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`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 1 of 74
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`EXHIBIT B
`EXHIBIT B
`
`
`
`

`

`(12) United States Patent
`YaWorski et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 8.492.359 B2
`*Jul. 23, 2013
`
`US008492359B2
`
`(54) LIPID FORMULATIONS FOR NUCLEICACID
`DELIVERY
`
`(75) 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)
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 59 days.
`This patent is Subject to a terminal dis-
`claimer
`
`(21) Appl. No.: 13/253,917
`
`(22) Filed:
`
`Oct. 5, 2011
`
`(65)
`
`CA
`CA
`
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`9/2001 Aneja
`6,287,591 B1
`9/2001 Semple et al.
`3. R
`383 E. tal
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`Continued
`Prior Publication Data
`(
`)
`US 2012/O183581 A1
`Jul 19, 2012
`FOREIGN PATENT DOCUMENTS
`ul. 19,
`2309.727 A1
`4f1999
`Related U.S. Application Dat
`2271582 A1 11, 1999
`U.S.
`O
`pp
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`(63) Continuation of application No. 12/424,367, filed on
`OTHER PUBLICATIONS
`Apr. 15, 2009, now Pat. No. 8,058,069.
`Arpicco, S., et al., “Preparation and Characterization of Novel
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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
`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.
`21 Claims, 24 Drawing Sheets
`
`

`

`US 8.492.359 B2
`Page 2
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`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 4 of 74
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`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 1 of 24
`
`US 8.492.359 B2
`
`
`
`G ?IduueS
`
`k k
`
`+
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 5 of 74
`
`(Seo peleelun
`
`) AqeA
`
`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 2 of 24
`
`US 8.492.359 B2
`
`
`
`
`
`
`
`
`
`
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 6 of 74
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`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 3 of 24
`
`US 8.492.359 B2
`
`Activity of SNALP Upon intravenous Administration in Mice
`group mean LSD (n=4)
`
`2. 5
`
`2. O
`
`O 5
`
`O.O
`
`w So o A & 9 NS N N.
`)
`-
`e
`& SS S S S S S S S S S S S
`cS ccS c. c.Sc. c. c9; c. c9 cc
`
`l
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 7 of 74
`
`FIG. 2
`
`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 4 of 24
`
`US 8.492.359 B2
`
`3.0
`
`2. 5
`
`2. O
`
`
`
`0. 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
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 8 of 74
`
`FIG. 3
`
`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 5 of 24
`
`US 8.492.359 B2
`
`Activity of SNALP Upon intravenous Administration in Mice
`group meant SD (n=4)
`
`112 O5O
`
`O 5 O.O al
`
`PBS Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 9 of 74
`
`FIG. 4
`
`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 6 of 24
`
`US 8.492.359 B2
`
`Activity of SNALP Upon intravenous Administration in Mice
`group meant SD (n=4)
`
`9 2.0
`1.
`CE
`2
`1.
`E 1.5
`
`cC
`(D
`g 1.0
`O
`CC
`5
`2
`O.5
`
`OO
`Nix No
`b
`Q NN N
`Q NS NN N. Nb NP N
`x 8 o 1,
`2
`?
`So
`a& cSoSoses essessssssssssssss
`cc cc cc cccsec.Sc. c9c.Sc.
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 10 of 74
`
`FIG. 5
`
`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 7 of 24
`
`US 8.492.359 B2
`
`140
`
`120
`
`1 OO
`
`Tolerability of IV 1:57 SNALP in Female BALB?c Mice, n
`168
`
`Alanine Aminotransferase
`Aspartate Aminotransferase
`Sorbital Dehydrogenase
`ALT levels indicate hepatocyte damage.
`Incr'd SDH,
`96
`
`4, SD error
`205
`
`
`
`
`
`
`
`
`
`
`
`2.
`
`Tolerability of IV 1:57 SNALP in Female BALB/c Mice,
`4, SD error
`Alanine Aminotransferase
`Aspartate Aminotransferase
`Sorbital Dehydrogenase
`&
`3xULN") is considered clinically
`fold increase (
`2
`significant
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 11 of 74
`
`Lipid dose
`siRNA dose
`
`F.G. 6B
`
`2 2 t
`2 2
`2 2.
`2
`e
`
`ºg ETFT (FEET)
`
`?XXXXXXXXXXX}} co
`Ñ SSSSSSSSSS: SN do
`
`Gear PB
`
`123
`9 mg/kg
`
`102
`
`7181920
`6
`Syringe Citrate
`ear PBS
`99
`
`79
`
`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 8 of 24
`
`US 8.492.359 B2
`
`O E 2.0
`FIG. 7A is
`CC
`Z 15
`Y
`E
`
`10
`
`0.5
`
`O.O
`
`<
`(D
`
`C
`KC
`
`2
`
`SNALP Activity From Different Manufacturing Processes
`IV, 48 h, ApoB-10048 U2/2 G1/2 (Dow),
`female BALB/C mice, n=4, SD error
`
`a.
`
`1:57
`1:57
`1:57
`PBS 157
`Syringe Gear Syringe Gear
`0.05mg 0.05mg 0.1mg/ 0.1mg/
`/kg
`/kg
`kg
`kg
`
`s
`
`Y
`
`FIG. 7E
`
`O6 SNALP Re-Formulation - Activity Assessment in BALB/C Mice
`48 h time point, n=4, SD error bars
`
`S. O.5
`ch
`8 0.4
`s
`5 E O3
`{ 0.2
`n
`$ 0.1
`2 -0.1
`
`1:57
`1:57
`Syringe Gear Syringe Gear
`Press Pump Press Pump
`0.05 mg/kg
`0.1 mg/kg
`
`FIG. 7C
`
`Efficacy of SNALP. Formulations
`Fresh Terminal Plasma, n=4 female Balb/c mice, SD Error Bars
`
`
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 12 of 74
`
`Press Pump Press Pump
`0.05 mg/kg
`0.1 mg/kg
`
`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 9 of 24
`
`US 8.492.359 B2
`
`Tolerability of 157 SNALP IV in BALB/c Mice, n=4 (Grp1-3 n=3), SD error
`
`6%
`
`
`
`9:1
`
`9:1
`
`6:1
`
`6:1
`
`6:1
`
`6:1
`
`9:1
`
`-2% - - - -
`
`- - - -
`
`- - - - - -
`
`- -
`
`- - - - -
`
`-
`
`- - -
`
`- -
`
`-
`
`- - - - .
`
`.
`
`.
`
`.
`
`PBS g1
`7mg/kg
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`. . . . . . . . .
`A . . . . . . . . . . . . . . . . . . . . . . . .
`-4%
`9:1 PBS
`11 mg/kg
`
`.
`
`.
`
`. - - - kg -
`
`- - /kg -
`
`-
`
`-
`
`- -/kg - - kg
`
`v -kg
`
`kg
`
`kg
`
`. . .
`
`.
`
`. . . . . . . . . . . . . . . . . . .
`
`. . . .
`
`. . . . . . . . . . . . . . .
`
`. . .
`
`-6%
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 13 of 74
`
`FIG. 8
`
`

