Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 1 of 107
`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 1 of 107
`
`EXHIBIT H
`EXHIBIT H
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 2 of 107
`Case n2zeovcz2cewi occNAFATAALAAA
`
`US009518272B2
`
`a2) United States Patent
`US 9,518,272 B2
`(0) Patent No.:
`*Dec. 13, 2016
`(45) Date of Patent:
`Yaworskiet al.
`
`(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 (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.: 15/153,487
`
`(22)
`
`Filed:
`
`May 12, 2016
`
`(65)
`
`Prior Publication Data
`
`US 2016/0251681 Al
`
`Sep. 1, 2016
`
`Related U.S. Application Data
`
`(63) Continuation of application No. 14/642,452, filed on
`Mar. 9, 2015, which is a 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.
`
`(51)
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2010.01)
`(2006.01)
`(2006.01)
`
`Int. Cl.
`CO7H 21/04
`CI2N 15/88
`AGIK 9/107
`AGIK 9/51
`AGIK 31/7088
`AGIK 31/7105
`AGIK 31/712
`AGIK 31/713
`AGIK 47/14
`CI2N 15/113
`AGIK 47/48
`AOLK W127
`(52) U.S. Cl.
`CPC wees CI2N 1548 (2013.01); A6LK 9/1075
`(2013.01); A61K 9/5123 (2013.01); A6LK
`31/7088 (2013.01); A6LK 31/712 (2013.01);
`AGIK 31/713 (2013.01); A6LK 31/7105
`(2013.01); A6IK 47/14 (2013.01), A61K
`47/48046 (2013.01); C12N 15/113 (2013.01);
`AOIK 9/1272 (2013.01); A6LK 9/1274
`(2013.01); CI2N 2310/14 (2013.01); C12N
`2310/321 (2013.01); CI2N 2310/3515
`(2013.01); CI2N 2320/32 (2013.01)
`(58) Field of Classification Search
`CPC vicecccscrssssecrsscnseecersetesenscnssenseeseonees CO7H 21/04
`
`See application file for complete search history.
`
`4,394,448 A
`4,438,052 A
`4,515,736 A
`4,598,051 A
`4,897,355 A
`5,013,556 A
`5,171,678 A
`5,208,036 A
`5,225,212 A
`5,264,618 A
`5,279,833 A
`5,283,185 A
`5,320,906 A
`5,334,761 A
`5,545,412 A
`5,578,475 A
`5,627,159 A
`5,641,662 A
`5,656,743 A
`5,674,908 A
`5,703,055 A
`5,705,385 A
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`(Continued)
`
`FOREIGN PATENT DOCUMENTS
`
`CA
`CA
`
`2309727 Al
`2271582 Al
`
`4/1999
`11/1999
`
`(Continued)
`
`OTHER PUBLICATIONS
`
`Arpicco, S., et al., “Preparation and Characterization of Novel
`Cationic Lipids Developed for Gene Transfection,” Proceed. Intl
`Symp. Control. Rel. Bioact. Mater. (Controlled Release Society,
`Inc.), 1999, vol. 26, pp. 759-760.
`Arpicco, S., et al., “Synthesis, characterization and transfection
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`Beale, G., et al., “Gene Silencing Nucleic Acids Designed by
`Scanning Arrays: Anti-EGFR Activity of siRNA, Ribozyme and
`DNAEnzymes Targeting a Single Hybridization-accessible Region
`using the Same Delivery System,” Journal of Drug Targeting, 2003,
`vol. 11, No. 7, pp. 449-456.
`(Continued)
`
`Primary Examiner — Kimberly Chong
`(74) Attorney, Agent, or Firm — Kilpatrick Townsend &
`Stockton LLP
`
`ABSTRACT
`(57)
`The present invention provides novel, stable lipid particles
`having a non-lamellar structure and comprising one or more
`active agents or therapeutic agents, methods of making such
`lipid particles, and methods of delivering and/or adminis-
`tering such lipid particles. More particularly, the present
`invention provides
`stable nucleic
`acid-lipid particles
`(SNALP) that have a non-lamellar structure and that com-
`prise a nucleic acid (such as one or moreinterfering RNA),
`methods of making the SNALP, and methods of delivering
`and/or administering the SNALP.
`22 Claims, 24 Drawing Sheets
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 3 of 107
`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 3 of 107
`
`US 9,518,272 B2
`
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`

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`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 4 of 107
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`

`

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`Sawada et al., “Microemulsions in supercritical CO, utilizing the
`polyethyleneglycol dialkylglycerol and their use for the solubiliza-
`tion of hydrophiles,” Dyes and Pigments, 2005, pp. 64-74, vol. 65.
