Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 1 of 74
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 1 of 74
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`EXHIBIT E
`EXHIBIT E
`
`
`
`

`

`(12) United States Patent
`YaWorski et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 9,364.435 B2
`*Jun. 14, 2016
`
`US009364435B2
`
`(54) LIPID FORMULATIONS FOR NUCLEICACID
`DELIVERY
`
`(71) Applicant: Protiva Biotherapeutics, Inc., Burnaby
`(CA)
`(72) Inventors: Edward Yaworski, Maple Ridge (CA);
`Kieu Lam, Surrey (CA); Lloyd Jeffs,
`Delta (CA); Lorne Palmer, Vancouver
`(CA); Ian MacLachlan, Mission (CA)
`
`(*) Notice:
`
`(73) Assignee: PROTIVA BIOTHERAPEUTICS,
`INC., Burnaby, BC (CA)
`s
`s
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`This patent is Subject to a terminal dis-
`claimer.
`
`(21) Appl. No.: 14/462,441
`9
`Aug. 18, 2014
`
`(22) Filed:
`
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`(65)
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`Prior Publication Data
`
`FOREIGN PATENT DOCUMENTS
`
`US 2015/O164799 A1
`
`Jun. 18, 2015
`
`Related U.S. Application Data
`(63) Continuation of application No. 13/928.309, filed on
`Jun. 26, 2013, OW Pat. No. 8,822,668, which is a
`continuation of application No. 13/253,917, filed O
`Oct. 5, 2011, now Pat. No. 8,492,359, which is a
`continuation of application No. 12/424,367, filed on
`Apr. 15, 2009, now Pat. No. 8,058,069.
`(60) Provisional application No. 61/045.228, filed on Apr.
`15, 2008.
`(51) Int. Cl.
`CI2N IS/II
`A 6LX 9/27
`A6 IK 4.8/00
`C7H 2L/00
`C07J 9/00
`CI2N IS/II3
`(52) U.S. Cl.
`CPC ............. A61K 9/1272 (2013.01); A61 K9/1271
`(2013.01); A61K 48/0025 (2013.01); C07H
`21/00 (2013.01); C07J 9/00 (2013.01); C12N
`15/III (2013.01); C12N 15/113 (2013.01);
`CI2N 2310/14 (2013.01); C12N 2320/32
`(2013.01)
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2010.01)
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 2 of 74
`
`(58) Field of Classification Search
`S. E. AR No. in N. 9/1271
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`(Continued)
`Primary Examiner — Brian Whiteman
`(74) Attorney, Agent, or Firm — Kilpatrick Townsend &
`Stockton LLP
`ABSTRACT
`(57)
`The present invention provides novel, stable lipid particles
`comprising one or more active agents or therapeutic agents,
`methods of making the lipid particles, and methods of deliv
`9.
`p1d p
`ering and/or administering the lipid particles. More particu
`larly, the present invention provides stable nucleic acid-lipid
`particles (SNALP) comprising a nucleic acid (such as one or
`more interfering RNA), methods of making the SNALP, and
`methods of delivering and/or administering the SNALP.
`20 Claims, 24 Drawing Sheets
`
`

