Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 1 of 30
`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 1 of 30
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`EXHIBIT G
`EXHIBIT G
`
`
`
`

`

`(12) United States Patent
`MacLachlan et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 9,504,651 B2
`Nov. 29, 2016
`
`USOO950.4651B2
`
`(54) LIPID COMPOSITIONS FOR NUCLEIC ACID
`DELIVERY
`
`(71) Applicant: PROTIVA BIOTHERAPEUTICS,
`INC., Burnaby (CA)
`
`(72) Inventors: Ian MacLachlan, Mission (CA); Lloyd
`Jeffs, Burnaby (CA); Lorne R. Palmer,
`Burnaby (CA); Cory Giesbrecht,
`Vancouver (CA)
`(73) Assignee: PROTIVA BIOTHERAPEUTICS,
`INC., Burnaby (CA)
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 15 days.
`(21) Appl. No.: 14/304,578
`
`(*) Notice:
`
`(52) U.S. Cl.
`CPC ............. A61K 9/1271 (2013.01); A61K 9/127
`(2013.01); A61K 9/1277 (2013.01); A61 K
`31/7084 (2013.01); A61 K3I/7088 (2013.01);
`A61K 47/10 (2013.01); A61K 47/24 (2013.01);
`A61K 47/44 (2013.01)
`(58) Field of Classification Search
`None
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`it. A
`E. SE t al.
`I - w
`a
`(Continued)
`FOREIGN PATENT DOCUMENTS
`
`2041075 A1 10, 1991
`2309.727 A1
`4, 1999
`(Continued)
`
`OTHER PUBLICATIONS
`
`1-1.
`(22) Filed:
`(65)
`
`Jun. 13, 2014
`Prior Publication Data
`
`CA
`CA
`
`Oct. 2, 2014
`US 2014/029.4937 A1
`Related U.S. Application Data
`(60) Continuati
`f
`lication No. 13/684,066, filed
`onunuauon OI appl1cauon No.
`Uoo, Illed on
`Nov. 21, 2012, which is a continuation of application
`No. 12/965,555, filed on Dec. 10, 2010, now Pat. No.
`8.329,070, which is a division of application No.
`10/611,274, filed on Jun. 30, 2003, now Pat No.
`7,901,708.
`(60) Provisional application No. 60/392,887, filed on Jun.
`28, 2002
`
`(51) Int. Cl
`A. iK 17/10
`A6 IK 47/24
`BOI. I3/04
`A 6LX 9/27
`3. fto
`A6 IK3I/7088
`A6 IK 47/44
`
`(2006.01)
`(2006.01)
`2006.O1
`(
`.01)
`(2006.01)
`3:08:
`(2006.01)
`(2006.01)
`
`
`
`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 2 of 30
`
`Lasic. D. D. "Novel applications of Liposomes' Tibtech, 1998, v.
`16, 307-321.*
`
`(Continued)
`Primary Examiner Mina Haghighatian
`Assistant Examiner — Erin Hirt
`(74) Attorney, Agent, or Firm — Kilpatrick Townsend &
`Stockton LLP
`
`ABSTRACT
`(57)
`The present invention provides apparatus and processes for
`producing liposomes. By providing a buffer Solution in a
`first reservoir, and a lipid solution in a second reservoir,
`continuously diluting the lipid solution with the buffer
`Solution in a mixing chamber produces a liposome. The lipid
`Solution preferably comprises an organic solvent, Such as a
`lower alkanol.
`
`14 Claims, 15 Drawing Sheets
`
`twixing
`Sieg
`
`ir
`Step
`
`i.igid
`ignors
`
`&a-Sass:
`&cs
`is 3:3:
`
`s
`Siasie
`SEs
`x2 sy
`
`

