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WO1998046208A1 - Systeme de delivrance d'adn de follicule pileux - Google Patents

Systeme de delivrance d'adn de follicule pileux Download PDF

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Publication number
WO1998046208A1
WO1998046208A1 PCT/US1998/007645 US9807645W WO9846208A1 WO 1998046208 A1 WO1998046208 A1 WO 1998046208A1 US 9807645 W US9807645 W US 9807645W WO 9846208 A1 WO9846208 A1 WO 9846208A1
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WIPO (PCT)
Prior art keywords
nucleic acid
delivery system
liposomal delivery
dna
wall material
Prior art date
Application number
PCT/US1998/007645
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English (en)
Inventor
Norman D. Weiner
Blake Roessler
Susan Niemiec
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The Regents Of The University Of Michigan
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Publication date
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Publication of WO1998046208A1 publication Critical patent/WO1998046208A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1796—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/14—Liposomes; Vesicles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/606—Nucleosides; Nucleotides; Nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Topical deliver- of gene vectors to cells within the skin is an attractive strategy for gene therapy of many human diseases, including a number of dermatological conditions thought to be mediated by abnormal regulation of soluble cytokines (Kondo et al. (1995) J Invest Dermatol 105:334-338; Hammerberg et al. (1992) J Clin Invest 90:571-583; Debets et al. (1995) J Invest Dermatol 105:480; Hoffmann et al. (1995) J Invest Dermatol 105:467; Wenzel et al. (1995) I Invest Dermatol 105:467).
  • an object of the invention is to provide a method for delivering a nucleic acid to a mammalian skin cell in vivo by topical admininstration of the nucleic acid and a Hposomal delivery system to a portion of skin having hair follicles.
  • a further object of the invention is to provide a Hposomal delivery system, containing a combination of non-ionic and cationic Iipids, for delivering a nucleic acid to a perifollicular cell in vivo.
  • Another object of the invention is to provide a method for treating a skin disorder by gene therapy using topical administration of a nucleic acid and a Hposomal delivery system to a skin having hair follicles.
  • the invention pertains to a method of delivering a nucleic acid to a mammalian cell in vivo.
  • the method comprises providing a Hposomal delivery system for the nucleic acid, wherein the Hposomal delivery sytem contains a lipid vesicle and a cationic lipid.
  • the preferred lipid vesicle has a primary wall material and a secondary wall material, wherein the primary wall material is preferably selected from the group consisting of C12- 18 glyceryl mono- and diesters and the secondary wall material is preferably selected from the group consisting of quaternary dimethyldiacyl amines, polyoxyethylene acyl alcohols, polygycerol fatty acids, and sorbitan fatty acid esters.
  • the method further includes incorporating the nucleic acid into the Hposomal delivery system to produce a nucleic acid/liposomal delivery system and topically administering the nucleic acid/liposomal delivery sytem to the mammalian cell at a location of the skin having hair follicles.
  • the invention is based at least in part on the discovery that a non-ionic Hposomal formulation containing a non-ionic Hposome such as one which form glyceryl dilaurate, cholesterol, and polyoxyethylene- 10-stearyl ether, and a cationic lipid such as 1,2- dioleoyloxy-3(trimethylammonio)propane could efficiently mediate transfection of a nucleic acid into perifollicular cells in vivo following topical administration.
  • a non-ionic Hposome such as one which form glyceryl dilaurate, cholesterol, and polyoxyethylene- 10-stearyl ether
  • a cationic lipid such as 1,2- dioleoyloxy-3(trimethylammonio)propane
  • the invention provides a method wherein a nucleic acid is delivered to cells which are in proximity of a hair follicle and the nucleic acid is introduced in these cells via topical administration of a non-ionic/cationic Hposomal delivery system and the nucleic acid.
  • the mammalian cell is associated with follicular units.
  • the mammalian cell can be associated with a pilosebaceous unit.
  • the mammalian cell can be a follicular cell, e.g., a perifollicular cell.
  • the invention further provides a nucleic acid/liposomal delivery system for delivering a nucleic acid topically to a mammal in vivo.
  • the nucleic acid/liposomal delivery system contains a nucleic acid and a Hposomal delivery system for the nucleic acid.
  • the Hposomal delivery sytem includes a lipid vesicle with a primary wall material, and a secondary wall material, and a cationic lipid.
  • the primary wall material is preferably selected from the group consisting of C12-C18 glyceryl mono-and diesters and the secondary wall material is preferably selected from the group consisting of quaternary dimethyldiacyl amines, polyoxyethylene acyl alcohols, polyglycerol fatty acids, and sorbitan fatty acid esters.
  • the preferred cationic lipid is selected from the group consisting of N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium bromide (DOTMA), l,2-dimyristyloxypropyl-N,N-dimethyl-hydroxyethyl ammonium bromide (DMRIE), [N-(N ⁇ N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Choi), dioctadecylamidoglycyl spermidine (DOGS), dimethyl dioctadecylammonium bromide (DDAB), dioleoyl phosphatidylethanolamine (DOPE), 2,3-dioleoyloxyl-N- [2(sperminecarbozamide-0-ethyl]-N,N-dimethyl- 1 -propanaminium trifluoroacetate
  • DOTMA N-[l-(
  • TAP l-[2-(oleoyloxy)-ethyl]-2-oleyl-3-(2hydroxyethyl) imidazolinium chloride
  • DOTIM l,2-dioleoyloxy-3-(trimethylammonio) propane
  • TAP 1,2-diacyl-3- trimethylammonium propane
  • DAP l,2-diacyl-3-dimethylammonium propane
  • TAP and DAP can have various chains (14:0, 16:0, 18:0, and 18:1) and various acyl groups (dimyristoyl, dipalmitoyl, distearoyl, and dioleoyl).
  • the cationic lipid is DOTAP.
  • the primary wall material of the lipid vesicle is selected from the group consisting of glyceryl dilaurate, glyceryl distearate, and their mixtures.
  • a more preferred secondary wall material of the lipid vesicle is polyoxyethylene- 10-stearyl ether.
  • the nucleic acid to be delivered to a mammalian cell can contain a transgene.
  • the transgene encodes a protein.
  • the protein can be a ligand, a receptor, an agonist of a ligand, an agonist of a receptor, an antagonist of a ligand, or an antagonist of a receptor.
  • the protein encoded by the transgene can be an IL-1 receptor antagonist.
  • the protein can be a transdominant negative hormone receptor, such as an androgen receptor.
  • the nucleic acid expresses an antisense RNA or a ribozyme.
  • the method of the invention and the nucleic acid/liposomal delivery system of the invention can be used to treat various conditions, such as dermatological disorders. Accordingly, the invention pertains to methods of treatment of disorders selected from the group consisting of infectious diseases, neoplastic diseases, autoimmune disorders, alopecia, aquired conditions, acne, baldness, atopic dermatitis, and eczema.
  • the autoimmune disorder can be psoriasis or alopecia areata.
  • kits comprising the Hposomal delivery system and instructions for use.
  • the invention provides a kit containing some or all of the materials for forming the Hposomal delivery system packaged separately and instructions explaining the preparation of the delivery system.
  • the kits can further comprise a nucleic acid, either together with the Hposomal delivery system, or in a separate container.
  • Figure I shows a Southern blot containing DNA from hamster ear treated with a single dose of pSG5IL-lra and sacrified 4 hours (4), 6 hours (6), 8 hours (8), 12 hours (12) and 24 hours (24) later, or treated with buffer alone (Cl) or with aqueuos DNA (C2), hybridized with a probe specific for human IL-lra.
  • Figure 2 are graphs reprenting the amount of IL-lra present in ventral skin (Panel A), auricular carticlage (Panel B), and dorsal skin (Panel C) of hamster ear from 1 to 8 days following twice daily topical application of NC liposomes with or without pSG5IL-lra DNA for three days to the ventral surface of the hamster ear.
  • Figure 3 is a graph showing the amount of IL-lra present in the ventral surface of hamster ear 24 hours following twice daily topical application of NC liposomes (NC),
  • NC + DNA NC liposomes with pSG5IL-lra DNA
  • PC phospholipidic/cationic liposomes
  • PC + DNA phospholipidic/cationic liposomes with pSG5IL-lra DNA
  • PC + DNA phospholipidic/cationic liposomes with pSG5IL-lra DNA
  • the invention pertains to a method for delivering a nucleic acid to a mammalian cell.
  • the cell is in vivo.
  • the method comprises providing a Hposomal delivery system for the nucleic acid, the Hposomal delivery system comprising a lipid vesicle having a primary wall material and a secondary wall material, and a cationic lipid.
