Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 1 of 222 PageID #: 18049
`
`Exhibit A
`
`
`
`
`
`
`
`
`
`

`

`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 2 of 222 PageID #: 18050
`
`USOO8058069B2
`
`(12) United States Patent
`YaWorski et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 8,058,069 B2
`Nov. 15, 2011
`
`(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)
`(73) Assignee: Protiva Biotherapeutics, Inc., Burnaby,
`B.C. (CA)
`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
`
`(*) Notice:
`
`(65)
`
`Prior Publication Data
`US 2010/O 130588A1
`May 27, 2010
`
`Related U.S. Application Data
`(60) Provisional application No. 61/045.228, filed on Apr.
`15, 2008.
`
`(51) Int. Cl.
`(2006.01)
`C07H 2L/04
`(2006.01)
`CI2N 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)
`
`References Cited
`
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`OTHER PUBLICATIONS
`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
`
`(57)
`ABSTRACT
`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
`
`

`

`US 8,058,069 B2
`Page 2
`
`FOREIGN PATENT DOCUMENTS
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`* cited by examiner
`
`

`

`U.S. Patent
`
`
`
`US 8,058,069 B2
`
`
`
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 4 of 222 PageID #: 18052
`
`

`

`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 2 of 24
`
`US 8,058,069 B2
`
`
`
`k k
`
`+ i +
`
`
`
`VNNIS ?e?O L Wu
`
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 5 of 222 PageID #: 18053
`
`(Seo pepeeun 96) AqeA
`
`

`

`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 3 of 24
`
`US 8,058,069 B2
`
`Activity of SNALP Upon intravenous Administration in Mice
`group mean + SD (n=4)
`
`2. 5
`
`2. O
`
`15
`
`OO1 O5O
`
`n
`
`so o v
`
`Q
`
`Q
`
`S NN
`
`N. Nb NX
`
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 6 of 222 PageID #: 18054
`
`FIG. 2
`
`

`

`U.S. Patent
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`Nov. 15, 2011
`
`Sheet 4 of 24
`
`US 8,058,069 B2
`
`3.0
`
`2. 5
`
`2. O
`
`
`
`
`
`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-00252-MSG Document 316-4 Filed 05/13/24 Page 7 of 222 PageID #: 18055
`
`FIG. 3
`
`

`

`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 5 of 24
`
`US 8,058,069 B2
`
`Activity of SNALP Upon intravenous Administration in Mice
`group meant SD (n=4)
`
`
`
`112 O5O
`
`
`
`O. 5
`
`O. O PBS Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8
`
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 8 of 222 PageID #: 18056
`
`FIG. 4
`
`

`

`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 6 of 24
`
`US 8,058,069 B2
`
`Activity of SNALP Upon Intravenous Administration in Mice
`group meant SD (n=4)
`
`112 O5.0
`O 5 0.0 I
`
`Nx No
`N
`NS NN N
`Q to
`so o 1,
`X
`e in
`<ssssss SSSSSS SQS SQS SQ
`S.
`S.
`S.
`ecce eccessessesses
`
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 9 of 222 PageID #: 18057
`
`FIG. 5
`
`

`

`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 7 of 24
`
`US 8,058,069 B2
`
`Tolerability of IV 1:57 SNALP in Female BALB/c Mice, n
`
`4, SD error
`
`2
`
`Alanine Aminotransferase
`Aspartate Aminotransferase
`Sorbital Dehydrogenase
`&
`ALT levels indicate hepatocyte damage.
`d SDH
`Incr
`96
`
`81
`
`140
`
`120
`
`1 OO
`
`8
`
`N
`
`Tolerability of IV 1:57 SNALP in Female BALB?c Mice,
`4, SD error
`8.7
`Alanine Aminotransferase
`Aspartate Aminotransferase
`Sorbital Dehydrogenase
`2-fold increase ("3xULN") is considered clinically
`significant
`
`7.9
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`B 2.
`2. 2
`2.
`1
`
`B
`
`ear PBS Syringe Citrate
`99
`79
`
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 10 of 222 PageID #: 18058
`
`Lipid dose
`siRNA dose
`
`F.G. 6B
`
`2. : : : 2. 2: 2: 2: 2: 2. 2:
`
`22222227
`Syringe PBS
`123
`9 mg/kg
`
`Gear PB
`102
`
`

