Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 1 of 104
`Case 1:22-cv-02229-MKV Document1-6 Filed 03/18/22 Page 1 of 104
`
`EXHIBIT F
`EXHIBIT F
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 2 of 104
`YN"TTTATAATTA
`
`US009404127B2
`
`a2) United States Patent
`US 9,404,127 B2
`(10) Patent No.:
`
` Yaworskiet al. (45) Date of Patent: *Aug. 2, 2016
`
`
`(54) NON-LIPOSOMAL SYSTEMS FOR NUCLEIC
`ACID DELIVERY
`
`(56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`(71) Applicant: PROTIVA BIOTHERAPEUTICS,
`INC., Burnaby (CA)
`
`(72)
`
`.
`.
`Inventors: Ed Yaworski, Maple Ridge (CA); Lloyd
`B. Jeffs, Delta (CA); Lorne R. Palmer,
`Vancouver (CA)
`
`(73) Assignee: PROTIVA BIOTHERAPEUTICS,
`INC., Burnaby, BC (CA)
`
`(*) Notice:
`
`Subject to any disclaimer, the term ofthis
`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.: 14/642,452
`°
`
`(22)
`(65)
`
`Filed:
`
`Mar.9, 2015
`Prior
`Publication Dat
`rior Fublication
`Data
`US 2016/0032320 Al
`Feb. 4. 2016
`7
`
`Related U.S. Application Data
`CA
`(63) Continuation of application No. 13/807,288,filed as
`application No. PCT/CA2011/000778 on Jun. 30, ©
`2011, now Pat. No. 9,006,417.
`
`(60) Provisional application No. 61/360,480, filed on Jun.
`30, 2010.
`
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`11/1999 Wheeleret al.
`(Continued)
`
`FOREIGN PATENT DOCUMENTS
`2309727 Al
`4/1999
`2271582 AL 11/1999
`(Continued)
`
`OTHER PUBLICATIONS
`
`(2006.01)
`.
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2010.01)
`(2006.01)
`
`Arpicco, S., et al., “Preparation and Characterization of Novel
`Cationic Lipids Developed. for Gene Transfection,” Proceed. Int’!
`
`Symp. Control. Rel. Bioact. Mater. (Controlled Release Society,
`Inc.), 1999, vol. 26, pp. 759-760.
`.
`(Continued)
`
`Primary Examiner — Kimberly Chong
`.
`:
`.
`(74) Attorney, Agent, or Firm — Kilpatrick Townsend &
`Stockton LLP
`
`(57)
`
`ABSTRACT
`
`(1) eeeLod
`CI2N 15/88
`AGIK 9/107
`AGIK 9/51
`AGIK 31/7088
`AGIK 31/7105
`AG6IK 31/712
`A6IK 31/713
`AGIK 47/14
`CI2QN 15/113
`AOLK 9/127
`The present invention provides novel, stable lipid particles
`(52) US.Cl
`“having a non-lamellarstructure and comprising one or more
`CPC ecscce CI2N 158 (2013.01); A61K 9/1975
`active agents or therapeutic agents, methods ofmaking such
`(2013.01); AGIK 9/5123 (2013 01); A6IK
`lipid particles, and methods of delivering and/or administer-
`31/7088 (2013.01); A61K 31/712 (2013.01):
`ing such lipid particles. More particularly, the present inven-
`A61K 31/713 (2013.01); A6IK 31/7105
`(2013.01); A6IK A7/A (2013 01); CN tion provides stable nucleic acid-lipid particles (SNALP) that
`15/113 (2013 01); AGIK 9/1272(2013 01);
`havea non-lamellar structure and that comprise a nucleic acid
`ABIK 9/1274 (2013 01); CI2N 2310/14
`(such as one or more interfering RNA), methods of making
`(2013.01); CI2N 310/321 (2013.01)
`the SNALP, and methods of delivering and/or administering
`(58) Field of Classificati
`S
`h
`.
`the SNALP.
`Classification Searc