  • the method further comprises incorporating the nucleic acid into the Hposomal delivery system to produce a nucleic acid/liposomal delivery system and topically administering the nucleic acid/liposomal delivery system to the mallian cell.
  • the mallian cell is at a location of the skin having hair follicles.
  • the primary wall material is preferably selected from the group consisting of C12-C18 glyceryl mono- and diesters.
  • the secondary cell wall material is preferably selected from the group consisting of quaternary dimethyldiacyl amines, polyoxyethylene acyl alcohols, polyglycerol fattly acids, and sorbitan fatty acid esters.
  • the invention further pertains to a nucleic acid/liposomal delivery system for delivering a nucleic acid topically to a mammal in vivo.
  • the nucleic acid/liposomal delivery system comprises a nucleic acid and a Hposomal delivery system for said nucleic acid.
  • the Hposomal delivery sytem comprises a lipid vesicle having a primary wall material, a secondary wall material, and a cationic lipid.
  • the primary wall material is preferably selected from the group consisting of C12-C18 glyceryl mono- and diesters.
  • the secondary wall material is preferably selected from the group consisting of quaternary dimethyldiacyl amines, polyoxyethylene acyl alcohols, polygycerol fatty acids, and sorbitan fatty acid esters.
  • Hposomal delivery system which is used interchangeably herein with the term “topical gene delivery system” is intended to include a system, which mediates or facilitates introduction of a macromolecule, e.g., a nucleic acid, into a cell using liposomes, lipid vesicles, or other lipid compositions.
  • nucleic acid/liposomal delivery system is intended to include a complex comprising the Hposomal Hposomal delivery system and a nucleic acid.
  • non-ionic/cationic Hposomal delivery system is used interchangeably herein with the terms “NC Hposomal delivery system”, “NC Hposomal suspension”, and “NC Hposomal formulation”, and is intended to include a Hposomal delivery system having non-ionic lipid vesicles and cationic Iipids.
  • the term "delivering a nucleic acid to a cell” is used interchangeably herein with the term “transfecting a cell with a nucleic acid” and "introducing a nucleic acid into a cell”. Accordingly, the invention pertains to a method for transfecting a mammalian cell with a nucleic acid.
  • the term "incorporating a nucleic acid into a Hposomal delivery system” is intended to include adding a nucleic acid to a Hposomal delivery system, such that the nucleic acid/liposomal delivery system formed is capable of delivering the nucleic acid to a cell in vivo.
  • the mammalian cell is a cell from a skin having hair follicles.
  • the skin containes several layers, including the epidermis, which is the outside layer of the skin, the dermis, and the subcutaneous layer.
  • the epidermis includes at least the following cell layers: the stratum corneum, which is the outside layer of the skin, the stratum granulosum, the stratum spinosum, and the stratum basale (germinativum).
  • the dermis is defined as the region between the epidermis and the subcutaneous layer and includes, but is not limited to, the papillary region or layer, the reticular region or layer, hair follicle roots, sensory nerves, sudoriferous glands and ducts, and sebaceous glands.
  • the subcutaneous layer includes, but is not limited to, arteries, autonomic motor nerves, veins, sudoriferous glands, and lamellated (Pacinian) corpuscules.
  • Hair follicles can be thought of as a self contained organ system that bridges dermis and epidermis. At the epidermal surface the follicular opening ranges from 15- 100 microns, while along the depth of the hair shaft the physical distance between hair shaft and perifollicular cells ranges from 3-10 microns.
  • the term "perifollicular cell" is intended to include a cell, e.g., follicular cell, located in close proximity to the hair shaft. Close proximity is intended to include within several cell layers.
  • a perifollicular cell can be a cell lining the perifollicular space, or a cell further away from the perifollicular space.
  • perifollicular space is intended to include the space between the hair shaft and the cells, e.g., perifollicular cells, lining the hair shaft.
  • a hair follicle is connected to pilosebaceous (oil) glands through pilosebaceous ducts.
  • pilosebaceous unit is intended to include pilosebaceous glands and ducts. The ducts of the pilosebaceous glands open directly into this perifollicular space.
  • the anatomical structure of the follicle is well suited as a receptacle for introduction into skin of exogenous liquids and microscopic particulates, such as non-ionic/cationic Hposomal formulations of the invention and their contents.
  • the invention provides methods for introducing a nucleic acid into cells proximate to the perifollicular space or ducts connected to the perifollicular cell, such as pilosebaceous ducts.
  • the invention provides methods for transfecting perifollicular cells, e.g., perifollicular fibroblasts along the interior length and base of the hair shaft.
  • the invention further provides methods for transfecting sebocytes (gland lining cells), such as sebocytes present within pilosebaceous glands, and keratinocytes, such as keratinocytes located near the opening of the follicle.
  • Other target cells include cells from any of the layers of the epidermis and the dermis which are close to a hair shaft.
  • the root of a hair follicle also termed hair root, includes the medulla, the cortex, and the cuticle of the hair. On the outside of the cuticle is the internal root sheath, followed by the external root sheath and the connective tissue sheath, containing blood vessels. Accordingly, the invention also provides methods for delivering nucleic acids to cells from these layers or tissues.
  • Non-ionic/cationic lipid formulations are described below.
  • Preferred non-ionic components include a lipid vesicle having a primary wall material and a secondary wall material.
  • the term "primary wall material” is used interchangeably herein with the term “primary lipid” and as used herein means that the lipid constitutes the greatest proportion (by weight) of any single lipid material forming the lipid bilayers or lipid vesicle, preferably 60% or greater.
  • the primary wall material is selected from the group consisting of C ⁇ 2-C ⁇ g glyceryl mono- and diesters and Ci 2-C ⁇ g fatty alcohols.
  • Preferred primary Iipids are C1 and C18 fatty alcohols, glycol stearate, glyceryl monostearate, glyceryl distearate, and glyceryl dilaurate.
  • Other primary Iipids are also within the scope of the invention, so long as they form a non-ionic lipid vesicle with a secondary lipid together or not with other reagents.
  • the primary lipid is glyceryl dilaurate.
  • secondary wall material and “secondary lipid” are used intechangeably herein and is preferably selected from the group consisting of quaternary dimethyldiacyl amines (including ammonium derivatives such as chlorides), polyoxyethylene acyl alcohols (tradename BRIJ), polygycerols, fatty acids, and sorbitan fatty acid esters (tradename SPAN).
  • Other secondary Iipids are also within the scope of the invention, so long as they form a non-ionic lipid vesicle with a primary lipid together or not with other reagents.
  • the secondary lipid is polyoxyethylene- 10-stearyl ether.
  • the lipid vesicle further contains additional components or agents, such as charge producing agents.
  • the lipid vesicle contains a sterol, such as cholesterol, a cholesterol salt or ester, phytocholesterol, or hyrdocortisone.
  • a sterol such as cholesterol, a cholesterol salt or ester, phytocholesterol, or hyrdocortisone.
  • Another preferred additive is ⁇ -tocopherol, preferably added at about 1% by weight of total Iipids.
  • a particularly preferred non-ionic vesicle of the invention contains glyceryl dilaurate as the primary lipid, polyoxyethylene- 10-stearyl ether as the secondary lipid, and cholesterol, preferably in a weight ratio of about 50:23:15, respectively.
  • a cationic lipid for the preparation of a Hposomal delivery system is dependent on its ability to transfect a wide variety of cells, the inherent toxicity of the lipid to the cells, and its compatibility with the non-ionic lipid vesicles. Accordingly, preferred cationic Iipids include bilayer-forming cationic Iipids containing unsaturated fatty acid chains. It has been shown previously that DOTMA, used alone, is capable of mediating transfection of specific cell types (Feigner et al. (1987) Proc Natl Acad Sci USA 84:7413-7417).
  • preferred cationic Iipids are selected from the NC Hposomal delivery system of the invention comprises a cationic lipid, which is preferentially selected from the group consisting of N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium bromide (DOTMA), l,2-dimyristyloxypropyl-N,N-dimethyl- hydroxy ethyl ammonium bromide (DMRIE), [N-(N, N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Choi), dioctadecylamidoglycyl spermidine (DOGS), dimethyl dioctadecylammonium bromide (DDAB), dioleoyl phosphatidylethanolamine (DOPE), 2,3-dioleoyloxyl-N-[2(sperminecarbozamide-0-ethyl]-N
  • TAP and DAP can have various chains (14:0, 16:0, 18:0, and 18:1) and various acyl groups (dimyristoyl, dipalmitoyl, distearoyl, and dioleoyl).
  • Any cationic lipid that does not degrade the NC Hposomal delivery system and is capable of delivering a nucleic acid into a cell, when used together with a non-ionic lipid vesicle can be used.
