Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 1 of 73
`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 1 of 73
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`E X HI BI T A
`EXHIBIT A
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`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 2 of 73
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`U S O O 8 0 5 8 0 6 9 B 2
`
`(12) U n i t e d States Patent
`Y a W o r s k i et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`U S 8,058,069 B 2
`Nov. 15, 2011
`
`(54) L I P I D F O R M U L A T I O N S F O R N U C L E I C A C I D
`D E L I V E R Y
`
`(75) Inventors: E d w a r d Yaworski, Maple Ridge (CA);
`Kieu L a m , Surrey (CA); Lloyd Jeffs,
`Delta (CA); Lorne Palmer, Vancouver
`(CA); Ian MacLachlan, Mission (CA)
`(73) Assignee: Protiva Biotherapeutics, Inc., Burnaby,
`B.C. (CA)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.: 12/424,367
`
`(22) Filed:
`
`Apr. 15, 2009
`
`(65)
`
`P r i o r Publication D a t a
`U S 2010/O 1 3 0 5 8 8 A 1
`M a y 27, 2 0 1 0
`
`Related U.S. Application Data
`(60) Provisional application No. 61/045.228, filed on Apr.
`15, 2008.
`
`(51) Int. Cl.
`(2006.01)
`C 0 7 H 2L/04
`(2006.01)
`C I 2 N 5/88
`(52) U.S. Cl. ....................................... 435/458:536/24.5
`(58) Field of Classification Search ................. 536/24.5;
`435/.458
`See application file for complete search history.
`
`(56)
`
`R e f e r e n c e s C i t e d
`
`U . S . P A T E N T D O C U M E N T S
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`7, 1986 Papahadjopoulos et al.
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`5/1993 Eppstein et al.
`5,225,212 A
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`1 1/1993 Felgner et al.
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`1/1994 R o s e
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`8/1996 Eppstein et al.
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`
`12/2001 Tousignant et al.
`2 0 0 1 / 0 0 4 8 9 4 0 A 1
`2 0 0 3 / O O 7 7 8 2 9 A 1
`4 / 2 0 0 3 M a c L a c h l a n
`7/2003 Fosnaugh et al.
`2 0 0 3 . 0 1 4 3 7 3 2 A 1
`2004, O O 6 3 6 5 4 A 1
`4 / 2 0 0 4 D a v i s et al.
`2 0 0 4 / O 1 4 2 8 9 2 A 1
`7 / 2 0 0 4 F i n n et al.
`2 0 0 4 / O 2 5 3 7 2 3 A 1
`1 2 / 2 0 0 4 T a c h a s et al.
`2 0 0 4 / O 2 5 9 . 2 4 7 A 1
`1 2 / 2 0 0 4 T u S c h l e t al.
`2 0 0 5 , O O 6 4 5 9 5 A 1
`3 / 2 0 0 5 M a c L a c h l a n et al.
`2005, 0 1 1 8 2 5 3 A 1
`6 / 2 0 0 5 M a c L a c h l a n et al.
`2006, O O O 8 9 1 . 0 A 1
`1 / 2 0 0 6 M a c L a c h l a n et al.
`2 0 0 7 / 0 0 4 2 0 3 1 A 1
`2 / 2 0 0 7 M a c L a c h l a n et al.
