WO1992006180A1 - Targeting viruses and cells for selective internalization by cells - Google Patents

Targeting viruses and cells for selective internalization by cells Download PDF

Info

Publication number
WO1992006180A1
WO1992006180A1 PCT/US1991/007103 US9107103W WO9206180A1 WO 1992006180 A1 WO1992006180 A1 WO 1992006180A1 US 9107103 W US9107103 W US 9107103W WO 9206180 A1 WO9206180 A1 WO 9206180A1
Authority
WO
WIPO (PCT)
Prior art keywords
virus
cell
modified
receptor
cells
Prior art date
Application number
PCT/US1991/007103
Other languages
French (fr)
Inventor
George Y. Wu
Catherine H. Wu
Original Assignee
University Of Connecticut
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Connecticut filed Critical University Of Connecticut
Priority to AU88603/91A priority Critical patent/AU660629B2/en
Priority to JP3517570A priority patent/JPH07500961A/en
Publication of WO1992006180A1 publication Critical patent/WO1992006180A1/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86Viral vectors
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/87Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00Reverse transcribing DNA viruses
    • C12N2730/00011Details
    • C12N2730/10011Hepadnaviridae
    • C12N2730/10111Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00Reverse transcribing RNA viruses
    • C12N2740/00011Details
    • C12N2740/10011Retroviridae
    • C12N2740/13011Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00Reverse transcribing RNA viruses
    • C12N2740/00011Details
    • C12N2740/10011Retroviridae
    • C12N2740/13011Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041Use of virus, viral particle or viral elements as a vector
    • C12N2740/13045Special targeting system for viral vectors
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00Vectors comprising a targeting moiety
    • C12N2810/10Vectors comprising a non-peptidic targeting moiety
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00Vectors comprising a targeting moiety
    • C12N2810/50Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00Vectors comprising a targeting moiety
    • C12N2810/50Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/859Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from immunoglobulins

Definitions

  • Viruses represent a natural and efficient means for the introduction of foreign genes into cells.
  • viruses are useful tools for the study of genes, and gene regulation in vitro and for gene therapy.
  • most viruses have broad cell specificity and can infect a wide variety of cell types. This can lead to foreign gene expression in many tissues, some of which may be undesirable, especially for clinical applications.
  • viral infection is mediated by interactions between viral envelopes and plasma membranes of target cells.
  • specific viral structures are recognized and bound by cellular receptors.
  • HIV employs envelope glycoproteins to bind to helper T lymphocytes via CD4 (T4) receptors.
  • T4 CD4
  • virus specificity can be redirected by attaching antibodies to viruses.
  • Goud, B., et aL. Virolo ⁇ v 161:251-254 (1988) linked anti-transferrin receptor antibodies to obtain delivery of a retrovirus to human cells bearing the transferrin receptor.
  • a means for targeting viral or other types of nucleic acid vectors containing foreign genes to a target cell and obtaining infection and replication of the virus would be useful in gene therapy.
  • the invention pertains to a method of targeting a virus or a cell to a target cell for selective internalization in vivo (or i vitro) by the cell and to modified viruses and cells which are targeted for selective internalization by a target cell.
  • a virus or cell is targeted to the target cell for internalization by introducing a receptor- specific molecule onto the surface of the virus or cell to produce a modified virus or cell which specifically binds to a receptor on the surface of the target cell.
  • the modified virus or cell can be administered to an organism where it binds selectively to the receptor of the target cell.
  • the receptor-binding results in internalization by the target cell.
  • the cellular receptor can be a receptor which mediates endocytosis of a bound ligand such as the asialoglycoprotein receptor of hepatocytes and the receptor-specific molecule can be a natural or synthetic ligand for the receptor.
  • the receptor-specific molecule can be introduced onto the surface of the virus or cell (e.g., onto a viral envelope or cellular membrane) by chemically coupling it, either directly or through bridging agents, to the surface or by treating the surface to expose the molecule for receptor recognition.
  • the method of this invention can be used to produce viral or cellular vectors for selective delivery of material such as nucleic acid (genes) to a target cell.
  • exogenous genes can be incorporated and expressed selectively in a target cell.
  • These vectors can be used in gene therapy and in other applications which call for selective genetic alteration of cells.
  • the method also provides a means for altering the natural tropism of an infective agent such as a virus or bacterium.
  • An infective agent can be modified so that it will infect a cell which, in unmodified form, it would not normally infect. In this way, animal models of human diseases which do not have adequate experimental animal counterparts can be developed for study of the diseases.
  • an ecotropic human pathogen such as the hepatitis or AIDS virus
  • Figure 1 shows in situ ⁇ -galactosidase expression in NIH 3T3, HepG2 and SK Hepl cells treated separately with unmodified or modified murine leukemia virus.
  • Figure 2 shows internalization of 3 5 S-biolabeled modified Moloney murine leukemia virus.
