Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 1 of 1274 PageID #: 8307
`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 1 of 1274 PagelD #: 8307
`
`JOINT APPENDIX 45
`JOINT APPENDIX 45
`
`

`

`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 2 of 1274 PageID #: 8308
`
`
`
`uii I IIM II 1111111111111111 11 uii
`
`US006734171B1
`
`(12) United States Patent
`Saravolac et al.
`
`(lo) Patent No.:
`(45) Date of Patent:
`
`US 6,734,171 B1
`*May 11, 2004
`
`5,631,237 A • 5/1997 Dzau et al.
`5,705,385 A
`1/1998 Bally et al.
`5,708,385 A * 1/1998 Shou et al.
`5,753,262 A * 5/1998 Wyse et al.
`5,820,873 A • 10/1998 Choi et al.
`5,827,703 A • 10/1998 Debs et al.
`5,885,613 A * 3/1999 Holland et al.
`5,976,567 A
`' 11/1999 Wheeler et al.
`5,981,501 A
`11/1999 Wheeler et al.
`2002/0192651 Al • 12/2002 Wheeler
`
`514/44
`
`435/320.1
`424/450
`424/283.1
`435/455
`424/450
`424/450
`
`435/6
`
`FOREIGN PATENT DOCUMENTS
`
`CA
`CA
`CA
`CA
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`GB
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`
`067133
`
`2067133
`2067178
`1305054
`0 118 316
`0 072 111
`0 220 797
`0 370 491
`0 422 543
`0 482 860
`526700
`546951
`572049
`0 445 131
`0 354 855
`0 496 813
`0 496 835
`2185397
`WO 88/04924
`WO 90/04384
`WO 91/05545
`WO 91/05546
`WO 93/19738
`WO 94/07466
`WO 94!21281
`WO 94/22429
`WO 94/26251
`WO 94/27580
`WO 95/31183
`WO 96/34598
`W096/37194
`W096/40964
`
`6/1990
`4/1991
`99 74//119921
`
`
`3/1983
`10/1985
`5/1987
`5/1990
`4/1991
`4/1992
`2/1993
`6/1993
`12/1993
`4/1994
`12/1994
`12/1994
`5/1995
`7/1987
`7/1988
`5/1990
`5/1991
`5/1991
`10/1993
`4/1994
`9/1994
`10/1994
`11/1994
`12/1994
`11/1995
`11/1996
`11/1996
`12/1996
`
`OTHER PUBLICATIONS
`
`Zelphati et al., Liposomes as a carrier for intracellular
`delivery of antisense oligonucicotidcs: a real magic bullet?,
`1996, Journal of Controlled Release, vol. 41, pp. 99-119.*
`
`(List continued on next page.)
`
`Primary Examiner—Dave T. Nguyen
`(74) Attorney; Agent, or Firm—Townsend & Townsend &
`Crew LLP
`
`(57)
`
`ABSTRACT
`
`The present invention relates to lipid-based formulations for
`nucleic acid delivery to cells, methods for the preparation of
`such formulations and, in particular, to lipid encapsulated
`plasmids. The compositions are safe and practical for clini-
`cal use. In addition, the present invention provides methods
`for introducing nucleic acids into cells and for inhibiting
`tumor growth in cells using such lipid-nucleic acid formu-
`lations.
`
`19 Claims, 31 Drawing Sheets-
`
`MRNA-GEN-00222371
`
`(54) METHODS FOR ENCAPSULATING
`NUCLEIC ACIDS IN LIPID BILAYERS
`
`(75)
`
`Inventors: Edward George Saravolac, Vancouver
`(CA); Yuan-Peng Zhang, Mountain
`View, CA (US); Jeffery J. Wheeler,
`Surrey (CA); Pieter R. Cullis,
`Vancouver (CA); Peter Scherrer,
`Vancouver (CA); Ljillona D. Kojic,
`Vancouver (CA); Olga Ludlcovski, Port
`Coquitlam (CA)
`
`(73) Assignee: Inex Pharmaceuticals Corp., Burnaby
`(CA)
`
`(* ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(h) by 0 days.
`
`This patent is subject to a terminal dis-
`claimer.
`
`(21) Appl. No.: 09/169,573
`
`(22) Filed:
`
`Oct. 9, 1998
`
`Related U.S. Application Data
`(60) Provisional application No. 60/063,473, filed on Oct. 10,
`1997.
`(51) Int. C1.7
`(52) U.S. Cl.
`
`
`
`A61K 48/00
`514/44; 424/450; 435/320.1;
`435/455; 435/458
`424/450; 435/325,
`435/320.1, 455, 458; 514/44
`
`(58) Field of Search
`
`(56)
`
`References Cited
`
` 252/316
` 424/1.1
` 424/38
` 424/1.1
` 424178
`568/621
` 428/402.2
` 264/43
` 264/4.6
` 424/450
` 260/403
` 424/450
` 424/85.91
` 528/301
` 424/450
`424/485
` 424/422
` 424/450
` 424/450
` 424/450
` 424/450
` 424/450
` 424/450
` 424/450
` 424/450
` 424/178.1
` 424/450
` 264/4.32
`
`U.S. PATENT DOCUMENTS
`
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`5,552,155 A
`9/1996 Bailey et al.
`5,593,622 A
`1/1997 Yoshioka et al.
`
`D
`
`JA001352
`
`