`ield
`of
`None
`
`See application file for complete search history.
`
`22 Claims, 24 Drawing Sheets
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 3 of 104
`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 3 of 104
`
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`DNAcomplexesandthe efficiency of gene delivery,” J. Biol. Chem.,
`276(50):476 15-47622, 2001.
`Songet al., “Characterization of the inhibitory effect of PEG-lipid
`conjugates on the intracellular delivery of plasmid and antisense
`DNA mediated by cationic lipid liposomes,” Biochimica et
`Biophysica Acta, 2002, 1558:1-13.
`Sorensen,et al., “Gene Silencing by Systemic Delivery of Synthetic
`siRNAsin Adult Mice”, J. Biol. Chem., 2003, V. 327, pp. 761-766.
`Spagnou,S., et al., “Lipidic Carriers of siRNA: Differences in the
`Formulation, Cellular Uptake, and Delivery with Plasmid DNA,”
`Biochemistry, 2004, vol. 43, pp. 13348-13356.
`Stamatatos, L., et al., “Interactions of Cationic Lipid Vesicles with
`Negatively Charged Phospholipid Vesicles and Biological Mem-
`branes,” Biochemistry, 1988, vol. 27, pp. 3917-3925.
`Szoka,F., et al., “Comparative Properties and MethodsofPreparation
`of Lipid Vesicles (Liposomes),” Ann. Rev. Biophys. Bioeng., 1980,
`vol. 9, pp. 467-508.
`Szoka,F., et al., “Procedure for preparation of liposomes with large
`internal aqueous space and high capture by reverse-phase evapora-
`
`tion,” Proc. Natl. Acad. Sci. USA, 1978, vol. 75, No. 9, pp. 4194-
`4198.
`Tabatt, K.et al., “Effect of cationic lipid and matrix lipid composition
`on solid lipid nanoparticle-mediated gene transfer,’ European J. of
`Pharmaceutics and Biopharmaceutics, 57:155-162, 2004.
`Teixeira, H. et al., “Characterization of oligonucleotide/lipid interac-
`tions in submicron cationic emulsions: influence ofthe cationic lipid
`structure and the presence of PEG-lipids,” Biophysical Chemistry,
`92:169-181, 2001.
`Tekmira Pharmaceuticals and Protiva Biotherapeutics Inc., Poster
`entitled “Manufacturing, Safety and Efficacy of SNALP Formulated.
`siRNA,” Presented at CHI—Discovery on Target—Oct. 23, 2008,
`Boston, MA, by Ian MacLachlan.
`Templeton, “Cationic Liposome-mediated Gene Delivery in vivo”,
`Bioscience Reports, 2002, vol. 22, No. 2, pp. 283-295.
`Van Der Woude,I., et al., “Parameters influencing the introduction of
`plasmid DNAinto cells by the use of synthetic amphiphiles as a
`carrier system,” Biochimica et Biophysica Acta, 1995, vol. 1240, pp.
`34-40.
`Wheeler, et al., “Stabilized Plasmid-lipid Particles: Constructions
`and Characterization,” Gene Therapy, V. 6, pp. 271-281.
`Wilson, R.,
`et
`al.
`“Counterion-Induced Condensation of
`Deoxyribonucleic Acid,” A Light-Scattering Study, Biochemistry,
`1979, vol. 18, No. 11, pp. 2192-2196.
`Woodle, M.C., et al., “Versatility in lipid compositions showing
`prolonged circulation with
`sterically stabilized liposomes,”
`Biochimica et Biophysica Acta, 1992, vol. 1105, pp. 193-200.
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`
`