`2 0 0 9 , 0 2 9 1 1 3 1 A 1 * 1 1 / 2 0 0 9 M a c l a c h l a n et al. .......... 4 2 4 / 4 5 0
`
`W O
`W O
`W O
`W O
`W O
`W O
`W O
`W O
`W O
`W O
`W O
`W O
`W O
`W O
`W O
`W O
`W O
`W O
`W O
`
`F O R E I G N P A T E N T D O C U M E N T S
`W O 9 1 ( 1 6 0 2 4 A 1
`10, 1 9 9 1
`W O 9 3 , 0 5 1 6 2 A 1
`3, 1 9 9 3
`W O 93/1224.0 A 1
`6, 1 9 9 3
`W O 9 3 , 1 2 7 5 6 A 2
`7, 1 9 9 3
`W O 9 3 / 2 4 6 4 0 A 2
`12, 1 9 9 3
`W O 9 3 , 2 5 6 7 3 A 1
`12, 1 9 9 3
`W O 9 5 / 0 2 6 9 8 A 1
`1, 1 9 9 5
`W O 9 5 / 1 8 8 6 3 A 1
`7, 1 9 9 5
`W O 9 5 / 3 5 3 0 1 A 1
`1 2 / 1 9 9 5
`W O 9 6 , 0 2 6 5 5 A 1
`2, 1 9 9 6
`W O 9 6 , 1 0 3 9 0 A 1
`4 f 1 9 9 6
`W O 9 6 . 4 0 9 6 4 A 2
`12, 1 9 9 6
`W O 9 6 , 4 1 8 7 3 A 1
`12, 1 9 9 6
`W O O 1 , 0 5 3 . 7 4 A 1
`1, 2 0 0 1
`W O 0 2 / 0 3 4 2 3 6 A 2
`5, 2 0 0 2
`W O O 2 / O 8 7 5 4 1 A 1
`11, 2 0 0 2
`W O O 3 , O 9 7 8 0 5 A 2
`1 1 / 2 0 0 3
`W O 2 0 0 4 / 0 6 5 5 4 6 A 2
`8, 2 0 0 4
`W O 2 0 0 4 / 1 1 0 4 9 9 A 1
`1 2 / 2 0 0 4
`(Continued)
`
`O T H E R P U B L I C A T I O N S
`
`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 quatemary a m m o n i u m deter
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`
`(Continued)
`
`Primary Examiner — Brian Whiteman
`(74) Attorney, Agent, or Firm — Kilpatrick Townsend &
`S t o c k t o n L L P
`
`(57)
`A B S T R A C T
`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
`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.
`
`22 Claims, 24 Drawing Sheets
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 3 of 73
`
`U S 8,058,069 B 2
`Page 2
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`W O
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`W O
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`W O
`W O
`W O
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`F O R E I G N P A T E N T D O C U M E N T S
`W O 2 0 0 5 / 0 0 7 1 9 6 A 2
`1, 2 0 0 5
`W O 2 0 0 5 / 0 2 6 3 7 2 A 1
`3, 2 0 0 5
`W O 2 0 0 5 / 1 2 0 1 5 2 A 2
`1 2 / 2 0 0 5
`W O 2 0 0 9 / 0 8 6 5 5 8 A 1
`T 2 0 0 9
`W O 2 0 0 9 / 1 1 1 6 5 8 A 2
`9, 2 0 0 9
`W O 2 0 1 0 / 0 4 2 8 7 7 A 1
`4 / 2 0 1 0
`W O 2 0 1 0 / 0 4 8 . 2 2 8 A 2
`4 / 2 0 1 0
`W O 2 0 1 0 / 0 8 8 5 3 7 A 2
`8, 2 0 1 0
`W O 2 0 1 0 / 1 0 5 2 0 9 A 1
`9, 2 0 1 0
`
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`* cited by examiner
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 4 of 73
`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 4 of 73
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`U.S. P a t e n t
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 1 of 24
`
`U S 8,058,069 B 2
`US 8,058,069 B2
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`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 5 of 73
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`U.S. P a t e n t
`
`Nov. 15, 2011
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`S h e e t 2 o f 2 4
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`U S 8,058,069 B 2
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`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 6 of 73
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`U . S . P a t e n t
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`Nov. 15, 2011
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`Sheet 3 of 24
`
`U S 8,058,069 B 2
`
`Activity of S N A L P U p o n intravenous Administration in Mice
`group m e a n + S D (n=4)
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`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 7 of 73
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`U . S . P a t e n t
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`Nov. 15, 2011
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`Sheet 4 of 24
`
`U S 8,058,069 B 2
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`3 . 0
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`
`F I G . 3
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`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 8 of 73
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`U . S . P a t e n t
`
`Nov. 15, 2011
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`Sheet 5 of 24
`
`U S 8,058,069 B 2
`
`Activity of S N A L P Upon intravenous Administration in Mice
`group m e a n t S D (n=4)
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`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 9 of 73
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`U . S . P a t e n t
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`Nov. 15, 2011
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`Sheet 6 of 24
`
`U S 8,058,069 B 2
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`Activity of S N A L P U p o n Intravenous Administration in Mice
`group m e a n t S D (n=4)
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`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 10 of 73
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`U.S. P a t e n t
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`Nov. 15, 2011
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`S h e e t 7 o f 2 4
`
`U S 8,058,069 B 2
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`Tolerability of IV 1:57 S N A L P in Female BALB/c Mice, n
`
`4, S D error
`
`2
`
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`Alanine Aminotransferase
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`A L T levels indicate hepatocyte damage.
