Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 1 of 695 PageID #: 6792
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 1 of 695 PagelD #: 6792
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`JOINT APPENDIX 01
`JOINT APPENDIX 01
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`GENV-00000004
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`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 2 of 695 PageID #: 6793
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`Case 1:22-cv-00252-MSG Document 18“
`
`Ke)
`“at
`of i
`/
`ANA
`
`TOALL,TOWHOMTHESE; PRESENTS; SHALL, COME:
`UNITED STATES DEPARTMENT OF COMMERCE
`United States Patent and Trademark Office
`
`July 13, 2022
`
`THIS IS TO CERTIFY THAT ANNEXED HERETOIS A TRUE COPY FROM
`THE RECORDSOF THIS OFFICE OF:
`PATENTNUMBER:8,058,069
`
`ISSUE DATE: November15, 2011
`
`ByAuthorityof the
`Under Secretary of Commercefor Intellectual Property
`and Director of the United States Patent and Trademark Office
`
`Rodney Glover
`Certifying Officer
`
`(ar
`Ae
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`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 3 of 695 PageID #: 6794
`Case 1:22-cv-00252-MSG DocumenteT aa 6794
`
`
`US008058069B2
`
`US 8,058,069 B2
`(10) Patent No.:
`a2) United States Patent
`Yaworskiet al.
`(45) Date of Patent:
`Nov. 15, 2011
`
`
`(54) LIPID FORMULATIONS FOR NUCLEIC ACID
`DELIVERY
`
`(75)
`
`Inventors: Edward Yaworski, Maple Ridge (CA);
`Kieu Lam, Surrey (CA); Lloyd Jeffs,
`Delta (CA); Lorne Palmer, Vancouver
`(CA); Ian MacLachlan, Mission (CA)
`
`(73) Assignee: Protiva Biotherapeuties, Inc., Burnaby,
`B.C. (CA)
`Subject to any disclaimer, the term ofthis
`patent is extended or adjusted under 35
`US.C. 154() by0days.
`
`(*) Notice:
`
`(65)
`
`2001/0048940 Al
`12/2001 ‘Tousignant et al.
`2003/0077829 Al
`4/2003 MacLachlan
`2003/0143732 Al
`7/2003 Fosnaugh etal.
`2004/0063654 Al
`4/2004 Davisetal.
`2004/0142892 Al
`7/2004: Finn etal.
`2004/0253723 Al
`12/2004 Tachasetal.
`2004/0259247 Al
`12/2004 Tuschletal.
`2005/0064595 Al
`3/2005 MacLachlan etal.
`2005/0118253 Al
`6/2005 MacLachlan etal.
`2006/0008910 Al
`1/2006 MacLachlanet al.
`2007/0042031 Al
`2/2007 Macl.achlanet al.
`2009/0291131 A1*
`11/2009 Maclachlan et al.
`.......... 424/450
`FOREIGN PATENT DOCUMENTS
`wo Oabetes “ 01993
`wo
`WO 93/12240 Al
`6/1993
`wo
`WO93/12756 A2
`7/1993
`WoO
`WO 93/24640 A2
`12/1993
`wo
`(21) Appl. No.: 12/424,367
`
`WO WO 93/25673 Al—12/1993
`Wo
`(22)
`Filed:
`Apr. 15, 2009
`WO 95/02698 Al
`1/1995
`Wo
`WO 95/18863 Al
`7/1995
`wo
`WO95/35301 AL
`12/1995
`Prior Publication Data
`WO
`WO 96/02655 Al
`2/1996
`WO
`WO 96/10390 Al
`4/1996
`US 2010/0130588 Al
`May 27, 2010
`
`WO WO 96/40964 A2=12/1996
`:
`.
`tcati
`,
`Wo
`WO96/41873 Al
`12/1996
`Related U.S. Application Data
`wo
`WO 01/05374 Al
`1/2001
`(60) Provisional application No. 61/045,228, filed on Apr.
`WO
`WO 02/034236 A2
`5/2002
`15. 2008
`WoO
`WO02/087541 Al
`11/2002
`° WO 03/097805 A2—11/2003. WO
`
`
`
`WO
`WO2004/065546 A2
`8/2004
`WO
`WO 2004/110499 AL
`12/2004
`+sContinued
`(
`)
`
`(51)
`
`Int. Cl.
`(2006.01)
`CO7H 21/04
`(2006.01)
`CI2N 15/88
`(52) US. C1. ccc ccccceeeeeteeeeeenes 435/458; 536/24.5
`(58) Field of Classification Search ................. 536/24.5;
`o
`.