`

`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 3 of 1274 PageID #: 8309
`
`US 6,734,171 B1
`Page 2
`
`OTHER PUBLICATIONS
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`Filion et al., Major limitations in the use of cationic lipo-
`somes for DNA delivery, 1998, International Journal of
`Pharmaceutics, vol. 192, pp. 159-170.*
`Branch, A good antisense molecule is hard to find, Feb.
`1998, TIBS, vol. 23, pp. 45-50.*
`Anderson, Human gene therpy, Apr. 30,1998, Nature, vol.
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`Verma et al. Gene therapy —promises, problems and prope-
`spects, Sep. 18,1997, Nature, vol. 389, pp. 239-242.*
`Abuchowski, ct al., "Treatment of 15178Y Tumor—Bearing
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`Chonn, et al., "Ganglioside Gm, and hydrophilic polymers
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`1147:185-193.
`Huang, et al., "Extravasation and Transcytosis of Liposomes
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`
`Liu, et al., "Role of liposome size and RES blockade in
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`stability of poly(ethylene glycol)—lipid conjugates incorpo-
`rated into large unilamellar vesicles," 1994, Biochimica et
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`
`* cited by examiner
`
`MRNA-GEN-00222372
`
`JA001353
`
`

`

`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 4 of 1274 PageID #: 8310
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 1 of 31
`
`US 6,734,171 B1
`
`MICELLE
`
`LIPID
`MOLECULE
`
`gliggni>gatigAgg
`
`BILAYER
`
`HEXAGONAL H11
`
`F/G
`
`MRNA-GEN-00222373
`
`JA001354
`
`

`

`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 5 of 1274 PageID #: 8311
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 2 of 31
`
`US 6,734,171 B1
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`
`JA001355
`
`

`

`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 6 of 1274 PageID #: 8312
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 3 of 31
`
`US 6,734,171 B1
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`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 7 of 1274 PageID #: 8313
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 4 of 31
`
`US 6,734,171 B1
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`JA001357
`
`

`

`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 8 of 1274 PageID #: 8314
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 5 of 31
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`US 6,734,171 B1
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`JA001358
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`

`

`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 9 of 1274 PageID #: 8315
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 6 of 31
`
`US 6,734,171 B1
`
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`
`MRNA-GEN-00222378
`
`JA001359
`
`

`

`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 10 of 1274 PageID #: 8316
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 7 of 31
`
`US 6,734,171 B1
`
`POOL % DODAC
`
`MRNA-GEN-00222379
`
`(001- N01181 +/-) NOIIVIISOON3 INnind
`
`JA001360
`
`

`

`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 11 of 1274 PageID #: 8317
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 8 of 31
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`US 6,734,171 B1
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`
`JA001361
`
`