`96
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`
`Tolerability of IV 1:57 S N A L P in Female BALB?c Mice,
`4, S D error
`8.7
`Alanine Aminotransferase
`Aspartate Aminotransferase
`Sorbital Dehydrogenase
`2-fold increase ("3xULN") is considered clinically
`significant
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`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 11 of 73
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`U . S . P a t e n t
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`Nov. 15, 2011
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`Sheet 8 of 24
`
`U S 8,058,069 B 2
`
`FIG. 7 A
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`IV, 48 h, ApoB-10048 U2/2 G1/2 (Dow),
`female B A L B / c mice, n=4, S D error
`
`P B S
`
`1.
`1:57
`1:57
`1:57
`1:57
`Syringe Gear Syringe Gear
`0.05mg 0.05mg 0.1mg/ 0.1mg/
`/kg
`/kg
`kg
`kg
`
`F I G . 7 B
`
`FIG. 7 C
`
`0.6 S N A L P Re-Formulation - Activity Assessment in BALB/c Mice
`-
`4 8 h time point, n=4, S D error bars
`a
`Sp 0.5
`ch
`3 0.4
`sC.
`
`0.3
`& O.2
`n
`$ 0.1
`2 -0.1
`
`1:57
`1:57
`Syringe Gear Syringe Gear
`Press P u m p
`Press P u m p
`0.05 mg/kg
`0.1 mg/kg
`
`S
`S
`9
`8
`
`s
`o
`
`O
`c
`
`
`
`-a, 25%
`
`Efficacy of S N A L P F o r m u l a t i o n s
`Fresh Terminal E
`.
` n=4 female Balb/c mice, S D Error Bars
`7 0
`60
`50
`40
`3 0
`2 0
`1O
`O
`
`Press P u m p Press P u m p
`0.05 mg/kg
`0.1 mg/kg
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 12 of 73
`
`U . S . P a t e n t
`
`Nov. 15, 2011
`
`Sheet 9 of 24
`
`U S 8,058,069 B 2
`
`Tolerability of 1 5 7 S N A L P IV in BALB/c Mice, n=4 (Grp1-3 n=3), S D error
`
`6 %
`
`
`
`4 %
`
`2 %
`
`- 2 %
`
`- 4 %
`
`- 6 %
`
`9:1
`6:1
`6:1
`6:1
`6:1
`9:1
`9mg/ 11 m g 11 m g 13mg/15mg/ 17mg/11 mg/
`k
`
`F I G . 8
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 13 of 73
`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 13 of 73
`
`U.S. Patent
`
`Nov.
