(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`12 January 2012 (12.01.2012) (10) International Publication Number
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`(43) International Publication Date
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`WO 2012/006369 A2
`
`
`G1)
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`Q1)
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`International Patent Classification:
`A61K 48/00 (2006.01)
`
`(81)
`
`International Application Number:
`PCT/US201 1/043096
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`(22)
`
`International Filing Date:
`
`Filing Language:
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`Publication Language:
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`6 July 2011 (06.07.2011)
`
`English
`
`English
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO,
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU,ID,IL,IN,IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NL
`NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD,
`SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR,
`TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`Priority Data:
`61/361,794
`
`6 July 2010 (06.07.2010)
`
`US
`
`(for all designated States except US): NO-
`Applicant
`VARTIS AG [CH/CH]; Lichtstrasse 35, CH-4056 Basel
`(CH),
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`Inventor; and
`(for US only): GEALL, Andrew
`Inventor/Applicant
`[US/US]; c/o Novartis Vaccines And Diagnostics, Inc.,
`P.O. Box 8097, Emeryville, CA 94662-8097 (US).
`
`Agents: LEE, Helen et al.; Novartis Vaccines And Diag-
`nostics, Inc., IP Services M/S X-100B, P.O. Box 8097,
`Emeryville, CA 94662-8097 (US).
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG,
`ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD,RU,TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FL FR, GB, GR, HR, HU, IE, IS, IT, LT, LU,
`LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SL SK,
`SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, ML, MR, NE,SN, TD, TG).
`Published:
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`without international search report and to be republished
`upon receipt ofthat report (Rule 48.2(g))
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`(25)
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`(26)
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`(30)
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`(7)
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`(72)
`(75)
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`(74)
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`(54) Title: IMMUNISATION OF LARGE MAMMALS WITH LOW DOSES OF RNA
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`(57) Abstract: RNA encoding an immunogenis delivered to a large mammal at a dose of between 2ug and 100g. Thus the in-
`vention provides a method ofraising an immuneresponse in a large mammal, comprising administering to the mammal a dose of
`between 2ug and 100ug of immunogen-encoding RNA.Similarly, RNA encoding an immunogen can be delivered to a large mam-
`mal at a dose of 3ng/kg to 150ng/kg. The delivered RNA canelicit an immuneresponsein the large mammal.
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`
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`WoO2012/006369A2[IIITIMATIONANITAIGLINAC
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`Page1 of 57
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`BioNTech-Pfizer Exhibit 1010
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`WO 2012/006369
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`PATENT APPT ICATION
`DOCKEPCT/US2011/043096 )-PCT
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`IMMUNISATION OF LARGE MAMMALS WITH LOW DOSES OF RNA
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`This application claims the benefit of US provisional application 61/361,794 (filed July 6, 2010), the
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`complete contents of which are hereby incorporated herein by reference for all purposes.
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`TECHNICAL FIELD
`
`This invention is in the ficld of non-viral delivery of RNA for immunisation.
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`BACKGROUND ART
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`The delivery of nucleic acids for immunising animals has been a goal for several years. Various
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`approaches have been tested, including the use of DNA or RNA,of viral or non-viral delivery
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`vehicles (or even no delivery vehicle, in a “naked” vaccine), of replicating or non-replicating vectors,
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`10
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`or of viral or non-viral vectors.
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`Various different doses of nucleic acids have been delivered in previous in vivo studies. Reference |
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`delivered 50ug of lipoplexed mRNA or DNA to mice,but also usedintraglossal lug and 10g doses
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`to analyse luciferase expression in tongue tissue. Reference 2 delivered 12ug of mRNA encoding
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`influenza virus nucleoprotein to mice. Reference 3 delivered 0.1ug, lug or 10ug of self-replicating
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`15
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`RNA encoding B-galactosidase to mice. Reference 4 delivered 10ug ofself-replicating RNA
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`encoding rabies virus glycoprotein to mice. Reference 5 delivered a total of 2ug or 4ug of DNA
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`encoding influenza haemagglutinin to humans, but did not deliver RNA.
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`Experience with DNA vaccines was encouraging in early work with small animals (e.g. mice) but as
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`the technology moved into large animals (e.g. humans) it became clear that potency decreased. Thus
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`very high doses would be required (e.g. milligrams rather than micrograms), but clinical-grade DNA
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`is expensive to manufacture.