`

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`U.S. Patent
`
`Aug.2, 2016
`
`Sheet 1 of 24
`
`US 9,404,127 B2
`
`Step One: Blending
`Lipids stock in 90% Ethanol
`
`Impingement
`zone
`
`RNSIRNA stock inEDTA=Ecistaltic Stabilized
`
`Pump
`NALP in 45%
`Ethanol
`
`Step Two: Diluting
`
`Stabilized NALP
`
`in 45% Ethanol
`
`Dilution Zone
`
` Peristaitic
`Warm
`Pump
`citrate/NaC!
`buffer
`
`Stabilized
`NALP in 22.5%
`Ethanol
`
`FIG. 1A
`
`

`

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`Aug.2, 2016
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`Sheet 2 of 24
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`US 9,404,127 B2
`
`
`
`Lipidsstockin100%Ethanol
`
`
`
`SIRNAstockinEDTA
`
`SNALPin50%Ethanol
`
`Impingement
`Zone
`
`Stabilized NALP in 17% Ethanol
`
`FIG. 1B
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 8 of 104
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`Aug.2, 2016
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`Sheet 3 of 24
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`US 9,404,127 B2
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`
`
`FIG. 2A
`
`

`

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`Sheet 4 of 24
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`US 9,404,127 B2
`
`
`
`
`
`Bare HPF Sample spanning_... and vitrified inSample drop placed ...andthinned
`
`
`on the grid
`by blotting
`holes in film
`liquid ethane
`v
`
`The cryo vitrification technique +(|)-
`
`
`
`hae
`
`EY: 2 =
`
`
`Grid with noley polymer film
`(HPF)
`
`FIG. 2B
`
`

`

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`Aug.2, 2016
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`Sheet 5 of 24
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`US 9,404,127 B2
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`
`
`Hole size: 1-6 yum
`
`Thickness of sample film:
`10-500 nm
`
`FIG. 2C
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 11 of 104
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`Aug.2, 2016
`
`Sheet 6 of 24
`
`US 9,404,127 B2
`
`Particle size (nm)
`
`Final encapsulation (%)
`
`Number-averaged
`Diameter size (nm)
`
`[lipid] mg/mL
`
`93 (0.12)
`
`46"
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 12 of 104
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`Aug.2, 2016
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`Sheet 7 of 24
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`US 9,404,127 B2
`
`{lipid] mg/mL
`
`Particle size
`(nm)
`
`Diameter size (nm)
`
`Final encapsulation (%)}
`
`Number-averaged
`
`FIG. 4
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 13 of 104
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`U.S. Patent
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`Aug.2, 2016
`
`Sheet 8 of 24
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`US 9,404,127 B2
`
`Number-averaged
`Final encapsulation (%)
`Particle size
`[lipid] mg/mL
`(nm) Diameter size (nm)
`
`15
`116 (0.06)
`
`64*
`
`FIG. 5
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 14 of 104
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`U.S. Patent
`
`Aug.2, 2016
`
`Sheet 9 of 24
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`US 9,404,127 B2
`
`Number-averaged
`Final encapsulation (%)
`Particle size
`[lipid] mg/mL
`(nm) Diameter size (nm)
`
`108 (0.09)
`15
`
`59°
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 15 of 104
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`Sheet 10 of 24
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`US 9,404,127 B2
`
`Presenceof lamellar particles using SDM
`
`25
`
`20
`
`= or
`
`= oO
`
`
`
`%lamellarparticles
`
`1:62 SNALP
`1:57 SNALP
`2:30 SNALP
`10:15 SNALP
`
`Numberof non-
`594 (77%)
`665 (91%)
`325 (95%)
`313 (99%)
`lameilar particles
`
`
`
`Number of
`lamellar particles
`
`173 (23%)
`
`67 (9%)
`
`16 (5%)
`
`4 (1%)
`
`FIG. 7
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 16 of 104
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`
`Aug.2, 2016
`
`Sheet 11 of 24
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`US 9,404,127 B2
`
`i
`
`Final encapsulation (%)
`
`Number-averaged
`Diameter size (nm)*
`
`[lipid] mg/mL
`
`Particle size
`(nm)
`58 (007)
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 17 of 104
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`Aug.2, 2016
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`Sheet 12 of 24
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`US 9,404,127 B2
`
`
`[lipid] mg/mL Particle size
`
`
`(nm)
`
`Number-averaged
`Diameter size (nm)*
`
`Finai encapsulation (%)
`
`& (019
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 18 of 104
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`U.S. Patent
`
`Aug.2, 2016
`
`Sheet 13 of 24
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`US 9,404,127 B2
`
`[lipid] mg/mL
`
`Particle size
`
`15
`
`78 (0.03)
`
`Final encapsulation (%)
`
`Number-averaged
`Diameter size (nm)
`PT
`
`FIG. 10
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 19 of 104
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`Aug.2, 2016
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`Sheet 14 of 24
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`US 9,404,127 B2
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`[lipid] mg/mL
`
`Particle size
`(nm)
`
`Final encapsulation (%)
`
`Number-averaged
`
`Diameter size (nm)
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 20 of 104
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`Aug.2, 2016
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`Sheet 15 of 24
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`US 9,404,127 B2
`
`Presence of lamellar particles using DDM
`
`particles
`
`%lamellar
`
`Po 2:30 SNALP
`Number of non-
`1386 (99%)
`lamellar particles
`
`2:40 SNALP
`1191 (99%)
`
`1:57 SNALP
`694 (>99%)
`
`4:62 SNALP
`707 (>99%)
`
`2 (<1%)
`
`lamellar particles
`
`Numberof
`
`14 (1%)
`
`10 (1%)
`
`2 (<1%)
`
`FIG, 12
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 21 of 104
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`U.S. Patent
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`Aug.2, 2016
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`Sheet 16 of 24
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`US 9,404,127 B2
`
`
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 22 of 104
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`U.S. Patent
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`Aug.2, 2016
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`Sheet 17 of 24
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`US 9,404,127 B2
`
`
`
`FIG. 14
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 23 of 104
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`Aug.2, 2016
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`Sheet 18 of 24
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`US 9,404,127 B2
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`
`
`FIG. 15
`
`

