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WO1994006923A1 - Modification d'un virus pour rediriger l'infectivite et amplifier l'apport cible de polynucleotides a des cellules - Google Patents

Modification d'un virus pour rediriger l'infectivite et amplifier l'apport cible de polynucleotides a des cellules Download PDF

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Publication number
WO1994006923A1
WO1994006923A1 PCT/US1993/009034 US9309034W WO9406923A1 WO 1994006923 A1 WO1994006923 A1 WO 1994006923A1 US 9309034 W US9309034 W US 9309034W WO 9406923 A1 WO9406923 A1 WO 9406923A1
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virus
cell
polynucleotide
carrier
modified
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PCT/US1993/009034
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English (en)
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George Y. Wu
Catherine H. Wu
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The University Of Connecticut
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    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10211—Aviadenovirus, e.g. fowl adenovirus A
    • C12N2710/10241—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10243—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Carriers comprised of a cell-specific binding agent, such as a ligand for a cellular receptor (e.g., an asialoglycoprotein for the asialoglycoprotein receptor and transferrin for the transferrin receptor), and a polynucleotide-binding agent, such as a polycation (e.g., polylysine) which can bind negatively-charged polynucleotides, can specifically target a polynucleotide to a receptor-bearing cell.
  • a cell-specific binding agent such as a ligand for a cellular receptor (e.g., an asialoglycoprotein for the asialoglycoprotein receptor and transferrin for the transferrin receptor)
  • a polycation e.g., polylysine
  • the cointernalized virus disrupts the endosome before it fuses with the lysosomal vesicles, thereby releasing the polynucleotide into the intracellular environment.
  • virus-addition studies, cited above showed enhancement of gene expression in vitro, the use of this technique in vivo is not likely to be practical because the non-targeted virus cannot be efficiently directed to the same endocytotic vesicles as the complex. For this reason, forced co-localization by direct chemical linkage of the complex to the virus has been used by Wagner et al. who chemically coupled adenovirus to polylysine and formed transferrin-based ternary complexed with foreign DNA.
  • This invention pertains to a modified virus having the capacity to disrupt cellular endosomal vesicles, useful for delivering a polynucleotide to a target cell for selective cellular internalization and to methods of preparing and using the modified virus.
  • the modified virus comprises a virus which has linked to its surface, a molecular complex comprised of a polynucleotide complexed with a carrier comprised of a cell-specific binding agent and a polynucleotide-binding agent. Coupling of the complex to the virus targets the virus to the target cell where it is internalized along with the polynucleotide through the same pathway.
  • the capacity of the virus to disrupt endosomes is directly linked to the polynucleotide being delivered to the cell.
  • Coupling of the complex to the virus can also block the natural specificity of the virus and results in targeted gene expression directed by the attached cell-specific binding agent.
  • the virus can be any virus which is capable of disrupting endosomes upon internalization by a receptor-bearing cell.
  • the polynucleotide delivered can be RNA or DNA.
  • the polynucleotides can be genes encoding a variety of proteins, including secretory proteins, cell surface proteins, and immunogenic proteins.
  • the polynucleotide can be a ribozyme or an antisense construct which inhibits the expression of a specific gene or genes of cellular (e.g., a cellular oncogene) or of noncellular origin (e.g., a viral oncogene or the genes of an infecting pathogen such as a virus).
  • the cell-specific binding agent of the carrier is specific for a cellular surface structure, typically a receptor, which mediates internalization of bound ligands by endocytosis into cellular endosomes, such as the asialoglycoprotein receptor of hepatocytes.
  • the cell-specific binding agent can be a natural or synthetic ligand (e.g., a protein, polypeptide, glycoprotein, carbohydrate, etc.) or it can be an antibody, or an analogue thereof, which specifically binds a cellular surface structure which then mediates internalization of the bound complex.