  • the preferred cationic lipid for use in the invention is l,2-dioleoyloxy-3-(trimethylammonio) propane (DOTAP).
  • DOTAP l,2-dioleoyloxy-3-(trimethylammonio) propane
  • a preferred NC Hposomal delivery system of the invention contains glyceryl dilaurate as the primary lipid, polyoxyethylene- 10-stearyl ether as the secondary lipid, cholesterol, and DOTAP as the cationic lipid, preferably in a weight ratio of about 50:23 : 15 : 12, respectively.
  • the non-ionic lipid vesicle is prepared and subsequently mixed with the cationic lipid.
  • the primary lipid and the secondary lipid of the non-ionic lipid vesicle are mixed together with the cationic lipid. Additional agents, e.g., Iipids or chemical reagents, can be added at an appropriate time during the preparation of the NC Hposomal delivery system.
  • a preferred method for preparing the Hposomal formulations of the invention consist of mixing appropriate amounts of the Iipids and melting them together. Such a method is further described herein in the Examples.
  • Hposome delivery system Various combinations and ratios of primary wall material, secondary wall material, cationic lipid and additional agents (if any) can be used for preparing a Hposome delivery system of the invention.
  • the composition and ratio of the different components of the Hposomal delivery system depends on the characteristics of each component. The ratio may also vary according to the particular use of the Hposomal delivery system and/or the contents of the Hposomal delivery system.
  • a preferred ratio between glyceryl dilaurate (GDL) as the primary lipid, cholesterol (CH), polyoxyethylene- 10-stearyl ether (POE) as the secondary lipid, and l,2-dioleoyloxy-3-(trimethylammonio) propane (DOTAP) as the cationic lipid is about 50:15:23: 12 by weight, respectively.
  • GDL glyceryl distearate
  • DOTAP l,2-dioleoyloxy-3-(trimethylammonio) propane
  • Yet other preferred formulations include formulations in which the ratio of GDS: CH: POE: DOTAP are X: 15: Y: 12, wherein X is the weight percent of GDS and Y is the weight percent of POE and X ranges from about 45%-55% and Y ranges from about 18% to 28%.
  • Another preferred formulation contains GDL:GDS:CH: POE: DOTAP in a ratio of 25:25:15:23:12 by weight.
  • Also within the scope of the invention are such formulations in which the ratio of GDL:GDS is varied, such that the penetration enhancer effects of the GDL-based formulation are combined with the reservoir effects of the GDS-based formulation.
  • Liposomal suspensions are preferably sonicated, e.g., for about 20 minutes at room temperature, to reduce their size.
  • Formulations having liposomes of uniform size can be obtained, as compared to formulations having liposomes of 500 ⁇ 100 nm for the unsonicated NC liposomes.
  • the DNA/NC Hposomal suspension is then preferably incubated at room temperature, e.g., for about 45 minutes, prior to topical administration.
  • Formulations having a mean particle size greater than 2 ⁇ m can be obtained using such methods.
  • Preferred ratios of nucleic acid to lipid weight ratio are 1 : 1 and 1 :2. However, ratios of about 1 :3, 1 :4, 1 :5, 1 :10, 1 :25, 1 :50, and intermediate ratios thereof, are also within the scope of the invention. Such ratios can vary according to various criteria, e.g., the type of constituents of the liposomal delivery system, the cell type to be transfected, and the type of nucleic acid. Preferred sizes of the liposomes (not including the nucleic acid) in the NC liposomal formulations include liposomes of about 25 to about 500 nm, with 100 to about 250 nm preferred.
  • the size of liposomes can be changed by various processes known to those skilled in the art. For example, the size of liposomes can be reduced by vortexing. Alternatively, the size of liposomes can be changed and/or rendered more uniform by extruding the liposomes through a series of straight-bore polycarbonate membranes of varying pore diameters at high pressure (250 psi) (Fleisher et al. supra).
  • a preferred ratio of nucleic acid to cationic lipid in the liposomal formulations of the invention is based on two factors: a) the maximum cationic lipid concentration that could be used without inducing toxic effects, and b) the amount of cationic lipid that would be required for near optimum complexation of the nucleic acid.
  • a preferred ratio of nucleic acid to cationic lipid is about 0.4 ⁇ g of DNA per nmol of cationic lipid.
  • a lipid formulation can contain 12% weight DOTAP and a DNA concentration of about 7 mg/ml or about 0.4 ⁇ g/nmol cationic lipid.
  • ratios of about 0.25 to about 1.5 ⁇ g/nmol cationic lipid are also within the scope of the invention, where the DNA component is expressed by weight and the lipid component is expressed in molar quantity.
  • the size distribution of the liposomes and the liposome-DNA complexes, as well as their integrity and quality can be determined by various techniques.
  • a Nikon Diaphot inverted light microscope can be used to visualize particles with diameters greater than 0.5 ⁇ m.
  • a NiComp 370 laser light scattering particle sizer can be used to visualize particles of smaller size.
  • highly pure lipid components for the preparation of the Hposome delivery systems of the invention.
  • the use of highly purified components also reduces the possibility of in vivo toxicity associated with contaminants present in the Iipids.
  • Such highly purified Iipids can be bought from manufacturers, such as ICI Americas or Sigma Chemicals.
  • the nucleic acid introduced into cells can be any nucleic acid, including DNA, single stranded DNA, e.g., oligonucleotides, and RNA, e.g., mRNA, tRNA, and ribozymes.
  • the nucleic acid can be small, containing a few nucleotides, or large, containing at least several kilobases.
  • the nucleic acid can be a yeast artificial chromosome (YAC).
  • YAC yeast artificial chromosome
  • the nucleic acid can be a natural nucleic acid or prepared chemically.
  • the term "nucleic acid", as used herein, is intended to include at least one nucleic acid molecule.
  • a nucleic acid can be one, two, or more nucleic acid molecules.
  • a nucleic acid corresponding to two expression vectors can be mixed with the liposomal delivery system and introduced into a target cell, e.g., a follicular cell.
  • the nucleic acid molecules can be linear or circular, depending on the type of nucleic acid.
  • a preferred nucleic acid is a DNA molecule with at least one expressible form of a transgene.
  • the term "expressible form of a transgene" is intended to include a transgene and all regulatory elements necessary for expression of the appropriate expression of the transgene.
  • regulatory elements include basic promoters (e.g., TATA box sequence), enhancers, silencers, polyadenylation signals, and the like. These are further defined below.
  • the terms "regulatory element”, “regulatory sequence”, and “regulatory nucleic acid element” are used interchangeably herein and are intended to include any nucleic acid fragment having a specific nucleic acid sequence which, alone or together with one or more other nucleic acid fragments is capable of modifying the expression of a gene to which the regulatory sequence is operably linked. Modifying expression of a gene can be upregulating the expression of a gene or downregulating the expression of a gene. Regulatory elements are known to those skilled in the art and are described, e.g., in Goeddel (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA.
  • promoter is intended to include a nucleic acid which is capable of controlling the expression of a gene to which it is operably linked.
  • a promoter typically contains at least one regulatory nucleic acid element.
  • the term “5' flanking sequence” is intended to include a nucleic acid sequence located 5', i.e., upstream, of the transcription initiation site of a gene.
  • the term 5" flanking sequence is used interchangeably herein with the term “promoter”.
  • 3' flanking sequence is intended to include a nucleic acid sequence located 3', i.e., downstream of the polyadenylation signal of a gene.
  • operably linked as applied to a transgene and regulatory element or promoter is intended to mean that the nucleotide sequence of a transgene, is linked to a regulatory sequence in a manner which allows expression of the transgene in a host cell.
  • the term "operably linked” is intended to include any linkage that allows the regulatory sequence to control expression of the transgene of interest.
  • a regulatory sequence has a chemical linkage to the gene of interest, such as natural linkage between two nucleic acid bases.
  • the regulatory sequence or promoter can be linked directly to the transgene.
  • the regulatory sequence can be linked indirectly to the transgene, such as by a linker. Such a linker can vary in length and may contain sequences that are recognized by restriction enzymes.
  • a regulatory element can be a portion of a promoter to which the RNA Polymerase binds and which contains a TATA box. Such a regulatory element is responsible for the basal or constitutive transcription of a gene and is classically referred to as a promoter. As used herein, however, the term “promoter” also refers to a larger fragment of DNA controlling gene expression.
  • a regulatory sequence can be an enhancer. Generally, an enhancer is an element which modifies the basal transcription of a gene which it controls. An enhancer element can be located several kilobases away from the gene whose expression it affects and it can be located in any part of a gene, such as in 3' or 5' non coding sequences and in introns. Regulatory elements also include silencers, i.e., DNA elements which will reduce the level of transcription of a gene.