  • Figure 3 shows a chromatogram of asialooro- mucoid-complexed Psi2 virus on Sephadex G150.
  • Figure 4 shows the ⁇ -galactosidase activity of various cells exposed to Psi2 virus-asialoglyco- protein conjugate.
  • a virus or cell is targeted for selective internalization into a target cell by modifying the surface of the virus or cell to introduce a molecule which specifically binds to a surface receptor of the target cell.
  • the cellular surface receptor is one which will mediate internalization of the targeted virus or cell.
  • the modified virus or cell binds to the receptor of the target cell in vivo and is internalized by the cell.
  • viruses can be modified to infect specific target cells. Such modified viruses can be used to selectively deliver exogenous, functional DNA to a target cell in order confer a new biological or biochemical property upon the cell or to abrogate an existing property.
  • the tropism of a virus can be altered or redirected to target infectivity to a cell type or types not normally infected by the virus in natural (or unaltered) form.
  • a variety of different enveloped viruses can be targeted by the method of this invention.
  • the viruses can be RNA (retroviruses) or DNA viruses (e.g., hepatitis virus, adenovirus).
  • the virus can be replication defective or otherwise defective in structure or function.
  • viral particles either essentially or completely devoid of genomic nucleic acid (e.g., "empty" viral envelope) can also be targeted.
  • the present method also provides a means of targeting cells. These include cellular organisms such as bacteria, protozoa or trypanosomes whose tropism can be altered.
  • mammalian cells can be targeted.
  • the receptor-specific molecule can be a ligand for the surface receptor of the target cell.
  • the molecule is a ligand for a cellular surface receptor which mediates internalization of the ligand by the process of endocytosis, such as the asialoglycoprotein receptor of hepatocytes.
  • Glycoproteins having certain exposed terminal carbohydrate groups can be used as receptor-specific molecules.
  • asialoglycoprotein (galactose-terminal) ligands are preferred.
  • asialoglycoproteins include asialoorosomucoid or asialofetuin.
  • Other useful galactose-terminal carbohydrates for hepatocyte targeting include carbohydrate trees obtained from natural glycoproteins, especially tri- and tetra-antennary structures that either contain terminal galactose residues or can be enzymatically treated to expose terminal galactose residues.
  • naturally occurring plant carbohydrates such as arabinogalactan can be used.
  • other types of carbohydrates can be used.
  • mannose and mannose-6 phosphate or carbohydrates having these terminal carbohydrate structures could used to target macrophages or endothelial cells.
  • receptor ligands such as peptide hormones could also be used to target viruses or cells to corresponding receptors. These include insulin, glucagon, gastrin polypeptides and their respective receptors.
  • the receptor-specific molecule can be a receptor or receptor-like molecule, such as an antibody, which binds a ligand (e.g., antigen) on the cell surface.
  • a ligand e.g., antigen
  • Antibodies specific for cellular surface receptors can be produced by standard procedures.
  • the receptor-specific molecule is introduced onto the surface of the virus or cell so that it will be recognized by the cognate cellular surface receptor.
  • the receptor-specific molecule can be introduced onto the envelope of a virus or the membrane of a cell.
  • the molecule will be coupled to (or exposed on) a proteinaceous component of the surface but other components may be used.
  • the receptor-specific molecule can be introduced onto the surface of the virus or cell by different means.
  • the receptor-specific molecule is chemically coupled to the surface.
  • galactose moieties ligand for the asialoglycoprotein receptor
  • the receptor-specific molecule can be chemically coupled to components of the surface of the virus or cell through bridging agents such as biotin and avidin.
  • a biotinylated receptor-specific molecule can be linked through avidin or streptavidin to a biotinylated surface component of the virus or cell.
  • the virus or cell can be chemically treated to expose a receptor-specific molecule on the surface.
  • Surface polycarbohydrates can be enzymatically cleaved to expose desired carbohydrate residues (e.g., galactose residues) as terminal residues for specific receptor recognition and binding.
  • desired carbohydrate residues e.g., galactose residues
  • neurominidase treatment of certain polycarbohydrates leaves exposed terminal galactose residues in a tri- or tetra-antennary arrangement.
  • the modified virus or cell is administered in vivo, generally in an amount sufficient to saturate receptors of the target cell and thereby maximize uptake by the cell. They can be administered parenterally (typically intravenously) in a physiologically acceptable vehicle such as normal saline.
  • the method of this invention can be used to selectively deliver nucleic acid (DNA or RNA) to a target cell in vivo (or in vitro) so that it is expressed in the cell.
  • the nucleic acid can be an exogenous gene, a genetic regulatory element or an antisense inhibitor of gene function.
  • the nucleic acid is incorporated into a viral vector which has been modified, according to the method of this invention, to target it to the cell.
  • Preferred viral vectors for delivery of foreign genes in vivo (or ex vivo) are retroviruses.
