Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 1 of 104
`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 1 of 104
`
`EXHIBIT D
`EXHIBIT D
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 2 of 104
`NNTTTATAATTA
`
`US009006417B2
`
`a2) United States Patent
`US 9,006,417 B2
`(10) Patent No.:
`
`(45) Date of Patent: Apr.14, 2015
`Yaworskiet al.
`
`(54) NON-LIPOSOMAL SYSTEMS FOR NUCLEIC
`ACID DELIVERY
`
`(75)
`
`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:
`
`(21) Appl. No.:
`
`(22)
`
`PCTFiled:
`
`(86) PCT No::
`
`Subject to any disclaimer, the term ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`13/807,288
`
`Jun. 30, 2011
`
`PCT/CA2011/000778
`
`§ 371 (©),
`(2), (4) Date:
`
`Apr. 18, 2013
`
`(87) PCT Pub. No.: WO2012/000104
`
`PCT Pub. Date: Jan. 5, 2012
`
`(65)
`
`Prior Publication Data
`
`US 2013/0303587 Al
`
`Nov. 14, 2013
`
`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
`5,736,392 A
`5,820,873 A
`5,877,220 A
`5,885,613 A
`5,958,901 A
`5,976,567 A
`5,981,501 A
`6,020,202 A
`6,020,526 A
`6,034,135 A
`6,051,429 A
`6,075,012 A
`6,165,501 A
`6,172,049 Bl
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`
`(Continued)
`
`FOREIGN PATENT DOCUMENTS
`
`Related U.S. Application Data
`
`CA
`CA
`
`2309727 Al
`2271582 Al
`
`4/1999
`11/1999
`
`(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)
`(2006.01)
`
`Int. Cl.
`CO7H 21/04
`AGIK 47/14
`AGIK 9/51
`AGIK 31/7088
`AGIK 31/7105
`AGIK 31/712
`AGIK 31/713
`CI2N 15/88
`AOIK 9/107
`AOIK 9/127
`(52) U.S.C.
`CPC veeceeceeeeeen AGIK 47/14 (2013.01); AOIK 9/1075
`(2013.01); A6IK 9/1272 (2013.01); A6IK
`9/1274 (2013.01); A6LK 9/5123 (2013.01);
`A61K 31/7088 (2013.01); A6LK 31/7105
`(2013.01); A6LK 31/712 (2013.01); A6LK
`31/713 (2013.01); CI2N 15/88 (2013.01)
`(58) Field of Classification Search
`None
`
`See application file for complete search history.
`
`(56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`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
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`OTHER PUBLICATIONS
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`Epandet al. (Chemistry and Physics of Lipids 135, 2005: 39-53).*
`Arpicco, S., et al., “Preparation and Characterization of Novel
`Cationic Lipids Developed. for Gene Transfection,” Proceed. Int’!
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`(Continued)
`
`Primary Examiner — Kimberly Chong
`(74) Attorney, Agent, or Firm — Kilpatrick Townsend &
`Stockton LLP
`
`(57)
`
`ABSTRACT
`
`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 administer-
`ing such lipid particles. More particularly, the present inven-
`tion provides stable nucleic acid-lipid particles (SNALP)that
`have a non-lamellarstructure and that comprise a nucleic acid
`(such as one or more interfering RNA), methods of making
`the SNALP, and methodsof delivering and/or administering
`the SNALP.
`
`24 Claims, 24 Drawing Sheets
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 3 of 104
`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 3 of 104
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`US 9,006,417 B2
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`

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`
`References Cited
`OTHER PUBLICATIONS
`
`et al., “Physico-chemical characterization of
`Rotenberg. M.
`Intralipid™emulsions,” Biochemica et Biophysica Acta, 1086:265-
`272, 1991.
`Rudolph, C. et al., “Application of novel solid lipid nanoparticle
`(SLN)-gene vector formulations based on a dimeric HIV-1 TAT-
`peptidein vitro andin vivo,” Pharmaceutical Research, 21 (9): 1662-
`1669, 2004.
`Smisterova, J. et al., “Molecular shape ofthe cationic lipid controls
`the structure of cationic lipid/dioleylphosphatidylethanolamine-
`DNAcomplexesandthe efficiency of gene delivery,” J. Biol. Chem.,
`276(50):476 15-47622, 2001.
