`YaWorski et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 9.404,127 B2
`* Aug. 2, 2016
`
`USOO9404127B2
`
`(54) NON-LIPOSOMAL SYSTEMS FOR NUCLEIC
`ACD DELVERY
`
`(56)
`
`(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 of this
`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) Filed:
`
`Mar. 9, 2015
`
`(65)
`
`Prior Publication Data
`US 2016/003232O A1
`Feb. 4, 2016
`
`Related U.S. Application Data
`(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.
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2010.01)
`(2006.01)
`
`(51) Int. Cl.
`C7H 2L/04
`CI2N 5/88
`A 6LX 9/07
`A 6LX 9/5
`A6 IK3I/7088
`A 6LX3L/705
`A6 IK3I/72
`A6 IK3I/713
`A6 IK 47/4
`CI2N IS/II3
`A61 K9/127
`(52) U.S. Cl.
`CPC .............. CI2N 15/88 (2013.01); A61K 9/1075
`(2013.01); A61 K9/5 123 (2013.01); A61 K
`3 1/7088 (2013.01); A61 K3I/712 (2013.01):
`A61 K3I/713 (2013.01); A61 K3I/7105
`(2013.01); A61 K47/14 (2013.01): CI2N
`15/113 (2013.01); A61 K9/1272 (2013.01);
`A61 K9/1274 (2013.01); C12N 23 10/14
`(2013.01); C12N 23.10/321 (2013.01)
`(58) Field of Classification Search
`None
`See application file for complete search history.
`
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`
`OTHER PUBLICATIONS
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`Arpicco, S., et al., “Preparation and Characterization of Novel
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`(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 administer
`ing Such lipid particles. More particularly, the present inven
`tion provides stable nucleic acid-lipid particles (SNALP) that
`have a non-lamellar structure and that comprise 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
`
`PROTIVA - EXHIBIT 2029
`Moderna Therapeutics, Inc. v. Protiva Biotherapeautics, Inc. - IPR2018-00739
`
`
`
`(56)
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`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
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`Szoka, F., et al., “Comparative Properties and Methods of Preparation
`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
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`Tabatt, K. et al., “Effect of cationic lipid and matrix lipid composition
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`Teixeira, H. et al., "Characterization of oligonucleotide? lipid interac
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`92: 169-181, 2001.
`Tekmira Pharmaceuticals and Protiva Biotherapeutics Inc., Poster
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`Bioscience Reports, 2002, vol. 22, No. 2, pp. 283-295.
`VanDerWoude, I., et al., “Parameters influencing the introduction of
`plasmid DNA into 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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`Wilson, R., et al., "Counterion-Induced Condensation of
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`1979, vol. 18, No. 11, pp. 2192-2196.
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`prolonged circulation with sterically stabilized liposomes.”
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`Xu, Y. et al., “Physicochemical characterization and purification of
`cationic lipoplexes.” Biophysical Journal, 77:341-353, 1999.
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`Delivery into Adult Mice.” Science, 1993, vol. 261, pp. 209-211.
`
`
`
`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 1 of 24
`
`US 9.404,127 B2
`
`Step One: Blending
`Lipids Stock in 90% Ethanol
`
`Impingement
`
`siRNA stock in EDTA
`
`Stabilized
`Peristaltic
`Pump NALP in 45%
`Ethano
`
`Step Two: Diluting
`
`Stabilized NALP
`in 45%. Ethano
`
`
`
`Dilution Zone
`
`Peristatic
`Pump
`
`Warm
`citrate/NaC
`buffer
`
`Stabilized
`NAP in 22.5%
`Ethanol
`
`FIG. 1A
`
`
`
`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 2 of 24
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`US 9.404,127 B2
`
`
`
`
`
`Impingement
`Zone
`
`
`
`Stabilized NALP in 17%. Ethanol
`
`FIG. 1B
`
`
`
`U.S. Patent
`U.S. Patent
`
`Aug. 2, 2016
`Aug. 2, 2016
`
`Sheet 3 of 24
`Sheet 3 of 24
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`US 9.404,127 B2
`US 9,404,127 B2
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`
`
` Climate Chamber
`
`FIG. 2A
`FIG. 2A
`
`
`
`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 4 of 24
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`US 9.404,127 B2
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`
`
`The Cryo vitrification technique
`
`
`
`- CD
`
`Bare HPF Sample drop placed ... and thinned Sample spanning
`on the grid
`by blotting
`holes in film
`
`... and vitrified in
`liquid ethane
`
`m 5um
`
`
`
`Grid with holey polymer film
`(HPF)
`
`FIG. 2B
`
`
`
`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 5 of 24
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`US 9.404,127 B2
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`
`
`Hole size: 1-6 pum
`
`Thickness of sample film:
`10-500 am
`
`FIG. 2C
`
`
`
`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 6 of 24
`
`US 9.404,127 B2
`
`
`
`lipid mg/mL.
