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`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 1 of 75
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`EXHIBIT C
`EXHIBIT C
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`
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`

`

`(12) United States Patent
`YaWorski et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 8,822,668 B2
`*Sep. 2, 2014
`
`USOO8822668B2
`
`(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
`s
`(CA); Ian MacLachlan, Mission (CA)
`(73) Assignee: Protiva Biotherapeutics, Inc., Burnaby
`BC (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.
`This patent is Subject to a terminal dis-
`claimer.
`
`(21) Appl. No.: 13/928,309
`9
`Jun. 26, 2013
`
`(22) Filed:
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`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
`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.
`
`-
`
`-
`
`-
`
`-
`
`-
`
`23 Claims, 24 Drawing Sheets
`
`

`

`US 8,822,668 B2
`Page 2
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`* cited by examiner
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`U.S. Patent
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`US 8,822,668 B2
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`(Seo peee Jun
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`US 8,822,668 B2
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`U.S. Patent
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`Sep. 2, 2014
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`Sheet 3 of 24
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`US 8,822,668 B2
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`Activity of SNALP Upon intravenous Administration in Mice
`group meani. SD (n=4)
`
`2. 5
`
`2. O -
`
`O. 5 -
`
`O. O
`?
`?o
`Ko
`Q S NY N.
`Go A Q
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`sesssssssssssss
`S.
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`S.
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`S.
`S.
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`S.
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`x
`SR
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`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 7 of 75
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`FIG 2
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`U.S. Patent
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`Sheet 4 of 24
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`US 8,822,668 B2
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`3.0
`
`2. 5
`
`2. O
`
`
`
`O. 5
`
`O. O
`
`-47% WS PBS Control
`
`-77% WS PBS COntrol
`
`PBS
`
`2:30 SNALP 5x1 mg/kg 1:57 SNALP 5x0.1 mg/kg
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`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 8 of 75
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`FIG. 3
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`U.S. Patent
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`Sep. 2, 2014
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`Sheet 5 of 24
`
`US 8,822,668 B2
`
`Activity of SNALP Upon intravenous Administration in Mice
`group meant SD (n=4)
`
`2.0
`
`11 O5
`
`O 5
`
`O.O PBS Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8
`
`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 9 of 75
`
`FIG. 4
`
`

`

`U.S. Patent
`
`Sep. 2, 2014
`
`Sheet 6 of 24
`
`US 8,822,668 B2
`
`Activity of SNALP Upon intravenous Administration in Mice
`group mean it SD (n=4)
`
`2. O
`
`1 5 r
`
`1 O
`
`O 5
`
`O.O lili
`
`NS NN N- N5 Nx No
`1 Q to
`co
`e & cS
`cS S s cS s
`CS
`oS s
`oSsssssssss
`CS CS CS
`GS GS GS CS c. GS GS GS GS
`CS
`
`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 10 of 75
`
`FIG. 5
`
`

`

`U.S. Patent
`
`Sep. 2, 2014
`
`Sheet 7 of 24
`
`US 8,822,668 B2
`
`Tolerability of IV 1:57 SNALP in Female BALB?c Mice, in
`168
`
`Alanine Aminotransferase
`Aspartate Aminotransferase
`Sorbital Dehydrogenase
`ALT levels indicate hepatocyte damage
`Incr'd SDH,
`96
`
`4, SD error
`
`81
`
``N
`
`?
`
`Tolerability of IV 1:57 SNALP in Female BALB/C Mice,
`SD error
`4
`n
`Alanine Aminotransferase
`2
`ASpartate Aminotransferase
`Sorbital Dehydrogenase
`3
`is considered clinically
`-fold increase ("3xULN"
`)
`2
`significant
`
`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 11 of 75
`
`
`
`ear PBS
`
`yringe Citrate
`
`79
`
`123
`9 mg/kg
`
`Lipid dose
`siRNA dose
`
`F.G. 6B
`
`)[XXXXXXXXXXXXXXX:S . EGOE!!!!!!!!!!!!!
`
`KXXXXXXXXXXXIEG
`Œ(
`
`SSSSSSSSSSS i CN (/)
`
`?&&&&&&&&&T? go
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`

`U.S. Patent
`
`Sep. 2, 2014
`
`Sheet 8 of 24
`
`US 8,822,668 B2
`
`S. 20, SNALP Activity From Different Manufacturing Processes
`FIG. 7A is -
`IV, 48 h, ApoB-10048 U2/2 G1/2 (Dow),
`<C
`female BALB/C mice, n=4, SD error
`Z 1.5
`?
`8
`5 1.0.
`CC
`(D
`
`0.5
`
`Ol
`CC
`as 0.0
`2
`
`M
`1
`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
`
`O6 SNALP Re-Formulation - Activity Assessment in BALB/C Mice
`48 h time point, n=4, SD error bars
`
`FIG. 7B a
`S 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 42-4 Filed 09/06/22 Page 12 of 75
`
`
`
`Efficacy of SNALP. Formulations
`3. Fresh Terminal Plasma, nP4 female Balb/c mice, SD Error Bars
`Q
`70
`O)
`S
`6O.
`S
`50
`92
`8
`40 -
`9
`30
`O
`st
`20
`o
`1 O
`cus
`O
`S
`f
`c
`?h
`
`Press Pump Press Pump
`0.05 mg/kg
`0.1 mg/kg
`
`