  • the targeted viral vector is administered in vivo, as described, where i is selectively taken up by the target cell.
  • the method of this invention can be used to alter the natural tropism of an infectious agent.
  • Ecotropic (species-restricted) agents can be made to infect species which they normally, in unmodified form, do not infect.
  • the ability to target the infectivity of an infectious agent can be used to develop new experimental systems for the study of human infectious diseases to produce cells that can correct genetic defects in vivo, or target a corrective gene in vivo.
  • Certain pathogenic viruses such as hepatitis virus or human immunodeficiency virus infect only human cells.
  • such viruses can be modified to enable them to infect experimental animals such as rodents.
  • the hepatitis virus which infects only human liver cells can be modified so that it will infect non-human liver cells.
  • a ligand for rodent asialoglycoprotein receptor e.g., galactose
  • galactose asialoglycoprotein receptor
  • This modified hepatitis virus which can infect a rodent and the infected rodent or rodent cells provides an experimental animal system for study of the hepatitis virus.
  • the invention is illustrated further by the following examples.
  • a model retroviral system was used.
  • the virus an ecotropic, replication-defective, Moloney murine leukemia virus containing the gene for bacterial ⁇ -galactosidase produced in a ⁇ ere cell line was kindly provided by Dr. James Wilson, University of Michigan. Wilson, J.M., e_£ al. Proc. Natl. Acad. Sci. USA 87:439-443 (1990). Under normal circumstances, this virus infects only rodent cells. Wilson, J.M. , e_£ al. Proc. Natl. Acad. Sci. USA £5_:3014-3018 (1988); Goud, B., et al. Virology JL__3.:251-254 (1988).
  • the producer cells were grown in Dulbecco's modified Eagle's medium (GIBCO Laboratories, Grand Island, NY) supplemented with 10% heat-inactivated calf serum (GIBCO) .
  • Dulbecco's modified Eagle's medium GIBCO Laboratories, Grand Island, NY
  • heat-inactivated calf serum GIBCO
  • producer cells were cultured in serum-free Dulbecco's modified Eagle's medium for 3 days.
  • two strategies were developed for the modification of the surface of the harvested virus: A) chemical coupling of galactose residues to the virus and B) chemical coupling of an asialoglycoprotein to the virus.
  • virus-containing medium was applied on a 10-20% sugar gradient in which ⁇ -lactose was substituted for sucrose (Sigma, St. Louis, MO) in 10 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, and was ultracentrifuged (LB-55, Beckman Instruments, San Ramon, CA) at 40,000 rpm in VTi 55 rotor (Beckman) at 4°C for 17 hours.
  • Fetal bovine serum (GIBCO) was added subsequently to make a 10% solution. Except for stability experiments, all samples were used immediately after preparation. Viability of unmodified virus preparations was determined by transfection assays in NIH 3T3 mouse fibroblasts using limiting dilutions of viral stock (Danos, 0. and Mulligan, R.C. Proc. Natl. Acad. Sci. USA &_>:6460-6464 (1988)) and quantitated by determination of positive cells stained with X-gal. Sanes, J.R., et al. EMBO J. 5:3133-3142 (1986).
  • virus was biosynthetically labeled by incubation of producer cells (5.0 x 10 6 cells) in 50% serum-free and 50% serum- and methionine-free Dulbecco's modified Eagle's medium containing 10 ⁇ Ci/ml 35s_methionine (Amersham, Arlington Heights, IL) for 3 days. Virus was isolated from supernatants and modified as described above followed by dialysis against minimum essential medium.
  • human hepatoma cell lines HepG2, asialoglycoprotein receptor (+) (Schwartz, A.L., e ⁇ al. J. Biol. Chem. 25.6:8878-8881 (1981)) obtained from B.B. Knowles, Wistar Institute, Philadelphia, PA; and SK Hepl, receptor (-) from D.A. Shafritz, Albert Einstein College, of Medicine, Bronx, NY; a rat hepatoma cell line, Morris 7777, receptor (-) (Wu. G.Y., ej al. J. Biol. Chem.
  • NIH 3T3 murine fibroblast cell line NIH 3T3 (Goud, B., et aj___. Virology 163.:251-254 (1988)) which is also asialoglycoprotein receptor (-) .
  • the latter two cell lines were purchased from American Type Culture Collection (Rockville, MD) . All were maintained in Eagle's minimum essential medium supplemented with 10% heat inactivated fetal bovine serum at 37°C under 5% C ⁇ 2-
  • RNA 0.5 mg viral protein
  • Dulbecco's modified Eagle's medium were added to the culture medium and exposed to cells for 5 days at 37°C under 5% CO2.
  • Results were expressed in U/mg of cellular protein according to the method by Norton, P.A. and Coffin, J.M. Mol. Cell. Biol. 1:281-290 (1985), using purified E___ coli ⁇ -galactosidase (Sigma) activity as a standard. Protein concentrations of the cellular samples were determined using a Bio-Rad Protein Assay Kit (Bio-Rad) following the manufacturer's instructions.