`

`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 12 of 1274 PageID #: 8318
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 9 of 31
`
`US 6,734,171 B1
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`
`JA001362
`
`

`

`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 13 of 1274 PageID #: 8319
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 10 of 31
`
`US 6,734,171 B1
`
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`
`JA001363
`
`

`

`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 14 of 1274 PageID #: 8320
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 11 of 31
`
`US 6,734,171 B1
`
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`JA001364
`
`

`

`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 15 of 1274 PageID #: 8321
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 12 of 31
`
`US 6,734,171 B1
`
`FIG. I2A.
`
`FIG. /28.
`
`FIG. 12C.
`
`MRNA-GEN-00222384
`
`JA001365
`
`

`

`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 16 of 1274 PageID #: 8322
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 13 of 31
`
`US 6,734,171 B1
`
`0.0%
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`
`5.0%
`
`10%
`
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`
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`
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`
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`15000
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`
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`
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`
`MRNA-GEN-00222385
`
`JA001366
`
`

`

`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 17 of 1274 PageID #: 8323
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 14 of 31
`
`US 6,734,171 B1
`
`FIG 144.
`
`Fa. /48.
`
`MRNA-GEN-00222386
`
`JA001367
`
`

`

`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 18 of 1274 PageID #: 8324
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 15 of 31
`
`US 6,734,171 B1
`
`14.2%
`FREE ONA REMOVED INEX32I
`
`A
`
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`
`5
`
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`
`20
`
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`
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`ISOLATED INEX321
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`B
`
`1200
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`MRNA-GEN-00222387
`
`JA001368
`
`

