-PC, MS
`Windows operating system, is hereby incorporated by refer
`ence in its entirety for all purposes.
`
`35
`
`BACKGROUND OF THE INVENTION
`
`2
`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);
`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 t