`435/458
`See application file for complete search history.
`.
`:
`References Cited
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`OTHER PUBLICATIONS
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`
`d
`Conti
`(Continued)
`
`Primary Examiner — Brian Whiteman
`(74) Attorney, Agent, or Firm — Kilpatrick Townsend &
`Ne
`Stockton LLP
`.
`(57)
`
`ABSTRACT
`
`The present invention provides novel, stable lipid particles
`comprising one or more active agents or therapeutic agents,
`methods of makingthelipid particles, and methodsofdeliv-
`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 ofmaking the SNALP, and
`methodsof delivering and/or administering the SNALP.
`
`22 Claims, 24 Drawing Sheets
`
`JA002
`GENV-00000002
`
`,
`
`........ 435/458
`....... 536/24.5
`....... 536/24.5
`
`JA002
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 4 of 695 PageID #: 6795
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 4 of 695 PagelD #: 6795
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`US 8,058,069 B2
`
`Page 2
`
`OTHER PUBLICATIONS
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`* cited by examiner
`
`JA003
`GENV-00000003
`
`JA003
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 5 of 695 PageID #: 6796
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 5 of 695 PagelD #: 6796
`
`Nov. 15, 2011
`
`Sheet 1 of 24
`
`U.S. Patent
`
`ActivityofSNALPonHT29CellLine
`
`US 8,058,069 B2
`
`100
`
`10
`
`FIG.1A
`
`
`
`nMTotalsiRNA
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`©
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`(sI9D peyeayun %) AMNGeIA
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`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 6 of 695 PageID #: 6797
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 6 of 695 PagelD #: 6797
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 2 of 24
`
`US 8,058,069 B2
`
`o =o
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`(s}19D peyeasun %) Ageia
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`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 7 of 695 PageID #: 6798
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 7 of 695 PagelD #: 6798
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 3 of 24
`
`US 8,058,069 B2
`
`N a
`
`N oO
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`LiverApoB:GAPDmRNARatio SoOoh
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`Activity of SNALP Upon Intravenous Administration in Mice
`group mean + SD (n=4)
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`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 8 of 695 PageID #: 6799
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 8 of 695 PagelD #: 6799
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 4 of 24
`
`US 8,058,069 B2
`
`2.0
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`FIG. 3
`
`JA007
`GENV-00000007
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`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 9 of 695 PageID #: 6800
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 9 of 695 PagelD #: 6800
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 5 of 24
`
`US 8,058,069 B2
`
`Activity of SNALP Upon Intravenous Administration in Mice
`group mean + SD (n=4)
`ee
`
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`PBS Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8
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`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 10 of 695 PageID #: 6801
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 10 of 695 PagelD #: 6801