`

`US 9,504,651 B2
`Page 2
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`5, 1985 Deamer
`4,515,736 A
`7, 1986 Papahadjopoulos et al.
`4,598,051 A
`8, 1987 Kikuchi et al.
`4,687,661 A
`3: El
`823. 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 11/1993 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.
`5,424,073. A
`6, 1995 Rahman et al.
`5,478,860 A 12/1995 Wheeler et al.
`5,545,412 A
`8/1996 Eppstein et al.
`5,552,157 A
`9/1996 Yagi et al.
`5,578.475 A 11/1996 Jessee
`5,593,622 A *
`1/1997 Yoshioka ............. A61K 9, 1271
`5,627,159 A
`5, 1997 Shih et al.
`264/43
`5,641,662 A
`6, 1997 Debs et al.
`5,653,996 A
`8, 1997 HSu
`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. ................ 435/320.1
`38. A 38 tly Nelson et al.
`5,830.430 A * 11/1998 Unger et al. ................. 424f1.21
`35. A
`3229 R. 1.
`595890. A
`9/1999 Dwyer eral
`5,976,567 A 11/1999 Wheeler et al.
`5,981,501 A 11/1999 Wheeler et al.
`6,007,838 A 12/1999 Alving et al.
`6,020,202 A
`2/2000 Jessee
`6,034,135 A
`3, 2000 Schwartz et al.
`6,051,429 A
`4/2000 Hawley-Nelson et al.
`6,075,012 A
`6/2000 Gebeyehu et al.
`6,093,348 A
`7/2000 Kowalski et al.
`6,165,501 A 12/2000 Tirosh et al.
`6,172,049 B1
`1/2001 Dwyer et al.
`6,251,939 B1
`6/2001 Schwartz et al.
`6,284,267 B1
`9/2001 Aneja
`6,287,591 B1
`9/2001 Semple et al.
`6,339,173 B1
`1/2002 Schwartz et al.
`6,376,248 B1
`4/2002 Hawley-Nelson et al.
`6,534,484 B1
`3/2003 Wheeler et al.
`
`7/2003 Edgerly-Plug
`6,596,305 B1
`6,638,529 B2 10/2003 Schwartz et al.
`
`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 3 of 30
`
`2/2004. Wheeler et al.
`6,696,424 B1
`6,734,171 B1* 5/2004 Saravolac .......... A61K 48,0008
`424/450
`
`6,815,432 B2 11/2004 Wheeler et al.
`6,835,395 B1
`12/2004 Semple et al.
`
`6,858,224 B2
`7,166,745 B1
`
`2/2005 Wheeler et al.
`1/2007 Chu et al.
`
`7,479,573 B2
`
`1/2009 Chu et al.
`
`3/2010 Gebeyehu et al.
`7,687,070 B2
`6/2010 Heyes et al.
`7,745,651 B2
`7,799,565 B2 * 9/2010 MacLachlan et al. ........ 435/458
`7,803,397 B2
`9/2010 Heyes et al.
`7,807,815 B2 10/2010 MacLachlan et al.
`7,901,708 B2 * 3/2011 MacLachlan .......... A61K 9,127
`424/450
`
`7,915.450 B2
`
`3/2011 Chu et al.
`
`
`
`OU
`
`a
`
`C. a.
`
`7/2011 MacLachlan et al.
`7,982,027 B2
`8,058,068 B2 11/2011 Hawley-Nelson et al.
`8,058,069 B2 * 1 1/2011 Yaworski et al. ............ 435/458
`8, 158,827 B2
`4/2012 Chu et al.
`8,227,443 B2
`7/2012 MacLachlan et al.
`8,283,333 B2 * 10/2012 Yaworski et al. .......... 514,44 A
`8,329,070 B2 * 12/2012 MacLachlan .......... A61's
`8,466,122 B2 * 6/2013 Heyes et al. ...
`... 514,44 A
`8.492.359 B2 * 7/2013 Yaworski et al. .......... 514,44 A
`9,005,654 B2
`4/2015 MacLachlan et al.
`9,006,417 B2
`4/2015 Yaworski et al.
`2001/0048940 Al 12/2001 Tousignant et al.
`2003, OO69.173 A1
`4/2003 Hawley-Nelson et al.
`2003/OO72794 A1
`4/2003 Boulikas
`2003/OO77829 A1
`4/2003 MacLachlan
`2003/0O83272 A1
`5/2003 Wiederholt et al.
`2003/O124033 A1
`7/2003 Baker et al.
`2003.0143732 A1
`7/2003 Fosnaugh et al.
`2004/0032037 A1
`2/2004 Katinger et al.
`2004/003.7874 A1
`2/2004 Hong et al.
`2004, OO63654 A1
`4/2004 Davis et al.
`38: 836 A.
`239: Matthian et al.
`a
`2004/0253723 A1 12/2004 Tachas et 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 Al
`11/2005 Gebeyehu et al.
`2006, OO58249 A1
`3/2006 Tong et al.
`3888, 3. A.
`1858.
`by hy t al.
`2007/0042031 A1
`2/2007 MacLachlan et al.
`38783 A.
`398,
`s s al
`2008/0200417 A1
`8/2008 Semple et al.
`2009.0143583 A1
`6, 2009 Chu et al.
`2009,019 1259 A1
`7, 2009 Li et al.
`2010. O159593 A1
`6, 2010 Chu et al.
`2011/0216622 A1
`9/2011 MacLachlan et al.
`2012/0136073 A1
`5/2012 Yang et al.
`2012fO238747 A1
`9/2012 Chu et al.
`2013/0303587 A1 11/2013 Yaworski et al.
`2014/0044772 A1
`2/2014 MacLachlan .......... A61K 9,127
`424/450
`
`2016,0032320 A1
`
`2/2016 Yaworski et al.
`
`FOREIGN PATENT DOCUMENTS
`
`A
`CA
`CA
`
`CA
`CA
`
`EP
`EP
`EP
`IE
`JP
`
`JP
`JP
`
`WO
`
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`
`if A HE
`2397O16 A1
`T 2001
`2457959 A1 12/2002
`
`2473135 A1
`2490983 A1
`
`6, 2003
`1, 2004
`
`O O55 576 A1
`1 013 268 A
`1 203 614 A1
`911350
`O3-126211
`
`06-080560
`O2-52.5063
`
`7, 1982
`6, 2000
`11, 2000
`4f1991
`5, 1991
`
`3, 1994
`8, 2002
`
`89/07929 A1
`
`9, 1989
`
`3, 1993
`93,05162 A1
`5, 1993
`93,09236 A1
`6, 1993
`93.1224.0 A1
`7, 1993
`93.12756 A2
`93.24640 A2 12, 1993
`93.25673 A1 12, 1993
`95,02698 A1
`1, 1995
`95, 18863 A1
`7, 1995
`
`