`

`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 14 of 74
`Page 14
`-3 Filed 09/06/22
`
`U.S. Patent
`
`US 8,492,359 B2
`
`ce<>o>>>>>olUlUCODlmlCUCUCOCOTCOCCDakecDeCOD+©SG©G©FTFA
`
`FIG. 9
`
`
`
`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 11 of 24
`
`US 8.492.359 B2
`
`Tolerability of IV 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).
`
`5
`
`1200
`
`1,000
`
`8OO
`
`S
`
`600
`
`400
`
`200
`
`
`
`PBS
`
`6:1
`6:1
`6:1
`6:1
`9:1
`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
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 15 of 74
`
`FIG 10A
`
`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 12 of 24
`
`US 8.492.359 B2
`
`
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 16 of 74
`
`euoNJO bul Jeddin po--X
`
`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 13 of 24
`
`US 8.492.359 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
`
`112 O5.0
`
`O. 5
`
`FIG. 11B
`
`0.40
`
`0.35
`
`0. 3 O
`
`O. 2 5
`
`0. 1 5
`
`2-tailed T-test: p=0.078
`
`SNALP Re-Formulation - Activity Assessment in BALB?c. Mice
`DOW ApoB lead siRNA, 48 h time point, n=4, SD error bars
`LLO =9%
`
`-53%
`1 excluded
`
`OO 12 OO
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 17 of 74
`
`0.05 -
`
`OOO
`
`
`
`kg
`kg
`mg/kg
`1:57 SNALP (10:1)
`
`0.1 mg/l 0.2 mg/l 0.4 mg/
`0.05
`Kg
`kg
`kg
`mg/kg
`New 1:57 SNALP (7:1)
`
`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 14 of 24
`
`CO LO <r çr) (N. ~
`O CD C C C c C
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 18 of 74
`
`LP (7
`:1)
`
`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 15 of 24
`
`US 8.492.359 B2
`
`120%
`
`11.5%
`
`1 10%
`
`10.5%
`
`100%
`
`95%
`
`90%
`8
`
`--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 42-3 Filed 09/06/22 Page 19 of 74
`
`F.G. 13
`
`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 16 of 24
`
`US 8.492.359 B2
`
`- A - Contro SNAP -()- Active SNALP
`
`100%
`
`80%
`
`60%
`
`40%
`
`20%
`
`O%
`15 20 25 30 35 40 45 50 55 6.O 65 7O 75 80 85 90
`Days after seeding
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 20 of 74
`
`FIG. 14
`
`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 17 of 24
`
`US 8.492.359 B2
`
`
`
`CC
`2.
`Y
`S
`?h
`?
`CC
`CD
`--
`Na
`-
`l
`--
`C
`(S
`CD
`s
`
`PBS
`
`LuC
`
`PLK1424
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 21 of 74
`
`FIG. 15
`
`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 18 of 24
`
`US 8.492.359 B2
`
`
`
`-- PLK1424 5'RACE
`product
`476bp
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 22 of 74
`
`FIG. 16
`
`