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet6 of 24
`
`US 8,058,069 B2
`
`Activity of SNALP UponIntravenous Administration in Mice
`group mean + SD (n=4)
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`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 11 of 695 PageID #: 6802
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 11 of 695 PagelD #: 6802
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 7 of 24
`
`US 8,058,069 B2
`
`4, SD error
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`
`
`{EWWONJoyur]Jaddppjo4-x
`
`RXRAMROOD
`Posseed
`
`
`FIG. 6B
`
`JA0010
`GENV-00000010
`
`JA0010
`
`
`
`
`
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 12 of 695 PageID #: 6803
`Document 181-1 Filed 01/03/24 Page 12 of 695 PagelD #: 6803
`Case 1:22-cv-00252-MSG
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 8 of 24
`
`US 8,058,069 B2
`
`SNALPActivity From Different Manufacturing Processes
`iV, 48 h, ApoB-10048 U2/2 G1/2 (Dow),
`female BALB/c mice, n=4, SD error
`
`d
`
`PBS
`
`1:57
`1:57
`1:57
`Syringe Gear Syringe Gear
`0.05mg 0.05mg 0.1mg/ 0.1mg/
`/kg
`kg
`kg
`!kg
`
`SNALP Re-Formulation - Activity Assessment in BALB/c Mice
`
`2.0
`
`1.0
`
`0.5
`
`0.0
`
`2
`FIG.7A &
`
`¢=w
`
`c EQa
`
`o¢S
`
`Ga
`
`Sx 7|
`
` 1.5
`48 h time point, n=4, SD error bars
`
`“0.1
`
`“4:57
`Syringe Gear
`Press
`Pump
`0.05 mg/kg
`
`Syringe Gear
`Press
`Pump
`0.1 mg/kg
`
`5S
`
`©a
`
`A5<w
`
`o EgO
`
`o $&o
`
`O
`aa
`
`FIG.7B
`
`FIG. 7C
`
`Efficacy of SNALP Formulations
`Fresh Terminal Plasma, n=4 female Balb/c mice, SD Error Bars
`70
`
`
`
`
`
`
`
`PiasmaTotalCholesterol(mg/dL)
`
`
`
`60
`
`50}
`40
`
`30
`
`20
`
`10
`
`0
`
`99,
`o%
`
`-15%
`
`717%
`
`19%
`
`
` Zs
`
`PBS
`
`J
`1:57 1 4:57 | 4:57 | 1:57
`Syringe Gear Syringe Gear
`Press
`Pump Press Pump
`0.05 mg/kg
`0.1 mg/kg
`
`JA0011
`GENV-00000011
`
`JA0011
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 13 of 695 PageID #: 6804
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 13 of 695 PagelD #: 6804
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 9 of 24
`
`US 8,058,069 B2
`
`Tolerability of 1|57 SNALP IV in BALB/c Mice, n=4 (Grp1-3 n=3), SD error
`
`
`
`
`
`
`
`
`
`
`
`
`9:1
`
`#91
`
`61
`
`61
`
`61
`
`6:1
`
`6%
`
`4%
`
`2%
`
`0%
`
`-2%
`
`-4%
`
`
`
`
`
`LossatHour24(vsPre-Dose) -6%
`BodyWeight
`
`Smg/ 11mg mgge yee kee
`
`kg
`
`k
`
`kg
`
`11img/kg
`
`
`FIG. 8
`
`JA0012
`GENV-00000012
`
`JA0012
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 14 of 695 PageID #: 6805
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 14 of 695 PagelD #: 6805
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 10 of 24
`
`US 8,058,069 B2
`
`
`
`VSiP26901
`
`(A Platelet Count
`
`
`
`Tolerability of lV 1]57 SNALP Prepared at 9:1 Lipid:Drug Ratio
`
`NEEa\\=ocog@fFoFOF8bfo092002
`Sooa668&8GSG&G+AS&S8G©FFSG
`
`
`
`Gear PBS InLine at
`) mg/kg
`7 (71
`
`Gear PBS inLine
`11 (112) mg/kg
`
`FIG. 9
`
`JA0013
`GENV-00000013
`
`JA0013
`
`
`
`
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 15 of 695 PageID #: 6806
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 15 of 695 PagelD #: 6806
`
`U.S. Patent
`
`Nov.15, 2011
`
`Sheet 11 of 24
`
`US 8,058,069 B2
`
`Tolerability of lV 1:57 Gear PBS In-Line SNALP in Female BALB/c
`Mice, n=4, SD error
`
`Alanine Aminotransferase
`Aspartate Aminotransferase
`Sorbital Dehydrogenase
`Samples taken at 24 h time point except
`
`for last grp (48 h).
`
`1.200
`’
`
`1,000
`
`S 800
`
`”
`
`‘oo
`
`i
`
`
`
`BIRR
`
`093) :
`y
`WA|
`q)
`Pye:
`WATE
`TWO
`oe
`456}
`a
`iy
`rr
`yy
`| |aA
`AA
`WA
`YAl|
`a.