`

`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 8 of 24
`
`US 8,058,069 B2
`
`SNALP Activity From Different Manufacturing Processes
`IV, 48 h, ApoB-10048 U2/2 G1/2 (Dow),
`female BALB/c mice, n=4, SD error
`
`S. 2.0
`FIG. 7A is
`<
`f 15
`E
`1.0
`
`C
`(D
`g 0.5
`O g
`
`0.0
`
`2
`
`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
`
`0.6 SNALP Re-Formulation - Activity Assessment in BALB/c Mice
`-
`48 h time point, n=4, SD error bars
`FIG. 7B 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 Pump Press Pump
`0.05 mg/kg
`0.1 mg/kg
`
`FIG. 7C
`
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 11 of 222 PageID #: 18059
`
`S
`S
`9
`8
`
`s
`o
`
`O
`c
`
`
`
`-a, 25%
`
`Efficacy of SNALP Formulations
`Fresh Terminal E. n=4 female Balb/c mice, SD Error Bars
`70
`60
`50
`40
`30
`20
`1O
`O
`
`Press Pump Press Pump
`0.05 mg/kg
`0.1 mg/kg
`
`

`

`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 9 of 24
`
`US 8,058,069 B2
`
`Tolerability of 157 SNALP IV in BALB/c Mice, n=4 (Grp1-3 n=3), SD error
`
`6%
`
`
`
`4%
`
`2%
`
`-2%
`
`-4%
`
`-6%
`
`9:1
`6:1
`6:1
`6:1
`6:1
`9:1
`9mg/ 11 mg 11 mg 13mg/15mg/ 17mg/11 mg/
`k
`
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 12 of 222 PageID #: 18060
`
`FIG. 8
`
`

`

`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 13 of 222 PageID #: 18061
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 13 of 222 PagelD #: 18061
`
`U.S. Patent
`
`Nov.15, 2011
`
`Sheet 10 of 24
`
`US 8,058,069 B2
`
`
`
`FIG. 9
`
`

`

`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 11 of 24
`
`US 8,058,069 B2
`
`Tolerability of IV 1:57 Gear PBS In-Line SNALP in Female BALB/c
`Mice, n=4, SD error
`
`1200
`
`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
`
`400
`
`200
`
`
`
`2
`
`4 5
`
`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
`
`2
`
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 14 of 222 PageID #: 18062
`
`F.G. 10A
`
`

`

`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 15 of 222 PageID #: 18063
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 15 of 222 PagelD #: 18063
`
`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 12 of 24
`
`US 8,058,069 B2
`
`
`
`
`
`
`
`
`
`10}11{12}13)14]15|16/17/18]19/20/21/22(23/24|25)26/27|28/29|30/31/3233343536/37/3839|40|44
`
`
`
`
`
`AlanineAminotransferase
`
`SS
`
`E]AspartateAminotransferase BS
`SorbitalDehydrogenase
`
`
`
`
`ee
`
`TolerabilityofIV1:57GearPBSIn-LineSNALPinFemaleBALB/cMice,n=4,SDerror
`
`
`
`
`
`
`
`
`
`
`
`
`
`by
`
`
`
`2-foldincrease("3xULN")isconsideredclinicallysignificant
`
`T
`oO
`op
`
`t
`oO
`N
`
`JBWWON fo yuusaddp pjoj-x
`
`9:1(10)
`
`FIG.10B
`
`112
`
`PBS
`
`
`EZIIIIILIPIITILIDIOPILLIILOEPILEDELIOILEPILLEDLLLLILELLEDRRR)
`PoA,
`PRA
`eeee
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`Led
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`BRO
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` SASESESESISTSRRTRSESEETEER
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`1img/kg 112
` 17mg/kg 121
`15mg/kg 1076:1SNALP(7)
`13mg/kg 93
`11mg/kg 78
`11mg/kg
`9:1SNALP(10)
`
`9mg/kg
`Vyoi0SeemSeesNy
`siRNAdose —T
`Lipiddose
`
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`
`
`
`
`Pye
`ED
`PAA
`ed
`VL
`Boe
`2777A
`PY
`
`

`

`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 13 of 24
`
`US 8,058,069 B2
`
`F.G. 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
`30
`O
`
`2.5
`s
`2.0 -
`
`?
`
`(p 15 -
`?
`O
`Cl
`<C 10 -
`
`2
`-
`
`0.5
`0.0
`
`FIG. 11B
`
`0.40
`
`O.35
`
`0. 2 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
`
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 16 of 222 PageID #: 18064
`
`
`
`o
`
`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)
`
`