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`There remains a need for further and improved nucleic acid vaccines.
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`DISCLOSURE OF THE INVENTION
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`According to a first aspect of the invention, RNA encoding an immunogen is delivered to a large
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`25
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`mammalat a dose of between 2g and 100ug. As shown below, a dose of 66g is immunogenic in
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`calves. An adult cow has a body weight ~10x that of an adult human and so the inventor has shown
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`that a humandoseof 5-10ug RNAis realistic.
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`According to a second aspect of the invention, RNA encoding an immunogenis delivered to a large
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`mammalat a dose of 0.1ug/kg to 1.5ug/kg. As shown below,a dose of ~0.94u¢/ke is immunogenic
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`30
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`in cattle. Prior art studies have used 100ng to 104g RNA in mice which, with a ~20g body weight,is
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`Sug/kg to 500ug/kg.
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`WO 2012/006369
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`PATENT APPT ICATION
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`Thus the invention provides a method of raising an immune response in a large mammal, comprising
`
`administering to the mammal a dose of between 2ug and 100ug of immunogen-encoding RNA.
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`The invention also provides an immunogen-encoding RNA for use in an in vive methodofraising an
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`immuneresponse in a large mammal, wherein the method comprises administering between 2ug and
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`100ug of the RNA to the mammal.
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`The invention also provides the use of an immunogen-encoding RNA in the manufacture
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`medicamentfor raising an in vivo immuneresponse in a large mammal, wherein the medicament has
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`between 2ug and 100ug of immunogen-encoding RNA perunit dose.
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`The invention also provides a pharmaceutical composition for a large mammal, comprising between
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`10
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`2ug and 100ug of immunogen-encoding RNAperunit dose. In a typical dosage volume of 0.5ml the
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`concentration of the immunogen-encoding RNA will thus be between 4ug/ml and 200ug/ml.
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`The invention also provides a unit dose of a pharmaceutical composition for administration to a large
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`mammal, wherein the unit dose comprises between 2ug and 100ug of immunogen-encoding RNA.
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`The invention also provides a delivery device (e.g. syringe, nebuliser, sprayer, inhaler, dermal patch,
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`15
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`etc.) containing a pharmaceutical composition for administration to a large mammal, wherein the
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`composition in the device contains between 2ug and 100ug of immunogen-encoding RNA.
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`The invention also provides a hermetically sealed container containing a pharmaceutical composition
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`for administration to a large mammal, wherein the composition in the container contains between
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`2ug and L00ug of inmunogen-encoding RNA.
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`20
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`The invention also provides a method of raising an immune response in a large mammal, comprising
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`administering to the mammal between 0.1ug and 1.5ug RNA per kg of the mammal’s body weight.
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`The invention also provides an immunogen-encoding RNA for use in an /n vive method ofraising an
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`immune response in a large mammal, wherein the method comprises administering between 0.1 ug
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`and 1.5ug RNAper kg of the mammal’s body weight.
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`25
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`The invention also provides the use of an immunogen-encoding RNA in the manufacture
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`medicamentfor raising an in vivo immuneresponse in a large mammal, wherein the medicament has
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`between 0.1 ug and 1.5ug of immunogen-encoding RNA per kg of the mammal’s body weight.
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`Administration
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`The invention involves administration of RNA to a large mammal. Thesite of administration will
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`30
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`usually be muscle tissue, such as skeletal muscle. Alternatives to intramuscular administration
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`include, but are not limited to:
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`intradermal, intranasal, intraocular, subcutaneous, intraperitoneal,
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`intravenous,
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`interstitial, buccal,
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`transdermal, or
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`sublingual administration.
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`Intradermal
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`and
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`intramuscular administration are two preferred routes.
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`Administration can be achieved in various ways. For instance,
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`injection via a needle (eg. a
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`hypodermic needle) can be used, particularly for
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`intramuscular,
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`subcutaneous,
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`intraocular,
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`intraperitoneal or intravenous administration. Needle-free injection can be used as an alternative.
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`Intramuscular injection is the preferred way of administering RNA according to the invention.
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`Injection into the upper arm, deltoid or thigh muscle (e.g. anterolateral thigh) is typical.