  • Preferred promoters include strong promoters, such as viral promoters, e.g., a cytomegalovirus (CMV) promoter such as the CMV IE promoters.
  • CMV cytomegalovirus
  • a preferred CMV promoter is the CMV early enhancer promoter, which is a powerful constitutive promoter.
  • An even more preferred promoter is a truncated version of the CMV early enhancer promoter, such as the promoter included in the expression plasmid pCVC103.
  • Other preferred viral promoters include SV40 promoters, e.g., the SV40 LTR promoter, the SV40 early promoter, and the SV40 later promoter.
  • Other promoters include the AD2 MLP, the HSV TK, and the MMTV LTR.
  • tissue specific regulatory element is intended to include a regulatory element that stimulates expression of a gene to which it is operably linked predominantly in a specific tissue or cell type.
  • a "tissue” as defined herein can be a -an organ, e.g., kidney, liver, heart or a population of cells making up a structure, such as an epithelium.
  • Tissue specific expression of a gene is also intended to include expression of the gene in a cell at a specific stage of differentiation and not in another stage of differentiation.
  • Preferred tissue specific regulatory elements include regulatory elements that predominantly stimulate expression of a gene to which it is operably linked in a specific type of perifollicular cell.
  • a preferred regulatory element stimulates gene expression predominantly in perifollicular fibroblasts.
  • Other preferred regulatory elements stimulate gene expression predominantly in sebocytes.
  • the invention provides methods for selectively expressing a gene in a particular type of perifollicular cell, even though the nucleic acid comprising the gene is delivered to all types of perifollicular cells.
  • Other preferred regulatory elements that can be used to control expression of the gene introduced into cells by the method of the invention include inducible regulatory elements. Such regulatory elements are responsive to specific agents, e.g., inducers. Accordingly, expression of an inducible element can be stimulated by an agent or repressed by an agent.
  • Inducers within the scope of the invention include agents which interact with a receptor on the surface of a cell or within a cell, such as hormones, cytokines, and chemicals.
  • Inducible regulatory systems for use in mammalian cells are known in the art, for example systems in which gene expression is regulated by heavy metal ions (Mayo et al. (1982) Cell 29:99-108; Brinster et al. (1982) Nature 296:39-42; Searle et al. (1985) Mol Cell Biol 5:1480-1489), heat shock (Nouer et al. (1991) In: Heat Shock Response Nouer L. Ed., CRC, Boca Raton , FL, ppl67-220), hormones (Lee et al.
  • An inducer can also be light of a certain wavelength, such as ultraviolet light. Accordingly, the invention provides methods for delivering a nucleic acid to skin cells in vivo and methods for controlling expression of the genes delivered.
  • regulatory _ elements which have been modified at least in part.
  • a regulatory element that is tissue specific sebocytes can be engineered to become inducible by a specific compound by, e.g., operably linking the tissue specific regulatory element to a synthetic element responsive to the said compound.
  • the efficiency of a promoter or regulatory element to control expression of a gene tissue specifically can be determined by transfection of tissue culture cells or skin cells as described herein.
  • methods of transfection other than by the liposomal delivery system of the invention can be used to determine efficiency of a promoter in tissue culture cells.
  • a promoter can be tested in tissue culture cells by transfection using calcium chloride methods, electroporation, or other methods known in the art.
  • the nucleic acid introduced into cells according to the method of the invention comprises at least one transgene.
  • transgene is intended to include a gene which is introduced into a cell.
  • the transgene can encode a protein or a peptide.
  • the transgene can be a nucleic acid that is transcribed into RNA, but does not encode a peptide.
  • a transgene can be a nucleic acid which upon transcription into an RNA molecule is an "antisense" strand of another nucleic acid in the cell or outside the cell, such that upon expression of the transgene and synthesis of antisense molecules, a function in the cell is affected.
  • the antisense nucleic acid inhibits or reduces expression of another nucleic acid, such as an endogenous nucleic acid.
  • the transgene can be any gene whose expression in perifollicular cells is beneficial to a specific condition.
  • the transgene can also be a gene encoding a protein which is secreted and which is beneficial to cells surrounding the hair shaft, e.g., for treating a specific condition.
  • the protein encoded by the transgene can be a secreted protein, e.g., a soluble protein, a membrane bound protein, or an intracellular protein.
  • Preferred transgenes encode therapeutic proteins.
  • a therapeutic protein can be a soluble protein, such as a ligand, a receptor, an agonist of a ligand, an agonist of a receptor, an antagonist of a ligand, and an antagonist of a receptor.
  • the ligand can be, e.g., a hormone, a growth factor, or a cytokine.
  • a preferred soluble protein is an IL-1 receptor antagonist (IL-lra).
  • Interleukin-1 receptor antagonist protein is a well characterized competitive inhibitor of IL-1 that is produced by several types of cells found within the skin, including monocytes, macrophages, fibroblasts, keratinocytes, and polymorphonuclear leukocytes (Bigler et al.
  • the invention provides methods for treating skin diseases, e.g, skin diseases involving aberrations in the expression of IL-1, IL-1 receptor, or IL-1 antagonists.
  • cDNA for human interleukin-1 receptor antagonist protein is described in Arend et al. (1989) J Immunol 143:1851-1858, Arend et al. ( ⁇ 990) J Clin Invest 85:1694-1697, and Seckinger et al. (1987) J Immunol 139:1541-1545.
  • Another preferred soluble protein is a soluble or transdominant negative hormone receptor, such as an androgen receptor. Follicular cells transfected with a transdominant negative androgen receptor can have special applicability to the treatment of acne and male pattern baldness.
  • the transgene encodes a suicide gene.
  • a suicide gene is intended to include a gene which induce cell death, e.g., when the cell is exposed to a specific condition.
  • suicide genes include genes encoding a protein involved in apoptosis, such as Fas. Apoptosis in Fas-bearing cells is induced upon binding of a Fas ligand with the Fas receptor on the Fas-bearing cells. (Nagata and Golstein (1995) Science 267:1449).
  • Other apoptotic genes include genes from the Bcl-2 family, such Bcl-Xs (Boise et al.
  • TK thymidine kinase
  • a cell expressing TK will be killed upon treatment with gancyclovir.
  • the suicide gene is a toxin, such as ricin. Accordingly, the method of the invention provides methods for eliminating or killing perifollicular cells. Such methods can be used, for example, to treat malignant or benign skin cancers or for cosmetic purposes.
  • the transgene comprises a nucleotide sequence containing one or more open reading frames, i.e., sequences that code for peptides, such that upon transfection into perifollicular cells according to the method of the invention, at least one protein or peptide is synthesized in the perifollicular cell.
  • the transgene can encode a single peptide or the transgene can encode several peptides.
  • the transgene is a nucleotide sequence, which is expressed into one or more functional RNA molecules (e.g., an antisense RNA molecule or a ribozyme).
  • the functional RNA molecule inhibits, or at least decreases, expression of one or more endogenous genes in the specific target cell.
  • the method of the invention is useful for decreasing expression of a selected gene in perifollicular cells.
  • the level of a hormone, or hormone receptor can be reduced by introducing into perifollicular cells a nucleic acid encoding an RNA molecule which is antisense to the hormone or hormone receptor RNA according to the methods of the invention, such that translation of the hormone or hormone receptor RNA is inhibited or reduced.
  • an “antisense” nucleic acid comprises a nucleotide sequence which is complementary to a "sense” nucleic acid, e.g., complementary to an mRNA sequence encoding a protein, constructed according to the rules of Watson and Crick base pairing. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense sequence complementary to a sequence of an mRNA can be complementary to a sequence found in the coding region of the mRNA or can be complementary to a 5' or 3' untranslated region of the mRNA.
  • an antisense nucleic acid is complementary to a region preceding or spanning the initiation codon or in the 3' untranslated region of an mRNA.
  • the transgene encodes a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • a ribozyme having specificity for a nucleic acid of interest can be designed based upon the nucleotide sequence of the nucleic acid.
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the base sequence of the active site is complementary to the base sequence to be cleaved in an mRNA of interest. See for example Cech et al. U.S. Patent No. 4,987,071 ; and Cech et al. U.S. Patent No. 5,116,742.
  • an antisense nucleic acid or ribozyme can be synthesized in vitro and introduced into perifollicular cells according to the method of the invention.
  • Methods for in vitro production of antisense nucleic acids and ribozymes are known in the art.
  • the transgene is contained in a plasmid or vector containing sequences or elements well known in the art for preparing the nucleic acid prior to transfection.
  • sequences include those that enable the nucleic acid to be replicated, such as a bacterial origin of replication.
  • Suitable plasmid expression vectors include CDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al.(1987) EMBO J 6:187-195).