`

`US 9,364.435 B2
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`
`

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`U.S. Patent
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`US 9,364.435 B2
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`U.S. Patent
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`Jun. 14, 2016
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`Sheet 2 of 24
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`US 9,364.435 B2
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`6 ?IduueS
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`k k
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`+
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`(seo peee Jun%) AqelA
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`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 3 of 24
`
`US 9,364.435 B2
`
`Activity of SNALP Upon intravenous Administration in Mice
`group mean + SD (n=4)
`
`2. O
`
`O. 5 w
`
`0. O
`«? SS s
`cS c. c.
`
`X
`SS s
`c.
`
`Nb N^
`NS NN N
`o
`o A Q
`o
`S S S S S S S S S
`c.c. c. c9; c.9 c. c9cs
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 7 of 74
`
`FIG. 2
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 4 of 24
`
`US 9,364.435 B2
`
`
`
`3.0
`
`2. 5
`
`2. O
`
`O15
`
`O. 5
`
`0. O
`
`-47%. VS PBS Control
`
`-77% WS PBS Control
`
`PBS
`
`2:30 SNALP 5x1 mg/kg 1:57 SNALP 5x0.1 mg/kg
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 8 of 74
`
`FIG. 3
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 5 of 24
`
`US 9,364.435 B2
`
`Activity of SNALP Upon Intravenous Administration in Mice
`group mean + SD (n=4)
`
`Int
`
`PBS Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8
`
`112 O-5O
`
`O 5 O.O
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 9 of 74
`
`FIG. 4
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 6 of 24
`
`US 9,364.435 B2
`
`Activity of SNALP Upon intravenous Administration in Mice
`group meant SD (n=4)
`
`112 O-5-O-
`
`O 5 OO
`
`Nix No
`NS NN N. N.
`Q to
`so o 1,
`b
`se in
`<ssssssss Sess’ssess.
`c. c c c c
`s c c c c c c c CS
`
`I
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 10 of 74
`
`FIG. 5
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 7 of 24
`
`US 9,364.435 B2
`
`Tolerability of IV 1:57 SNALP in Female BALB/c Mice, n=4, SD error
`
`ALT levels indicate hepatocyte damag
`96
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Tolerability of
`IV 1:57 SNALP in Female BALB?c Mice,
`4, SD error
`n
`Alanine Aminotransferase
`Aspartate Aminotransferase
`Sorbital Dehydrogenase
`is considered clinically
`2-fold increase ("3XULN")
`significant
`
`çN? P |(-)-(');
`
`**********************j CN ·
`-XXXXXXXXXXXXXXXXXX?
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 11 of 74
`
`Lipid dose
`siRNA dose
`
`F.G. 6B
`
`9 mg/kg
`
`102
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 8 of 24
`
`US 9,364.435 B2
`
`S. 2.0
`FIG. 7A is -
`CC
`É 1.5
`8
`1.0
`
`CC
`(D
`g 0.5
`C.
`CC
`2
`
`0.0
`
`SNALP Activity From Different Manufacturing Processes
`IV, 48 h, ApoB-10048 U2/2 G1/2 (Dow),
`female BALB/C mice, n=4, SD error
`
`1:57
`1:57
`1:57
`PBS 1:57
`Syringe Gear Syringe Gear
`0.05mg 0.05mg 0.1mg/ 0.1mg/
`/kg
`/kg
`kg
`kg
`
`0.6 SNALP Re-Formulation - Activity Assessment in BALB/c Mice
`48 h time point, n=4, SD error bars
`
`FIG. 7B as