`
`Tabatt. K.etal., “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.
`Xu, Y. et al., “Physicochemical characterization and purification of
`cationic lipoplexes,” Biophysical Journal, 77:341-353, 1999.
`
`* cited by examiner
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 6 of 104
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`U.S. Patent
`
`Apr. 14, 2015
`
`Sheet 1 of 24
`
`US 9,006,417 B2
`
`Step One: Blending
`
`Lipids stock in 90% Ethanol
`
`Impingement
`Zone
`
`SIRNA stock inEDTA
`
`=istaltic
`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. 1A
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 7 of 104
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`U.S. Patent
`
`Apr. 14, 2015
`
`Sheet 2 of 24
`
`US 9,006,417 B2
`
`
`
`Lipidsstockin100%Ethanol
`
` siRNA
`stockinEDTA
`Zone SNALPin50%Ethanol
`
` Impingement
`
`Stabilized NALP in 17% Ethanol
`
`FIG. 1B
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 8 of 104
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`U.S. Patent
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`Apr. 14, 2015
`
`Sheet 3 of 24
`
`US 9,006,417 B2
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`
`
`FIG. 2A
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 9 of 104
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`U.S. Patent
`
`Apr. 14, 2015
`
`Sheet 4 of 24
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`US 9,006,417 B2
`
`The cryovitrification technique
`
`Bare HPF Sample spanning_... and vitrified inSample drop placed ..andthinned
`
`
`
`
`
`
`
`
`byblotting
`onthegrid
`t
`
`YY@eee
`
`Bort2 [ALF
`
`ms ——
`
`woes 5M
`
`
`
`‘
`
`poe
`
`ie
`
`4
`
`“45
`
`holesinfilm
`
`liquidethane
`
`Grid with holey polymer film
`
`(HPF)
`
`10X
`
`120 X
`
`
`
`FIG. 2B
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 10 of 104
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`U.S. Patent
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`Apr. 14, 2015
`
`Sheet 5 of 24
`
`US 9,006,417 B2
`
`
`
`Hole size: 1-6 xm
`
`Thickness of sample film:
`10-500 nm
`
`FIG. 2C
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 11 of 104
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`U.S. Patent
`
`Apr. 14, 2015
`
`Sheet 6 of 24
`
`US 9,006,417 B2
`
`[lipid] mg/mL
`
`Particle size (nm)
`
`Final encapsulation (%)
`
`Number-averaged
`
`Diametersize (nm)
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 12 of 104
`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 12 of 104
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`U.S. Patent
`
`Apr. 14, 2015
`
`Sheet 7 of 24
`
`US 9,006,417 B2
`
`[pid] mg/mL
`
`Particle size
`(nm)
`
`Diametersize (nm)
`
`Final encapsulation (%)
`
`Number-averaged
`
`FIG. 4
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 13 of 104
`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 13 of 104
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`U.S. Patent
`
`Apr. 14, 2015
`
`Sheet 8 of 24
`
`US 9,006,417 B2
`
`Number-averaged
`Final encapsulation (%)
`Particle size
`[lipid] mg/mL
`(nm) Diameter size (nm)
`
`15
`116 (0.06)
`97
`
`64*
`
`FIG. 5
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 14 of 104
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`U.S. Patent
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`Apr. 14, 2015
`
`Sheet 9 of 24
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`US 9,006,417 B2
`
`[lipid] mg/mL
`
`Final encapsulation (%)
`
`
`Number-averaged
`Particle size
`Diameter size (nm)
`(nm)
`
`rosooy [ae
`.09)
`
`
`ae.