  • Other preferred elements which can be included in the expression vector or plasmid include elements allowing episomal maintenance and/or autonomous replication of the plasmid containing the transgene (see below) or elements favouring chromosomal integration of the transgene.
  • the expressible form of a transgene introduced into perifollicular cells is in a viral vector.
  • Viral vectors include recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus- 1. Nucleic acids introduced into cells using these vectors become stably integrated into the chromosomal DNA of the host cell.
  • a major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type virus in the cell population.
  • Defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller (1990) Blood 76:271).
  • retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) is replaced by a gene of interest rendering the retrovirus replication defective.
  • retroviral coding sequence gag, pol, env
  • Other viral vectors which can be used include adenovirus-derived vectors (See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155).
  • adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA).
  • Expression of the transgene of interest can be under control of, for example, the E1A promoter, the major late promoter (MLP) and associated leader sequences, the E3 promoter, or exogenously added promoter sequences.
  • Adeno-associated virus exhibits a high frequency of stable integration (see for example Flotte et al. (1992) Am J Respir Cell Mol Biol 7:349- 356; Samulski et al. (1989) J Virol 63:3822-3828; and McLaughlin et al. (1989) J Virol 62:1963-1973).
  • a variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc N ⁇ tl Ac ⁇ d Sci USA 81 :6466-6470; Tratschin et al. (1985) Mol Cel.
  • purified nucleic acids for use in the liposomal delivery system of the invention.
  • contamination of plasmid DNA with bacterial Iipids and or polysaccharides is preferably avoided.
  • contaminants can also contribute to the potential toxicity of the liposomal formulations. Therefore, it is preferable to use highly purified plasmid DNA, e.g., pharmaceutical grade manufactured under Food and Drug Administration standards of Good Laboratory Practices, (GLP) for the method of the invention.
  • GLP Good Laboratory Practices
  • nucleic acid having an appropriate physical conformation.
  • the physical state of plasmid DNA (supercoiled, closed circular, nicked, linear) can effect the amount of DNA per unit volume, the amount of DNA that can associate in an appropriate physicochemical orientation with charged liposomes of a defined size, and probably can effect the 'bioavailability' of the plasmid (partially degraded plasmid is unavailable for transcription). Accordingly, it is preferable to use nucleic acids which are supercoiled in the liposomal delivery system of the invention. Supercoiling of DNA is controlled by the regulated activity of topoisomerases.
  • Topoisomerase II in an ATP dependent process enzymatically converts DNA into negatively supercoiled double helices.
  • Purified DNA gyrase can be used as a biochemical reagent for induction of supercoiling in plasmid DNA (Bates and Maxwell (1989) EMBO J 8:1861-1866; Cozzarelli (1980) Science 207:953-960; Kreuzer and Cozzarelli (1980) Cell 20:245-254; Marini et al. (1980) J Biol Chem 255:4976-4979; Maxwell and Gellert (1986) Adv Protein Chem 38:69-107).
  • the expression plasmid DNA is treated with a DNA gyrase prior to final FPLC (fast performance liquid chromatography) or equivalent chromatographic purification in order to achieve a uniform physical structure within the plasmid DNA.
  • plasmid DNA can be incubated with DNA gyrase (Gibco BRL) in the presence of 1 mM ATP in order to induce formation of negative supercoils.
  • DNA gyrase Gibco BRL
  • a preferred plasmid or vector containing the transgene has been gyrase treated and chromatographically sized purified.
  • Final composition of the liposomal formulations are preferably performed in a laminar flow environment using sterile techniques immediately before use of the reagents in vivo.
  • Molecules such as macromolecules, other than nucleic acids, can also be introduced into cells according to the method of the invention.
  • the liposomal delivery system can be used to deliver a proteinous compound.
  • a proteinous compound is intended to include any compound which has at least one amino acid.
  • Preferred proteinous compounds include compounds which are hydrophilic and anionic compounds.
  • the liposomal formulations of the invention and the nucleic acid can be administered topically to a skin having hair follicles.
  • the liposomal formulations are in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the liposomal formulation, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • the liposomal formulations and the nucleic acid are topically applied to the subjects in a biologically compatible form suitable for pharmaceutical administration in vivo.
  • biologically compatible form suitable for administration in vivo is meant a form of the liposomal formulations in which any toxic effects are outweighed by the therapeutic effects of the agent.
  • subject is intended to include living organisms in which an immune response can be elicited, e.g., mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
  • Administration of a therapeutically active amount of a nucleic acid/liposomal formulation of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result.
  • a therapeutically active amount of a nucleic acid/liposomal formulation may vary according to factors such as the condition to be treated, disease state, the density of hair follicles on the skin, the type of skin, and the ability of the liposomal formulation to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • the liposomal preparations may contain a preservative to prevent the growth of microorganisms or other agent preventing degradation of liposomes.
  • the ability to deliver expression plasmid DNA or other nucleic acid to follicular skin cells via topical application of a liposomal formulation allows the treatment of many dermatologic diseases.
  • dermatologic diseases Several categories of dermatologic diseases could be treated according to the method of the invention: diseases of a follicular nature, infectious diseases, neoplastic diseases, autoimmune diseases, and acquired conditions.
  • diseases of a follicular nature infectious diseases, neoplastic diseases, autoimmune diseases, and acquired conditions.
  • the general strategy is similar; transfection of accessible cells in order to mediate the expression of a transgenic protein having biological effects proximal to the site of application.
  • transgenic secreted proteins the biological effects can extend regionally beyond the immediate microenvironment of the transduced cells.
  • the invention provides a method for the modulation of cell adhesion molecules and pro-inflammatory cytokines via the expression of soluble receptors or receptor antagonist proteins, such as for the treatment of autoimmune skin diseases including psoriasis and alopecia areata.
  • the expression of soluble or transdominant negative androgen receptors by transfected follicular cells can be used for treating acne and male pattern baldness.
  • the method of the invention further provides methods for treating skin diseases, such as alopecia areata, psoriasis, atopic dermatitis and eczema. Since these conditions have been associated with aberrations in the expression of the proinflammatory cytokine IL-1 as well as IL-1 receptors and naturally occurring IL-1 antagonists (Kondo et al. supra; Hammerberg et al. supra; Debets et al. supra; Hoffmann et al. supra; Wenzel et al. supra), the invention provides methods for modulating the amount of these proteins according to the disease, such that the skin disease is improved or cured.
  • skin diseases such as alopecia areata, psoriasis, atopic dermatitis and eczema. Since these conditions have been associated with aberrations in the expression of the proinflammatory cytokine IL-1 as well as IL-1 receptors and naturally occurring IL-1 antagonists (Kondo et al. supra; Hammerberg e
  • liposomal gene delivery systems which are administered systemically. Accordingly, the invention provides methods for treating diseases for which systemic administration of a nucleic acid is preferable.
  • Expression plasmid used in these studies were derived from the eukaryotic expression vector pSG5 (Stratagene), and utilizes the SV40 LTR as a promoter and the SV40 polyadenylation sequence.
  • the cDNA for human interleukin-1 receptor antagonist protein was cloned into the BamHI site of pSG5 to yield pSG5IL-lra.
  • the gene for E. coli ⁇ -galactosidase was cloned into the BamHI site of pSG5 to yield pSG51acZ.
  • the orientation of the transgene within recombinant plasmids was confirmed using a combination of restriction endonuclease mapping and dideoxynucleotide sequencing.
  • Plasmid DNA was prepared from the DH5- ⁇ strain of Escherichia coli transformed with the recombinant plasmids and grown in LB broth containing ampicillin (50 ⁇ g/ml). Plasmid DNA was purified using a modification of the alkaline lysis method followed by two separate bandings in cesium chloride equilibrium gradients (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Ethidium bromide was extracted from the DNA with butanol and the batches were then dialyzed extensively against purified water (Millipore) and ethanol precipitated.
  • pSG5IL-lra In the case of pSG5IL-lra, all batches of purified DNA were tested for the presence of recombinant human IL-lra protein by ELISA. None of the DNA used for the in vivo studies contained detectable levels of human IL-lra (detection limits less than 29 pg/ml). Fluorescently labeled pSG5IL-lra plasmid was produced in an identical fashion except that ethidium bromide intercalated into the DNA during the purification process was retained and excess non-incorporated ethidium bromide was removed by extensive dialysis in Tris-EDTA buffer.
  • Nonionic/Cationic-based Liposomal Formulations used in the experiments contained glyceryl dilaurate (GDL; Sigma Chemicals, >90% 1,3, isomer), cholesterol (CH; Sigma Chemicals), polyoxyethylene- 10-stearyl ether (POE- 10; ICI Americas), and l,2-dioleoyloxy-3-(trimethylammonio) propane (DOTAP; Avanti Polar Lipids) at a weight percent ratio of 50:15:23:12.