  • the polynucleotide-binding agent of the carrier is a compound such as a polycation which stably complexes the polynucleotide under extracellular conditions and releases the polynucleotide under intracellular conditions in functional form.
  • the molecular complex can be introduced onto the surface of the virus by chemical coupling either directly or through bridging agents.
  • the modified virus can be used to deliver polynucleotides to cells in vivo, in vitro, or ex vivo.
  • Figure 1 is photographs of cells following exposure to complexes of plasmid DNA containing the gene for nuclear localizing ⁇ -galactosidase.
  • Panels A-C, Huh7 are photographs of cells following exposure to complexes of plasmid DNA containing the gene for nuclear localizing ⁇ -galactosidase.
  • a virus which disrupts cellular endosomes can be used to enhance expression of polynucleotides delivered to cells.
  • a molecular complex which is targeted to a specific cell and which contains a polynucleotide to be delivered to the cell is coupled to the virus. Coupling of the molecular complex to the virus targets the virus for cointernalization into the target cell along with the polynucleotide and through the same pathway. The coupling of the complex also results in alteration of viral infectivity by interfering with the virus' interaction with its natural receptor. Targeted expression of the polynucleotide is exclusively directed by the attached cell-specific binding agent.
  • the cointernalized virus disrupts the endosomes containing the polynucleotide, thereby releasing the polynucleotide into the cell and avoiding lysosomal degradation to result in increased expression of the polynucleotide by the cell.
  • Viruses useful in this invention are those which are capable of disrupting endosomal vesicles. Generally, these are viruses with exposed capsid proteins (nonenveloped viruses). However, some enveloped viruses, such as influenza virus, may be used. A preferred virus is adenovirus. Particularly preferred is Type 5 adenovirus, available from American Type Culture Collection, which is noncarcinogenic. The virus can be replication defective or otherwise defective or truncated in structure or function as long as it retains or contains the component(s) necessary to disrupt endosomes (e.g., inactivated intact adenovirus).
  • the polynucleotide can be RNA or DNA.
  • targeted polynucleotides can be genes encoding secretory proteins (see U.S. Patent Application Serial No. 710,558, filed on June 5, 1991, and Serial No. 893,736, filed on June 5, 1992), such as clotting factors and other blood proteins; cell surface proteins (see U.S. Patent Application Serial No. 695,598, filed on May 3, 1991), such as cell surface receptors for low density lipoproteins, for growth factors, or for hormones; immunogenic proteins (seeU.S. Patent Application Serial No.
  • the polynucleotide can be an antisense polynucleotide (U.S. Patent Application Serial Number 864,003, filed on April 3, 1992).
  • the polynucleotide can be an RNA molecule which has catalytic activity (e.g., a ribozyme).
  • a polynucleotide is complexed with a carrier comprised of a cell-specific binding agent and a polynocleotide-binding agent.
  • the cell- specific binding agent is a molecule which specifically binds a cellular surface structure which mediates its internalization by, for example, the process of endocytosis.
  • the surface structure can be a protein, polypeptide. carbohydrate, lipid, or a combination thereof. It is typically a surface receptor which mediates endocytosis of the ligand.
  • the cell-specific binding agent can be a natural or synthetic ligand which binds the receptor.
  • the ligand can be a protein, polypeptide, glycoprotein or glycopeptide, carbohydrate, glycolipid, or a combination thereof which has functional groups that are exposed sufficiently to be recognized by the cell surface structure. It can also be component of a biological organism such as a virus or a cell (e.g., mammalian, bacterial, protozoan).
  • the cell-specific ligand can also be an antibody, or an analogue of an antibody such as a single chain antibody, which binds the cell surface structure.
  • Ligands useful in forming the carrier will vary according to the particular cell to be targeted.
  • galactose-terminal carbohydrates such as carbohydrate trees obtained from natural glycoproteins, especially structures that either contain terminal galactose residues or can be enzymatically treated to expose terminal galactose residues, can be used although other ligands such as polypeptide hormones may also be employed.