`
`
`
`
`
`
`
`FIG. 6
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 15 of 104
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`U.S. Patent
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`Apr. 14, 2015
`
`Sheet 10 of 24
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`US 9,006,417 B2
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`Presenceof lamellar particles using SDM
`
`particles
`
`%lamellar
`
`P| 14045 SNALP|2:30 SNALP 1:57 SNALP 1:62 SNALP
`
`
`
`
`
`
`
`
`lamellar particles
`
`Number of non-
`lamellar particles
`Number of
`
`594 (77%)
`
`473 (23%)
`
`665 (91%)
`
`325 (95%)
`
`313 (39%)
`
`67 (9%)
`
`16 (5%)
`
`4 (1%)
`
`FIG. 7
`
`

`

`
`
`
`
`Final encapsulation (%)
`
`Number-averaged
`Diameter size (nm)*
`
`
`
`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 16 of 104
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`U.S. Patent
`
`Apr. 14, 2015
`
`Sheet 11 of 24
`
`US 9,006,417 B2
`
`
`
`[lipid] mg/mL
`
`Particle size
`(nm)
`
`
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 17 of 104
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`Apr. 14, 2015
`
`Sheet 12 of 24
`
`US 9,006,417 B2
`
`
` Particle size
`
`
`
`a[
`
`Number-averaged
`Diameter size (nm)*
`
`lipid] mg/mL
`
`(nm)
`
`Final encapsulation (%)
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 18 of 104
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`U.S. Patent
`
`Apr. 14, 2015
`
`Sheet 13 of 24
`
`US 9,006,417 B2
`
`Number-averaged
`Particle size
`Diametersize (nm)
`(nm)
`707|edd
`
`
`
`{lipid] mg/mL
`
`
`
`
`
`Final encapsulation (%)
`
`
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 19 of 104
`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 19 of 104
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`U.S. Patent
`
`Apr. 14, 2015
`
`Sheet 14 of 24
`
`US 9,006,417 B2
`
`Final encapsulation (%)
`
`Number-averaged
`Diametersize (nm)
`
`
`
`
`(lipid) mg/mL
`
`
`
`
`15
`
`Particle size
`(nm)
`7008
`
`
`
`Ll ee Se Ow
`
`88 86ee ee
`
`
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 20 of 104
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`U.S. Patent
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`Apr. 14, 2015
`
`Sheet 15 of 24
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`US 9,006,417 B2
`
`Presence of lamellar particles using DDM
`
`particles
`
`%lamellar
`
`
`po 9:30 SNALP
`
`
`1386 (99%)
`
`9:40 SNALP
`
`1:57 SNALP
`
`41191 (99%)
`
`694 (>99%)
`
`Numberof non-
`lamellar particles
`40 (1%)
`14 (1%)
`Numberof
`
`lamellar particles
`
`4:62 SNALP
`
`707 (>99%)
`
`
`
`
`2 (<1%)
`
`2 (<1%)
`
`FIG. 12
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 21 of 104
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`Apr. 14, 2015
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`Sheet 16 of 24
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`US 9,006,417 B2
`
`FIG. 13
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 22 of 104
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`U.S. Patent
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`Apr. 14, 2015
`
`Sheet 17 of 24
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`US 9,006,417 B2
`
`
`
`FIG. 14
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 23 of 104
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`U.S. Patent
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`Apr. 14, 2015
`
`Sheet 18 of 24
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`US 9,006,417 B2
`
`
`
`FIG. 15
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 24 of 104
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`U.S. Patent
`
`Apr. 14, 2015
`
`Sheet 19 of 24
`
`US 9,006,417 B2
`
`
`
`2a2203-|esfoBeeG86elhU6mBlUB
`
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 25 of 104
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`U.S. Patent
`
`Apr. 14, 2015
`
`Sheet 20 of 24
`
`US 9,006,417 B2
`
`3.0 7
`
`2.54
`
`
`
`
`
`-47% vs PBS Control -77% vs PBS Control
`
`2:30 SNALP 5 x 1 mg/kg
`
`4:57 SNALP 5 x 0.1 mg/kg
`
`FIG. 17
`
`2.0 -
`
`
`
`
`
`LiverApoB:GAPDmRNARatio
`
`0.5
`
`0.0 +
`
`PBS
`
`
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 26 of 104
`
`U.S. Patent
`
`US 9,006,417 B2
`
`
`
`
`
`
`oneyWNWddvo:dody
`
`re)°19--a
`
`
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 27 of 104
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`U.S. Patent
`
`Apr. 14, 2015
`
`Sheet 22 of 24
`
`US 9,006,417 B2
`
`ones ddV9 : LA 1d
`
`FIG.19
`
`PBS
`
`
`
`1:57SNALP7:54SNALP
`®
`
`oO
`—!