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`The administration site can include both immune cells (such as macrophages e.g. bone marrow
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`derived macrophages), dendritic cells (e.g. bone marrow derived plasmacytoid dendritic cells and/or
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`10
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`bone marrow derived myeloid dendritic cells), monocytes (e.g. human peripheral blood monocytes),
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`etc.) and non-immunecells (such as muscle cells, which may be multinucleated and may be arranged
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`into fascicles, and/or fibroblasts). The immunecells can be present at the time of administration, but
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`will usually infiltrate the site after administration. For example, the tissue damage caused by invasive
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`administration (e.g. caused by a needle at the administration site) can cause immunecells to infiltrate
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`15
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`the damagedarea.
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`RNA enters the cytoplasm of the immune cells and/or the non-immunecells. Entry can be via
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`endocytosis. Inside the endosomes of immune cells the RNA can bind to TLR7 (ssRNA), TLR8
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`(ssRNA) or TLR3 (dsRNA), thereby triggering innate immune pathways. When RNA escapes from
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`the endosomesinto the cytoplasm of immune and non-immune cells it can bind to RNA helicases
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`(e.g.
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`in the RIG-I-like receptor family i.e. RLRs) such as RIG-I (RLR-1), MDAS (RLR-2) and/or
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`LGP2 (RLR-3), also triggering innate immune pathways. The RNA can also be translated in the
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`immune and/or non-immune cells,
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`leading to expression of the immunogen, and ultimately to
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`presentation of the expressed immunogen via the MHC system. The cells can also secrete type I
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`interferons and/or pro-inflammatory cytokines to provide a local adjuvant effect.
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`The RNA can be delivered as naked RNA (e.g. merely as an aqueous solution of RNA) but, to
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`enhance both entry to immune and non-immunecells and also subsequent intercellular effects, and
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`also to reduce the amount of RNA required for a good immunogenic effect, the RNA is preferably
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`administered in combination with a delivery system, such as a particulate or emulsion delivery
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`system. Three useful delivery systems of interest are (i) liposomes(ii) non-toxic and biodegradable
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`30
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`polymer microparticles (ii) cationic submicron oil-in-water emulsions. Liposomes are a preferred
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`delivery system.
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`According to a first aspect of the invention, RNA encoding an immunogenis delivered to a large
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`mammalat a dose of between 2ug and 100g. For instance, the dose can be between 5yg and 75yg,
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`between 6ug and 50g, between 7ug and 25g, between 8ug and 20ug, or between 9ug and |5ug.
`-3-
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`PATENT APPT ICATION
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`Specific doses can be Sug, 6ug, 7ug, Sug, 9ug, 1Oug, llug, I2ug, I3ug, l4ug, l5yg, 20ug, 25ug,
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`30ug, 35ug, 40ug, 45ug, 50ug, 60ug, 70ug, 80ug, 90ug, or 100g. A human dose may be 5-10ug.
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`According to a second aspect of the invention, RNA encoding an immunogenis delivered to a large
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`mammal at a dose of between 0.lug RNA per kg of body weight to 1.5ug RNA per kg of body
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`weight. For instance, the dose can be between 0.2u9/kg to 1.2u¢/kg, between 0.3ug/kg to 1.1ug/kg,
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`between 0.4u¢/kg to 1.0ug/ke, between 0.5ug/kg to 1.0ug/kg, or between 0.5ug/ke to 1.5ug/kg.
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`Specific doses can be 0.lug/kg, 0.15ug/kg, 0.2ug/kg, 0.25ug/kg, 0.3pug9/kg, O.4ug/kg, 0.5ug/ke,
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`lug/kg, or 1.2ug/kg.
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`Liposomes
`Various amphiphilic lipids can form bilayers in an aqueous environment to encapsulate a RNA-
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`10
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`containing aqueous core as a liposome. These lipids can have an anionic, cationic or zwitterionic
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`hydrophilic head group. Formation of liposomes from anionic phospholipids dates back to the 1960s,
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`and cationic liposome-forming lipids have been studied since the 1990s. Some phospholipids are
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`anionic whereas other are zwitterionic and others are cationic. Suitable classes of phospholipid
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`15
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`include,
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`but
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`are
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`not
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`limited
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`to,
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`phosphatidylethanolamines,
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`phosphatidylcholines,
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`phosphatidylserines, and phosphatidyl-glycerols, and some useful phospholipids are listed in Table 1.