`

`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 19 of 1274 PageID #: 8325
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 16 of 31
`
`US 6,734,171 B1
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`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 20 of 1274 PageID #: 8326
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`U.S. Patent
`
`May 11, 2004
`
`Sheet 17 of 31
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`US 6,734,171 B1
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`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 21 of 1274 PageID #: 8327
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`U.S. Patent
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`May 11, 2004
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`Sheet 18 of 31
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`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 22 of 1274 PageID #: 8328
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`U.S. Patent
`
`May 11, 2004
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`Sheet 19 of 31
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`US 6,734,171 B1
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`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 23 of 1274 PageID #: 8329
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 20 of 31
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`US 6,734,171 B1
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`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 24 of 1274 PageID #: 8330
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`U.S. Patent
`
`May 11, 2004
`
`Sheet 21 of 31
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`US 6,734,171 B1
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`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 25 of 1274 PageID #: 8331
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`U.S. Patent
`
`may 11, 2004
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`Sheet 22 of 31
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`US 6,734,171 B1
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`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 26 of 1274 PageID #: 8332
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`U.S. Patent
`
`May 11, 2004
`
`Sheet 23 of 31
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`US 6,734,171 B1
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`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 27 of 1274 PageID #: 8333
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`U.S. Patent
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`May 11, 2004
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`Sheet 24 of 31
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`US 6,734,171 B1
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`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 28 of 1274 PageID #: 8334
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 25 of 31
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`US 6,734,171 B1
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`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 29 of 1274 PageID #: 8335
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`U.S. Patent
`
`May 11, 2004
`
`Sheet 26 of 31
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`US 6,734,171 B1
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`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 30 of 1274 PageID #: 8336
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 27 of 31
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`US 6,734,171 B1
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`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 31 of 1274 PageID #: 8337
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 28 of 31
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`US 6,734,171 B1
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`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 32 of 1274 PageID #: 8338
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 29 of 31
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`US 6,734,171 B1
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`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 33 of 1274 PageID #: 8339
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 30 of 31
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`US 6,734,171 B1
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`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 34 of 1274 PageID #: 8340
`
`U.S. Patent
`
`May 11, 2004
`
`Sheet 31 of 31
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`US 6,734,171 B1
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`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 35 of 1274 PageID #: 8341
`
`US 6,734,171 B1
`
`1
`METHODS KM ENCAPSULATING
`NUCLEIC ACIDS IN LIPID BILAYERS
`
`CROSS-REFERENCE TO RELATED
`APPLICATION
`This application claims priority under 119(e) to U.S.
`Provisional Patent Application Serial No. 60/063,473, filed
`on Oct. 10, 1997, which is hereby incoporated by reference.
`
`FIELD OF TILE INVENTION
`This invention relates to lipid-based formulations for
`nucleic acid delivery to cells, methods for the preparation of
`such formulations and, in particular, to lipid encapsulated
`plasmids. The compositions are safe and practical for clini-
`cal use.
`
`BACKGROUND OF THE INVENTION
`Gene therapy is an area of current interest which involves