`O 0.5
`ch
`8. 0.4
`sC.
`0.3
`& 02
`n
`$ 0.1
`c
`2 -0.1
`
`Syringe Gear Syringe Gear
`Press Pump Press Pump
`0.05 mg/kg
`0.1 mg/kg
`
`FIG. 7C
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 12 of 74
`
`Efficacy of SNALP. Formulations
`1. Fresh Terminal Plasma, n=4 female Balb/c mice, SD Error Bars
`g
`70
`co)
`5 60
`S
`50
`92 8
`40
`30
`20
`10
`O
`
`
`
`st
`o
`
`Syringe Gear 'Syringe Gear
`Press Pump Press Pump
`0.05 mg/kg
`0.1 mg/kg
`
`r
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 9 of 24
`
`US 9,364.435 B2
`
`Tolerability of 157 SNALP IV in BALB/c Mice, n=4 (Grp1-3 n=3), SD error
`
`6%
`
`d g
`G 4%
`92
`
`W
`
`3t 2% . . .
`
`.
`
`. .
`
`.
`
`.
`
`. . . .
`
`.
`
`. . . . .
`
`. . .
`
`. . . . . .
`
`. . . . . . .T. . . . . .
`
`. . . . .
`
`.
`
`. . . . . . . . . . . . . .
`
`. . . . . . . . .
`
`. . . . .
`
`. . . . . . . 3% H
`
`km
`
`7
`
`TT 4
`
`W. %
`N
`3% H
`: o, Illir. 2. H%.1% 3% E
`91 on 61 sh 6:1
`6:1
`9:1
`W2
`|
`9mg/ 11 mg 11 mg 13mg/15mg/ 17mg/11mg/
`
`:
`E
`
`- -2%
`
`-
`
`-
`
`- - -
`
`- -
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`. - - -
`
`-
`
`-
`
`- -
`
`-
`
`-
`
`- - - -
`
`- - - -
`
`-
`
`-
`
`-
`
`- kg
`
`/kg
`
`/kg
`
`kg
`
`kg
`
`kg
`
`kg
`
`PBS 91
`7mg/kg
`-40A . . . . . . . . . . . . . . . . . . . . . . . .
`. . . . . . . . .
`9.1 PBS
`11 mg/kg
`
`. . . . . .
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`. .
`
`. . . .
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`. . . . . . . . . . . . . . .
`
`is 4%
`S
`f
`
`-6%
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 13 of 74
`
`FIG. 8
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 10 of 24
`
`US 9,364.435 B2
`
`Tolerability of IV 157 SNALP Prepared at 9:1 Lipid:Drug Ratio
`
`813
`
`828
`
`No.
`NOEN 794
`
`C) O C O o co o
`<t. CN o co co < c\!
`O O CO O C O o CD
`
`
`
`
`
`9
`8
`7
`Gear PBS InLine at
`11 (112) mg/kg
`
`ine at
`7 (71) mg/kg
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 14 of 74
`
`FIG. 9
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 11 of 24
`
`US 9,364.435 B2
`
`Tolerability of IV 1:57 Gear PBS in-Line SNALP in Female BALB/c
`Mice, n=4, SD error
`
`Alanine Aminotransferase
`Aspartate Aminotransferase
`E3 Sorbital Dehydrogenase
`Samples taken at 24 h time point except
`for last grp (48 h).
`
`2
`
`6 8 8
`
`6
`
`1200
`s
`
`1,000
`
`s
`
`800
`
`600
`
`400
`
`200
`
`%
`:
`::Kx.
`sia
`orza::125, 22:EX), 12:33, P7:
`6:1
`:1
`6:1
`PBS
`9:1
`9:1
`6:1
`SNALP SNALP SNALP SNALP SNALP SNALP SNALP
`9mg/ 11 mg/ 11 mg/ 13mg/ 15mg/ 17mg/ 11 mg/kg
`kg
`kg
`kg
`kg
`kg
`kg
`48h
`
`: 2
`
`al
`
`*
`
`*
`
`
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 15 of 74
`
`FIG. 10A
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 12 of 24
`
`US 9,364.435 B2
`
`
`
`Zaaya
`X.
`-xxxx
`
`XXX SS
`X
`
`SS
`
`YYYYYYZ
`
`77,777-??
`Sassass
`22
`
`N
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 16 of 74
`
`09
`
`07
`
`09
`
`|euONJO -ul Jeddin plo--X
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 13 of 24
`
`US 9,364.435 B2
`
`FIG. 11A
`157 Gear PBS In-Line SNALP Activity From Different input Lipid: Drug Ratios
`IV, 48 h, ApoB-10048 U2/2 G1/2 (Dow), female BALB/cmice, n=4, SD error
`30
`
`2-tailed T-test: p=0.078
`
`N
`
`N
`N
`Y N
`QNS)
`N
`Q
`'.
`N
`.N
`SS SSNS SS SS R&S SSS's
`
`SNALP Re-Formulation - Activity Assessment in BALB/c Mice