  • virus was added to the cell media together with a 100-fold molar excess of a natural asialoglycoprotein, asialoorosomucoid, prepared by desialylation (Oka, J.A., and Weigel, P.H. J. Biol. Chem. 258: 10253-10262 (1983)) of orosomucoid as previously described by Whitehead., D.H., and Sam ons, H.G. Biochim. Biophys. Acta 124:209-211 (1966). Background enzyme activity was determined in corresponding untreated cells and subtracted from the values of viral-treated samples. All assays were performed in triplicate and the results expressed as means + S.E.
  • Table 1 shows that unmodified virus did not produce enzymatic activity in human HepG2 or SK Hepl cells as expected from the ecotropism of the virus. Also, modified virus did not produce ⁇ -galactosidase activity in SK Hepl, asialoglycoprotein receptor (-) cells. However, modified virus did produce high ⁇ -galactosidase activity, 71.2 ⁇ 4.8U/mg of cellular protein, in human HepG2, asialoglycoprotein receptor (+) cells. Furthermore, this enzymatic activity was completely suppressed by addition of a large molar excess of asialoorosomucoid, supporting the notion that the transfection by modified virus was, in fact, mediated by asialoglycoprotein receptors.
  • ⁇ -galactosidase activity was high, 50.6 ⁇ 5.2. in Morris 7777 rat cells after exposure to unmodified virus.
  • ⁇ -galactosidase activity in these same cells was significantly lower when exposed to the same amount of modified virus.
  • the same tendency was seen in originally susceptible murine NIH 3T3 cell as enzymatic activity after exposure to unmodified virus, 56.7 + 1.8, was more than double that following exposure to modified virus 27.0 ⁇ 0.9.
  • the coupling reaction linking lactose to protein has been shown to be enhanced under alkaline conditions. Schwartz, B.A. and Gray, G.R. Arch. Biochem. Biophys. 181:542-549 (1977).
  • virus modified at different pHs were administered to He ⁇ G2 cells, and ⁇ -galactosidase activity measured.
  • Table 2 shows that enzymatic activity rose from 50.3 + 1.2, for virus modified at pH 7.4; to 71.2 + 4.8, for virus modified at pH 8.0. However, activity was significantly lower, 25.1 + 2.4, in cells treated with virus modified at pH 8.4.
  • + virus was modified at pH 8.0 then incubated with cells for 5 days. * calculated as the difference in activity between treated and untreated cells.
  • HepG2, SK Hepl and Morris 7777 cells were incubated at 37°C in serum-free Dulbecco's modified Eagle's medium containing 3 5s-biolabeled, modified virus, 3.3 ⁇ g viral RNA (98 ⁇ g viral protein) (Watanabe, N., e£ al. Cancer Immunol. Immunother. 28:157-163 (1989)) with a specific activity of 6.1x10 ⁇ cpm/mg viral RNA.
  • Figure 2 shows that, of the two human and one rodent cell lines, only the human HepG2 asialoglycoprotein receptor (+) cells demonstrated significant specific uptake of labeled virus.
  • Specific ⁇ -galactosidase activity was calculated as the difference between samples treated with virus alone, and samples treated with modified virus plus a 100-fold molar excess of asialoorosomucoid.
  • the coupling of lactose to proteins to target artificial asialoglycoproteins is based on the specificity of sodium cyanoborohydride to reduce Schiff's bases formed between aldehyde and amino groups to render the bonds irreversible.
  • Treatment of viruses with aldehydes is not always similarly benign. For example formaldehyde has been used to inactivate viruses in the production of vaccines. Buynak, E.B., et al. J. Am. Med. Assoc. 235: 2832-2834 (1976).
  • the data presented here indicate that under the conditions described, the modification process results not only in altered specificity of infection, but also results in preservation of viral gene expression. Furthermore, the data indicate that the production of modified yet functional virus increased with increasing pH of the modification reaction up to a limit of approximately 8.0, beyond which the function of the virus became compromised. Many retroviruses have been shown to enter cells normally via endocytosis and are thought to introduce their genetic material during an acidification step in the pathway. Andersen, K.B. and Nexo, P.A. Virology 125:85-98 (1983). Although the asialoglycoprotein endocytotic pathway is ultimately degradative with delivery of ligands to lysosomes (Tolleshaug, H., ei al. Biochim.
  • asialoglycoprotein receptor (+) cells conjugated virus was incubated for 10 days with each of five cell lines: Hep G2, receptor (+); Huh-7, receptor (+); SK Hepl, human hepatoma receptor (-); Mahlavu, receptor (-) and Morris 7777, rat hepatoma receptor (-) cells.