`
`15, 2011
`
`Sheet 10 of 24
`
`US 8,058,069 B2
`
`Tolerability of !V 157 SNALP Preparedat 9:1
`
`Lipid:Dru
`
`g Ratio
`
`o.lmUC<“CMDWmUCDUmDmCCUCUCOCCTCSBPeslei>|+NSNOSOUMOUlUUTSHLUUNN
`
`Platelet Count
`
`Gear PBSInLine at
`7 (71) mg/kg
`
`nLine at
`Gear
`411 (112) mg/kg
`
`FIG. 9
`
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 14 of 73
`
`U . S . P a t e n t
`
`Nov. 15, 2011
`
`Sheet 11 of 24
`
`U S 8,058,069 B 2
`
`Tolerability of IV 1:57 Gear P B S In-Line S N A L P in Female BALB/c
`Mice, n=4, S D error
`
`1 2 0 0
`
`1,000
`
`Alanine Aminotransferase
`Aspartate Aminotransferase
`Sorbital Dehydrogenase
`Samples taken at 24 h time point except
`for last grp (48 h).
`
`9
`
`6 O O
`
`4 0 0
`
`2 0 0
`
`
`
`2
`
`4 5
`
`2
`
`%
`
`P B S
`
`6:1
`6:1
`6:1
`6:1
`9:1
`9:1
`S N A L P S N A L P S N A L P S N A L P S N A L P S N A L P S N A L P
`9mg/
`11 mg/ 11 mg/ 13mg/ 15mg/
`17mg/ 11 mg/kg
`kg
`kg
`kg
`kg
`kg
`kg
`48h
`
`F . G . 1 0 A
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 15 of 73
`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 15 of 73
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 12 of 24
`
`US 8,058,069 B2
`
`
`
`P ODIABPTTIPEDIIIZEDSESIETSA
`
`proPTEPITIPTTPEPTIDEIPDPITOTITPOOPIE
`
`
`
`By/bup,|ByG6wz,|By/Bug,|Sybue,|ByBup,|BybuypyBy/6wg|asopYNUIS
`
`
`
`Lvlovee|ee4¢|9€|9¢|ve|€¢|2eLeo€6z|8¢)22|92|9¢|re|e2|Zeb2|0e/6bBi}ZbjorGLvbebzhHbOLHAENBENENSENEENSvy4NENRERN
`
`
`
`(OL)L:6(2)dIVNS1:9(OL)dIVNS1:6Sad
`
`
`ZLLLZLZOL€6glZLL26esoppidiy
`
`ENEENNNENNSENBENNNyN.NNNaha.LOL
`
`
`JOSQS‘p=U‘SOIavgeeWEyUldTWNSEUIT-UlSadJeAD2G:1AlJoAyiqesajo1
`
` ~ENN4NNENAh|N4N:vlNe+NBranneneenettertoateecedeneeteerseeenencetentnaeeLOZANdeedneeEeeendeed
`
`
`ENBENEEN
`SSOr
`
`
`
`
`
`jueoyiuBisAjjeoyul[opasepisuodSi(,NTNXE,,)@SE2J9U!PjOj-Z
`esejajsuejouiulyayepedsy=fF]
`
`QSEISJSUBJJOUIWWYSUIURlYKY
`
`
`eseuabolpAyaq|eWqiosfe
`
`Gob“Dla
`
`
`
`0¢
`
`JEWWON JO pul saddp pjo4-x
`
`
`
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 16 of 73
`
`U.S. P a t e n t
`
`Nov. 15, 2011
`
`Sheet 13 of 24
`
`U S 8 , 0 5 8 , 0 6 9 B 2
`
`F.G. 1 1 A
`1 5 7 Gear P B S In-Line S N A L P Activity From Different input Lipid:Drug Ratios
`IV, 48 h, ApoB-10048 U2/2 G1/2 (Dow), female BALB/c mice, n=4, SD error
`
`2-tailed T-test: p=0.078
`
`S N A L P Re-Formulation - Activity Assessment in BALB/c Mice
`D O W A p o B lead siRNA, 4 8 h time point, n=4, S D error bars
`L L O = 9 %
`
`- 5 3 %
`1 e x c l u d e d
`
`
`
`o
`
`kg
`kg
`mg/kg
`1:57 S N A L P (10:1)
`
`0.1 m g / 0.2 m g / l 0.4 m g /
`kg
`kg
`kg
`mg/kg
`N e w 1:57 S N A L P (7:1)
`
`30
`O 2.5
`s
`
`2 . 0 -
`
`?