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`Useful cationic lipids include, but are not
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`limited to, dioleoyl
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`trimethylammonium propane
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`(DOTAP), 1,2-distearyloxy-N,N-dimethy]-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,Ndimethyl-
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`3-aminopropane (DODMA), §1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-
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`20
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`dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA). Zwitterionic lipids include, but are not
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`limited to, acyl zwitterionic lipids and ether zwitterionic lipids. Examples of useful zwitterionic
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`lipids are DPPC, DOPC and dodecylphosphocholine. The lipids can be saturated or unsaturated. The
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`use of at least one unsaturated lipid for preparing liposomes is preferred. If an unsaturated lipid has
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`twotails, both tails can be unsaturated, or it can have one saturated tail and one unsaturatedtail.
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`25
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`Liposomes can be formed from a single lipid or from a mixture oflipids. A mixture may comprise
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`(i) a mixture of anionic lipids (ii) a mixture of cationic lipids (iii) a mixture of zwitterionic lipids
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`(iv) a mixture of anionic lipids and cationic lipids (v) a mixture of anionic lipids and zwitterionic
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`lipids (vi) a mixture of zwitterionic lipids and cationic lipids or (vii) a mixture of anionic lipids,
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`cationic lipids and zwitterionic lipids. Similarly, a mixture may comprise both saturated and
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`30
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`unsaturated lipids. For example, a mixture may comprise DSPC (zwitterionic, saturated), DiinDMA
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`(cationic, unsaturated), and/or DMG(anionic, saturated). Where a mixture oflipids is uscd, notall of
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`the componentlipids in the mixture need to be amphiphilic e.g. one or more amphiphilic lipids can
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`be mixed with cholesterol.
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`The hydrophilic portion of a lipid can be PEGylated (i.e. modified by covalent attachment of a
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`35
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`polyethylene glycol). This modification can increase stability and prevent non-specific adsorption of
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`WO 2012/006369
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`PATENT APPT ICATION
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`the liposomes. For instance,
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`lipids can be conjugated to PEG using techniques such as those
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`disclosed in reference 6 and 7. Various lengths of PEG can be used e.g. between 0.5-8kDa.
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`A mixture of DSPC, DlinDMA, PEG-DMGandcholesterol is used in the examples.
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`Liposomes are usually divided into three groups: multilamellar vesicles (MLV); small unilamellar
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`vesicles (SUV); and large unilamellar vesicles (LUV). MLVs have multiple bilayers in each vesicle,
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`forming several
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`separate aqueous compartments. SUVs and LUVs have a single bilayer
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`encapsulating an aqueous core; SUVstypically have a diameter <S50nm, and LUVs have a diameter
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`>50nm. Liposomes useful with of the invention are ideally LUVs with a diameter in the range of
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`50-220nm. For a composition comprising a population of LUVs with different diameters: (i) at least
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`80% by number should have diameters in the range of 20-220nm,(ii) the average diameter (Zav, by
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`intensity) of the population is ideally in the range of 40-200nm,and/or(iii) the diameters should have
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`a polydispersity index <0.2. The liposome/RNA complexes of reference 1 are expected to have a
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`diameter in the range of 600-800nm andto have a high polydispersity.
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`Techniques for preparing suitable liposomes are well known in the art e.g. see references 8 to 10.
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`One useful method is described in reference |] and involves mixing (i) an ethanolic solution of the
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`lipids (ii) an aqueoussolution of the nucleic acid and(iii) buffer, followed by mixing, equilibration,
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`dilution and purification. Preferred liposomes ofthe invention are obtainable by this mixing process.
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`10
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`15
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`RNA is preferably encapsulated within the liposomes, and so the liposome forms a outer layer
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`around an aqueous RNA-containing core. This encapsulation has been found to protect RNA from
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`20
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`RNase digestion. The liposomes can include some external RNA (e.g. on the surface of the
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`liposomes), but at least half of the RNA (andideally all of it) is encapsulated.
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`Polymeric microparticles
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`Various polymers can form microparticles to encapsulate or adsorb RNA. The use of a substantially
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`non-toxic polymer means that a recipient can safely receive the particles, and the use of a
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`biodegradable polymer meansthat the particles can be metabolised after delivery to avoid long-term
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`persistence. Useful polymers are also sterilisable,
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`to assist
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`in preparing pharmaceutical grade
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`formulations.