`Shin, et al. “Acid-triggered release via dePEGylation of DOPE
`liposomescontaining acid-labile vinyl ether PEG-lipids,” Journal of
`Controlled Release, 2003, vol. 91, pp. 187-200.
`Smisterova,J. et al., “Molecular shape of the cationic lipid controls
`the structure of cationic lipid/dioleylphosphatidylethanolamine-
`DNA complexes and the efficiency of gene delivery,” J. Biol.
`Chem., 276(50):47615-47622, 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 Methods of Prepa-
`ration 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 evapo-
`ration,” Proc. Natl. Acad. Sci. USA, 1978, vol. 75, No. 9, pp.
`4194-4198.
`Tabatt, K. et al., “Effect of cationic lipid and matrix lipid compo-
`sition 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 inter-
`actions in submicron cationic emulsions: influence of the cationic
`lipid structure and the presence of PEG-lipids,” Biophysical Chem-
`istry, 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
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`prolonged circulation with
`sterically
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`
`* cited by examiner
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 6 of 107
`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 6 of 107
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`U.S. Patent
`
`Dec. 13, 2016
`
`Sheet 1 of 24
`
`US 9,518,272 B2
`
`Step One: Blending
`
`Lipids stock in 90% Ethanol
`
`Impingement
`Zone
`
`SIRNA
`IRNA
`
`stock in
`kin EDTA
`
`Peristaltic
`Pump
`
`Stabilized
`NALP in 45%
`Ethanol
`
`Step Two: Diluting
`
`Stabilized NALP
`in 45% Ethanol
`
`Dilution Zone
`
` Peristaltic
`Warm
`Pump
`citrate/NaCl
`buffer
`
`Stabilized
`NALP in 22.5%
`Ethanol
`
`FIG. TA
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 7 of 107
`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 7 of 107
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`U.S. Patent
`
`Dec. 13, 2016
`
`Sheet 2 of 24
`
`US 9,518,272 B2
`
`
`
`Lipidsstockin100%Ethanol
` siRNA
`stockinEDTA
`
`SNALPin50%Ethanol
`
`Impingement
`Zone
`
`Stabilized NALP in 17% Ethanol
`
`FIG. 1B
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 8 of 107
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`
`Dec. 13, 2016
`
`Sheet 3 of 24
`
`US 9,518,272 B2
`
`aman
`
`Climate Chamber
`
`FIG. 2A
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 9 of 107
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`U.S. Patent
`
`Dec. 13, 2016
`
`Sheet 4 of 24
`
`US 9,518,272 B2
`
`SeTorsz3
`Se.
`
`“a
`
`The cryo vitrification technique
`=
`
`etoctaee. MA
`
`
`‘Bare HPF«6Sample drop placed... and thinned «Sample spanning _... and vitrified in
`
`on the grid
`by blotting
`holes in film
`liquid ethane
`
`
`t
`v
`eawenonneneB11
`
`
`
`
`ies tg) opti
`£2 9 trPa
`
`
`tA ee
`
`Grid with holey polymerfilm
`(HPF)
`
`eeeeertea
`
`FIG. 2B
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 10 of 107
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`Dec. 13, 2016
`
`Sheet 5 of 24
`
`US 9,518,272 B2
`
` =I
`
`
`
` °
`Hole size: 1-6 pm
`
`.