`

`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 17 of 222 PageID #: 18065
`ment 316-4 Filed 05/13/24 Page 17
`of 222 PagelD
`Case
`1:22-cv-00252-MSG Docu
`#: 18065
`
`U.S. Patent
`
`US 8,058,069 B2
`
`
`
`
`
`olmUC<“CW]WSCCLlUDlDlmlClCUCDOlUlmCUCDOCOCOOD—SDSOMOSFDONS
`
`
`
`LVe
`
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`
`FIG. 12
`
`aAGG
`
`,
`
`|
` a .i
`
`aL
`
`
`
`
`
`
`

`

`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 15 of 24
`
`US 8,058,069 B2
`
`O
`120%
`
`11.5%
`5. 11.0%
`
`105%
`
`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-00252-MSG Document 316-4 Filed 05/13/24 Page 18 of 222 PageID #: 18066
`
`FIG. 13
`
`

`

`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 16 of 24
`
`US 8,058,069 B2
`
`- A - Control SNALP -<>- Active SNALP
`
`
`
`O%
`15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90
`Days after seeding
`
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 19 of 222 PageID #: 18067
`
`FIG. 14
`
`

`

`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 17 of 24
`
`US 8,058,069 B2
`
`
`
`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
`
`PBS
`
`Luc
`
`PLK1424
`
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 20 of 222 PageID #: 18068
`
`FIG. 15
`
`

`

`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 18 of 24
`
`US 8,058,069 B2
`
`
`
`-- PLK1424.5'RACE
`product
`476bp
`
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 21 of 222 PageID #: 18069
`
`FIG. 16
`
`

`

`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 19 of 24
`
`US 8,058,069 B2
`
`
`
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 22 of 222 PageID #: 18070
`
`FIG. 17
`
`

`

`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 20 of 24
`
`US 8,058,069 B2
`
`
`
`6x2mg/kg Mean tumor volume
`
`-0- LUC-DMA
`-- PLK-DMA
`- A - PLK-DSA
`
`8
`
`10
`
`12
`
`14
`
`16
`Days
`
`18
`
`20
`
`22
`
`24
`
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 23 of 222 PageID #: 18071
`
`FIG. 18
`
`

`

`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 21 of 24
`
`US 8,058,069 B2
`
`PLK mRNA silencing in scid/beige mice treated with 2mg/kg 1:57 SNALP against
`subcutaneous Hep3B tumors
`
`0.70
`
`-37%
`
`-52%
`
`-65%
`0.
`
`N.
`SNS
`&S
`
`0.60-
`
`100%
`
`.C.
`t
`
`0.50 E
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`2 0.40 E.
`
`-15%
`0.
`
`
`
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`R
`
`0.20-
`
`|
`
`0.10- E
`
`0.00–
`
`&
`
`SNS)
`8x8
`
`n
`
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 24 of 222 PageID #: 18072
`
`FIG. 19
`
`

`

`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 22 of 24
`
`US 8,058,069 B2
`
`6x2mg/kg Mean tumor volume
`
`Crossover dosing of Luc
`cDMA group with 6 x 2
`mg/kg PLK cDSA SNALP
`
`
`
`-- Luc-DMA
`-H PLK-DMA
`-A - PLK-DSA
`
`Initial 6 x 2 mg/kg
`
`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-00252-MSG Document 316-4 Filed 05/13/24 Page 25 of 222 PageID #: 18073
`
`FIG. 20
`
`

`

`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 23 of 24
`
`US 8,058,069 B2
`
`Mean hPLK (1:4) : hCGAPDH (1:40) minus "background"
`
`120
`
`
`
`1.00
`
`0.80
`
`0.60
`
`0.40
`
`0.20
`
`0.00
`
`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-00252-MSG Document 316-4 Filed 05/13/24 Page 26 of 222 PageID #: 18074
`
`FIG. 21
`
`

`

`U.S. Patent
`
`Nov. 15, 2011
`
`Sheet 24 of 24
`
`US 8,058,069 B2
`
`-0- 1:57 PEG-CDSA SNALP
`
`- A - 1:57 PEG-CDMA SNALP
`
`
`
`70
`
`5 O
`
`4 O
`
`30
`
`20
`
`10
`
`O
`O
`
`1
`
`2
`
`3
`
`5
`4
`Time (h)
`
`6
`
`7
`
`8
`
`9
`
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 27 of 222 PageID #: 18075
`
`FIG, 22
`
`