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`Suitable non-toxic and biodegradable polymers include, but are not
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`limited to, poly(a-hydroxy
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`acids), polyhydroxy butyric acids, polylactones (including polycaprolactones), polydioxanones,
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`30
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`polyvalerolactone,
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`polyorthoesters,
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`polyanhydrides,
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`polycyanoacrylates, —tyrosine-derived
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`polycarbonates, polyvinyl-pyrrolidinones or polyester-amides, and combinationsthereof.
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`In some embodiments,
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`the microparticles are formed from poly(a-hydroxy acids), such as a
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`poly(lactides) (“PLA”), copolymers of lactide and glycolide such as a poly(D,L-lactide-co-glycolide)
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`5.
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`(“PLG”), and copolymers of D,L-lactide and caprolactone. Useful PLG polymers include those
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`having a lactide/glycolide molar ratio ranging, for example, from 20:80 to 80:20 e.g. 25:75, 40:60,
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`45:55, 50:50, 55:45, 60:40, 75:25. Useful PLG polymers include those having a molecular weight
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`between, for example, 5,000-200,000 Da e.g. between 10,000-100,000, 20,000-70,000, 30,000-
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`40,000, 40,000-50,000 Da.
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`The microparticles ideally have a diameter in the range of 0.02um to 8um. For a composition
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`comprising a population of microparticles with different diameters at least 80% by number should
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`have diameters in the range of 0.03-7um.
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`Techniques for preparing suitable microparticles are well known in the art e.g. see references 10, 12
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`10
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`(in particular chapter 7) and 13. To facilitate adsorption of RNA, a microparticle may include a
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`cationic surfactant and/orlipid e.g. as disclosed in references 14 & 15. An alternative way of making
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`polymeric microparticles is by molding and curing e.g. as disclosed in reference 16.
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`Microparticles of the invention can have a zeta potential of between 40-100 mV.
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`One advantage of microparticles over liposomesis that they are readily lyophilised for stable storage.
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`15
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`RNA can be adsorbed to the microparticles, and adsorption is facilitated by including cationic
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`materials (e.g. cationic lipids) in the microparticle.
`
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`Oil-in-water cationic emulsions
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`Oil-in-water emulsions are known for adjuvanting influenza vaccines e.g. the MF59™ adjuvant in
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`the FLUAD™ product, and the ASO3 adjuvant in the PREPANDRIX™ product. RNA delivery
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`20
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`according to the present invention can utilise an oil-in-water emulsion, provided that the emulsion
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`includes one or more cationic molecules. For instance, a cationic lipid can be included in the
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`emulsion to provide a positive droplet surface to which negatively-charged RNA canattach.
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`The emulsion comprises one or more oils. Suitable oil(s) include those from, for example, an animal
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`(such as
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`fish) or a vegetable source. The oil
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`is
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`ideally biodegradable (metabolisable) and
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`25
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`biocompatible. Sources for vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil,
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`coconut oil, and olive oil, the most commonly available, exemplify the nut oils. Jojoba oil can be
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`used e.g. obtained from the jojoba bean. Seed oils include safflower oil, cottonseed oil, sunflower
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`seed oil, sesame seed oil and the like. In the grain group, corn oil is the most readily available, but
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`the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale and the like mayalso be used.
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`30
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`6-10 carbon fatty acid esters of glycerol and 1,2-propanediol, while not occurring naturally in seed
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`oils, may be prepared by hydrolysis, separation and esterification of the appropriate materials starting
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`from the nut and seed oils. Fats and oils from mammalian milk are metabolisable and so may be
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`used. The procedures for separation, purification, saponification and other means necessary for
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`obtaining pure oils from animal sources are well knownin theart.
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`Most fish contain metabolisable oils which may be readily recovered. For example, cod liver oil,
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`shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils which may be
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`used herein. A number of branched chain oils are synthesized biochemically in 5-carbon isoprene
`
`units and are generally referred to as terpenoids. Preferred emulsions comprise squalene, a shark liver
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`oil which is a branched, unsaturated terpenoid (C39Hs9; [(CH3)2C[=CHCH2CH2C(CH3)]2=CHCHp-]2;
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`2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene; CAS RN 7683-64-9). Squalane, the
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`saturated analog to squalene, can also be used. Fish oils, including squalene and squalane, are readily
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`available from commercial sources or may be obtained by methods knownin theart.