`
`|
`Thickness of sample film:
`10-500 nm
`
`FIG. 2C
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 11 of 107
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`U.S. Patent
`
`Dec. 13, 2016
`
`Sheet 6 of 24
`
`US 9,518,272 B2
`
`[lipid] mg/mL
`
`Particle size (nm)
`
`Final encapsulation (%)
`
`Number-averaged
`Diametersize (nm)
`
`(08)
`
`FIG. 3
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 12 of 107
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`U.S. Patent
`
`Dec. 13, 2016
`
`Sheet 7 of 24
`
`US 9,518,272 B2
`
`[lipid] mg/mL
`
`Particle size
`(nm)
`
`90.1)|96a
`
`
`Final encapsulation (%)
`
`Number-averaged
`Diametersize (nm)
`
`
`
`
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 13 of 107
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`U.S. Patent
`
`Dec. 13, 2016
`
`Sheet 8 of 24
`
`US 9,518,272 B2
`
`flipid] mg/mL
`
`Particle size
`(nm)
`
`
`
`
`Final encapsulation (%)
`
`Number-averaged
`Diameter size (nm)
`
`
`
`pe Pe05)ee
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 14 of 107
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`U.S. Patent
`
`Dec. 13, 2016
`
`Sheet 9 of 24
`
`US 9,518,272 B2
`
`
`
`[lipid] mg/mL
`
`
`
`Final encapsulation (%)
`
`Number-averaged
`Particle size
`Diametersize (nm)
`(nm)
`10.09) [|
`
`
`
`
`
`
`
`FIG. 6
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 15 of 107
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`Dec. 13, 2016
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`Sheet 10 of 24
`
`US 9,518,272 B2
`
`Presenceof lamellar particles using SDM
`
`particles
`
`%lamellar
`
`
`pe 10:15 SNALP
`
`
`594 (77%)
`
`173 (23%)
`
`Numberofnon-
`lamellar particles
`
`Numberof
`lamellarparticles
`
`2-30 SNALP
`
`665 (91%)
`
`4:57 SNALP
`
`325 (95%)
`
`4:62 SNALP
`
`313 (99%)
`
`
`
`
`4 (1%)
`
`
`
`67 (9%)
`
`16 (5%)
`
`FIG. 7
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 16 of 107
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`U.S. Patent
`
`Dec. 13, 2016
`
`Sheet 11 of 24
`
`US 9,518,272 B2
`
`Number-averaged
`Diameter size (nm)*
`
`sa(007)
`
`[lipid] mg/mt
`
`Particle size
`(nm)
`
`Final encapsulation (%)
`
`FIG. 8
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 17 of 107
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`U.S. Patent
`
`Dec. 13, 2016
`
`Sheet 12 of 24
`
`US 9,518,272 B2
`
`
`
`
`[lipid] mg/mL
`
`Particle size
`(nm)
`
`65 0.10
`
`Final encapsulation (%)
`
`Number-averaged
`Diameter size (nm)*
`
`
`
`
`
`
`
`FIG. 9
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 18 of 107
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`
`Dec. 13, 2016
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`Sheet 13 of 24
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`US 9,518,272 B2
`
` {lipid} mg/mL
`
`
`
`Number-averaged
`Particle size
`Diametersize (nm)
`(am)
`7g(0.03) |
`
`Final encapsulation (%)
`
`ce
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 19 of 107
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`Dec. 13, 2016
`
`Sheet 14 of 24
`
`US 9,518,272 B2
`
`{lipid] mg/mL
`
`Particle size
`(nm)
`
`78008
`
`s
`
`Final encapsulation (%)
`
`Number-averaged
`Diameter size (nm)
`
`FIG. 11
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 20 of 107
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`Dec. 13, 2016
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`Sheet 15 of 24
`
`US 9,518,272 B2
`
`Presence of lamellar particles using DDM
`
`particles
`
`%lamellar
`
`P| 2:30 SNALP
`
`Numberof non-
`
`1386 (99%)
`
`2:40 SNALP
`
`41194 (99%)
`
`4:57 SNALP
`
`694 (>99%)
`
`4:62 SNALP
`
`707 (>99%)
`
`lamellar particles Numberof
`
`lamellar particles
`
`14 (1%)
`
`10 (1%)
`
`2 (<1%)
`
`2 (<1%)
`
`FIG. 12
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 21 of 107
`22-CV-02229-MKV Document 42-9 Filed 09/06/22 Page 21 of 107
`Case 1
`
`Sheet 16 of 24
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`US 9,518,272 B2
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`Dec. 13, 2016
`
`U.S. Patent
`
`FIG. 13
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 22 of 107
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`Case 1
`
`U.S. Patent
`
`Dec. 13, 2016
`
`Sheet 17 of 24
`
`US 9,518,272 B2
`
`
`
`FIG. 14
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 23 of 107
`22-CV-02229-MKV Document 42-9 Filed 09/06/22 Page 23 of 107
`Case 1
`
`U.S. Patent
`
`Dec. 13, 2016
`
`Sheet 18 of 24
`
`US 9,518,272 B2
`
`
`
`FIG. 15
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 24 of 107
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`U.S. Patent
`
`Dec. 13, 2016
`
`Sheet 19 of 24
`
`
`
`
`02OQ2©a9@SONNS
`
`
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 25 of 107
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`Dec. 13, 2016
`
`Sheet 20 of 24
`
`US 9,518,272 B2
`
`BO pennennenannsnnnnnnnsnnnsennnunnuinnsinnsnnnntcunsnncennn
`
`-77% vs PBS Contra!