`22077
`
`52-37 (Y} (60 42-0749 Bay AY
`9 eee BAea AAS tenet Ves VAAee
`9:1
`6:1
`6:1
`6:1
`6:1
`9:1
`9:1
`PBS
`snaLP SNALP SNALP SNALP SNALP SNALP SNALP
`9mg/
`1img/
`11img/
`13mg/
`15mg/
`17mg/ 11mg/kg
`kg
`kg
`Kg
`kg
`kg
`kg
`48h
`
`(VY
`
`VA
`
`688
`
`
`
`
`FIG. 10A
`
`JA0014
`GENV-00000014
`
`JA0014
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 16 of 695 PageID #: 6807
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 16 of 695 PagelD #: 6807
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 12 of 24
`
`US 8,058,069 B2
`
`
`
`1img/kg
`RARE)
`PPELoeocd
`Rese
`CZZIZLIIITTEA
`
` ie:
` 17mg/kg 121
`:
`PSOBO
`POOFPTEOTee
`RRSreeae
`ed
`4
`
`ZI
`(LILILILIIITILIILILITTLVIIIIIEELITILLITE:
`Roo
` 15mg/kg 107
`RapialBeer46:1SNALP(7)
`13mg/kg 93
`FIG.10B
`11rma/kg 78
`
`
`AlanineAminotransferase
`1img/kg
`9:1SNALP(10)
`
`9mg/kg
`
`SEAiceFERRE
`siRNAdose |
`Lipiddose
`
`
`
`
`
`
`2-foldincrease("3xULN")isconsideredclinicallysignificant
`
`112 9:1(10)
`
`112
`
`PBS
`
`—=©+aO
`
`o©6
`
`9 ~o
`
`y co
`
`O
`iO
`wo
`stcD
`oo
`oy
`WN
`
`~-o2c
`
`9QNc
`
`eNaN
`
`
`
`ELL,RERRAS
`PPLeLL.eeeee
`ROCCEECRTES
`
`eR
`:
`» EPLLLLA
`:
`
`POI £0
`
`NW
`
`wN w
`
`xNo
`
`oNN“
`
`l=N
`
`S©“o7 c
`
`oo
`a=
`©o
`wwod
`wv-
`“=
`No
`
`Gi
`
`RY
`
`ta
`Oy
`eeetd
`ea
`cece
`EMA
`RES
`F724
`
`-- o
`
`O-
`
`0
`
`T
`Q
`fap)
`
`F
`Q
`N
`
`Tv
`Oo
`™
`
`
`
`
`
`DehydrogenaseAspartateAminotransferaseEESorbital
`
`SY Es]
`
`
`
`
`
`
`
`Tolerabilityof'V1:57GearPBSIn-LineSNALPinFemaleBALB/cMice,n=4,SDerror
`
`
`
`
`
`JEWWORN Jo yilur seddp pjo4-x
`
`JA0015
`GENV-00000015
`
`JA0015
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 17 of 695 PageID #: 6808
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 17 of 695 PagelD #: 6808
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 13 of 24
`
`US 8,058,069 B2
`
`FIG. 11A
`
`1|57 Gear PBS In-Line SNALP Activity From Different Input Lipid:Drug Ratios
`IV, 48h, ApoB-10048 U2/2 G1/2 (Dow}, female BALB/c mice, n=4, SD error
`
`3.0)
`
`
`
`2-tailed T-test: p=0.078
`
`
`
`,
`x
`x
`N
`vhy® Sao Tho yd ts
`S Wn Wag
`REQS™ WKN
`QOGQS™
`IQ’ ROQN go So:
`
`84%
`
`\
`
`2.5
`
`2©
`
`3
`X20
`
`a c
`
`f6
`
`co
`o
`< 1.0
`
`©Q
`
`1.5 4
`
`0.5
`
`0.0
`
`G2—
`
`0.40
`
`0.35
`
`So w °
`
`2 bo on
`
`So Ny Qo
`
`0.15
`
`0.10
`
`0.05
`
`
`
`
`
`RelativePlasma[ApoB-100]
`
`FIG. 11B
`
`SNALP Re-Formulation - Activity Assessment in BALB/c Mice
`DOW ApoBlead siRNA,48 h time point, n=4, SD error bars
`LLQ =9%
`
`-28%
`
`
`
`0.00leEEee etoe
`
`.