  • Figure 4 lane 1 shows that Hep G2 receptor (+) cells treated with conjugate had beta-galactosidase activity at a level of 2.3 units/mg of cell protein which is approximately 50% of the activity of the producer cell line, BAG shown in lane 11.
  • Hep G2 cells without treatment were at a level of 1.81 units/mg.
  • Huh-7 receptor (+) cells treated with conjugate had higher levels of beta-galactosidase, 3.8 units/mg as shown in lane 3 compared to those cells treated with biotinylated virus without asialoorosomucoid present in a complex shown in lane 4. This was similar to the levels obtained from these cells that were not treated at all as seen in lane 5. Lane 6 shows that Mahlavu receptor (-) cells treated with conjugate did not have any significant beta-galactosidase activity compared to those same cells that were untreated shown in lane 7.
  • lanes 8 and 9 show that Morris 7777 cells treated with other conjugate or biotinylated virus without asialoorosomucoid, lanes 8 and 9 respectively, showed no significant beta-galactosidase activity compared to those same cells that were untreated shown in lane 10.
  • SK HEPL cells responded similarly to the receptor (-) Morris 7777 cells.
  • Hepatitis B virus is a human pathogen that possesses very narrow host (species) and organ (liver) specificities, in vitro, the virus is also very fastidious as evidenced by the fact that human hepatocytes or hepatoma cells in culture cannot be infected by HBV without unusual and highly artificial conditions such as high concentrations of corticosteroids.
  • Hepatitis B virus was obtained from Hep G2 producer cells chronically infected with HBV as described by Sells et. ai. Proc. Natl. Acad. Sci. :1005-1009 (1987), and maintained in Dulbecco's modified Eagle's medium (MEM) containing G418 as 380 mg/ml, supplemented with 10% heat inactivated fetal bovine serum.
  • MEM Dulbecco's modified Eagle's medium
  • Huh7 human hepatoma cell line which possesses asialoglycoprotein receptors and IMR-90 fibroblasts which do not possess asialoglycoprotein receptors were maintained in Dulbecco's modified Eagle's minimum essential medium supplemented with 10% fetal bovine serum (FBS) .
  • FBS fetal bovine serum
  • He ⁇ G2 cells were cultured in serum free media for three days. The medium was centrifuged at 2000 rpm to remove debris and the supernatant applied on 10-20% lactose gradient, pH 7.4, 8.0 or 8.4, and ultracentrifuged at 40000 rpm in VTi55 rotor at 4°C for 16 hours to pellet and isolate the virus.
  • HBV HBV HBV obtained (3.0 mg of protein) was lactosaminated in a similar fashion to that described in Example 1 using 10 mg of sodium cyanoborohydride for 3 hours at 25°C.
  • the modified virus was sterilized by filtration through 0.45 ⁇ m membranes and then dialyzed against MEM through membranes with a 12-14000 molecular weight exclusion limit followed by dialysis against MEM plus 10% FBS.
  • Huh7 and IMR-90 cells were plated at 25-50% confluence in 35 or 100 mm diameter plastic dishes. Cell medium was removed and replaced with medium containing modified or unmodified virus and incubated at 37°C. Cells were washed and changed to fresh medium every three days and at regular intervals cells were studied for the presence of HBV DNA and medium analyzed for the presence of hepatitis B surface antigen (HBsAg) .
  • HBsAg hepatitis B surface antigen
  • DNA was extracted from cells according to the method by Blin, N. and Stafford, D.W. Nucleic Acid Res. 2:2303-2312 (1976), in which the cells were washed twice with 10 ml of cold Tris-buffered saline (TBS), scraped off into TBS and centrifuged at 200 rpm. The cell pellet was resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, was added to the same buffer containing 20 mg/ml RNase, 0.5% SDS, and then treated with proteinase K. Cellular DNA was isolated by ethanol precipitation after phenol extraction.
  • TBS cold Tris-buffered saline
  • the DNA was analyzed by Southern blot using a ⁇ 32 P-ATP labeled cDNA probe specific for HBV sequences (a Bam HI restriction fragment of plasmid adw HTD carrying the HBV genome, obtained from Dr. Jake Liang, Massachusetts General Hospital).
  • the background color absorbance was approximately 0.121 in untreated Huh7 cells and there was no significant difference between day 1 and day 7. Unmodified HBV did not result in significant production of HBsAg. Absorbance here was approximately 0.180. Similarly, the color absorbance reflecting HBV levels in IMR-90 cells did not exceed 0.110. However, Huh7 cells treated with modified HBV released HBsAg into their supernatants, with absorbance ranging from 0.760 to 0.865. Table 4
  • HBsAg Hepatitis B Surface Antigen

Abstract

Viruses or cells are targeted for selective internalization into a target in vivo. A molecule specific for a receptor on the surface of the target cell is introduced onto the surface of the virus or cell. The modified virus or cell binds the receptor in vivo and is internalized by the target cell. The method provides vectors for selective delivery of nucleic acids to specific cell types in vivo and a means to alter the tropism of an infectious agent.