`
`Particle size (nm)
`
`Final encapsulation (%)
`
`15
`
`93 (0.12)
`
`
`
`Number-averaged
`Diameter size (nm)
`46*
`
`
`
`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 7 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)
`
`
`
`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 8 of 24
`
`US 9.404,127 B2
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`
`
`lipid) mg/mL.
`
`15
`
`Particle size
`(nm)
`116 (0.06)
`
`Final encapsulation (%)
`
`Number-averaged
`Diameter size (nm)
`64*
`
`
`
`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 9 of 24
`
`US 9.404,127 B2
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`
`
`lipid mg/mL
`
`15
`
`Particle Size
`(nm)
`108 (0.09)
`
`Final encapsulation (%)
`
`Number-averaged
`Diameter size (nm)
`59*
`
`
`
`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 10 of 24
`
`US 9.404,127 B2
`
`25
`
`2O
`
`3.
`.9
`E 15
`s
`C
`e
`
`10
`
`SS
`
`5 -
`
`O
`
`Presence of lamellar particles using SDM
`
`O:15
`
`2:30
`
`157
`
`13
`2
`82
`
`
`
`Number of non-
`lamellar particles
`Number Of
`lamellar particles
`
`10:15 SNAP
`
`2:30 SNAP
`
`1:57 SNALP
`
`162 SNALP
`
`594 (77%)
`
`665 (91%)
`
`325 (95%)
`
`313 (99%)
`
`173 (23%)
`
`67 (9%)
`
`16 (5%)
`
`4 (1%)
`
`FIG. 7
`
`
`
`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 11 of 24
`
`US 9.404,127 B2
`
`
`
`lipid mg/mL
`
`Particle size
`(nm)
`58 (0.07)
`
`Final encapsulation (%)
`
`Number-averaged
`Diameter size (nm)
`37
`
`
`
`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 12 of 24
`
`US 9.404,127 B2
`
`lipid mg/ml.
`
`
`
`
`
`Particle size
`(nm)
`65 (O. 11)
`
`
`
`Final encapsulation (%)
`
`Number-averaged
`Diameter size (nm)
`
`
`
`
`
`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 13 of 24
`
`US 9.404,127 B2
`
`
`
`lipid) mg/mL.