  • naturally occurring plant carbohydrates such as arabinogalactan can be used as ligands.
  • Other useful ligands for hepatocyte targeting include glycoproteins having exposed terminal carbohydrate groups such as asialoglycoproteins (galactose-terminal).
  • galactose-terminal ligands can be formed by coupling galactose-terminal carbohydrates such as lactose or arabinogalactan to nongalactose-bearing proteins by reductive lactosamination.
  • galactose-terminal carbohydrates such as lactose or arabinogalactan
  • additional asialoglycoproteins include, but are not limited to, asialoorosomucoid, asialofetuin and desialylated vesicular stomatitis virus.
  • Such ligands can be formed by chemical or enzymatic desialylation of glycoproteins that possess terminal sialic acid and penultimate galactose residues.
  • the cell-specific ligand can be a receptor or receptor-like molecule, such as an antibody which binds a ligand (e.g., an antigen which when bound is internalized) on the cell surface.
  • a ligand e.g., an antigen which when bound is internalized
  • Such antibodies can be produced by standard procedures.
  • the polynucleotide-binding agent of the carrier complexes the polynucleotide to be delivered. Complexation with the polynucleotide must be sufficiently stable (either in vivo or in vitro) to prevent significant uncoupling of the polynucleotide extracellularly prior to internalization by the target cell. However, the complex must be cleavable under appropriate conditions within the cell so the polynucleotide is released in functional form within the cell.
  • the binding between the polynucleotide-binding agent and the polynucleotide is based on electrostatic attraction which provides sufficient extracellular stability, but is releasable intracellularly.
  • Preferred polynucleotide-binding agents are polycations that bind negatively charged polynucleotides. These positively charged proteins can bind noncovalently with the polynucleotide to form a targetable molecular complex which is stable extracellularly but releasable intracellularly.
  • Suitable polycations are polylysine, polyarginine, polyomithine, basic proteins such as histones, avidin, protamines and the like.
  • a preferred polycation is polylysine.
  • noncovalent bonds that can be used to releasably link the expressible polynucleotide include hydrogen bonding, hydrophobic bonding, electrostatic bonding alone or in combination such as, anti-polynucleotide antibodies bound to polynucleotide, and streptavidin or avidin binding to polynucleotide- containing bio tiny lated nucleotides.
  • the carrier of the molecular complex can be formed by chemically linking the cell- specific binding agent to the polynucleotide-binding component.
  • the chemical linkage is typically covalent.
  • a preferred linkage is a peptide bond. This can be formed with a water soluble carbodiimide as described by Jung, G. et al. (1981) Biochem. Biophys. Res. Commun. 101: 599-606.
  • Alternative linkages are disulfide bonds or strong noncovalent linkages as in avidin-biotin coupling.
  • the chemical linkage can be optimized for the particular cell-specific binding agent and polynucleotide-binding agent used to form the carrier. Reaction conditions can be designed to maximize linkage formation but to minimize the formation of aggregates of the carrier components.
  • the optimal ratio of cell-specific binding agent to polynucleotide- binding agent can be determined empirically. When polycations are used, the molar ratio of the components will vary with the size of the polycation and the size of the polynucleotide. In general, this ratio ranges from about 10:1 to 1:1, preferably about 5:1. Uncoupled components and aggregates can be separated from the carrier by molecular sieve or ion exchange chromatography (e.g., Aquapore ⁇ M cation exchange, Rainin).
  • the carrier is coupled to virus to form a modified virus.
  • the coupling can be done chemically.
  • the chemical coupling is performed under conditions which preserve the virus' ability to disrupt endosomes.
  • the amount of carrier coupled to the virus is sufficient to target the virus to the desired cell receptor and in certain embodiments, sufficient to inhibit the specificity of the virus in unmodified form.
  • the coupling is performed by linking a carbohydrate on the viral surface to amino groups of the carrier.