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`Other useful oils are the tocopherols, particularly in combination with squalene. Where the oil phase
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`of an emulsion includes a tocopherol, any ofthe a, B, y, 5, © or & tocopherols can be used, but
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`a-tocopherols are preferred. D-o-tocopherol and DL-a-tocopherol can both be used. A preferred
`
`a-tocopherol is DL-a-tocopherol. An oil combination comprising squalene and a tocopherol (e.g.
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`15
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`DL-a-tocopherol) can be used.
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`The oil in the emulsion may comprise a combinationofoils e.g. squalene and at least one furtheroil.
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`The aqueous component of the emulsion can be plain water (e.g. w.fi.) or can include further
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`components e.g. solutes. For instance, it may include salts to form a buffer e.g. citrate or phosphate
`
`salts, such as sodiumsalts. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer;
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`20
`
`a succinate buffer; a histidine buffer; or a citrate buffer. A buffered aqueous phase is preferred, and
`
`buffers will typically be included in the 5-20mM range.
`
`The emulsion also includes a cationic lipid. Preferably this lipid is a surfactant so that it can facilitate
`
`formation andstabilisation of the emulsion. Uscful cationic lipids generally contains a nitrogen atom
`
`that is positively charged under physiological conditions e.g. as a tertiary or quaternary amine. This
`
`25
`
`nitrogen can be in the hydrophilic head group of an amphiphilic surfactant. Useful cationic lipids
`
`include, but are not
`
`limited to: 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 3'-[N-
`
`(N'.N'-Dimethylaminoethane)-carbamoyl]Cholesterol
`
`(DC
`
`Cholesterol),
`
`dimethyldioctadecyl-
`
`ammonium (DDA e.g.
`
`the bromide), 1,2-Dimyristoyl-3-Trimcthyl-AmmoniumPropane (DMTAP),
`
`dipalmitoyl(C16:0)trimethyl ammonium propane (DPTAP), distearoyltrimethylammonium propane
`
`30
`
`(DSTAP). Other useful cationic lipids are: benzalkonium chloride (BAK), benzethonium chloride,
`
`cetramide (which contains tetradecyltrimethylammonium bromide and possibly small amounts of
`
`dedecyltrimethylammonium bromide and hexadecyltrimethyl ammonium bromide), cetylpyridinium
`
`chloride (CPC), cetyl
`
`trimethylammonium chloride (CTAC), N,N',N'-polyoxyethylene (10)-N-
`
`tallow-1,3 -diaminopropane, dodecyltrimethylammonium bromide, hexadecyltrimethyl-ammonium
`
`-7-
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`WO 2012/006369
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`PATENT APPT ICATION
`DOCKEPCT/US2011/043096 )-PCT
`
`bromide, mixed alkyl-trimethyl-ammonium bromide, benzyldimethyldodecylammonium chloride,
`
`benzyldimethylhexadecyl-ammonium
`
`chloride,
`
`benzyltrimethylammonium
`
`methoxide,
`
`cetyldimethylethylammonium bromide,
`
`dimethyldioctadecyl
`
`ammonium bromide
`
`(DDAB),
`
`methylbenzethonium chloride, decamethonium chloride, methyl mixed trialkyl ammonium chloride,
`
`methyl
`
`trioctylammonium chloride), N,N-dimethyl-N-[2
`
`(2-methyl-4-(1,1,3,3tetramethylbutyl)-
`
`phenoxy]-ethoxy)ethyl]-benzenemetha-naminium chloride
`
`(DEBDA), dialkyldimetylammonium
`
`salts,
`
`[1-(2,3-dioleyloxy)-propyl]-N,N,N,trimethylammonium
`
`chloride,
`
`1,2-diacyl-3-
`
`(trimethylammonio) propane (acyl group=dimyristoyl, dipalmitoyl, distearoyl, dioleoyl), 1,2-diacyl-3
`
`(dimethylammonio)propane (acyl group=dimyristoyl, dipalmitoyl, distearoyl, dioleoyl), 1,2-dioleoyl-
`
`10
`
`3-(4'-trimethyl-ammonio)butanoyl-sn-glycerol, 1,2-dioleoyl 3-succinyl-sn-glycerol choline ester,
`
`cholesteryl (4'-trimethylammonio) butanoate, N-alkyl pyridiniumsalts (e.g. cetylpyridinium bromide