  • GDL glyceryl dilaurate
  • CH cholesterol
  • POE- 10 polyoxyethylene- 10-stearyl ether
  • DOTAP l,2-dioleoyloxy-3-(trimethylammonio) propane
  • the lipid mixture also contained ⁇ -tocopherol (1% by weight of total lipids; Eastman Kodak).
  • lipids were mixed and melted at 70°C in a sterile polystyrene centrifuge tube.
  • the lipid melt was then filtered through a 0.22 ⁇ m filter (Nuclepore®) and the filtrate was reheated in a water-bath at 70°C prior to being drawn into a sterile syringe.
  • a second syringe containing sterile, autoclaved, double-distilled water was preheated to 65°C and connected via a 3-way sterile stopcock to the lipid phase syringe.
  • the aqueous phase was then slowly injected into the lipid phase syringe.
  • the mixture was rapidly passed back and forth between the two syringes while being cooled under cold tap water until the mixture was at room temperature and stored at 4°C until use.
  • the total lipid concentration in the suspension was 100 mg/ml.
  • the resulting NC liposomal suspensions were examined using a Nikon Diaphot light microscope to assure integrity and quality of the liposomal preparations.
  • the liposomal suspension was sonicated for 20 minutes at room temperature and an equal volume of aqueous pSG5IL- lra or pSG51acZ DNA solution (7 mg DNA/ml) was added by inversion mixing followed by incubation at room temperature for 45 minutes. Control formulations contained no DNA.
  • LUV Large unilamellar liposomes
  • PC egg phosphatidylcholine
  • ⁇ -tocopherol 1% by weight of total lipids; Eastman Kodak
  • a reverse-phase evaporation method based on that reported by Szoka and Papahadjopoulos (Szoka F., Jr. and Papahadjopoulos D. (1978) PNAS USA 75:4194-4198) was used in the preparation of the LUVs.
  • the lipids were dissolved in a chloroform-methanol mixture (2:1 [v/v]).
  • Isotonic 0.05 M HEPES buffer (Sigma Chemical Co.), pH 7.4, was added to form a clear mixture.
  • the solvent-to-buffer ratio was 6:1 (v/v).
  • the solvents were removed using a rotoevaporator (Buchli) maintained at 45°C.
  • the total lipid concentration in the suspension was 75 mg/ml.
  • the resulting liposomal suspensions were examined using a Nikon Diaphot light microscope to assure integrity and quality of the liposomal preparations.
  • the liposomes were then sonicated for 20 minutes at room temperature before the addition of an equal volume of aqueous pSG5IL-lra DNA solution (7 mg/ml).
  • the liposomal DNA mixture was then allowed to incubate at room temperature for 45 minutes prior to topical application to the ventral side of hamster ears.
  • the total amount of lipid applied per ear of NC based liposomes was 15 mg (2.5 mg/dose), and the total lipid applied per ear with the PC-based liposomes was 11.25 mg (1.875 mg/dose).
  • the total amount of DNA applied was 1.05 mg (0.175 mg/dose).
  • Ears of untreated animals were also used as negative controls. All experiments were carried out under non-occluded conditions. At the time of sacrifice the ears were isolated by sharp dissection, weighed and measured along each border (in order to calculate the surface area exposed to treatment), then processed as described below. One ml of blood was also collected from each animal immediately following sacrifice and centrifuged at 14,000 rpm (Beckman) for 10 minutes at 4°C. The serum was collected and assayed for IL-lra content.
  • the surface of the ventral ear was swabbed several times again with PBS soaked tissue before the ventral ear was separated from the underlying cartilage.
  • the ventral ear was placed on a 60 mm polystyrene dish with the epidermal side facing down.
  • 500 ⁇ l of a 0.05 M Isotonic HEPES, pH 7.4 was placed on the ear and the sebaceous glands were carefully scraped using a sterile, dull scalpel. This procedure was carried out twice. Tissues were then minced in 1 ml of homogenate buffer and collected in sterile Eppendorf tubes along with the gland scrapings.
  • Genomic DNA was isolated from the tissue homogenates using DNAzolTM reagent (Life Technologies, Inc., Gaithersburg, MD) following the manufacturer's protocol. The concentration of DNA isolated from each sample was determined spectrophotometrically. Samples (10 ⁇ g of total DNA) were loaded onto 0.8% agarose gels, electrophoresed at 20 V for 21 hours, and transferred to a Nytran membrane. Controls included lanes containing known amounts of pSG5IL-lra (15 or 150 ⁇ g), both undigested and linearized by digestion with EcoRl .
  • the membrane was probed with [32p]-labeled oligonucleotide probes specific for human IL-lra generated from a 650 bp fragment of human IL-1 ra cDNA that was isolated from pSG51L-lra using random hexanucleotide primers, [32p]-dCTP (Amersham), and Klenow fragment (BRL).
  • Hybridization buffer included 10% dextran sulfate, 35% formamide, 5X SSC, 0.1% SDS and 100 ⁇ g/ml salmon sperm DNA, and were performed overnight at 42°C.
  • Membranes were washed twice for 10 min at room temperature in 2X SSC/0.1% SDS, twice for 10 minutes at 65°C in lXSSC/0.1% SDS, and once for 10 minutes at 65°C in O.lXSSC/0.1% SDS. Membranes were wrapped in plastic and exposed to film (Kodak X-AR5) at -70°C using intensifying screens.
  • IL-lra Detection of IL-lra by Immunohistochemistry Treated ears were embedded in OCT medium (Miles, Inc., Eikhart, IN) and frozen in isopropyl alcohol containing dry ice. The frozen samples were stored at -80°C before sectioning. Serial sections (5 ⁇ m) were obtained using a cryostat (Tissue-Tee II Model 4553, Lab-Tec Products, Miles Laboratories, Inc., Naperville, IL) and placed on poly-L-lysine double-coated slides. The tissue sections were then immediately fixed in 100% acetone for 10 minutes at 4°C and processed using a Histostain-SP AEC kit (Zymed Lab, Inc., South San Francisco, CA) according to the manufacturer's recommendations.
  • OCT medium Miles, Inc., Eikhart, IN
  • isopropyl alcohol containing dry ice The frozen samples were stored at -80°C before sectioning. Serial sections (5 ⁇ m) were obtained using a cry
  • the sections were reacted with primary murine anti-human IL-lra (29 ng/ml, Bachem Bioscience, Inc., King of Prussia, PA) for 30 minutes. After completion of the protocol, slides were counterstained with hematoxylin, rinsed a final time and mounted with 100 ⁇ l of GVA-mount before being examined and photographed using a Nikon Optiphot microscope (Nikon, Tokyo, Japan).
  • Each ear strata (ventral skin, auricular cartilage and dorsal skin) was isolated from the ear using sharp dissection as follows. Following sacrifice, the ears were cut at the base and pinned to a board with the ventral side facing up. The ventral side was swabbed several times with PBS soaked tissue and stripped with tape until all hair was removed and the surface looked shiny indicating complete removal of the stratum corneum. The surface of the ventral ear was then swabbed several times again with PBS soaked tissue before the ventral ear was separated from the underlying cartilage. The ventral ear was then placed on a sialinized glass slide with the epidermal side facing down.
  • a homogenate buffer (0.05 M Isotonic HEPES, pH 7.4, 0.1% PMSF, 0.1% HSA) was placed on the ear and the sebaceous glands were carefully scraped using a sterile, dull scalpel. This procedure was carried out twice.
  • the ventral skin strata was then minced in 1 ml of homogenate buffer and collected in sterile Eppendorf tubes along with the gland scrapings.
  • the cartilage and dorsal skin were minced in 1 ml of homogenate buffer each.
  • the samples were then sonicated on ice for 2 minutes and centrifuged at 15,000 rpm for 10 minutes at 4°C. The supernatants were collected immediately and analyzed for human IL-lra protein.
  • Human IL-lra protein was measured using a human specific IL-lra protein immunoassay (Research and Diagnostics Systems, Minneapolis, MN). Tissue homogenates were assayed in triplicate and the optical density of each sample was determined using a spectrophotometer set to 450 nm with the correction wavelength set to 570 nm. A standard curve to 2000 pg/ml was also prepared using the homogenate buffer as the diluent and negative control. The detection limit of this ELISA was 29 pg/ml. The test samples were compared to the standard curve to determine IL-lra concentration. The results were expressed as 10"9 gm human IL-lra/cm ⁇ and/or 10"° gm human IL-lra/gm tissue.