`the introduction of genetic material into a cell to facilitate
`expression of a deficient protein. Plasmid DNA has been
`encapsulated or complexed with lipid-based carriers by a
`number of methods including reverse phase evaporation
`(Fraley, et al., J. Biol. Chem., 255:10431-10435 (1980);
`Soriano, et al., Proc. Natl. Acad. Sci. USA, 80:7128-7131
`(1983); Nakanishi, et al., Exper. Cell Res., 159:399-409
`(1985); Nandi, et al., J. BioL Chem., 261:16722-16726
`(1986); and Alin°, et al., Biochem. Biophys. Res. Commun.,
`192:174-181 (1993)); Cat* EDTA chelation (Szelei, et al.,
`Biochem. J., 259:549-553 (1989)); detergent dialysis
`(Wang, et al., Proc. Natl. Acad. Sci. USA, 84:7851-7855
`(1987)); lipid hydration (Lurquin, Nucleic Acids Res.,
`6:3773-3784 (1979); Yagi, et al., Biochem. Mol. Biol.
`International, 32:167-171 (1994)); ether injection (Fraley,
`et al., Proc. Natl. Acad. Sci., 76:3348-3352 (1979); Nicolau,
`et al., Biochem. Biophys. Res. Comm., 108:982-986 (1982));
`and sonication (Jay, et al., Bioconj. Chem., 6:187-194
`(1987) and Puyal, et al., Eur. J. Biochem., 228:697-703
`(1993)).
`Reverse phase techniques typically encapsulate only
`about 10 to 20% of DNA in solution and the final DNA to
`lipid ratio is quite low. For example, Nakanishi, et al. (Exper
`Cell Res., 159:399-409 (1985)) reported a final DNA to lipid
`ratio of 1.5 Ng DNA to 2.5 mg lipid, while Soriano, et al.
`(Proc. Natl. Acad. Sci. USA, 80:7128-7131 (1983)) reported
`a DNA to lipid ratio of about 14 pg DNA to 60 µmol of
`lipids. The maximum theoretical encapsulation efficiency
`expected by reverse phase is only about 40%. Other
`methods, such as rehydration of freeze dried vesicles with
`DNA, have been shown to yield trapping efficiencies
`between 30 and 40% (Baru, et al., Gene, 161:143-150
`(1995)). Others have sought to increase the entrapment of
`DNA by the inclusion of cationic lipids in the lipid suspen-
`sion (Stavridis, et al., 1986; Puyal, et al., Eur. J. Biochem.,
`228:697-703 (1995)), or by rendering the DNA positively
`charged by 10 coating it with basic proteins such as
`lysozymes (Jay, et al., Proc. Natl. Acad. Sci. USA,
`84:1978-1980 (1987)). Although trapping efficiencies as
`high as 50% were achieved by the lysozyme method, the
`amount of DNA loaded per mg of lipid was low (5 pg/mg
`lipid) and the largest DNA molecule tested was only 1 kb.
`Trapping efficiencies as high as 60-90% were achieved by
`Puyal, et al. (Eur. J. Biochem., 228:697-703 (1995)) with a
`higher DNA to lipid ratio (13 pg,,,,umole lipid) using a 6.3 kb
`ssDNA (M13 phage). The major drawback of this technique
`and the one described by Jay, et al., Bioconj. Chem.,
`6:187-194 (1987)) is that sonication was used. Sonication of
`DNA typically leads to some degradation of the lipid vesicle.
`
`2
`Detergent dialysis is a method of encapsulation which has
`no deleterious effects on the DNA. Wang, et al., Proc. Nail.
`Acad. Sci. USA, 84:7851-7855 (1987) applied a detergent
`dialysis technique followed by extrusion through a 0.2 pm
`5 polycarbonate filter. A 4.6 kb plasmid was entrapped in
`vesicles approximately 200 nm in diameter with a trapping
`efficiency of about 14-17%, giving a DNA to lipid ratio of
`about 26 pg DNA to 10 pmole lipid.
`Ideally, a delivery vehicle for a nucleic acid or plasmid
`10 will have the following characteristics: a) small enough and
`long lived enough to distribute from local injection sites
`when given intravenously, b) capable of carrying a large
`amount of DNA per particle to enable transfection of all
`sizes of genes and to reduce the volume of injection, c)
`homogeneous, d) reproducible, e) protective of DNA from
`extracellular degradation and f) capable of transfecting
`target cells in such a way that the DNA is not digested
`intracellularly.
`The present invention provides such compositions and
`20 methods for their preparation and use.
`
`15
`
`25
`
`35
`
`SUMMARY OF THE INVENTION
`In one aspect, the present invention provides composi-
`tions which arc nucleic acid (e.g., plasmid)-lipid composi-
`tions. In these compositions, a nucleic acid (e.g., plasmid or
`an antisense molecule) is encapsulated in a self-assembling
`lipid vesicle in an amount of from about 20 pg nucleic
`acid/mg lipid to about 400 pg nucleic acid/mg lipid. The
`lipid vesicle will typically be a liposome or lipid particle (a
`30 bilayer vehicle coating the plasmid and having little or no
`aqueous interior). The lipid vesicle can be prepared from a
`wide variety of lipids or combinations of lipids. The com-
`positions can also include targeting groups and modified
`lipids (e.g., ATTA-lipids, gangliosides, such as ganglioside
`GM!), PEG-lipids, such as PEG-ceramides, and lipids hav-
`ing reactive functional groups for the attachment of targeting
`groups or circulation stabilizers). Preferably, the lipid
`vesicles will comprise cationic lipids and fusogenic lipids.
`Additionally, the nucleic acid (e.g., plasmid)-lipid compo-
`sitions described herein can he prepared having a narrow
`size distribution (typically 50 nm to about 150 nm) without
`the use of sizing methods, such as extrusion and sonication
`methods.
`in another aspect, the present invention provides methods
`for the encapsulation of nucleic acids, antisense, ribozymes
`and, particularly, plasmids in a lipid bilayer carrier. Such
`methods are related to a detergent dialysis method using
`cationic lipids of any desired concentration in combination
`50 with a dialysis buffer of an ionic strength (salt concentration,