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`US 9,504,651 B2
`Page 3
`
`(56)
`
`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
`
`References Cited
`
`FOREIGN PATENT DOCUMENTS
`
`95.35301 A1 12/1995
`96,02655 A1
`2, 1996
`96,10390 A1
`4f1996
`96.40964 A2 12, 1996
`96.41873 A1 12, 1996
`98/51278 A2 it 11/1998
`98.51285 A2 11/1998
`WO9851278 A2 * 11, 1998 .......... A61K 9.00
`99.14346 A2
`3, 1999
`OOO3683 A2
`1, 2000
`00.15820 A1
`3, 2000
`00, 29.103 A1
`5, 2000
`OO62813 A2 10, 2000
`O1,05373 A1
`1, 2001
`O1,053.74 A1
`1, 2001
`01.05873 A1
`1, 2001
`01,75164 A2 10, 2001
`01.93836 A1 12/2001
`O2/34236 A2
`5, 2002
`O2/43699
`6, 2002
`O2/43699 A2
`6, 2002
`02/085434 A1
`10, 2002
`02/087541 A1
`11 2002
`O3,059381 A2
`T 2003
`O3,O97805 A2 11/2003
`2004/002453 A1
`1, 2004
`2004/065546 A2
`8, 2004
`2004/110499 A1 12/2004
`2005/007 196 A2
`1, 2005
`2005/026372 A1
`3, 2005
`2005/120152 A2 12/2005
`2015, 011633 A1
`1, 2015
`
`OTHER PUBLICATIONS
`
`Meyer, O. et al. “Cationic liposomes coated with polyethylene
`glycol as carriers for oligonucleotides.” J. Biol. Chem. 1998, 273,
`15621-15627.*
`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
`activity of new Saturated and unsaturated cationic lipids.” IL
`Farmaco, 2004, vol. 59, pp. 869-878.
`Ballas, N., et al., "Liposomes bearing a quaternary ammonium
`detergent 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
`Transfer.” Science, 1994, vol. 266, p. 1326.
`Bass, “The short answer,” Nature, 2001, vol. 411, pp. 428-429.
`Batzri et al. “Single Bilayer Liposomes Prepared without Sonica
`tion.” Biochimica et Biophysica acta 1973, vol. 298, pp. 1015-1019.
`Beale, G., et al., “Gene Silencing Nucleic Acids Designed by
`Scanning 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, 1989.
`Brummelkamp et al., “A System for Stable Expression of Short
`Interfering RNAs in Mammalian Cells,” Science, 2002, vol. 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.
`
`Chonn et al., “Recent advances in liposomal drug-delivery sys
`tems.” Current Opinion in Biotechnology, 1995, pp. 698-708, vol.
`6.
`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, S. et al., “Duplexes of 21-nucleotide RNAs mediate RNA
`interference in cultured mammalian cells,” Nature, 411(24):494-498
`(May 2001).
`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.
`Georgopapadakou, N. H., ed., “Drug Transport in Antimicrobial and
`Anticancer Chemotherapy,” CRC Press, Marcel Dekker, Inc.
`(1995).
`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.
`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
`delivery 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 gene transfer.” J. Med. Chem.
`2002, vol. 45, pp. 99-114.
`Hirota et al., “Simple Mixing Device to Reproducibly Prepare
`Catonic Lipid-DNA Complexes (Lipoplexes)”, BioTechniques,
`Aug. 1999, vol. 27, No. 2, pp. 286-290.
`Hyde, S. et al., “Correction of the ion transport defect in cystic
`fibrosis transgenic mice by gene therapy,” Nature, 1993, vol. 362,
`pp. 250-255.
`Isele et al., "Large-Scale Production of Liposomes Containing
`Monomeric Zinc Phthalacyanine by Controlled Dilution of Organic
`Solvents' Journal of Pharmaceutical Sciences, Nov. 1994, vol. 83,
`No. 11, pp. 1608-1616.
`Jiang, L., et al., “Comparison of protein precipitation methods for
`sample preparation prior to proteomic analysis,” Journal of Chro
`matography 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.
`
`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 4 of 30
`
`