`<x
`za
`”
`st
`
`é
`oO
`3
`
`wo
`
`_
`
`Y
`
`ones GdV5 : gody
`
`
`PLK-1silencing
`
`
`
`
`inHep3Blivertumor
`
`Cc
`
`OD &
`Cc
`OM
`Oo .2
`
`5 y
`
`s
`
`Y £
`mo
`Oo
`Cc
`oO.
`<
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 28 of 104
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`U.S. Patent
`
`Apr. 14, 2015
`
`Sheet 23 of 24
`
`US 9,006,417 B2
`
`Anti-tumor efficacy of SNALP in subcutaneous Hep3B tumor-bearing
`mice after 6 x 3 mg/kg dosesintravenously adminstered twice weekly
`for 3 weeks
`
`
`—
`
`1600 }
`
`—@-PBS
`
`1800
`
`
`
`Tumorvolume
`
`1400
`
`1200
`
`1000
`
`800
`
`600
`
`400
`
`200
`
`0
`
`
`
`—™- 1:57 DLinDMA (PEG2000-C-DMA)
`
`—t#— 7:54 DLinDMA (PEG750-C-DMA)
`
`T
`
`T
`
`4
`
`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 1-4 Filed 03/18/22 Page 29 of 104
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`U.S. Patent
`
`Apr. 14, 2015
`
`Sheet 24 of 24
`
`US 9,006,417 B2
`
`aseud
`
`
`
`be‘Old
`
`(jo1alsajoyo
`
`
`
`SOPIMEdJEPIO|]OD
`
`
`
`s}l2}d1Iqoydo:pAH
`
`
`
`
`
`d1qnd40("H)jeuobexayasiaAu|
`‘sjuejepnspid)Aqpazillqeys)
`(pidijoydsoydJopidi-93adBe
`
`
`aquiuoVebalbbysnosuejuods
`~t
`
`
`(YN)PloeD19j9NNJOua}S9JOUD=pidiqs1uOHeD
`SIN‘spidi,pedeys-auosAqpaydope)
`
`
`
`
`uonpinsdeauasdnosSpeeyopydopAp
`
`puepidi|sJuoNedJopasodwios
`sueliquieweAqpayejnsdeoua
`
`a1eBaibbyajoolWpoweAul
`
`
`
`
`
`YN40)JBODjolalsajouD
`
`
`
`sapiydiyduiypidiq
`
`
`
`
`

`

`Case 1:22-cv-02229-MKV Document 1-4 Filed 03/18/22 Page 30 of 104
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`US 9,006,417 B2
`
`1
`NON-LIPOSOMAL SYSTEMS FOR NUCLEIC
`ACID DELIVERY
`
`2
`382. Cationic liposome complexesare also disclosed in U.S.
`Patent Publication No. 20030073640.
`
`CROSS-REFERENCE TO RELATED
`APPLICATION
`
`This application claims the benefit of U.S. Provisional
`Application No. 61/360,480,filed Jun. 30, 2010, which appli-
`cation is 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 (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 neutrallipid, and a detergent for
`transfecting insect cells are disclosed in U.S. Pat. No. 6,458,
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`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, particularly the lungs (Huanget al., Nature Biotech-
`nology, 15:620 (1997); Templetonet al., Nature Biotechnol-
`ogy, 15:647 (1997); Hofland et al., Pharmaceutical Research,
`14:742 (1997)).
`Other liposomaldelivery systems include, for example, the
`use ofreverse micelles, anionic liposomes, and polymerlipo-
`somes. Reverse micelles are disclosed in U.S. Pat. No. 6,429,
`200. Anionic liposomesare disclosed in U.S. Patent Publica-
`tion No. 20030026831. Polymer liposomes that incorporate
`dextrin or glycerol-phosphocholine polymersare disclosed in
`U.S.
`Patent
`Publication Nos.
`20020081736
`and
`20030082103, respectively.
`A genedelivery 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 thecir-
`culation for an extended period of time in order to achieve
`delivery to affected tissues. Th