`DOWApoB lead siRNA, 48 h time point, n=4, SD error bars
`LLO =9%
`
`-53%
`excluded
`
`.9
`
`2.5
`s
`2.0
`
`?
`
`9. (D 15
`
`O gd.
`CC 10
`al
`2
`-
`
`0.5
`
`O.O
`
`FIG. 11B
`
`0.40
`
`0.35 -
`
`0.30.
`
`0. 2 5
`
`O 2 O
`
`0.10
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 17 of 74
`
`0.05 -
`
`O.OO
`
`kg
`kg
`mg/kg
`1:57 SNALP (10:1)
`
`0.1 mg/ 0.2 mg/l 0.4 mg/
`kg
`kg
`kg
`mg/kg
`New 1:57 SNALP (7:1)
`
`
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 14 of 24
`
`US 9,364.435 B2
`
`N
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 18 of 74
`
`FIG. 12
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 15 of 24
`
`US 9,364.435 B2
`
`O
`120%
`
`11.5%
`
`5. 110%-
`
`10.5%
`
`100%-
`
`95%-
`
`90%-
`8
`
`-0- Luc - A - PLK1424
`
`12 16 20 24 28 32 36 40 44 48 52 56 60
`Study Day
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 19 of 74
`
`FIG. 13
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 16 of 24
`
`US 9,364.435 B2
`
`
`
`- A - Control SNALP -d- Active SNALP
`
`15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90
`Days after seeding
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 20 of 74
`
`FIG. 14
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 17 of 24
`
`US 9,364.435 B2
`
`
`
`2. O
`
`1 6
`
`1 2
`
`0. 8
`
`0. 4
`
`CC
`Z
`Y
`E
`?
`d
`CC
`CD
`C
`Na
`-
`n
`C
`C
`c
`CD
`
`D 0. O
`
`PBS
`
`Luc
`
`PLK1424
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 21 of 74
`
`FIG. 15
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 18 of 24
`
`US 9,364.435 B2
`
`
`
`-- PLK1424 5'RACE
`product
`476bp
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 22 of 74
`
`FIG, 16
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 19 of 24
`
`US 9,364.435 B2
`
`
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 23 of 74
`
`FIG. 17
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 20 of 24
`
`US 9,364.435 B2
`
`6x2mg/kg Mean tumor volume
`
`--Luc-DMA
`-- PLK-DMA
`- A - PLK-DSA
`
`
`
`100
`
`800
`
`CD 60
`C
`c
`d
`
`40
`
`20
`
`O
`8
`
`T
`10
`
`12
`
`--
`14
`
`-Thor
`18
`20
`22
`24
`
`16
`Days
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 24 of 74
`
`FIG. 18
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 21 of 24
`
`US 9,364.435 B2
`
`
`
`
`
`PLK mRNA silencing in scid/beige mice treated with 2mg/kg 1:57 SNALP against
`subcutaneous Hep3B tumors
`
`0.60
`
`100%
`
`
`
`0.30
`
`0.20
`
`0.10
`
`S s NS)
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 25 of 74
`
`FIG. 19
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 22 of 24
`
`US 9,364.435 B2
`
`6x2mg/kg Mean tumor volume
`
`Crossover dosing of Luc
`cDMA group with 6 x 2
`mg/kg PLK cDSA SNALP
`
`2200
`2000
`1800
`1600-
`E
`1400
`O
`> is 1200
`
`
`
`H
`
`-0- LUC-DMA
`-- PLK-DMA
`- A - PLK-DSA
`
`Initial 6 x 2 mg/kg
`
`8 10 12 14 16 18 20 22 24 26 2. 30 32 34 36 38 40 42 44 46 48 50
`ays
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 26 of 74
`
`FIG. 20
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 23 of 24
`
`US 9,364.435 B2
`
`Mean hPLK (1:4): hCGAPDH (1:40) minus "background"
`
`120
`
`
`
`1.OO
`
`O.80
`
`O.60
`
`0.40
`
`0.20
`
`O.00
`
`24h Luc 1:57 24h PLK 1:57 24h PLK 1:57 96h PLK 1:57 96h PLK 1.57
`CDMA
`CDMA
`CDSA
`CDMA
`CDSA
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 27 of 74
`
`FIG. 21
`
`