`

`US 9,504,651 B2
`Page 4
`
`(56)
`
`References Cited
`
`OTHER PUBLICATIONS
`
`Krichevsky, A. et al., “RNAi functions in cultured mammalian
`neurons.” 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
`dispersions containing novel metabolizable cationic amphiphiles.”
`Biochem. Biophys. Acta, 1990, vol. 1023, pp. 124-132.
`Liu et al., "Cationic liposome-mediated intravenous gene delivery,”
`J. Biol. Chem. 1995, vol. 270, pp. 24864-24870.
`MacLachlan, "Liposomal Formulations for Nucleic Acid Delivery.”
`Antisense Drug Technologies, Second Edition; 2007: pp. 237-270.
`Marshall, E., “Gene Therapy's Growing Pains,” Science, 1995, vol.
`269, pp. 1050-1055.
`Maurer et al. “Spontaneous Entrapment of Polynucleotides upon
`Electrostatic Interaction with Ethanol-Destabilized Cationic
`Liposomes.” Biological Journal, May 2001, vol. 80, pp. 2310-2326.
`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.
`Parr et al., Factors influencing the retention and chemical stability
`of poly(ethylene glycol)-lipid conjugates incorporated into large
`unilamellar vesicles, Biochimica et Biophysica Acta, 1994,
`11.95:21-30.
`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 CO2 utilizing the
`polyethyleneglycol dialkylglycerol and their use for the solubiliza
`tion of hydrophiles.” Dyes and Pigments, 2005, pp. 64-74, vol. 65.
`Semple et al. “Efficient encapsulation of antisense oligonucleotides
`in lipid vesicles using ionizable aminolipids: formation of novel
`Small multilamellar vesicle structures,” Biochim. Biophys. Acta,
`2001, vol. 1510, No. 1-2, pp. 152-166.
`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
`
`s
`
`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. Mol. Biol. Apr. 4, 2003, vol. 4, pp.
`T61-766.
`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 Prepara
`tion 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 acqueous space and high capture by reverse-phase evapo
`ration.” Proc. Natl. Acad. Sci. USA, 1978, vol. 75, No. 9, pp.
`4194-4198.
`Templeton, "Cationic liposome-mediated intravenous gene delivery
`in vivo.” Bioscience Reports, 2002, vol. 22, No. 2, pp. 283-295.
`Van Der Woude, 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.
`Wagner et al., “The Crossflow Injection Technique: an Improvement
`of the Ethanol Injection Method.” Journal of Liposome Research,
`Sep. 2002, vol. 12, No. 3, pp. 259-270.
`Wheeler et al., "Stabilized plasmid-lipid particles: construction and
`characterization.” Gene Ther. 1999, vol. 6, No. 2, pp. 271-281.
`Wilson, R. et al., "Counterion-Induced Condensation of Deoxyri
`bonucleic 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.
`Zelphati et al., “Stable and Monodisperse Lipoplex Formulations
`for Gene Delivery,” Gene Therapy, 1998, vol. 5, pp. 1272-1282.
`Zhu, N. et al., “Systemic Gene Expression. After Intravenous DNA
`Delivery into Adult Mice.” Science, 1993, vol. 261, pp. 209-211.
`Global Newswire, retrieved from http://globalnewswire.com on
`Feb. 27, 2013, Tekmira Sues Alnylam Pharmaceuticals for repeated
`misuse of trade secrets and confidential information, Mar. 16, 2011,
`pp. 1-3.
`Jeffs et al. "A Scalable, Extrusion-Free Method for Efficient
`Liposomal Encapsulation of Plasmid DNA” Pharmaceutical
`Research, 2005, vol. 22, No. 3 pp. 362-372.
`Kee, J., “e-Study guide for: Clinical calculations,” Just the Facts
`101, Textbook Key Facts, 6th Ed., 2015, 1 page.
`Omega Engineering, Inc., “Omegaflex(R) Peristaltic Pump Model
`No. FPU500.” retrieved online at <https://web.archive.org/web/
`20010715124628/http://www.omega.com/pdf/tubing/pumps/
`fpu500/fpu500.asp. cached Jul. 15, 2001, 2 pages.
`
`* cited by examiner
`it cited by third party
`
`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 5 of 30
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`