`

`U.S. Patent
`
`Sep. 2, 2014
`
`Sheet 9 of 24
`
`US 8,822,668 B2
`
`Tolerability of 157 SNALP IV in BALB/c Mice, n=4 (Grp 1-3 n=3), SD error
`
`
`
`6%
`
`4%
`
`2%
`
`-6%
`
`9:1
`6:1
`6:1
`6:1
`6:1
`9:1
`9mg/ 11 mg 11 mg 13.9/1591.9/19
`
`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 13 of 75
`
`FIG. 8
`
`

`

`U.S. Patent
`
`US 8,822,668 B2
`
`CO ÈN
`
`+
`
`CN
`
`
`
`
`
`
`
`
`
`
`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 14 of 75
`
`%
`
`7 (7
`
`)
`
`FIG. 9
`
`

`

`U.S. Patent
`
`Sep. 2, 2014
`
`Sheet 11 of 24
`
`US 8,822,668 B2
`
`Tolerability of IV 1:57 Gear PBS In-Line SNALP in Female BALB/c
`Mice, n=4, SD error
`
`9
`
`Z2:: % W: :
`W.
`% :: % 2.
`5
`% % 2 2
`
`688
`3.
`
`s
`
`3
`
`:X
`:X 2::::x:
`:: 2. :::
`3.
`9:1
`6:1
`6:1
`9:1
`6:1
`6:1
`9:1
`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
`
`Alanine Aminotransferase
`Aspartate Aminotransferase
`83 Sorbital Dehydrogenase
`Samples taken at 24 h time point except
`for last grp (48 h).
`
`1200
`
`1,000
`
`800
`
`s
`
`6OO
`
`400
`
`200
`
`
`
`
`
`PBS
`
`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 15 of 75
`
`FIG. 10A
`
`

`

`U.S. Patent
`
`Sep. 2, 2014
`
`Sheet 12 of 24
`
`US 8,822,668 B2
`
`ZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZ77 ZZZZZZZZZZZZZZZZ44
`KXXX
`ZZZZZZZZ
`::
`ZZYZZYZZZZZZ
`SxYxxxS
`ZZZZZZZZZZZZZZZZZZZZZZZ
`XXXXX
`277277-222
`XXXXXXXXXXXXXXXXX
`EZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZ
`XXXXXXXX
`s
`ZZZZZZZZZZZZZZZ
`SXXX
`...E.
`777.2777ZZZZZ
`XXXXXXXXXXX
`ZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZ
`XX
`
`
`
`XXXXX EA
`: 7272.27727
`SXX
`
`XXX
`Yaaaa.
`X
`:::::
`22222
`XXY
`:::::
`222222
`X
`.
`
`Z :
`
`2
`xx
`
`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 16 of 75
`
`g
`
`S.
`
`O
`cyd
`
`O
`CN
`
`O
`v
`
`|euON Olu Jeddn plo--X
`
`S s
`i s
`
`s
`
`i 8
`
`S f
`
`CN
`
`6
`
`

`

`U.S. Patent
`
`Sep. 2, 2014
`
`Sheet 13 of 24
`
`US 8,822,668 B2
`
`FIG. 1 1A
`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/c mice, n=4, SD error
`
`3.0
`
`2. 5
`
`2. O
`
`1 5
`
`1.O
`
`O. 5
`
`FIG 11B
`
`0.40
`
`O.35
`
`0.30
`
`0.25
`
`0.20
`
`0.15
`
`2-tailed T-test: p=0.078
`
`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
`h)
`
`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 17 of 75
`
`O. 10
`
`0.05
`
`O.OO
`
`
`
`Y77
`
`:::::::::::::::::::::::::::::::::
`
`:::::::::::::::::::::::::::::::
`
`:::::::::::::::::::::::::::::
`
`:::::::::::::::::::::::::::::::
`
`kg
`Kg
`mg/kg
`1:57 SNALP (10:1)
`
`0.1 mg/l 0.2 mg/l 0.4 mg/
`kg
`kg
`kg
`mg/kg
`New 1:57 SNALP (7:1)
`
`

`

`U.S. Patent
`
`Sep. 2, 2014
`
`Sheet 14 of 24
`
`CO LO <r çO ÇON -
`Q C C co o co C
`
`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 18 of 75
`
`