`
`( p 1 5 -
`?
`O
`C l
`<C 1 0 -
`
`2
`
`- 0.5
`
`0.0
`
`F I G . 1 1 B
`
`0.40
`
`O . 3 5
`
`0. 2 5
`
`
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 17 of 73
`
`U.S. Patent
`
`US 8,058,069 B2
`
`ES£3
`
`
`o,rmUmCUCO]FTlrCCOOTa=EeCOSTSPEOSCaestst—“‘
`MOWDSSOFMN=
`RA5
`
`FIG. 12
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 18 of 73
`
`U . S . P a t e n t
`
`Nov. 15, 2011
`
`Sheet 15 of 24
`
`U S 8,058,069 B 2
`
`O
`1 2 0 %
`
`1 1 . 5 %
`
`5. 1 1 . 0 %
`
`1 0 5 %
`
`1 0 0 %
`
`9 5 %
`
`9 0 %
`
`- 0 - L u c
`
`- A - P L K 1 4 2 4
`
`8
`
`1 2
`
`16
`
`2 0
`
`2 4
`
`2 8
`
`3 6
`3 2
`Study D a y
`
`4 0
`
`4 4
`
`4 8
`
`5 2
`
`5 6
`
`6 0
`
`FIG. 1 3
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 19 of 73
`
`U . S . P a t e n t
`
`Nov. 15, 2011
`
`Sheet 16 of 24
`
`U S 8,058,069 B 2
`
`- A - Control S N A L P
`
`-<>- Active S N A L P
`
`
`
`O %
`1 5
`
`2 0
`
`2 5
`
`3 0
`
`3 5
`
`6 0
`5 5
`5 0
`4 5
`4 0
`D a y s after seeding
`
`6 5
`
`7 0
`
`7 5
`
`8 0
`
`8 5 9 0
`
`F I G . 1 4
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 20 of 73
`
`U . S . P a t e n t
`
`Nov. 15, 2011
`
`Sheet 17 of 24
`
`U S 8,058,069 B 2
`
`
`
`n 5 4 0.8
`
`CC
`Z
`1.
`E
`?
`
`Na
`-
`n
`C
`C
`c
`CD
`s
`
`2. O
`
`1 6
`
`1 2
`
`O. 4
`
`O. O
`
`P B S
`
`L u c
`
`P L K 1 4 2 4
`
`F I G . 1 5
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 21 of 73
`
`U . S . P a t e n t
`
`Nov. 15, 2011
`
`Sheet 18 of 24
`
`U S 8,058,069 B 2
`
`
`
`- - P L K 1 4 2 4 . 5 ' R A C E
`product
`476bp
`
`F I G . 1 6
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 22 of 73
`
`U.S. P a t e n t
`
`N o v . 15, 2 0 1 1
`
`Sheet 19 of 24
`
`U S 8,058,069 B 2
`
`
`
`F I G . 1 7
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 23 of 73
`
`U . S . P a t e n t
`
`Nov. 15, 2011
`
`Sheet 20 of 24
`
`U S 8,058,069 B 2
`
`
`
`6x2mg/kg M e a n tumor volume
`
`- 0 - L U C - D M A
`- - P L K - D M A
`- A - P L K - D S A
`
`8
`
`1 0
`
`1 2
`
`1 4
`
`1 6
`Days
`
`1 8
`
`2 0
`
`2 2
`
`2 4
`
`F I G . 1 8
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 24 of 73
`
`U . S . P a t e n t
`
`Nov. 15, 2011
`
`Sheet 21 of 24
`
`U S 8,058,069 B 2
`
`PLK m R N A silencing in scid/beige mice treated with 2mg/kg 1:57 SNALP against
`subcutaneous Hep3B tumors
`
`0.70
`
`0.60-
`
`1 0 0 %
`
`.C.
`t
`
`0.50
`
`2 0.40
`
`|
`
`0.30
`
`0 . 2 0 -
`
`0.10-
`
`E
`E
`E
`
`E
`
`E
`
`E
`
`.