`

`U.S. Patent
`
`Jun. 14, 2016
`
`Sheet 24 of 24
`
`US 9,364.435 B2
`
`
`
`-0 - 1:57 PEG-CDSA SNALP
`
`-A - 1:57 PEG-CDMA SNALP
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 28 of 74
`
`FIG. 22
`
`

`

`US 9,364,435 B2
`
`1.
`LPID FORMULATIONS FOR NUCLECACD
`DELIVERY
`
`CROSS-REFERENCES TO RELATED
`APPLICATIONS
`
`The present application is a continuation of U.S. applica
`tion Ser. No. 13/928.309, filed Jun. 26, 2013, which applica
`tion is a continuation of Ser. No. 13/253,917, filed Oct. 5,
`2011, now U.S. Pat. No. 8,492.359, which application is a
`continuation of Ser. No. 12/424,367 filed Apr. 15, 2009, now
`U.S. Pat. No. 8,058,069, which application claims priority to
`U.S. Provisional Application No. 61/045.228, filed Apr. 15,
`2008, the disclosures of which are herein incorporated by
`reference in their entirety for all purposes.
`
`STATEMENT REGARDING FEDERALLY
`SPONSORED RESEARCH ORDEVELOPMENT
`
`Not applicable.
`
`NAMES OF PARTIES TO AJOINT RESEARCH
`AGREEMENT
`
`Not applicable.
`
`REFERENCE TO A “SEQUENCE LISTING. A
`TABLE, ORACOMPUTER PROGRAM LISTING
`APPENDIX SUBMITTED AS ANASCII TEXT
`FILE
`
`5
`
`10
`
`15
`
`25
`
`30
`
`The Sequence Listing written in file-77-3.TXT, created on
`Aug. 22, 2013, 8,192 bytes, machine format IBM-PC, MS
`Windows operating system, is hereby incorporated by refer
`ence in its entirety for all purposes.
`
`35
`
`BACKGROUND OF THE INVENTION
`
`2
`result, nonviral gene delivery systems are receiving increas
`ing attention (Worgall et al., Human Gene Therapy, 8:37
`(1997); Peeters et al., Human Gene Therapy, 7:1693 (1996):
`Yei et al., Gene Therapy, 1:192 (1994); Hope et al., Molecular
`Membrane Biology, 15:1 (1998)). Furthermore, viral systems
`are rapidly cleared from the circulation, limiting transfection
`to “first-pass' organs Such as the lungs, liver, and spleen. In
`addition, these systems induce immune responses that com
`promise delivery with Subsequent injections.
`Plasmid DNA-cationic liposome complexes are currently
`the most commonly employed nonviral gene delivery
`vehicles (Feigner, Scientific American, 276:102 (1997);
`Chonn et al., Current Opinion in Biotechnology, 6:698
`(1995)). For instance, cationic liposome complexes made of
`an amphipathic compound, a neutral lipid, and a detergent for
`transfecting insect cells are disclosed in U.S. Pat. No. 6,458,
`382. Cationic liposome complexes are also disclosed in U.S.
`Patent Publication No. 20030073640.
`Cationic liposome complexes are large, poorly defined
`systems that are not Suited for systemic applications and can
`elicit considerable toxic side effects (Harrison et al., Biotech
`niques, 19:816 (1995); Lietal. The Gene, 4:891 (1997); Tam
`etal, Gene Ther, 7:1867 (2000)). As large, positively charged
`aggregates, lipoplexes are rapidly cleared when administered
`in vivo, with highest expression levels observed in first-pass
`organs, particularly the lungs (Huang et al., Nature Biotech
`nology, 15:620 (1997); Templeton et al., Nature Biotechnol
`ogy, 15:647 (1997); Hofland et al., Pharmaceutical Research,
`14:742 (1997)).
`Other liposomal delivery systems include, for example, the
`use of reverse micelles, anionic liposomes, and polymer lipo
`somes. Reverse micelles are disclosed in U.S. Pat. No. 6,429,
`200. Anionic liposomes are disclosed in U.S. Patent Publica
`tion No. 20030026831. Polymer liposomes that incorporate
`dextrinor glycerol-phosphocholine polymers are disclosed in
`U.S.
`Patent
`Publication
`Nos. 20020081736 and
`20030082103, respectively.
`A gene delivery system containing an encapsulated nucleic
`acid for systemic delivery should be small (i.e., less than
`about 100 nm diameter) and should remain intact in the cir
`culation for an extended period of time in order to achieve
`delivery to affected tissues. This requires a highly stable,
`serum-resistant nucleic acid-containing particle that does not
`interact with cells and other components of the vascular com
`partment. The particle should also readily interact with target
`cells at a disease site in order to facilitate intracellular delivery
`of a desired nucleic acid.
`Recent work has shown that nucleic acids can be encapsu
`lated in small (e.g., about 70 nm diameter) “stabilized plas
`mid-lipid particles' (SPLP) that consist of a single plasmid
`encapsulated within a bilayer lipid vesicle (Wheeler et al.,
`Gene Therapy, 6:271 (1999)). These SPLPs typically contain
`the “fusogenic' lipid dioleoylphosphatidylethanolamine
`(DOPE), low levels of cationic lipid, and are stabilized in
`aqueous media by the presence of a poly(ethylene glycol)
`(PEG) coating. SPLPs have systemic application as they
`exhibit extended circulation lifetimes following intravenous
`(i.v.) injection, accumulate preferentially at distal tumor sites
`due to the enhanced vascular permeability in Such regions,
`and can mediate transgene expression at these tumor sites.
`The levels of transgene expression observed at the tumor site
`following i.v. injection of SPLPs containing 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
`
`Case 1:22-cv-02229-MKV Document 42-6 Filed 09/06/22 Page 29 of 74
`
`45
`
`RNA interference (RNAi) is an evolutionarily conserved
`process in w