  • An adenovirus for example, has glycoprotein fibers on its outer surface.
  • the glycoprotein fibers contain the saccharide N-acetyl-glucosamine, which is absent from other parts of the virus.
  • the carrier can be covalently bound specifically to these fibers through the N-acetyl-glucosamine.
  • Viral N-acetyl-glucosamine is oxidized to an aldehyde which reacts with the amino groups on, for example, the polylysine component of the molecular complex to form a Schiff s base.
  • the Schiff s base is stabilized by reduction using a reducing agent such as sodium cyanoborohydride (NaCN(BH3). Coupling the complex to the viral fibers redirects the virus to cells targeted by the receptor- specific ligand component of the complex and it abolishes the natural specificity of the adenovirus.
  • a reducing agent such as sodium cyanoborohydride (NaCN(BH3). Coupling the complex to the viral fibers redirects the virus to cells targeted by the receptor- specific ligand component of the complex and it abolishes the natural specificity of the adenovirus.
  • the polynucleotide can be complexed to the carrier by a stepwise dialysis procedure.
  • the dialysis procedure begins with a 2M NaCl dialyzate and ends with a 0.15M solution.
  • the gradually decreasing NaCl concentrations results in binding of the polynucleotide to the carrier.
  • dialysis may not be necessary; the polynucleotide and carrier are simply mixed and incubated.
  • the molecular complex can contain more than one copy of the same polynucleotide or one or more different polynucleotides.
  • the molar ratio of polynucleotide to the carrier can range from about 100(or more):l to about 1 :150 (or more), depending on the type and size of carrier and polynucleotide.
  • the modified virus of this invention can be used to selectively deliver a polynucleotide(s) to a target cell under a variety of conditions.
  • the modified virus is administered to an organism in a physiologically acceptable vehicle.
  • the modified virus can be administered parenterally. Preferably, it is injected intravenously.
  • cultured cells can be incubated with the modified virus in an appropriate medium under conditions conducive to endocytotic uptake by the cells.
  • the modified viruses can also be used ex vivo to enhance polynucleotide delivery to cells or tissues which have been removed from an organism and will subsequently be returned to that organism.
  • the reactants were coupled by addition l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Pierce Chemical Co., Rockville. IL) in a 140-fold molar excess over AsOR and stirred for 16 hrs at 25°C.
  • reaction mixture was then dialyzed against deionized water at 4°C for 72 hrs, lyophilized and purified by cation exchange chromatography using a high pressure liquid chromatographic system (Rainin Instrument Co., Woburn, MA) employing an Aquapore CX-300 1.0 cm X 25 cm cation exchange column (Rainin) with stepwise elution at a flow rate of 4.0 ml/min with 0.1 M sodium acetate, pH 5.0, 12 min; pH 2.5, 24 min; pH 2.25, 12 min; and pH 2.0, 14 min.
  • This fraction was further purified by electrophoresis using an apparatus similar to the Bio-Rad Model 491 Prep Cell as described by that manufacturer, but with an acid-urea gel system as described previously by Panyim and Chalkley (Arch. Biochem. Biophys. (1969) 130:337-346). Bands were eluted with 0.35 M ⁇ - alanine-acetic acid, pH 4.8, and checked for UV absorption at 230 nm. The second peak eluted was identified and confirmed as conjugate purified to homogeneity by an analytical acid-urea polyacrylamide gel electrophoresis. The purified conjugate was dialyzed against 0.025 M Tris-0.001 M EDTA (T-E) buffer through membranes with 12-14 kD exclusion limits.
  • T-E Tris-0.001 M EDTA
  • Type 5 adenovirus kindly provided by Dr. Hamish Young, Columbia University, NY, was propagated and amplified in HeLa S3 cells as described previously (Green M. and M. Pina (1963) Virology 20: 909-207). To avoid potential cytotoxic effects of wild-type virus in studies on targeted gene expression, replication defective dl312 adenovirus were also studied after propagation in 293 cells as described previously (Jones. N., and Schenk, T. (1979) Proc. Natl. Acad. Sci USA 76:3665-3669).