`

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`U.S. Patent
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`US 9,404,127 B2
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`
`
`
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 25 of 104
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`Aug.2, 2016
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`Sheet 20 of 24
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`US 9,404,127 B2
`
`
`
`3.0 eee to ereeats
`
`
`
`
`
`
`
`
`
`LiverApoB:GAPDmRNARatio
`
`-47% vs PBS Control
`
`-77% vs PBS Control
`
`PBS
`
`2:30 SNALP 5 x 1 mg/kg
`
`1:57 SNALP 5 x 0.1 mg/kg
`
`FIG. 17
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 26 of 104
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`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 21 of 24
`
`US 9,404,127 B2
`
`
`
`
`
`
`
`OnlyWNHWQavo-:gody
`
`wawoooa
`
`2So
`
`A
`
`
`
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 27 of 104
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`U.S. Patent
`
`Aug.2, 2016
`
`Sheet 22 of 24
`
`US 9,404,127 B2
`
`a~
`
`©W
`
`e
`
`oJ $a
`
`s
`~
`a
`2
`
`NS
`Wn
`
`K
`a
`
`o—t
`
`xS
`
`s
`~
`o
`ea
`$
`a
`
`Y
`m
`
`onel GdVO: bW1d
`
`
`
`
`ones ddyvD : gody
`
`& 6
`OE
`= SD
`© _
`5 3
`= 2
`OQ —
`
`+¥
`
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`—! ©
`Qo.
`
`&
`mH
`££
`Oo.
`Cc
`DS
`OE
`mo
`oO &
`QQ
`<<
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 28 of 104
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`U.S. Patent
`
`Aug.2, 2016
`
`Sheet 23 of 24
`
`US 9,404,127 B2
`
`Anti-tumorefficacy of SNALP in subcutaneous Hep3B tumor-bearing
`mice after 6 x 3 mg/kg dosesintravenously adminstered twice weekly
`for 3 weeks
`
`1800 -
`
`1600
`
`—#- PBS
`
`200 0
`
`—™@— 1:57 DLinDMA (PEG2000-C-DMA)
`
`hk 7:54 DLinNDMA (PEG750-C-DMA)
`
`
`
`Tumorvolume
`
`1400
`
`1200
`
`1000
`
`800
`
`600 -
`
`400 -
`
`16
`
`18
`
`20
`
`22
`
`24
`
`26
`
`28
`
`30
`
`32
`
`34
`
`36
`
`38
`
`40 42
`
`44
`
`46
`
`48
`
`50
`
`Study day
`
`FIG. 20
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 29 of 104
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`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 24 of 24
`
`US 9,404,127 B2
`
`
`
`
`
`2IgndJo("})jeuobexapyessaAu|sapdiyduiypidry
`
`
`
`,aselidjoJaysajoyD=pidig(VN)pIseD9;onNotuoljye5
`
`
`
`
`uoneinsdeauesdnosBpesyopydoipAy
`
`beOld
`
`
`
`SIYN‘spidi]pedeys-suosAqpaydope)
`
`sueiquieueBAqpayeinsdeoue
`
`
`
`puepidiya1uayesJopasoduoo
`
`(joJa}Sa]OYD
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`ayeBoibhyajjaoipypoweau]
`
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`
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`
`YNJO}[BONSjOI@ISajOYD
`
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`
`owluoebaliiysnoauejuods
`
`
`
`SIPIME fEployod
`
`
`
`syeyaigoydoupALy
`
`
`
`
`