`
`
`
`
`
`LiverApoB:GAPDmRNARatio
`
`PBS
`
`~47% vs PBS Conirol
`
`2:30 SNALP 5 x 4 mg/kg
`
`4:57 SNALP 5x 0.1 mg/kg
`
`FIG. 17
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 26 of 107
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`-9 Filed 09/06/22
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`
`
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 27 of 107
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`Dec. 13, 2016
`
`Sheet 22 of 24
`
`US 9,518,272 B2
`
`
`PLK-7silencing
`
`
`
`
`inHep3elivertumor
`
`
`
`
`
`ApoBsilencingin
`
`
`
`normalliver
`
`0.8
`
`©
`Oo
`
`=
`oO
`
`CQao
`
`0.0+
`
`PBS
`1:57SNALP7:54SNALP
`
`
`PBS
`
`1:57SNALP7:54SNALP
`
`OHEd QdVO : LW 1d
`
`FIG.19
`
`OEJ GdY¥D : gody
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 28 of 107
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`
`Dec. 13, 2016
`
`Sheet 23 of 24
`
`US 9,518,272 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
`
`1400 -
`
`—#-— 1:57 DLINDMA (PEG2000-C-DMA)
`
`~~ 7:54 DLINDMA (PEG750-C-DMA)
`
`
`
`Tumorvolume
`
`1200 -
`
`1000 -
`
`800 -
`
`600 -
`
`400
`
`
`200
`
`
`
`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 42-9 Filed 09/06/22 Page 29 of 107
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`Dec. 13, 2016
`
`Sheet 24 of 24
`
`US 9,518,272 B2
`
`signue(4)puoGexayasiaut
`
`
`
`
`
`
`SSEudtyN}pisesiayony
`
`
`JOLBISSJOUS prdsyayuoHeD
`
`
`
`saydiydurgpidry
`
`
`
`‘sjuepeLinspidyAgpezitqeys)
`
`
`
`(pidyoydsoud40pidy-ngd‘Be
`
`SouuoyebeiSby
`srmauejucls
`
`BPRILEofFEPIOYOD
`
`
`
`WN408RRQEERSjosmaseauey
`
`uy
`
`seneneeanensesaneseeeeliite
`
`
`scriniinwetscinyecupan
`8x
`
`beOld
`
`suBiqueu@Aqpeipinedeaus
`
`RUEDIdHGHOneS3OpesoduOS
`
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`
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`
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`
`
`ayeBaiiiyayoonypovaAu]
`
`
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`
`
`
`
`
`

`

`Case 1:22-cv-02229-MKV Document 42-9 Filed 09/06/22 Page 30 of 107
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`US 9,518,272 B2
`
`1
`NON-LIPOSOMAL SYSTEMS FOR NUCLEIC
`ACID DELIVERY
`
`CROSS-REFERENCE TO RELATED
`APPLICATION
`
`This application is a continuation of U.S. application Ser.
`No. 14/642,452, filed Mar. 9, 2015, which is a continuation
`of U.S. application Ser. No. 13/807,288,filed Apr. 18, 2013,
`and which issued on Apr. 14, 2015, as U.S. Pat. No.
`9,006,417 B2, which application is a National Phase appli-
`cation 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 refer-
`ence for all purposes.
`
`REFERENCE TO A “SEQUENCELISTING,” A
`TABLE, OR A COMPUTER PROGRAM LISTING
`APPENDIX SUBMITTED AS 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., Elbashir et 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 could efficiently silence gene expres-
`sion.
`
`Although the precise mechanism is still unclear, RNAi
`provides a potential new approach to downregulate or
`silence the transcription and translation of a geneofinterest.
`For example, it is desirable to modulate (e.g., reduce) the
`expression 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 athero-
`sclerosis and its manifestations, e.g., hypercholesterolemia,
`myocardial infarction, 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);
`Yeiet al., Gene Therapy, 1:192 (1994); Hopeet al., Molecu-
`lar 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,
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`2
`these systems induce immune
`In addition,
`and spleen.
`responses that compromise delivery with subsequent injec-
`tions.
`
`Plasmid DNA-cationic liposome complexesare currently
`the most commonly employed nonviral gene delivery
`vehicles (Felgner, Scientific American, 276:102 (1997);
`Chonn et al., Current O