  • Adenovirus samples 1.0 X 10 ⁇ particles, each in a total volume of lOO ⁇ l 0.025 M Tris-0.001 M EDTA (T-E) were reacted with 100 ⁇ l 0.02 M NaIO4 (Sigma) for 30 min at 25 °C in the dark. Then, 100 ⁇ l 0.20 M NaAs ⁇ 2 (Sigma), in T-E buffer was added for an additional 60 min.
  • AsOR-PL conjugate was radiolabeled with Na- ⁇ I by a chloramine-T method (Greenwood, F.C., et al. (1963) Biochem. J. 89:114-123) to a specific activity of 215 cpm/ng protein.
  • the number of modified viral particles in each fraction was determined by quantisation of DNA by UV absorption (Precious, B. and Russell, W.C. (1985) A Practical Approach In Virology, Mahey, B. W. j., editor IRL Press/Oxford, England 193-205) after proteinase K-phenol-chloroform extraction (Rowe, D.W., et al. (1978) Biochemistry 17:1582- 1590).
  • purified modified virus was treated with SDS and proteinase K to make final concentrations of 0.1 and 100 ⁇ g/ml, respectively.
  • the samples were incubated at 37°C for 1 hr followed by phenol-chloroform extraction, chloroform extraction and precipitation with 2.5 volumes of ethanol at -20°C overnight.
  • the nucleic acid was collected by centrifugation at 10,000 rpm for 20 min at 4°C and the DNA concentration determined by measurement of the UV absorption at 260 ran.
  • the maximum amount of AsOR-PL bound to virus was obtained with starting ratios of 20 ⁇ g of conjugate to 1 X 10* ⁇ viral particles. At this ratio, it was calculated that approximately 24 molecules of conjugate were bound to each viral particle.
  • Total protein content of the modified virus was determined by Bio-Rad assay as instructed by the manufacturer. Purified modified virus was filtered through 0.45 ⁇ membranes (Millipore) and remained stable in T-E buffer at 4°C for at least 2 weeks without loss of activity.
  • plasmid DNA 0.5 mg in 1 ml of 2 M NaCl was added in a ratio of 2 ⁇ g DNA to 0.4 ⁇ g modified virus, with respect to total protein. This ratio was determined optimal using an agarose gel retardation assay as described previously (Wu, G.Y. and Wu, C.H. (1987) J. Biol. Chem. 262:4429-4432).
  • the sample was then placed in dialysis tubing with an exclusion limit of 12-14 kD (Spectrapore), and step-wise dialyzed successively at 4°C for 0.5 hr against 1 1 of NaCl in each of the following concentrations: 1.5 M, 1.0 M, 0.5 M, 0.25 M and 0.15 M. After the final dialysis, the complex was filtered through 0.45 ⁇ membranes prior to use in subsequent studies.
  • HeLa S3 [asialoglycoprotein receptor (-)], human hepatoma, SK Hepl [asialoglycoprotein receptor (-)] cells, and human hepatoma Huh 7 [asialoglycoprotein receptor (+)] cells were cultured in plastic dishes. Cells were seeded at densities of 5 X ⁇ 0 ⁇ - cells in 35 mm plastic dishes containing minimal essential medium (MEM) (GIBCO) and 5% fetal calf serum (GIBCO) under 5% CO2 at 37°C.
  • MEM minimal essential medium
  • GIBCO minimal essential medium
  • GIBCO minimal fetal calf serum
  • Asialoglycoprotein receptor (+) and (-) cells all seeded at 5.0 X 10* ⁇ cells/dish, were transfected separately, 24 hrs later, with DNA 2 ⁇ g in 2 ml medium as DNA complex alone.