`

`U.S. Patent
`
`Nov. 29, 2016
`
`Sheet 1 of 15
`
`US 9,504,651 B2
`
`i
`
`Nia
`
`Syriesis
`
`x
`
`
`
`Tipic seiuixation T Nucleic Acid Dissolution
`ir assaic
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`
`130
`
`Mixing of Lipid & Plasma Sciutions to
`For i?sories
`
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`
`5
`
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`(trafiltratiot)
`
`17.
`
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`Ethario Renova
`(traitration) .
`
`195- Series
`
`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 6 of 30
`
`G. i
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`

`

`U.S. Patent
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`Nov. 29, 2016
`
`Sheet 2 of 15
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`US 9,504,651 B2
`
`
`
`Mixing
`
`Distic
`Step
`
`A.
`i.gic
`hionerners
`
`3.
`Sea-Saxe
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`K. 288
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`Ethario:
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`low
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`a 23
`
`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 7 of 30
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`is
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`

`

`U.S. Patent
`
`Nov. 29, 2016
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`Sheet 3 of 15
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`US 9,504,651 B2
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`
`
`
`
`
`
`
`
`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 8 of 30
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`

`

`U.S. Patent
`
`Nov. 29, 2016
`
`Sheet 4 of 15
`
`US 9,504,651 B2
`
`
`
`
`
`/*Citrate Buffer storagg
`
`8.
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`
`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 9 of 30
`
`
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`
`
`
`
`
`
`
`

`

`U.S. Patent
`
`Nov. 29, 2016
`
`Sheet 5 Of 15
`
`US 9,504,651 B2
`
`
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`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 10 of 30
`
`is is
`
`

`

`U.S. Patent
`
`Nov. 29, 2016
`
`Sheet 6 of 15
`
`US 9,504,651 B2
`
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`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 11 of 30
`
`ife. S
`
`

`

`U.S. Patent
`
`Nov. 29, 2016
`
`Sheet 7 of 15
`
`US 9,504,651 B2
`
`-------au---------a-Y--------------------------------------
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`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 12 of 30
`
`F.
`
`

`

`U.S. Patent
`
`Nov. 29, 2016
`
`Sheet 8 of 15
`
`US 9,504,651 B2
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`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 13 of 30
`
`... 8
`
`

`

`U.S. Patent
`
`Nov. 29, 2016
`
`Sheet 9 of 15
`
`US 9,504,651 B2
`
`EPCiChoi (55.45)
`r 80 % Ethar
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`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 14 of 30
`
`F. A
`
`