`

`US 8,058,069 B2
`
`1.
`LPID FORMULATIONS FOR NUCLECACD
`DELIVERY
`
`CROSS-REFERENCES TO RELATED
`APPLICATIONS
`
`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.
`
`STATEMENT REGARDING FEDERALLY
`SPONSORED RESEARCH ORDEVELOPMENT
`
`Not applicable.
`
`NAMES OF PARTIES TO AJOINT RESEARCH
`AGREEMENT
`
`Not applicable.
`
`REFERENCE TO A “SEQUENCE LISTING”
`Not applicable.
`
`BACKGROUND OF THE INVENTION
`
`10
`
`15
`
`25
`
`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 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
`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
`Such as cancer and atherosclerosis. The present invention
`addresses these and other needs.
`
`BRIEF SUMMARY OF THE INVENTION
`
`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
`
`RNA interference (RNAi) is an evolutionarily conserved
`process in which recognition of double-stranded RNA
`(dsRNA) ultimately leads to posttranscriptional Suppression
`30
`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
`45
`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):
`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.
`65
`Plasmid DNA-cationic liposome complexes are currently
`the most commonly employed nonviral gene delivery
`
`35
`
`40
`
`50
`
`55
`
`60
`
`Case 1:22-cv-00252-MSG Document 316-4 Filed 05/13/24 Page 28 of 222 PageID #: 18076
`
`

`

`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 protease. In other preferred
`embodiments, the lipid particles are substantially non-toxic
`to mammals such as humans.
`In one aspect, the present invention provides lipid particles
`comprising: (a) one or more active agents or therapeutic
`agents; (b) one or more cationic lipids comprising from about
`50 mol % to about 85 mol % of the total lipid present in the
`particle; (c) one or more non-cationic lipids comprising from
`about 13 mol % to about 49.5 mol% of the total lipid present
`in the particle; and (d) one or more conjugated lipids that
`inhibit aggregation of particles comprising from about 0.5
`mol % to about 2 mol % of the total lipid present in the
`particle.
`More particularly, the present invention provides serum
`stable nucleic acid-lipid particles (SNALP) comprising a
`nucleic acid (e.g., one or more interfering RNA molecules
`such as siRNA, aiRNA, and/or miRNA), methods of making
`the SNALP and methods of delivering and/or administering
`the SNALP (e.g., for the treatment of a disease or disorder).
`In certain embodiments, the nucleic acid-lipid particle
`(e.g., SNALP) comprises: (a) a nucleic acid (e.g., an interfer
`ing RNA); (b) a cationic lipid comprising from about 50 mol
`% to about 85 mol% of the total lipid present in the particle:
`(c) a non-cationic lipid comprising from about 13 mol % to
`about 49.5 mol% of the total lipid present in the particle; and
`(d) a conjugated lipid that inhibits aggregation of particles
`comprising from about 0.5 mol% to about 2 mol% of the total
`lipid present in the particle.
`In one preferred embodiment, the nucleic acid-lipid par
`ticle (e.g., SNALP) comprises: (a) an siRNA; (b) a cationic
`lipid comprising from about 56.5 mol % to about 66.5 mol%
`of the total lipid present in the particle; (c) cholesterol or a
`derivative thereof comprising from about 31.5 mol% to about
`42.5 mol% of the total lipid present in the particle; and (d) a
`PEG-lipid conjugate comprising from about 1 mol% to about
`2 mol% of the total lipid present in the particle. This preferred
`embodiment of nucleic acid-lipid particle is generally
`referred to herein as the “1:62 formulation.
`In another preferred embodiment, the nucleic acid-lipid
`particle (e.g., SNALP) comprises: (a) an siRNA; (b) a cat
`ionic lipid comprising from about 52 mol% to about 62 mol
`% of the total lipid present in the particle; (c) a mixture of a
`phospholipid and cholesterol or a derivative thereof compris
`ing from about 36 mol % to about 47 mol% of the total lipid
`present in the particle; and (d) a PEG-lipid conjugate com
`prising from about 1 mol % to about 2 mol% of the total lipid
`present in the particle. This preferred embodiment of nucleic
`acid-lipid particle is generally referred to herein as the “1:57
`formulation.
`The present invention also provides pharmaceutical com
`positions comprising a lipid particle described herein (e.g.,
`SNALP) and a pharmaceutically acceptable carrier.
`In another aspect, the present invention provides methods
`for introducing an active agent or therapeutic agent (e.g.,
`nucleic acid) into a cell, the method comprising contacting
`the cell with a lipid particle described herein such as a nucleic
`acid-lipid particle (e.g., SNALP).
`In yet another aspect, the present invention provides meth
`ods