  • DNA complexed to modified dl312 adenovirus at 2,000 particles/cell DNA complexed to modified dl312 adenovirus plus a 1000-fold excess of AsOR as described above. After 24 hrs of incubation, cells were fixed and washed (Beddington, R.S.P. et al. (1989) Development 106:34-46), and then stained with X-gal (Sanes, J.R. et al. (1986) EMBOJ.
  • Fig. 1 shows representative fields of cells following exposure to modified adenovirus or controls complexed to plasmid DNA containing the gene for nuclear localizing ⁇ -galactosidase.
  • Huh 7 asialoglycoprotein receptor (+) cells complex alone, panel A, produced 0.5 ⁇ 1%, but no positive cells in SK Hep 1 or HeLa S3, either receptor (-) cells, panels D and G, respectively.
  • modified dl312 virus complex exposed to Huh 7 cells produced 22 ⁇ 2.1% positive cells, panel B, but none in SK Hepl or HeLa S3, panels E and F, repsectively.

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Abstract

On amplifie l'apport ciblé de polynucléotides à des cellules par couplage à un virus capable de rompre des endosomes cellulaires d'un complexe moléculaire redirigeant la spécificité virale vers la cellule ciblée et portant le polynucléotide. Les virus efficaces dans le cadre de l'invention, tels que des adénovirus, sont généralement ceux qui possèdent des protéines capsides exposées et qui sont capables de rompre les vacuoles endosomes lors de l'internalisation par une cellule porteuse de récepteurs. On peut utiliser le virus modifié in vivo, in vitro, ou ex vivo, de façon à rediriger la spécificité de fixation à des cellules virales et à amplifier l'apport sélectif de polynucléotides à des cellules cibles.
PCT/US1993/009034 1992-09-24 1993-09-23 Modification d'un virus pour rediriger l'infectivite et amplifier l'apport cible de polynucleotides a des cellules WO1994006923A1 (fr)

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WO1994024299A1 (fr) * 1993-04-08 1994-10-27 Boehringer Ingelheim International Gmbh Adenovirus pour le transfert d'adn etranger dans des cellules eucariotes superieures
WO1995010624A1 (fr) * 1993-10-14 1995-04-20 Boehringer Ingelheim International Gmbh Particules a activite endosomolytique
WO1996029423A1 (fr) * 1995-03-20 1996-09-26 Baylor College Of Medicine Compositions et methodes pour induire une infection par des vecteurs retroviraux en dehors de leur gamme d'hotes
US5728399A (en) * 1994-06-29 1998-03-17 University Of Conn. Use of a bacterial component to enhance targeted delivery of polynucleotides to cells
US6074850A (en) * 1996-11-15 2000-06-13 Canji, Inc. Retinoblastoma fusion polypeptides
US6379927B1 (en) 1996-11-15 2002-04-30 Canji, Inc. Retinoblastoma fusion proteins
US6392069B2 (en) 1996-01-08 2002-05-21 Canji, Inc. Compositions for enhancing delivery of nucleic acids to cells
US6849399B1 (en) 1996-05-23 2005-02-01 Bio-Rad Laboratories, Inc. Methods and compositions for diagnosis and treatment of iron misregulation diseases
US7002027B1 (en) 1996-01-08 2006-02-21 Canji, Inc. Compositions and methods for therapeutic use
US7026116B1 (en) 1996-04-04 2006-04-11 Bio-Rad Laboratories, Inc. Polymorphisms in the region of the human hemochromatosis gene
US7067255B2 (en) 1996-04-04 2006-06-27 Bio-Rad Laboratories, Inc. Hereditary hemochromatosis gene
WO2011101031A1 (fr) 2010-02-19 2011-08-25 Université de Liège Polynucléotide pour utilisation dans le traitement de maladies induites par l'influenzavirus a, codant pour une protéine mx modifiée, ladite protéine mx modifiée, et animal transgénique exprimant un gène codant pour une protéine mx modifiée
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