`
`
`
`
`
`
`mo/kg
`kg
`kg
`mg/kg
`kg
`kg
`4:57 SNALP (10:1)
`New 1:57 SNALP (7:1)
`
`g
`
`
`0.4 ma|
`
`kg
`
`JA0016
`GENV-00000016
`
`JA0016
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 18 of 695 PageID #: 6809
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 18 of 695 PagelD #: 6809
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 14 of 24
`
`US 8,058,069 B2
`
`>>>©>7OOODSbOON
`
`
`
`2a
`
`AE
`
`1:57 SNALP
`
`(10:1)
`
`FIG. 12
`
`JA0017
`GENV-00000017
`
`JA0017
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 19 of 695 PageID #: 6810
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 19 of 695 PagelD #: 6810
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 15 of 24
`
`US 8,058,069 B2
`
`—t- PLK1424
`
`BodyWeight
`
`8
`
`12
`
`16
`
`20
`
`24
`
`28
`
`36
`32
`Study Day
`
`40
`
`44
`
`48
`
`52
`
`56
`
`60
`
`FIG. 13
`
`JA0018
`GENV-00000018
`
`JA0018
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 20 of 695 PageID #: 6811
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 20 of 695 PagelD #: 6811
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 16 of 24
`
`US 8,058,069 B2
`
`-&- ControlkSNALP
`
`-—<-— Active SNALP
`
`Survival
`
`20
`
`25
`
`30
`
`35
`
`60
`55
`50
`45
`40
`Days after seeding
`
`65
`
`70
`
`75
`
`80
`
`85
`
`90
`
`FIG. 14
`
`JA0019
`GENV-00000019
`
`JA0019
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 21 of 695 PageID #: 6812
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 21 of 695 PagelD #: 6812
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 17 of 24
`
`US 8,058,069 B2
`
`PLK1424
`
`<=aE ao
`
`u
`<<
`Qo
`
`= ¥_ o
`
`Ox c©o
`
`O =
`
`FIG. 15
`
`JA0020
`GENV-00000020
`
`JA0020
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 22 of 695 PageID #: 6813
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 22 of 695 PagelD #: 6813
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 18 of 24
`
`US 8,058,069 B2
`
`476bp
`
`~@— PLK1424 5'RACE
`product
`
`FIG. 16
`
`JA0021
`GENV-00000021
`
`JA0021
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 23 of 695 PageID #: 6814
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 23 of 695 PagelD #: 6814
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 19 of 24
`
`US 8,058,069 B2
`
`x200 mag
`
`
`x400 mag
`
`
`x400 mag
`
`FIG. 17
`
`JA0022
`GENV-00000022
`
`JA0022
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 24 of 695 PageID #: 6815
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 24 of 695 PagelD #: 6815
`
`U.S. Patent
`
`Nov.15, 2011
`
`Sheet 20 of 24
`
`US 8,058,069 B2
`
`6x2mg/kg Mean tumor volume
`
`volume
`
`
`—@ Luc-DMA
`
`~@- PLK-DMA
`—#- PLK-DSA
`
`Tumor
`
`10
`
`12
`
`14
`
`16
`Days
`
`18
`
`20
`
`22
`
`24
`
`FIG. 18
`
`JA0023
`GENV-00000023
`
`JA0023
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 25 of 695 PageID #: 6816
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 25 of 695 PagelD #: 6816
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 21 of 24
`
`US 8,058,069 B2
`
`PLK mRNAsilencingin scid /beige mice treated with 2mg/kg 1:57 SNALPagainst
`subcutaneous Hep3B tumors
`
`0.705
`060.
`050.
`0.40,
`
`0.30
`
`0.20
`
`0.10
`
`0.00-
`
`
`
`
`
`
`
`52%
`
`
`
`
`
`iH ii
`
`
`
`
`
`MeanhPLK1/hGAPDHratio
`
`
`
`FIG. 19
`
`JA0024
`GENV-00000024
`
`JA0024
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 26 of 695 PageID #: 6817
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 26 of 695 PagelD #: 6817
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 22 of 24
`
`US 8,058,069 B2
`
`oO
`
`Kk
`
`
`
`umorvolum
`
`6x2mg/kg Mean tumor volume
`
`Crossover dosing of Luc
`cDMA group with 6 x 2
`mg/kg PLK cDSA SNALP
`
`-* Luc-DMA
`= PLK-DMA
`-#- PLK-DSA
`
`initial 6 x 2 mg/kg
`SNALP
`
`pm, 2
`
`ada
`
`2200
`
`2000
`
`1800
`
`1600
`
`=okpo-sOQ2Oo©
`
`1000
`
`800
`
`600
`
`400
`
`200
`
`0
`
`8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
`Days
`
`FIG. 20
`
`JA0025
`GENV-00000025
`
`JA0025
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 27 of 695 PageID #: 6818
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 27 of 695 PagelD #: 6818
`
`US.
`
`Patent
`
`Nov. 15, 2011
`
`Sheet 23 of 24
`
`US 8,058,069 B2
`
`
`
`
`
`MeanhPLK(1:4):hGAPDH(1:40)
`
`1.20
`
`1.00
`
`0.80
`
`0.60
`
`0.40
`
`0.20
`
`0.00
`
`Mean hPLK (1:4) : hGAPDH (1:40) minus "background"
`
`
`
`
`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
`
`FIG. 21
`
`JA0026
`GENV-00000026
`
`JA0026
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 28 of 695 PageID #: 6819
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 28 of 695 PagelD #: 6819
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 24 of 24
`
`US 8,058,069 B2
`
`
`
`%injecteddose
`
`—— 1:57 PEG-cDSA SNALP
`
`—t— 1:57 PEG-cDMA SNALP
`
`
`
`Time (h)
`
`FIG, 22
`
`JA0027
`GENV-00000027
`
`JA0027
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 29 of 695 PageID #: 6820
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 29 of 695 PagelD #: 6820
`
`US 8,058,069 B2
`
`1
`LIPID FORMULATIONS FOR NUCLEIC ACID
`DELIVERY
`
`CROSS-REFERENCES TO RELATED
`APPLICATIONS
`
`The present application claimspriority to U.S. Provisional
`Application No. 61/045,228, filed Apr. 15, 2008, the disclo-
`sure of which is herein incorporated by reference in its
`entirety for all purposes.