`

`U.S. Patent
`
`Sep. 2, 2014
`
`Sheet 15 of 24
`
`US 8,822,668 B2
`
`O
`120%
`
`11.5%
`5. 1 10%
`
`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 42-4 Filed 09/06/22 Page 19 of 75
`
`FIG. 13
`
`

`

`U.S. Patent
`
`Sep. 2, 2014
`
`Sheet 16 of 24
`
`US 8,822,668 B2
`
`- A - Control SNAP -C)- Active SNALP
`
`100%
`
`80%
`
`60%
`
`40%
`
`20%
`
`O%
`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 42-4 Filed 09/06/22 Page 20 of 75
`
`FIG. 14
`
`

`

`U.S. Patent
`
`Sep. 2, 2014
`
`Sheet 17 of 24
`
`US 8,822,668 B2
`
`
`
`PBS
`
`Luc
`
`PLK1424
`
`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 21 of 75
`
`FIG. 15
`
`

`

`U.S. Patent
`
`Sep. 2, 2014
`
`Sheet 18 of 24
`
`US 8,822,668 B2
`
`
`
`-- PLK1424.5'RACE
`product
`476bp
`
`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 22 of 75
`
`FIG 16
`
`

`

`U.S. Patent
`
`Sep. 2, 2014
`
`Sheet 19 of 24
`
`US 8,822,668 B2
`
`x200 mag
`
`x400 mag
`
`
`
`
`
`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 23 of 75
`
`x400 mag
`
`FIG. 17
`
`

`

`U.S. Patent
`
`Sep. 2, 2014
`
`Sheet 20 of 24
`
`US 8,822,668 B2
`
`
`
`6x2mg/kg Mean tumor volume
`
`-- PLK-DMA
`-A-PLK-DSA
`
`8
`
`1 O
`
`12
`
`14
`
`16
`Days
`
`18
`
`20
`
`22
`
`24
`
`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 24 of 75
`
`FIG. 18
`
`

`

`U.S. Patent
`
`Sep. 2, 2014
`
`Sheet 21 of 24
`
`US 8,822,668 B2
`
`PLK mRNA silencing in scid/beige mice treated with 2mg/kg 1:57 SNALP against
`subcutaneous Hep3B tumors
`
`100%
`
`
`
`0.70
`0.60
`
`0.50
`
`0.40
`
`SwS
`s
`-
`
`x
`
`-52%
`
`&Q
`S
`
`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 25 of 75
`
`FIG. 19
`
`

`

`U.S. Patent
`
`Sep. 2, 2014
`
`Sheet 22 of 24
`
`US 8,822,668 B2
`
`6x2mg/kg Mean tumor volume
`
`Crossover dosing of Luc
`cDMA group with 6 x 2
`mg/kg PLK cDSA SNALP
`
`-0- LuC-DMA
`-H PLK-DMA
`- A - PLK-OSA
`
`Initial 6 x 2 mg/kg
`SNALP
`
`2200
`
`2000
`1800
`1600
`E
`1400
`O
`> 5 1200
`E
`1000
`8OO
`
`600
`
`400
`
`2OO
`
`8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
`Days
`
`
`
`--
`
`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 26 of 75
`
`FIG. 20
`
`

`

`U.S. Patent
`
`Sep. 2, 2014
`
`Sheet 23 of 24
`
`US 8,822,668 B2
`
`Mean hPLK (1:4): hCGAPDH (1:40) minus "background"
`
`120
`
`
`
`1.OO
`
`0.80
`
`0.60
`
`0.40
`
`0.20
`
`O.OO
`
`24h LUC 1:57 24h PLK 1.57 24h PK 1:57 96h PLK 1:57 96h PLK 1.57
`CDMA
`CDMA
`CDSA
`CDMA
`CDSA
`
`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 27 of 75
`
`FIG 21
`
`

`

`U.S. Patent
`
`Sep. 2, 2014
`
`Sheet 24 of 24
`
`US 8,822,668 B2
`
`
`
`-- 1:57 PEG-CDSA SNALP
`
`-A - 1:57 PEG-CDMA SNALP
`
`Case 1:22-cv-02229-MKV Document 42-4 Filed 09/06/22 Page 28 of 75
`
`FIG. 22
`
`

`

`US 8,822,668 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. 13/253,917, filed Oct. 5, 2011, now U.S. Pat. No.
`8.492.359, which application is a continuation of 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
`
`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
`
`RNA interference (RNAi) is an evolutionarily conserved
`process in which recognition of double-stranded RNA
`(dsRNA) ultimately leads to posttranscriptional Suppression
`40
`of gene expression. This Suppression is mediated by short
`dsRNA, also called small interfering RNA (siRNA), which
`induces specific degradation of mRNA through complemen
`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); Hammond et 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
`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 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
`the wild-type as well as immune response concerns. As a
`result, nonviral gene delivery systems are receiving increas
`
`2
`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 (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 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 expressio