`

`Case 1:22-cv-02229-MKV Document 1-6 Filed 03/18/22 Page 30 of 104
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`US 9,404,127 B2
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`1
`NON-LIPOSOMAL SYSTEMS FOR NUCLEIC
`ACID DELIVERY
`
`CROSS-REFERENCE TO RELATED
`APPLICATION
`
`This application is a continuation of U.S. application Ser.
`No. 13/807,288, filed Apr. 18, 2013, which application is a
`National Phase application under 35 U.S.C. §371 of PCT/
`CA2011/000778, filed Jun. 30, 2011, which application
`claims the benefit of U.S. Provisional Application No.
`61/360,480,filed Jun. 30, 2010, the disclosures of which are
`incorporated herein by reference for all purposes.
`
`REFERENCETO A “SEQUENCELISTING,” A
`TABLE, OR A COMPUTER PROGRAMLISTING
`APPENDIX SUBMITTEDAS AN ASCII TEXT
`FILE
`
`The Sequence Listing written in file -100-1.TXT, created
`on May 15, 2013, 4,096 bytes, machine format IBM-PC,
`MS-Windowsoperating system, is hereby incorporated by
`reference in its entirety for all purposes.
`
`BACKGROUND OF THE INVENTION
`
`RNAinterference (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); Hammondetal., Nat. Rev. Genet.,
`2:110-119 (2001)). More recently,
`it was discovered that
`introducing synthetic 21-nucleotide dsRNA duplexes into
`mammalian cells couldefficiently 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 genesfor the treatmentof neoplastic disorders
`such as cancer.It is also desirableto 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 RNAito 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 etal., Gene Therapy, 1:192 (1994); Hopeet al., Molecular
`MembraneBiology, 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 immuneresponses that com-
`promise delivery with subsequentinjections.
`Plasmid DNA-cationic liposome complexesare currently
`the most commonly employed nonviral gene delivery
`vehicles (Feigner, Scientific American, 276:102 (1997);
`
`25
`
`30
`
`40
`
`45
`
`50
`
`55
`
`2
`Chonn et al., Current Opinion in Biotechnology, 6:698
`(1995)). For instance, cationic liposome complexes made of
`an amphipathic compound, a neutrallipid, and a detergent for
`transfecting insectcells are disclosed in U.S. Pat. No. 6,458,
`382. Cationic liposome complexesare also disclosed in U.S.
`Patent Publication No. 20030073640.
`Cationic liposome complexes are large, poorly defined
`systemsthat 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)). Aslarge, positively charged
`aggregates, lipoplexes are rapidly cleared when administered
`in vivo, with highest expression levels observedin first-pass
`organs,