  • Example 1 Hybrid non-ionic/cationic (NC) liposome-DNA complexes mediate gene transfer in vitro
  • This Example compares the efficiency of non-ionic lipids, cationic lipids, e.g., LIPOFECTINTM, and combinations thereof to mediate transfection of DNA into cells in culture.
  • Five Hposomal formulations, listed in Table I, and plasmid DNA alone were used for in vitro transfections.
  • the DNA used in these transfections was the eukaryotic expression plasmid DNA (pSG51acZ), containing the gene for E. coli ⁇ -galactosidase. Successful transfectants were detected by histochemical staining with the chromogenic substrate X- gal.
  • Non-ionic liposomes glyceryl dilaurate:cholesterol:polyoxyethylene-l 0-stearyl ether (57:15:28)
  • X-gal solution is 5 mM K 3 Fe(CN) 6 , 5 mM K 4 Fe(CN)6, 2 mM MgCl 2 , 0.01% sodium deoxycholate, 0.02% NP-40, and 1 mM 5-bromo-4-chloro-3-indolyl- ⁇ -D- galactoside in PBS).
  • the number of transfectants resulting from each formulation was determined by visual examination of monolayer cultures for the presence of blue staining (lacZ+) cells using inverted light microscopy.
  • the relative transfection efficiency observed for each formulation was determined as the ratio of blue cells per test formulation versus the number of blue cells observed using the Lipofectin formulation (positive control). These results were converted to a linear scale and recorded in Table II.
  • Table II - Hybrid non-ionic/cationic liposomal formulations can mediate transfection of DNA in vitro.
  • GDL glyceryl dilaurate
  • CH cholesterol
  • POE polyoxyerhylene-10-stearyl ether
  • DOTAP l,2-dioleoyloxy-3-(trimethylammonio) propane
  • DODAP 1,2-dioleoyloxy- 3-(dimethylammonio) propane.
  • formulations consisting of mixtures of nonionic/cationic liposomes or the addition of particular cationic lipids (to facilitate transfection) to nonionic liposomes (to facilitate targeting) can effectively deliver plasmid DNA in an expressible form into cells in vitro.
  • This Example shows how different methods for the preparation of a topical Hposome-DNA formulation can affect the integrity of the expression plasmid DNA.
  • the particle size of the plasmid DNA-NC Hposome formulation had a mean particle size greater than 2 ⁇ m.
  • Example 3 NC liposomal formulations efficiently deliver plasmid DNA into the hair follicles
  • NC nonionic-cationic
  • the ventral surface of the hamster ear was treated with a single dose of NC Hposome containing pSG5IL-lra DNA (0.175 mg) that had been fluorescently labeled with ethidium bromide. Controls were treated with fluorescently labeled pSG5IL-lra DNA alone (0.175 mg).
  • the animals were sacrificed and the ears were isolated then snap frozen in OCT.
  • Serial cryosections (20 ⁇ m) were placed onto glass slides, mounted with Vecta-Shield mounting media (Vector Laboratories, Burlingame, CA). The sections were examined using a Bio-Rad MRO 600 laser scanning confocal microscope (Bio-Rad Microscience Div., Cambridge, MA), using an argon-krypton laser and analyzed with CoMOS program.
  • Plasmid DNA was visualized along the epidermal surface and within the perifollicular regions of the skin 6 hours after topical application of a single dose of the NC liposomal formulation. Although some of the plasmid DNA continued to be visualized on the skin surface 6 hours after application, the delivery of the labeled DNA into the hair follicles and perifollicular glands appeared to be complete by 24 hours post administration. Control animals treated with an aqueous formulation containing an equivalent dose of the fluorescently labeled plasmid failed to show evidence of DNA beyond the superficial epidermis after 24 hours of observation. This indicates that perifollicular delivery is a physicochemical property specific to NC Hposomal formulations.
  • Example 4 NC liposomal formulations deliver intact DNA to in vivo tissues
  • Example 3 shows that the plasmid DNA observed in vivo in the follicle following topical application of a NC-plasmid complex to skin, as described in Example 3, is mostly intact plasmid DNA.
  • the ventral surface of the hamster ear was treated with a single dose of NC Hposome containing pSG5IL-lra DNA (0.175 mg), or Hposome alone.
  • Duplicate animals treated with NC liposomes + DNA were sacrificed at 6, 12 and 24 hours after treatment.
  • a pair of animals treated with the NC liposomes alone were examined 24 hours after topical treatment.
  • the ears were isolated, depilated, minced, and total genomic DNA was isolated from the tissue homogenates. Ten ⁇ g of total genomic DNA was electrophoresed in a 0.8% agarose gel, then transferred to a Nytran membrane.
  • Controls included a lanes containing 15 and 150 pg of purified pSG5IL-lra (pi 5, pi 59 respectively), and duplicate amounts of pSG5IL-lra linearized by digestion with EcoRl.
  • the membrane was probed with [32p]-labeled oligonucleotide probes specific for human IL-lra, washed and exposed to film (Kodak X-AR5) at -70°C using intensifying screens.
  • the Southern blot hybridization shows the presence of bands specific for pSG5IL-lr in the NC Hposome treated skin beginning 6 hours after administration. Qualitatively, the amount of pSG5IL-lra present within the skin remained constant from 12 to 24 hours after treatment. Although some of the plasmid (p) DNA was identified in the form of supercoiled (sc) plasmid at 6 hours post-treatment, from 12 to 24 hours post-treatment most of the plasmid DNA was in the form of closed circular (cc) or linearized plasmid (linear). No bands were observed in the genomic DNA obtained from animals treated with liposomes alone.
  • results indicate that most of the plasmid DNA present within the skin was in the form of closed circular or linearized plasmid.
  • results further show that for the first 24 hours post-administration, plasmid DNA was not subjected to progressive degradation and suggests that some of the plasmid was delivered intracellularly and may have been protected from digestion by extracellular nucleases.
  • NC liposomal formulations function as transfecting agents in vivo
  • the ventral surface of the hamster ear was treated twice daily for three consecutive days with NC liposomes containing plasmid DNA. Controls were treated with an equivalent dose of NC liposomes without DNA. 24 hours after application of the last dose the ears were isolated and snap frozen in OCT. Serial cryosections (5 ⁇ m) were then obtained from the treated ears and placed onto glass slides. Sections were fixed in ice cold acetone blocked and reacted with monoclonal antibodies against human IL-lra (murine IgGl, 29 ng/ml) or irrelevant murine IgGl (29 ng/ml). Sections were counterstained with hematoxylin. Sections were examined by light microscopy (Nikon UFX-DX) for the presence of IL-lra expressing cells (red staining cells) and representative photomicrographs were obtained.
  • NC liposomal formulation also functioned as a transfecting agent.
  • Transfected human IL-lra expressing cells were identified within the follicles in the proximal third of the hair shaft and at the base of the hair shaft.
  • Negative controls treated with aqueous formulations of expression plasmid DNA, or liposomes alone failed to show evidence for IL-lra expressing perifollicular cells.
  • NC liposomal formulations efficiently mediated transfection of cells in vivo.
  • Example 6 Kinetics of expression of DNA transfected with NC liposomal formulations
  • This Example shows the kinetics of hlL-lra expression within treated skin over an 8 day period following a multiple dose (twice daily for three days) topical application protocol.
  • Formulations were applied to the ventral surface of hamster ears twice daily for 3 days (total dose of pSG5IL-lra DNA was 1 mg).
  • concentration of human IL-lra present in whole tissue homogenates obtained from different strata on days 1, 3, 5, and 8 post treatment was determined using ELISA. Assays were performed in triplicate and results expressed as pg hlL-lra surface area of treated skin.
  • NC liposome formulations are more efficient transfection agents than phospholipidic/cationic Hposome formulations
  • NC liposome formulation were then compared with those associated with a conventional PC liposome carrier system. Accordingly, this Example compares expression of transgenic human IL-lra in the ventral ear of the hamster following topical in vivo application of nonionic/cationic (NC) and phospholipid/cationic (PC) liposome formulations with and without pSG5IL-lra plasmid DNA. Formulations were applied to the ventral surface of the ear twice daily for 3 days
  • total dose of DNA was 1 mg.
  • concentration of transgenic human IL-lra present in whole tissue homogenates obtained from the ventral ear was determined using ELISA. Assays were performed in triplicate and results are expressed as pg IL-lra/cm ⁇ of treated skin surface area. Although the charged Hpid-DNA ratios were different for the two formulations, the total dose of DNA applied to the hamster ears was identical for the two systems. Under these conditions, it was found that the NC+DNA formulation mediated levels of transgene expression that were approximately four times higher than those obtained with the PC+DNA formulation (pO.OOl ) ( Figure 3).