`
`1
`
`STATEMENT REGARDING FEDERALLY
`SPONSORED RESEARCH OR DEVELOPMENT
`
`
`
`Not applicable.
`
`NAMES OF PARTIES TO A JOINT RESEARCH
`AGREEMENT
`
`Notapplicable.
`
`REFERENCETO A “SEQUENCELISTING”
`
`Not applicable.
`
`BACKGROUNDOF THE INVENTION
`
`2
`vehicles (Felgner, 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 insectcells are disclosed in U.S. Pat. No. 6,458,
`382. Cationic liposome complexesare also disclosed in U.S.
`Patent Publication No. 20030073640.
`Cationic liposome complexes are large, poorly defined
`systemsthat are not suited for systemic applications and can
`9 elicit considerable toxic side effects (Harrisonet al., Biotech-
`niques, 19:816 (1995), Lietal., The Gene, 4:891 (1997); Tam
`etal, Gene Ther, 7:1867 (2000)). Aslarge, positively charged
`aggregates,lipoplexes are rapidly cleared when administered
`in vivo, with highest expression levels observedinfirst-pass
`organs, particularly the lungs (Huanget al., Nature Biotech-
`nology, 15:620 (1997); Templetonet al., Nature Biotechnol-
`ogy, 15:647 (1997); Hoflandet al., Pharmaceutical Research,
`14:742 (1997)).
`Otherliposomaldelivery systems include, for example, the
`use ofreverse micelles, anionic liposomes, and polymerlipo-
`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
`dextrin or glycerol-phosphocholine polymersare 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 ofthe vascular com-
`partment. The particle shouldalso readily interact with target
`cells at a diseasesite in orderto facilitate intracellular delivery
`of a desired nucleic acid.
`Recent work has shownthat 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 (Wheeleret al.,
`Gene Therapy, 6:271 (1999)). These SPLPstypically 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 tumorsites
`due to the enhanced vascular permeability in such regions,
`and can mediate transgene expression at these tumorsites.
`The levels of transgene expression observedat the tumorsite
`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 siRNAinto cells. In addition, there is a
`needin the art for methods of downregulating the expression
`of genes ofinterest to treat or prevent diseases and disorders
`such as cancer and atherosclerosis. The present invention
`addresses these and other needs.
`
`40
`
`45
`
`RNAinterference (RNAj) 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 mRNAthrough complemen-
`tary base pairing. In several model systems, this natural
`response has been developed into a powerful tool for the
`investigation ofgene function(see,e.g., Elbashir et al., Genes
`Dev., 15:188-200 (2001); Hammondet 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
`providesa potential new approach to downregulate or silence
`the transcription and translation of a gene ofinterest. For
`example, it is desirable to modulate (e.g., reduce) the expres-
`sion of certain genes forthe treatment ofneoplastic disorders
`such as cancer.It is also desirable to silence the expression of
`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 5
`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 etal., Gene Therapy, 1:192 (1994); Hopeet al.,Molecular
`MembraneBiology, 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 immuneresponses that com-
`promise delivery with subsequent injections.
`Plasmid DNA-cationic liposome complexes are currently
`the most commonly employed nonviral gene delivery
`
`65
`
`BRIEF SUMMARY OF THE INVENTION
`
`The present invention provides novel. serum-stable lipid
`particles comprising one or more active agents or therapeutic
`agents, methods ofmakingthe lipid particles, and methods of
`
`JA0028
`GENV-00000028
`
`JA0028
`
`

`

`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 30 of 695 PageID #: 6821
`Case 1:22-cv-00252-MSG Document 181-1 Filed 01/03/24 Page 30 of 695 PagelD #: 6821
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`US 8,058,069 B2
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`4
`ing to a mammalian subjecta lipid particle des