Description

TARGETIHC VIRUSES AND CELLS FOR SELECTIVE INTERNALIZATION BY CELLS
Background of the Invention
Viruses represent a natural and efficient means for the introduction of foreign genes into cells.
For this reason, they are useful tools for the study of genes, and gene regulation in vitro and for gene therapy. However, most viruses have broad cell specificity and can infect a wide variety of cell types. This can lead to foreign gene expression in many tissues, some of which may be undesirable, especially for clinical applications.
Generally, viral infection is mediated by interactions between viral envelopes and plasma membranes of target cells. In many cases, specific viral structures are recognized and bound by cellular receptors. For example, HIV employs envelope glycoproteins to bind to helper T lymphocytes via CD4 (T4) receptors. Dalgleich, A.G., et al. Nature 312:763-767 (1984). These interactions have been shown to be responsible for the observed species and organ specificity.
Some investigators have shown that virus specificity can be redirected by attaching antibodies to viruses. For example, Goud, B., et aL. Viroloσv 161:251-254 (1988) linked anti-transferrin receptor antibodies to obtain delivery of a retrovirus to human cells bearing the transferrin receptor. However, while binding and internalization occurred, infection and replication did not. A means for targeting viral or other types of nucleic acid vectors containing foreign genes to a target cell and obtaining infection and replication of the virus would be useful in gene therapy.
Summary of the Invention The invention pertains to a method of targeting a virus or a cell to a target cell for selective internalization in vivo (or i vitro) by the cell and to modified viruses and cells which are targeted for selective internalization by a target cell. A virus or cell is targeted to the target cell for internalization by introducing a receptor- specific molecule onto the surface of the virus or cell to produce a modified virus or cell which specifically binds to a receptor on the surface of the target cell. The modified virus or cell can be administered to an organism where it binds selectively to the receptor of the target cell. The receptor-binding results in internalization by the target cell. The cellular receptor can be a receptor which mediates endocytosis of a bound ligand such as the asialoglycoprotein receptor of hepatocytes and the receptor-specific molecule can be a natural or synthetic ligand for the receptor. The receptor-specific molecule can be introduced onto the surface of the virus or cell (e.g., onto a viral envelope or cellular membrane) by chemically coupling it, either directly or through bridging agents, to the surface or by treating the surface to expose the molecule for receptor recognition.
The method of this invention can be used to produce viral or cellular vectors for selective delivery of material such as nucleic acid (genes) to a target cell. For example, exogenous genes can be incorporated and expressed selectively in a target cell. These vectors can be used in gene therapy and in other applications which call for selective genetic alteration of cells. The method also provides a means for altering the natural tropism of an infective agent such as a virus or bacterium. An infective agent can be modified so that it will infect a cell which, in unmodified form, it would not normally infect. In this way, animal models of human diseases which do not have adequate experimental animal counterparts can be developed for study of the diseases. For example, an ecotropic human pathogen (such as the hepatitis or AIDS virus) can be modified to infect a non-human host to produce an experimental system for study of the pathogen and the disease. Brief Description of the Figures
Figure 1 shows in situ β-galactosidase expression in NIH 3T3, HepG2 and SK Hepl cells treated separately with unmodified or modified murine leukemia virus.
Figure 2 shows internalization of 35S-biolabeled modified Moloney murine leukemia virus.
Figure 3 shows a chromatogram of asialooro- mucoid-complexed Psi2 virus on Sephadex G150.
Figure 4 shows the β-galactosidase activity of various cells exposed to Psi2 virus-asialoglyco- protein conjugate.
Detailed Description of the Invention A virus or cell is targeted for selective internalization into a target cell by modifying the surface of the virus or cell to introduce a molecule which specifically binds to a surface receptor of the target cell. The cellular surface receptor is one which will mediate internalization of the targeted virus or cell. The modified virus or cell binds to the receptor of the target cell in vivo and is internalized by the cell.
According to the method of this invention, viruses can be modified to infect specific target cells. Such modified viruses can be used to selectively deliver exogenous, functional DNA to a target cell in order confer a new biological or biochemical property upon the cell or to abrogate an existing property. In addition, the tropism of a virus can be altered or redirected to target infectivity to a cell type or types not normally infected by the virus in natural (or unaltered) form. A variety of different enveloped viruses can be targeted by the method of this invention. The viruses can be RNA (retroviruses) or DNA viruses (e.g., hepatitis virus, adenovirus). The virus can be replication defective or otherwise defective in structure or function. For example, viral particles either essentially or completely devoid of genomic nucleic acid (e.g., "empty" viral envelope) can also be targeted. The present method also provides a means of targeting cells. These include cellular organisms such as bacteria, protozoa or trypanosomes whose tropism can be altered. In addition, mammalian cells can be targeted. The receptor-specific molecule can be a ligand for the surface receptor of the target cell. Preferably, the molecule is a ligand for a cellular surface receptor which mediates internalization of the ligand by the process of endocytosis, such as the asialoglycoprotein receptor of hepatocytes.