`
`and cetylpyridintum chloride), N-alkylpiperidinium salts, dicationic bolaform electrolytes (Cj2Meg;
`
`CyBugs), dialkylglycetylphosphorylcholine,
`
`lysolecithin, L-o, dioleoyl-phosphatidylethanolamine,
`
`cholesterol
`
`hemisuccinate
`
`choline
`
`ester,
`
`lipopolyamines,
`
`including
`
`but
`
`not
`
`limited
`
`to
`
`15
`
`dioctadecylamidoglycylspermine
`
`(DOGS),
`
`dipalmitoyl
`
`phosphatidylethanol-amidospermine
`
`(DPPES),
`
`lipopoly-L (or D)-
`
`lysine (LPLL, LPDL), poly (L (or D)-lysine conjugated to N-
`
`group
`amino
`pendant
`ester with
`glutamate
`didodecyl
`glutarylphosphatidylethanolamine,
`(CyGluPhC,N'), ditetradecyl glutamate ester with pendant amino group (C12GluPhC,N'), cationic
`
`derivatives
`
`of
`
`cholesterol,
`
`including
`
`but
`
`not
`
`limited
`
`to
`
`cholesteryl-3
`
`B-oxysuccinamidoethylenetrimethylammonium salt,
`
`cholesteryl-3
`
`B-oxysuccinamidoethylene-
`
`dimethylamine, cholesteryl-3 B-carboxyamidoethylenetrimethylammonium salt, and cholesteryl-3
`
`B-carboxyamidoethylenedimethylamine. Other useful cationic lipids are described in refs. 17 & 18.
`
`The cationic lipid is preferably biodegradable (metabolisable) and biocompatible.
`
`In addition to the oil and cationic lipid, an emulsion can include a non-ionic surfactant and/or a
`
`25
`
`zwitterionic surfactant. Such surfactants include, but are not limited to: the polyoxyethylene sorbitan
`
`esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 and polysorbate
`
`80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold
`
`under the DOWFAX™tradename, such as linear EO/PO block copolymers; octoxynols, which can
`
`vary in the numberof repeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100,
`
`or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol
`
`(IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin); polyoxyethylene
`
`fatty ethers derived fromlauryl, cetyl, stearyl and oleyl alcohols (knownas Brij surfactants), such as
`
`triethyleneglycol monolauryl ether (Brij 30); polyoxyethylene-9-lauryl ether; and sorbitan esters
`
`(commonly known as the Spans), such as sorbitan trioleate (Span 85) and sorbitan monolaurate.
`
`Preferred surfactants for including in the emulsion are polysorbate 80 (Tween 80; polyoxyethylene
`
`30
`
`35
`
`sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100.
`-8-
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`PATENT APPT ICATION
`DOCKEPCT/US2011/043096 )-PCT
`
`Mixtures of these surfactants can be included in the emulsion e.g. Tween 80/Span 85 mixtures, or
`
`Tween 80/Triton-X100 mixtures. A combination of a polyoxyethylene sorbitan ester such as
`
`polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxy-
`
`polyethoxyethanol (Triton X-100) is also suitable. Another useful combination comprises laureth 9
`
`plus a polyoxyethylene sorbitan ester and/or an octoxynol. Useful mixtures can comprise a surfactant
`
`with a HLB value in the range of 10-20 (e.g. polysorbate 80, with a HLB of 15.0) and a surfactant
`
`with a HLBvaluein the range of 1-10 (e.g. sorbitan trioleate, with a HLB of 1.8).
`
`Preferred amounts of oil (% by volume)in the final emulsion are between 2-20% e.g. 5-15%, 6-14%,
`
`7-13%, 8-12%. A squalene content of about 4-6% or about 9-11% is particularly useful.
`
`10
`
`15
`
`Preferred amounts of surfactants (% by weight) in the final emulsion are between 0.001% and 8%.
`
`For example: polyoxyethylene sorbitan esters (such as polysorbate 80) 0.2 to 4%,
`
`in particular
`
`between 0.4-0.6%, between 0.45-0.55%, about 0.5% or between 1.5-2%, between 1.8-2.2%, between
`
`1.9-2.1%, about 2%, or 0.85-0.95%, or about 1%; sorbitan esters (such as sorbitan trioleate) 0.02 to
`
`2%, in particular about 0.5% or about 1%; octyl- or nonylphenoxy polyoxyethanols (such as Triton
`
`X-100) 0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to
`
`8%, preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%.