`type of ions) specific for the given cationic lipid concentra-
`tion. With the dialysis buffer of appropriate ionic strength,
`the methods provide encapsulation of 40-80% of the nucleic
`acid solution. The compositions above, and those formed by
`55 the methods described below, exhibit preferably less than
`about 30% degradation, more preferably, less than about
`15% degradation and, even more preferably, less than about
`5% degradation when digested with 0.1 to 10 U and, more
`preferably, 1 U of a nuclease after 30 minutes at 37° C.
`In particular, the invention provides a method for encap-
`sulating a nucleic acid in a lipid bilayer carrier, comprising:
`(a) combining a nucleic acid with a lipid-detergent mix-
`ture comprising an aggregation-preventing agent (e.g.,
`an ATTA-lipid, a PEG-lipid, such as a PEG-ceramide,
`a ganglioside, etc.) in an amount of about 5 mol % to
`about 20 mol %, cationic lipids in an amount of about
`0.5 mol % to about 50 mol % by weight, neutral or
`
`40
`
`45
`
`60
`
`65
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`MRNA-GEN-00222404
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`JA001385
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`Case 1:22-cv-00252-MSG Document 181-3 Filed 01/03/24 Page 36 of 1274 PageID #: 8342
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`US 6,734,171 B1
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`fusogenic lipids in an amount of from about 30 mol %
`to about 70 mol % and a detergent, to provide a nucleic
`acid-lipid-detergent mixture; and
`(b) dialyzing the nucleic acid-lipid-detergent mixture
`against a buffered salt solution and to encapsulate the
`nucleic acid in a lipid bilayer carrier. In these methods,
`the ionic strength (salt concentration) is adjusted for the
`cationic lipid concentration used in the lipid mixture
`and when necessary for the polynucleotide selected for
`encapsulation to entrap from about 40% to about 80%
`of the nucleic acid for any given concentration of
`cationic lipid.
`In another aspect, the present invention provides methods
`for introducing nucleic acids into cells and for inhibiting
`tumor growth in cells using the lipid-nucleic acid formula-
`tions described above.
`Other features, objects and advantages of the invention
`and its preferred embodiments will become apparent from
`the detailed description which follows.
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`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 provides an illustration of the phase properties of
`lipids.
`FIG. 2 illustrates the conical form of one fusogenic lipid
`and further illustrates how fusion with another membrane
`can occur.
`FIG. 3 provides an illustration of the detergent dialysis
`procedure for entrapping nucleic acids in fusogenic lipid
`vesicles.
`FIG. 4 illustrates the structures of three PEG-Ceramide
`conjugates and also provides half-times for their dissociation
`from a lipid vesicle.
`FIG. 5. Encapsulation of pINEXL018 plasmid using
`DODAC/DOPE/PEG-Cer-C8 (30:55:15 mol %) by deter- 35
`gent dialysis in citrate buffer. Effect of varying NaCl con-
`centration with constant citrate concentration (100 mM Na
`citrate, 5 mM HEPES, pH:7.2) is illustrated. The encapsu-
`lation efficiency and polydispersity, x2 (a measure of for-
`mulation homogeneity), of formulations are plotted as func-
`tions of NaCI. Successful formulations are indicated by the
`high encapsulation and low x 2.
`FIG. 6. Encapsulation of pINEX L018 plasmid using
`DODAC/DOPE/PEG-Cer-C8 system by detergent dialysis
`in citrate buffer containing constant NaCl concentration (150
`mM) and 5 mM HEPES, pH:7.2. The relationship between
`varying DODAC mol % and the optimal citrate concentra-
`tion and the effect upon encapsulation efficiency is demon-
`strated. Each point represents a formulation of acceptable
`size and encapsulation efficiency. The optimal range of
`citrate and DODAC concentrations are indicated by the solid
`line. Typically, those preparations prepared below the opti-
`mal citrate concentration have large sizes or aggregate, and
`those formulations prepared above the optimal citrate con-
`centration have low encapsulation efficiencies (0-30%).
`FIG. 7. Effect of DODAC concentration on plasmid
`encapsulation. In this study, the effect of small (1 mol %)
`changes of DODAC concentration were tested at constant
`lipid (10 mg/mL), plasmid (400 Jug/mL) and buffer concen-
`trations. Encapsulation efficiency dropped significantly with
`a decrease in DODAC concentration, indicating that care
`must be taken to precisely deliver DODAC at a given NaC1
`concentration. pINEXL002 was formulated in 150 mM
`NaPO4, 175 mM NaCl, pH 7.4, and pINEXP005 was
`formulated in 150 mM
`150 mM NaCI pH 7.4.
`HG. 8. Encapsulation of pINEXP005. Effect of varying
`NaC1 with constant NaPO4 concentration. The relationship
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`between varying the salt concentration on the encapsulation
`of plasmid over a range of INEX TCS DODAC concentra-
`tions is illustrated. Negative NaCl concentrations indicate
`where the buffer concentration was decreased to an extent
`where no NaCI was included in the dialysis buffer, and the
`phosphate buffer concentration alone was decreased to
`achieve encapsulation. Formulations were prepared contain-
`ing 10 mg/mL total lipid, 400 pg/mL plasmid DNA. In each
`1.0 ml formulation, the PEG-C8 concentration was main-
`tained at 15 mol %, the DODAC