`

`U.S. Patent
`
`Nov. 29, 2016
`
`Sheet 10 of 15
`
`US 9,504,651 B2
`
`
`
`
`
`34 is
`Citrate
`pit 4
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`Citrate
`pit &
`
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`
`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 15 of 30
`
`Citect fascii r S if the coast
`
`F
`
`is
`
`

`

`U.S. Patent
`
`Nov. 29, 2016
`
`Sheet 11 of 15
`
`US 9,504,651 B2
`
`ye; i.ipid Ratio for Safraine loaded E°CiChoi JW's
`
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`
`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 16 of 30
`
`fi.
`
`

`

`U.S. Patent
`
`Nov. 29, 2016
`
`Sheet 12 of 15
`
`US 9,504,651 B2
`
`
`
`TSpg.cg.e. s
`pl.G:popMA (20:55:10:15) | Mix
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`
`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 17 of 30
`
`TCciect the iiii fraction and neasure the fuorescence with and
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`Therefore, Caicein is: {i} encapsiiated within the vesicle
`(2) seif quenching at the current concentration
`fig. if
`
`

`

`U.S. Patent
`
`Nov. 29, 2016
`
`Sheet 13 of 15
`
`US 9,504,651 B2
`
`
`
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`
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`
`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 18 of 30
`
`fries. 2
`
`

`

`U.S. Patent
`
`US 9,504,651 B2
`
`dTdS
`
`
`
`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 19 of 30
`
`

`

`U.S. Patent
`
`Nov. 29, 2016
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`Sheet 15 of 15
`
`US 9,504,651 B2
`
`
`
`*---------|-~~~~~~
`
`-------------------
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`
`Case 1:22-cv-02229-MKV Document 42-8 Filed 09/06/22 Page 20 of 30
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`