`
`The absolute amounts of oil and surfactant, and their ratio, can be varied within wide limits while
`
`still forming an emulsion. A skilled person can easily vary the relative proportions of the components
`
`to obtain a desired emulsion, but a weight ratio of between 4:1 and 5:1 for oil and surfactant is
`
`20
`
`typical (excessoil).
`
`An important parameter for ensuring immunostimulatory activity of an emulsion, particularly in
`
`large animals, is the oil droplet size (diameter). The most effective emulsions have a droplet size in
`
`the submicron range. Suitably the droplet sizes will be in the range 50-750nm. Most usefully the
`
`average droplet size is less than 250nm e.g. less than 200nm, less than 150nm. The average droplet
`
`25
`
`size is usefully in the range of 80-180nm. Ideally, at least 830% (by number) of the emulsion’s oil
`
`droplets are less than 250 nm in diameter, and preferably at least 90%. Apparatuses for determining
`
`the average droplet size in an emulsion, and the size distribution, are commercially available. These
`
`these typically use the techniques of dynamic light scattering and/or single-particle optical sensing
`
`e.g. the Accusizer™ and Nicomp™series of instruments available from Particle Sizing Systems
`
`30
`
`(Santa Barbara, USA), or the Zetasizer™ instruments from Malvern Instruments (UK), or the
`
`Particle Size Distribution Analyzer instruments from Horiba (Kyoto, Japan).
`
`Ideally, the distribution of droplet sizes (by number) has only one maximum i.e. there is a single
`
`population of droplets distributed around an average (mode), rather than having two maxima.
`
`Preferred emulsions have a polydispersity of <0.4 e.g. 0.3, 0.2, orless.
`
`-9-
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`WO 2012/006369
`
`PATENT APPT ICATION
`DOCKEPCT/US2011/043096 )-PCT
`
`Suitable emulsions with submicron droplets and a narrow size distribution can be obtained by the use
`
`of microfluidisation. This technique reduces average oil droplet size by propelling streams of input
`
`components through geometrically fixed channels at high pressure and high velocity. These streams
`
`contact channel walls, chamber walls and each other. The results shear, impact and cavitation forces
`
`cause a reduction in droplet size. Repeated steps of microfluidisation can be performed until an
`
`emulsion with a desired droplet size average and distribution are achieved.
`
`As an alternative to microfluidisation, thermal methods can be used to cause phase inversion, as
`
`disclosed in reference 19. These methods can also provide a submicron emulsion with a tight particle
`
`size distribution.
`
`10
`
`Preferred emulsions can befiltersterilised 7.e. their droplets can pass through a 220nmfilter. As well
`
`as providingasterilisation, this procedure also removesany large droplets in the emulsion.
`
`In certain embodiments, the cationic lipid in the emulsion is DOTAP. The cationic oil-in-water
`
`emulsion may comprise from about 0.5 mg/ml to about 25 mg/ml DOTAP. For example, the cationic
`
`oil-in-water emulsion may comprise DOTAPat from about 0.5 mg/ml to about 25 mg/ml, from about
`
`15
`
`0.6 mg/ml to about 25 mg/ml, from about 0.7 mg/ml to about 25 mg/ml, from about 0.8 mg/ml to
`
`about 25 mg/ml, from about 0.9 mg/ml to about 25 mg/ml, from about 1.0 mg/ml to about 25 mg/ml,
`
`from about 1.1 mg/ml to about 25 mg/ml, from about 1.2 mg/ml to about 25 mg/ml, from about 1.3
`
`mg/ml to about 25 mg/ml, from about 1.4 mg/ml to about 25 mg/ml, from about 1.5 mg/ml to about
`
`25 mg/ml, from about 1.6 mg/ml to about 25 mg/ml, from about 1.7 mg/ml to about 25 mg/ml, from
`
`20
`
`about 0.5 mg/ml to about 24 mg/ml, from about 0.5 mg/ml to about 22 mg/ml, from about 0.5 mg/ml
`
`to about 20 mg/ml, from about 0.5 mg/ml to about 18 mg/ml, fr