`
`R
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`0.00–
`
`S N S )
`8x8
`
`&
`
`n
`
`-15%
`0.
`
`- 3 0 %
`
`- 3 7 %
`
`-65%
`0.
`
`-52%
`
`N.
`S N S
`&S
`
`F I G . 1 9
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 25 of 73
`
`U . S . P a t e n t
`
`Nov. 15, 2011
`
`Sheet 22 of 24
`
`U S 8,058,069 B 2
`
`6x2mg/kg M e a n tumor volume
`
`Crossover dosing of Luc
`c D M A group with 6 x 2
`mg/kg P L K c D S A S N A L P
`
`
`
`- - L u c - D M A
`- H P L K - D M A
`-A - P L K - D S A
`
`Initial 6 x 2 mg/kg
`
`8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6 2 8 3 0 3 2 3 4 3 6 3 8 4 0 4 2 4 4 4 6 4 8 5 0
`D a y s
`
`F I G . 2 0
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 26 of 73
`
`U . S . P a t e n t
`
`Nov. 15, 2011
`
`Sheet 23 of 24
`
`U S 8,058,069 B 2
`
`M e a n h P L K (1:4) : h C G A P D H (1:40) m i n u s "background"
`
`1 2 0
`
`
`
`1 . 0 0
`
`0 . 8 0
`
`0 . 6 0
`
`0 . 4 0
`
`0 . 2 0
`
`0 . 0 0
`
`2 4 h L u c 1:57 2 4 h P L K 1:57 2 4 h P L K 1:57 9 6 h P L K 1:57 9 6 h P L K 1.57
`C D M A
`C D M A
`C D S A
`C D M A
`C D S A
`
`F I G . 2 1
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 27 of 73
`
`U . S . P a t e n t
`
`Nov. 15, 2011
`
`Sheet 24 of 24
`
`U S 8,058,069 B 2
`
`- 0 - 1 : 5 7 P E G - C D S A S N A L P
`
`- A - 1 : 5 7 P E G - C D M A S N A L P
`
`
`
`7 0
`
`5 O
`
`4 O
`
`3 0
`
`2 0
`
`1 0
`
`O
`O
`
`1
`
`2
`
`3
`
`5
`4
`Time (h)
`
`6
`
`7
`
`8
`
`9
`
`F I G , 2 2
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 28 of 73
`
`U S 8,058,069 B 2
`
`1.
`L P I D F O R M U L A T I O N S F O R N U C L E C A C D
`D E L I V E R Y
`
`C R O S S - R E F E R E N C E S T O R E L A T E D
`A P P L I C A T I O N S
`
`The present application claims priority 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.
`
`S T A T E M E N T R E G A R D I N G F E D E R A L L Y
`S P O N S O R E D R E S E A R C H O R D E V E L O P M E N T
`
`Not applicable.
`
`N A M E S O F P A R T I E S T O A J O I N T R E S E A R C H
`A G R E E M E N T
`
`Not applicable.
`
`R E F E R E N C E T O A “ S E Q U E N C E L I S T I N G ”
`Not applicable.
`
`B A C K G R O U N D O F T H E I N V E N T I O N
`
`R N A interference (RNAi) is an evolutionarily conserved
`process in which recognition of double-stranded R N A
`(dsRNA) ultimately leads to posttranscriptional Suppression
`of gene expression. This Suppression is mediated by short
`dsRNA, also called small interfering R N A (siRNA), which
`induces specific degradation of m R N A through c o m p l e m e n
`tary base pairing. In several model systems, this natural
`response has been developed into a powerful tool for the
`investigation of gene function (see, e.g., Elbashiret al., Genes
`Dev., 15:188-200 (2001); H a m m o n d et al., Nat. Rev. Genet.,
`2:110-119 (2001)). More recently, it was discovered that
`introducing synthetic 21-nucleotide d s R N A duplexes into
`mammalian cells could efficiently silence gene expression.