`

`US 9,504,651 B2
`
`1.
`LPD COMPOSITIONS FOR NUCLEC ACID
`DELIVERY
`
`CROSS-REFERENCES TO RELATED
`APPLICATIONS
`
`This application is a continuation of U.S. application Ser.
`No. 13/684,066, filed Nov. 21, 2012, which is a continuation
`of U.S. application Ser. No. 12/965,555, filed Dec. 10, 2010,
`which is a divisional application of U.S. patent application
`Ser. No. 10/611.274, filed Jun. 30, 2003, which application
`claims priority to U.S. Provisional Application Ser. No.
`60/392,887, filed Jun. 28, 2002, the disclosures of which are
`hereby incorporated by reference in their entirety.
`
`10
`
`15
`
`BACKGROUND OF THE INVENTION
`
`2
`ronment (e.g., Reynolds numbers>2000). Therapeutic
`agents may then be loaded after vesicle formation
`Despite the apparent advances of U.S. Pat. No. 5,478.860
`and WO05373, there exists a need for processes and appa
`ratus for formulating and producing lipid vesicles, and in
`particular lipid vesicles encapsulating a therapeutic agent
`such as nucleic acid. The present invention fulfills these and
`other needs.
`
`BRIEF SUMMARY OF THE INVENTION
`
`The present invention provides processes and apparatus
`for making lipid vesicles that optionally contain a therapeu
`tic agent. The therapeutic agent can include, for example, a
`protein, a nucleic acid, an antisense nucleic acid, a drug, or
`the like. The present invention can be used to form lipid
`vesicles that contain encapsulated plasmid DNA or small
`molecule drugs. In one aspect, the lipid vesicles are prepared
`rapidly at low pressure and the approach is fully scalable. In
`certain preferred embodiments, the process does not involve
`a static mixer or specialized extrusion equipment.
`As such, in one embodiment, the present invention pro
`vides a process for producing a liposome. The process
`typically includes providing an aqueous Solution in a first
`reservoir, the first reservoir in fluid communication with an
`organic lipid solution in a second reservoir, and mixing the
`aqueous Solution with the organic lipid solution, wherein the
`organic lipid solution undergoes a continuous stepwise dilu
`tion to produce a liposome.
`In certain aspects, the aqueous solution Such as a buffer,
`comprises a therapeutic product, such that the therapeutic
`product is encapsulated in the liposome. Suitable therapeutic
`products include, but are not limited to, a protein, a nucleic
`acid, an antisense nucleic acid, a ribozyme, tRNA. SnRNA,
`siRNA (small interfering RNA), pre-condensed DNA, and
`an antigen. In certain preferred aspects, the therapeutic
`product is nucleic acid.
`In another embodiment, the present invention provides a
`process for producing a liposome encapsulating a therapeu
`tic product. The process typically includes providing an
`aqueous solution in a first reservoir, and providing an
`organic lipid solution in a second reservoir, wherein one of
`the aqueous solution and the organic lipid solution includes
`a therapeutic product. The process also typically includes
`mixing the aqueous Solution with the organic lipid solution,
`wherein the organic lipid solution mixes with the aqueous
`Solution so as to Substantially instantaneously produce a
`liposome encapsulating the therapeutic product. In certain
`aspects, the therapeutic product is a nucleic acid included in
`the aqueous solution. In certain aspects, the therapeutic
`product is lipophilic and is included in the organic lipid
`Solution. In certain aspects, the initial therapeutic product
`encapsulation efficiency is as high as about 90%.
`In still yet another embodiment, the present invention
`provides apparatus for producing a liposome encapsulating
`atherapeutic product. The apparatus typically includes a first
`reservoir for holding an aqueous solution, and a second
`reservoir for holding an organic lipid solution, wherein one
`of the aqueous solution and the organic lipid solution
`includes a therapeutic product. the apparatus also typically
`includes a pump mechanism configured to pump the aque
`ous and the organic lipid solutions into a mixing region at
`Substantially equal flow rates. In operation, the organic lipid
`Solution mixes with the aqueous Solution in the mixing
`region to Substantially instantaneously form a therapeutic
`product encapsulated liposome.
`
`25
`
`30
`
`35
`
`40
`
`45
`
`Many systems for administering active Substances into
`cells are already known, Such as liposomes, nanoparticles,
`polymer particles, immuno- and ligand-complexes and
`cyclodextrins (see, Drug Transport in antimicrobial and
`anticancer chemotherapy. G. Papadakou Ed., CRC Press,
`1995). Liposomes are typically prepared in the laboratory by
`Sonication, detergent dialysis, ethanol injection or dilution,
`French press extrusion, ether infusion, and reverse phase
`evaporation. Liposomes with multiple bilayers are known as
`multilamellar lipid vesicles (MLVs). MLVs are candidates
`for time release drugs because the fluids entrapped between
`layers are only released as each membrane degrades. Lipo
`Somes with a single bilayer are known as unilamellar lipid
`vesicles (UV). UVs may be made small (SUVs) or large
`(LUVs).
`Some of the methods above for liposome production
`impose harsh or extreme conditions which can result in the
`denaturation of the phospholipid raw material and encapsu
`lated drugs. In addition, these methods are not readily
`Scalable for mass production of large Volumes of liposomes.
`Further, lipid vesicle formation by conventional ethanol
`dilution, involves the injection or dropwise addition of lipid
`in an aqueous buffer. The resulting vesicles are typically
`heterogenous in size and contain a mixture of unilamellar
`and multilamellar vesicles.
`Conventional liposomes are formulated to carry therapeu
`tic agents either contained within the aqueous interior space
`(water-soluble drugs) or partitioned into the lipid bilayer(s)
`(water-insoluble drugs). Active agents which have short
`half-lives in the bloodstream are particularly suited to deliv
`ery via liposomes. Many anti-neoplastic agents, for
`example, are known to have a short half-life in the blood
`50
`stream such that their parenteral use is not feasible. How
`ever, the use of liposomes for site-specific delivery of active
`agents via the bloodstream is severely limited by the rapid
`clearance of liposomes from the blood by cells of the
`reticuloendothelial system (RES).
`U.S. Pat. No. 5,478,860, which issued to Wheeler et al.,
`on Dec. 26, 1995, and which is incorporated herein by
`reference, discloses microemulsion compositions for the
`delivery of hydrophobic compounds. Such compositions
`have a variety of uses. In one embodiment, the hydrophobic
`compounds are therapeutic agents including drugs. The
`patent also discloses methods for in vitro and in vivo
`delivery of hydrophobic compounds to cells.
`PCT Publication WO01/05373 to Knopov, et al., which is
`incorporated by reference herein, discloses techniques for
`preparing lipid vesicles using an ethanol injection-type
`process with a static mixer that provides a turbulent envi
`
`5