`
`15
`
`Particle size
`(nm)
`78 (0.03)
`
`Final encapsulation (%)
`
`96
`
`Number-averaged
`Diameter size (nm)
`55
`
`
`
`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 14 of 24
`
`US 9.404,127 B2
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`
`
`lipid mg/mL
`
`Particle size
`(nm)
`
`Final encapsulation (%)
`
`Number-averaged
`Diameter size (nm)
`
`
`
`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 15 of 24
`
`US 9.404,127 B2
`
`Presence of lamellar particles using DDM
`
`
`
`Number of non-
`Iamellar particles
`Number of
`lamellar particles
`
`2:3O SNAP
`
`24O SNAP
`
`1:57 SNAP
`
`162 SNAP
`
`1386 (99%)
`
`1191 (99%)
`
`694 (>99%)
`
`707 (>99%)
`
`14 (1%)
`
`1 O (1%)
`
`2 (<1%)
`
`2 (<1%)
`
`FIG, 12
`
`
`
`U.S. Patent
`U.S. Patent
`
`Aug. 2, 2016
`Aug.2, 2016
`
`Sheet 16 of 24
`Sheet 16 of 24
`
`US 9.404,127 B2
`US 9,404,127 B2
`
`
`
`
`
`i:3: SE
`
`FIG. 13
`
`
`
`U.S. Patent
`U.S. Patent
`
`Aug. 2, 2016
`Aug.2, 2016
`
`Sheet 17 of 24
`Sheet 17 of 24
`
`US 9.404,127 B2
`US 9,404,127 B2
`
`
`
`
`
`F.G. 14
`FIG. 14
`
`
`
`U.S. Patent
`U.S. Patent
`
`Aug. 2, 2016
`Aug.2, 2016
`
`Sheet 18 of 24
`Sheet 18 of 24
`
`US 9.404,127 B2
`US 9,404,127 B2
`
`
`
`
`
`FIG. 15
`FIG. 15
`
`
`
`U.S. Patent
`U.S. Patent
`
`US 9.404,127 B2
`
`
`
`2
`
`
`
`
`
`
`
`
`
`
`
`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 20 of 24
`
`US 9.404,127 B2
`
`3.0 --m-m-my------- -----------. v.--------8
`
`-----
`
`2.5
`
`
`
`47% vs. PBS Control
`
`-77% vs. PBS Contro
`
`
`
`PBS
`
`2:30 SNALP 5 x 1 mg/kg
`
`1:57 SNALP 5 x 0.1 mg/kg
`
`FIG. 17
`
`
`
`U.S. Patent
`U.S. Patent
`
`Aug.2, 2016
`
`Sheet 21 of 24
`Sheet 21 of 24
`
`US 9.404,127 B2
`US 9,404,127 B2
`
`
`
`
`
`
`
`2o
`
`A
`
`2
`
`oneyWNWdavo-:gody
`
`woQowoo=a
`
`
`
`
`
`
`
`
`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 22 of 24
`
`US 9.404,127 B2
`
`O)e. OdVSD Xc
`
`
`
`o
`N
`Ole J Old We : godw
`
`C
`
`wer
`
`
`
`U.S. Patent
`
`Aug. 2, 2016
`
`Sheet 23 of 24
`
`US 9.404,127 B2
`
`Anti-tumor efficacy of SNALP in subcutaneous Hep38 tumor-bearing
`mice after 6 x 3 mg/kg doses intravenously adminstered twice weekly
`for 3 Weeks
`
`18OO -
`
`
`
`16OO
`
`-- PBS
`
`-- 1:57 DLinDMA (PEG2000-C-DMA)
`
`-A-7:54 DLinDMA (PEG750-C-DMA)
`
`1400
`
`12OO
`
`1 OOO
`
`6OO -
`
`4OO -
`
`2OO
`
`O
`16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
`Study day
`
`FIG. 20
`
`
`
`U.S. Patent
`U.S. Patent
`
`Aug. 2, 2016
`Aug. 2, 2016
`
`Sheet 24 of 24
`Sheet 24 of 24
`
`US 9.404,127 B2
`US 9,404,127 B2
`
`
`
`
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`
`
`1.
`NON-LPOSOMAL SYSTEMIS FOR NUCLEC
`ACD DELIVERY
`
`US 9,404,127 B2
`
`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. S371 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.
`
`REFERENCE TO A “SEQUENCE LISTING. A
`TABLE, ORACOMPUTER PROGRAM LISTING
`APPENDIX SUBMITTED AS ANASCII TEXT
`FILE
`
`10
`
`15
`
`The Sequence Listing written in file -100-1.TXT, created
`on May 15, 2013, 4,096 bytes, machine format IBM-PC,
`MS-Windows operating system, is hereby incorporated by
`reference in its entirety for all purposes.
`
`BACKGROUND OF THE INVENTION
`
`25
`
`30
`
`35
`
`40
`
`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
`45
`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.
`Plasmid DNA-cationic liposome complexes are currently
`the most commonly employed nonviral gene delivery
`vehicles (Feigner, Scientific American, 276:102 (1997);
`
`50
`
`55
`
`60
`
`65
`
`2
`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