Glycoproteins having certain exposed terminal carbohydrate groups can be used as receptor-specific molecules. For specific targeting to hepatocytes, asialoglycoprotein (galactose-terminal) ligands are preferred. Examples of asialoglycoproteins include asialoorosomucoid or asialofetuin. Other useful galactose-terminal carbohydrates for hepatocyte targeting include carbohydrate trees obtained from natural glycoproteins, especially tri- and tetra-antennary structures that either contain terminal galactose residues or can be enzymatically treated to expose terminal galactose residues. In addition, naturally occurring plant carbohydrates, such as arabinogalactan can be used. For targeting other receptors, other types of carbohydrates can be used. For example, mannose and mannose-6 phosphate or carbohydrates having these terminal carbohydrate structures could used to target macrophages or endothelial cells.
Other receptor ligands such as peptide hormones could also be used to target viruses or cells to corresponding receptors. These include insulin, glucagon, gastrin polypeptides and their respective receptors.
Alternatively, the receptor-specific molecule can be a receptor or receptor-like molecule, such as an antibody, which binds a ligand (e.g., antigen) on the cell surface. Antibodies specific for cellular surface receptors can be produced by standard procedures.
The receptor-specific molecule is introduced onto the surface of the virus or cell so that it will be recognized by the cognate cellular surface receptor. For example, the receptor-specific molecule can be introduced onto the envelope of a virus or the membrane of a cell. In general, the molecule will be coupled to (or exposed on) a proteinaceous component of the surface but other components may be used.
The receptor-specific molecule can be introduced onto the surface of the virus or cell by different means. Preferably, the receptor-specific molecule is chemically coupled to the surface. For example, galactose moieties (ligand for the asialoglycoprotein receptor) can be covalently coupled to viral or cellular surface proteins by lactosamination, reductive amination, or via iminomethoxyethyl derivatives. In other embodiments, the receptor-specific molecule can be chemically coupled to components of the surface of the virus or cell through bridging agents such as biotin and avidin. For instance, a biotinylated receptor-specific molecule can be linked through avidin or streptavidin to a biotinylated surface component of the virus or cell.
Alternatively, the virus or cell can be chemically treated to expose a receptor-specific molecule on the surface. Surface polycarbohydrates can be enzymatically cleaved to expose desired carbohydrate residues (e.g., galactose residues) as terminal residues for specific receptor recognition and binding. For example, neurominidase treatment of certain polycarbohydrates leaves exposed terminal galactose residues in a tri- or tetra-antennary arrangement.
The modified virus or cell is administered in vivo, generally in an amount sufficient to saturate receptors of the target cell and thereby maximize uptake by the cell. They can be administered parenterally (typically intravenously) in a physiologically acceptable vehicle such as normal saline.
The method of this invention can be used to selectively deliver nucleic acid (DNA or RNA) to a target cell in vivo (or in vitro) so that it is expressed in the cell. The nucleic acid can be an exogenous gene, a genetic regulatory element or an antisense inhibitor of gene function. The nucleic acid is incorporated into a viral vector which has been modified, according to the method of this invention, to target it to the cell. Preferred viral vectors for delivery of foreign genes in vivo (or ex vivo) are retroviruses. The targeted viral vector is administered in vivo, as described, where i is selectively taken up by the target cell.
The method of this invention can be used to alter the natural tropism of an infectious agent. Ecotropic (species-restricted) agents can be made to infect species which they normally, in unmodified form, do not infect. The ability to target the infectivity of an infectious agent can be used to develop new experimental systems for the study of human infectious diseases to produce cells that can correct genetic defects in vivo, or target a corrective gene in vivo.
Certain pathogenic viruses such as hepatitis virus or human immunodeficiency virus infect only human cells. By the method of this invention, such viruses can be modified to enable them to infect experimental animals such as rodents. For example, the hepatitis virus which infects only human liver cells, can be modified so that it will infect non-human liver cells. To develop rodent models of hepatitis, for example, a ligand for rodent asialoglycoprotein receptor (e.g., galactose) can be introduced onto the surface of the hepatitis virus. This yields a modified hepatitis virus which will infect rodent liver cells. This modified hepatitis virus which can infect a rodent and the infected rodent or rodent cells, provides an experimental animal system for study of the hepatitis virus. The invention is illustrated further by the following examples.