  • ears treated with the NC liposomes + DNA expressed a mean of 165.5 ⁇ 15.6 pg/cm ⁇ human IL-lra, those treated NC liposomes alone expressed a mean of 31.4 ⁇ 24.0 pg/cm ⁇ human IL-lra.
  • Ears treated with PC liposomes + DNA expressed a mean of 40.8 ⁇ 8.1 pg/cm ⁇ human IL-lra and those treated with PC liposomes expressed mean levels of human IL-lra below the limits of detection by ELISA ( ⁇ 29 pg/cm2 ).
  • Controls treated with DNA alone expressed a mean of 32.5 ⁇ 1 1.0 pg/cm ⁇ of human IL-lra.
  • NC liposome formulations are more efficient in mediating in vivo transfection of cells than PC liposome formulations.

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Abstract

L'invention concerne un nouveau procédé et un nouveau système de délivrance d'acides nucléiques dans une cellule mammifère in vivo, par une application topique dudit acide nucléique et dudit système de délivrance. Ce procédé et ce système de délivrance utilisent une composition renfermant des liposomes, des lipides non ioniques, et un lipide cationique. Ce procédé et ce système consistant notamment en une administration topique, ils sont donc particulièrement adaptés à la thérapie génique de dysfonctionnements dermatologiques, par exemple la maladie néoplasique et l'alopécie.
PCT/US1998/007645 1997-04-17 1998-04-14 Systeme de delivrance d'adn de follicule pileux WO1998046208A1 (fr)

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DE19831043A1 (de) * 1998-07-13 2000-01-27 Deutsches Krebsforsch Hemmung von Alopezie
WO2000024895A2 (fr) * 1998-10-27 2000-05-04 Anticancer, Inc. Methode et modele de pigmentation capillaire
WO2000062813A2 (fr) * 1999-04-20 2000-10-26 The University Of British Columbia Lipides peg cationiques et méthodes d'utilisation
EP1060732A2 (fr) * 1999-05-27 2000-12-20 JOHNSON & JOHNSON CONSUMER PRODUCTS, INC. Nouvelles formulations topiques contenant des systèmes d'administration vésiculaires
WO2001042424A2 (fr) * 1999-11-29 2001-06-14 The Collaborative Group, Ltd. Liposomes cationiques, contenant des sphingolipides, destines a une administration topique de materiau bio-actif y compris de materiau genetique
WO2001042449A2 (fr) * 1999-12-10 2001-06-14 Anticancer, Inc. Procede permettant l'implantation de genes dans un mammifere
WO2001080900A2 (fr) * 2000-04-20 2001-11-01 The University Of British Columbia Procedes permettant d'ameliorer la transfection a mediation splp (particule plasmide-lipide stabilisee) au moyen de destabilisateurs de la membrane endosomale
US6372489B1 (en) 1998-10-27 2002-04-16 Anticancer, Inc. Method and model for hair pigmentation
WO2003020227A1 (fr) * 2001-09-03 2003-03-13 Richard Voellmy Compositions et methodes permettant de prevenir l'alopecie induite par une chimiotherapie
US6852334B1 (en) 1999-04-20 2005-02-08 The University Of British Columbia Cationic peg-lipids and methods of use
US7037499B1 (en) 1996-11-14 2006-05-02 The United States Of America As Represented By The Secretary Of The Army Adjuvant for transcutaneous immunization
US7189705B2 (en) 2000-04-20 2007-03-13 The University Of British Columbia Methods of enhancing SPLP-mediated transfection using endosomal membrane destabilizers
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US7405080B2 (en) 2000-03-23 2008-07-29 Voellmy Richard W Compositions and methods relating to prevention of chemotherapy-induced alopecia
US7527802B2 (en) 2001-02-13 2009-05-05 The United States Of America As Represented By The Secretary Of The Army Vaccine for transcutaneous immunization
US8911742B2 (en) 1996-11-14 2014-12-16 The United States Of America As Represented By The Secretary Of The Army Transcutaneous immunization without heterologous adjuvant

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Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7037499B1 (en) 1996-11-14 2006-05-02 The United States Of America As Represented By The Secretary Of The Army Adjuvant for transcutaneous immunization
US7378097B2 (en) 1996-11-14 2008-05-27 The United States Of America As Represented By The Secretary Of The Army Use of penetration enhancers and barrier disruption methods to enhance the immune response of antigen and adjuvant
US8911742B2 (en) 1996-11-14 2014-12-16 The United States Of America As Represented By The Secretary Of The Army Transcutaneous immunization without heterologous adjuvant
DE19831043A1 (de) * 1998-07-13 2000-01-27 Deutsches Krebsforsch Hemmung von Alopezie
WO2000024895A3 (fr) * 1998-10-27 2000-11-30 Anticancer Inc Methode et modele de pigmentation capillaire
US6372489B1 (en) 1998-10-27 2002-04-16 Anticancer, Inc. Method and model for hair pigmentation
WO2000024895A2 (fr) * 1998-10-27 2000-05-04 Anticancer, Inc. Methode et modele de pigmentation capillaire
WO2000062813A3 (fr) * 1999-04-20 2001-08-09 Univ British Columbia Lipides peg cationiques et méthodes d'utilisation
US6852334B1 (en) 1999-04-20 2005-02-08 The University Of British Columbia Cationic peg-lipids and methods of use
WO2000062813A2 (fr) * 1999-04-20 2000-10-26 The University Of British Columbia Lipides peg cationiques et méthodes d'utilisation
EP1060732A2 (fr) * 1999-05-27 2000-12-20 JOHNSON & JOHNSON CONSUMER PRODUCTS, INC. Nouvelles formulations topiques contenant des systèmes d'administration vésiculaires
EP1060732A3 (fr) * 1999-05-27 2001-12-12 JOHNSON & JOHNSON CONSUMER PRODUCTS, INC. Nouvelles formulations topiques contenant des systèmes d'administration vésiculaires
WO2001042424A2 (fr) * 1999-11-29 2001-06-14 The Collaborative Group, Ltd. Liposomes cationiques, contenant des sphingolipides, destines a une administration topique de materiau bio-actif y compris de materiau genetique
WO2001042424A3 (fr) * 1999-11-29 2001-12-13 Collaborative Group Ltd Liposomes cationiques, contenant des sphingolipides, destines a une administration topique de materiau bio-actif y compris de materiau genetique
WO2001042449A2 (fr) * 1999-12-10 2001-06-14 Anticancer, Inc. Procede permettant l'implantation de genes dans un mammifere
US7067496B2 (en) 1999-12-10 2006-06-27 Anticancer, Inc. Methods for introducing genes into mammalian subjects
WO2001042449A3 (fr) * 1999-12-10 2002-02-07 Anticancer Inc Procede permettant l'implantation de genes dans un mammifere
US7405080B2 (en) 2000-03-23 2008-07-29 Voellmy Richard W Compositions and methods relating to prevention of chemotherapy-induced alopecia
US7976826B2 (en) 2000-03-23 2011-07-12 Hsf Pharmaceuticals S.A. Compositions and methods relating to prevention of chemotherapy-induced alopecia
US8257686B2 (en) 2000-03-23 2012-09-04 Hsf Pharmaceuticals S.A. Compositions and methods relating to prevention of chemotherapy-induced alopecia
JP2004508012A (ja) * 2000-04-20 2004-03-18 ザ　ユニバーシティ　オブ　ブリティッシュ　コロンビア エンドソーム膜不安定剤を用いたｓｐｌｐ媒介性トランスフェクションの強化方法
WO2001080900A3 (fr) * 2000-04-20 2003-04-24 Univ British Columbia Procedes permettant d'ameliorer la transfection a mediation splp (particule plasmide-lipide stabilisee) au moyen de destabilisateurs de la membrane endosomale
US7189705B2 (en) 2000-04-20 2007-03-13 The University Of British Columbia Methods of enhancing SPLP-mediated transfection using endosomal membrane destabilizers
WO2001080900A2 (fr) * 2000-04-20 2001-11-01 The University Of British Columbia Procedes permettant d'ameliorer la transfection a mediation splp (particule plasmide-lipide stabilisee) au moyen de destabilisateurs de la membrane endosomale
US7527802B2 (en) 2001-02-13 2009-05-05 The United States Of America As Represented By The Secretary Of The Army Vaccine for transcutaneous immunization
WO2003020227A1 (fr) * 2001-09-03 2003-03-13 Richard Voellmy Compositions et methodes permettant de prevenir l'alopecie induite par une chimiotherapie
AU2001282400B2 (en) * 2001-09-03 2009-04-23 Richard Voellmy Compositions and methods relating to prevention of chemotherapy-induced alopecia

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