`Although the precise mechanism is still unclear, R N A i
`provides a potential new approach to downregulate or silence
`the transcription and translation of a gene of interest. For
`example, it is desirable to modulate (e.g., reduce) the expres
`sion of certain genes for the treatment of neoplastic disorders
`Such as cancer. It is also desirable to silence the expression of
`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 R N A i to be therapeutically useful. Viral vectors
`are relatively efficient gene delivery systems, but suffer from
`a variety of limitations, such as the potential for reversion to
`the wild-type as well as i m m u n e response concerns. As a
`result, nonviral gene delivery systems are receiving increas
`ing attention (Worgall et al., H u m a n Gene Therapy, 8:37
`(1997); Peeters et al., H u m a n 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 i m m u n e responses that c o m
`promise delivery with Subsequent injections.
`Plasmid DNA-cationic liposome complexes are currently
`the most commonly employed nonviral gene delivery
`
`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 insect cells are disclosed in U.S. Pat. No. 6,458,
`382. Cationic liposome complexes are also disclosed in U.S.
`Patent Publication N o . 2 0 0 3 0 0 7 3 6 4 0 .
`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); T a m
`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
`N o s .
`2 0 0 2 0 0 8 1 7 3 6
`a n d
`20030082103, respectively.
`A gene delivery system containing an encapsulated nucleic
`acid for systemic delivery should be small (i.e., less than
`about 100 n m 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 c o m
`partment. The particle should also readily interact with target
`cells at a disease site in order to facilitate intracellular delivery
`o f a desired nucleic acid.
`Recent work has shown that nucleic acids can be encapsu
`lated in small (e.g., about 70 n m diameter) “stabilized plas
`mid-lipid particles' (SPLP) that consist of a single plasmid
`encapsulated within a bilayer lipid vesicle (Wheeler et al.,
`Gene Therapy, 6:271 (1999)). These SPLPs typically contain
`the “fusogenic' lipid dioleoylphosphatidylethanolamine
`(DOPE), low levels of cationic lipid, and are stabilized in
`aqueous media by the presence of a poly(ethylene glycol)
`(PEG) coating. SPLPs have systemic application as they
`exhibit extended circulation lifetimes following intravenous
`(i.v.) injection, accumulate preferentially at distal tumor sites
`due to the enhanced vascular permeability in Such regions,
`and can mediate transgene expression at these tumor sites.
`The levels of transgene expression observed at the tumor site
`following i.v. injection of SPLPs containing the luciferase
`marker gene are Superior to the levels that can be achieved
`employing plasmid DNA-cationic liposome complexes (li
`poplexes) or naked D N A .
`Thus, there remains a strong need in the art for novel and
`more efficient methods and compositions for introducing
`nucleic acids such as s i R N A into cells. In addition, there is a
`need in the art for methods of downregulating the expression
`of genes of interest to treat or prevent diseases and disorders
`Such as cancer and atherosclerosis. The present invention
`a d d r e s s e s these a n d other needs.
`
`B R I E F S U M M A R Y O F T H E I N V E N T I O N
`
`1 0
`
`1 5
`
`2 5
`
`3 0
`
`3 5
`
`4 0
`
`4 5
`
`5 0
`
`5 5
`
`6 0
`
`6 5
`
`The present invention provides novel, serum-stable lipid
`particles comprising one or more active agents or therapeutic
`agents, methods of making the lipid particles, and methods of
`
`

`

`Case 1:22-cv-02229-MKV Document 42-2 Filed 09/06/22 Page 29 of 73
`
`U S 8,058,069 B 2
`
`3
`delivering and/or administering the lipid particles (e.g., for
`the treatment of a disease or disorder).
`In preferred embodiments, the active agent or therapeutic
`agent is fully encapsulated within the lipid portion of the lipid
`particle Such that the active agent or therapeutic agent in the
`lipid particle is resistant in aqueous solution to enzymatic
`degradation, e.g., by a nuclease or