`

`Patent
`
`Jul. 23, 2013
`
`Sheet 19 of 24
`
`US 8.492.359 B2
`
`
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 23 of 74
`
`FIG. 17
`
`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 20 of 24
`
`US 8.492.359 B2
`
`
`
`6x2mg/kg Mean tumor volume
`
`--Luc-DMA
`-HPLK-DMA
`- A - PLK-DSA
`
`8
`
`10
`
`12
`
`14
`
`16
`Days
`
`18
`
`20
`
`22
`
`24
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 24 of 74
`
`F.G. 18
`
`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 21 of 24
`
`US 8.492.359 B2
`
`PLK mRNA silencing in scid Ibeige mice treated with 2mg/kg 1:57 SNALP against
`subcutaneous Hep3B tumors
`
`100%
`
`-15%
`
`-30%
`
`
`
`-37%
`
`-52%
`
`-65%
`
`0.70
`
`0.60
`
`osol
`
`0.40
`
`0.30
`
`0.20
`
`O. 10
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 25 of 74
`
`FIG. 19
`
`

`

`-0- Luc-OMA
`-H PLK-DMA
`PLK-DSA
`
`--
`
`-
`
`2200
`2000
`1800
`1600
`8
`2 1400
`O
`o 5 1200
`E
`1000
`800
`600
`-:
`400
`200 use
`O 1TA---
`8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
`Days
`
`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 22 of 24
`
`US 8.492.359 B2
`
`6x2mg/kg Mean tumor volume
`
`Crossover dosing of Luc
`cDMA group with 6 x 2
`mg/kg PLK cDSA SNALP
`
`Initial 6 x 2 mg/kg
`SNAP
`
`? 4.
`D
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 26 of 74
`
`FIG. 20
`
`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 23 of 24
`
`US 8.492.359 B2
`
`120
`
`1.00
`
`O.80
`
`O.60
`
`O40
`
`0.20
`
`0.00
`
`Mean hPLK (1:4): hdAPDH (1:40) minus "background"
`
`0.99
`
`0.61
`
`O42
`
`0.23
`
`0.23
`
`24h Luc 1:57 24h PLK 1:57 24h PLK 1.57 96h PLK 1:57 96h PLK 1.57
`CDSA
`CDMA
`CDSA
`CDMA
`CDMA
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 27 of 74
`
`FIG 21
`
`

`

`U.S. Patent
`
`Jul. 23, 2013
`
`Sheet 24 of 24
`
`US 8.492.359 B2
`
`
`
`-- 1:57 PEG-CDSA SNAP
`
`-A- 1:57 PEG-CDMA SNAP
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 28 of 74
`
`FIG. 22
`
`

`

`US 8,492,359 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. 12/424,367 filed Apr. 15, 2009, now U.S. Pat.
`No. 8,058,069, which application claims priority to U.S. Pro
`visional Application No. 61/045,228, filed Apr. 15, 2008, the
`disclosures of which are herein incorporated by reference in
`their entirety for all purposes.
`
`10
`
`STATEMENT REGARDING FEDERALLY
`SPONSORED RESEARCH ORDEVELOPMENT
`
`15
`
`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
`
`25
`
`The Sequence Listing written in file-77-2.TXT, created on
`Mar. 13, 2012, 8,192 bytes, machine format IBM-PC, MS
`30
`Windows operating system, is hereby incorporated by refer
`ence in its entirety for all purposes.
`
`BACKGROUND OF THE INVENTION
`
`2
`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
`et al., 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 Biotechnology, 15:620 (1997); Templeton et al.,
`Nature Biotechnology, 15:647 (1997); Hofland et al., Phar
`maceutical 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
`nucleic acids such as siRNA 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
`
`RNA interference (RNAi) 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 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
`ing attention (Worgall et al., Human Gene Therapy, 8:37
`(1997); Peeters et al., Human Gene Therapy, 7:1693 (1996):
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Case 1:22-cv-02229-MKV Document 42-3 Filed 09/06/22 Page 29 of 74
`
`

`

`US 8,492,359 B2
`
`3
`Such as cancer and atherosclerosis. The present invention
`addresses these and other needs.
`
`BRIEF SUMMARY OF THE INVENTION
`
`10
`
`15
`
`25
`
`30
`
`35
`
`40
`
`50
`
`55
`
`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
`delivering and/or administering the lipid particles (e.g., for
`the treatment of a disease or disorder).
`In preferred embodiments, the act