EXAMPLE 1
Chemical Modification and Alteration of Host Cell Specificity of a Retrovirus
A model retroviral system was used. The virus, an ecotropic, replication-defective, Moloney murine leukemia virus containing the gene for bacterial β-galactosidase produced in a ψ ere cell line was kindly provided by Dr. James Wilson, University of Michigan. Wilson, J.M., e_£ al. Proc. Natl. Acad. Sci. USA 87:439-443 (1990). Under normal circumstances, this virus infects only rodent cells. Wilson, J.M. , e_£ al. Proc. Natl. Acad. Sci. USA £5_:3014-3018 (1988); Goud, B., et al. Virology JL__3.:251-254 (1988). The producer cells were grown in Dulbecco's modified Eagle's medium (GIBCO Laboratories, Grand Island, NY) supplemented with 10% heat-inactivated calf serum (GIBCO) . To prepare virus with as little contamination as possible from serum proteins, producer cells were cultured in serum-free Dulbecco's modified Eagle's medium for 3 days. Using this viral preparation, two strategies were developed for the modification of the surface of the harvested virus: A) chemical coupling of galactose residues to the virus and B) chemical coupling of an asialoglycoprotein to the virus. A. LACTOSAMINATION OF RETROVIRUSES
Virus was isolated from the culture medium according to the method of Goud, B., e£ al. Virology 163:251-254 (1988), but modified to permit coupling of lactose during the isolation procedure. In brief, virus-containing medium was applied on a 10-20% sugar gradient in which α-lactose was substituted for sucrose (Sigma, St. Louis, MO) in 10 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, and was ultracentrifuged (LB-55, Beckman Instruments, San Ramon, CA) at 40,000 rpm in VTi 55 rotor (Beckman) at 4°C for 17 hours. Samples were adjusted to various pHs, 7.4-8.4, prior to centrifugation in order to determine optimal conditions for modification. After centrifugation, the bottom fraction of the lactose gradient containing 3.0 mg of protein (0.1 mg viral RNA) was reacted with sodium cyanoborohydride (Sigma) as described previously. Goud, B., e_£ al. Virology 163:251-254 (1988). Following dialysis against minimum essential medium at 4°C for 24 hours, the samples were sterilized by passage through 0.45μ filters (Gelman Science Co., Ann Arbor, MI). Quantitation of the amount of virus present in samples prior to exposure to cells was determined by protein assay (Bio-Rad, Los Angeles, CA) according to the manufacturer's instructions, and confirmed by RNA assay (Nunro, H.N. and Fleck, A. Meth. Biochem. Anal. 14.:113-176 (1966)) after RNA extraction. ° Chomczynski, P. and Sacci, N. , Anal. Biochem.
162:156-159 (1987). Fetal bovine serum (GIBCO) was added subsequently to make a 10% solution. Except for stability experiments, all samples were used immediately after preparation. Viability of unmodified virus preparations was determined by transfection assays in NIH 3T3 mouse fibroblasts using limiting dilutions of viral stock (Danos, 0. and Mulligan, R.C. Proc. Natl. Acad. Sci. USA &_>:6460-6464 (1988)) and quantitated by determination of positive cells stained with X-gal. Sanes, J.R., et al. EMBO J. 5:3133-3142 (1986).
For uptake studies, virus was biosynthetically labeled by incubation of producer cells (5.0 x 106 cells) in 50% serum-free and 50% serum- and methionine-free Dulbecco's modified Eagle's medium containing 10 μCi/ml 35s_methionine (Amersham, Arlington Heights, IL) for 3 days. Virus was isolated from supernatants and modified as described above followed by dialysis against minimum essential medium.
Cells and Cell Culture
To evaluate the effects of chemical modification on viral infection specificity, several cell lines were employed: human hepatoma cell lines, HepG2, asialoglycoprotein receptor (+) (Schwartz, A.L., e± al. J. Biol. Chem. 25.6:8878-8881 (1981)) obtained from B.B. Knowles, Wistar Institute, Philadelphia, PA; and SK Hepl, receptor (-) from D.A. Shafritz, Albert Einstein College, of Medicine, Bronx, NY; a rat hepatoma cell line, Morris 7777, receptor (-) (Wu. G.Y., ej al. J. Biol. Chem. 263: 4719-4723 (1988)); and a murine fibroblast cell line NIH 3T3 (Goud, B., et aj___. Virology 163.:251-254 (1988)) which is also asialoglycoprotein receptor (-) . The latter two cell lines were purchased from American Type Culture Collection (Rockville, MD) . All were maintained in Eagle's minimum essential medium supplemented with 10% heat inactivated fetal bovine serum at 37°C under 5% Cθ2-
Assays for Viral Infection and Functional Gene Expression
In order to determine whether virus remained infectious and functional after chemical modification, the two human and two rodent cell lines were exposed to modified and unmodified virus followed by measurement of cellular β-galactosidase activity. Target cells were plated at a density of 0.5-2.0 x 105 cells/ml in 60 mm plastic dishes (Falcon Scientific Co, Lincoln Park, NJ) . Equal amounts, 16.7 μg RNA, (0.5 mg viral protein) of modified and unmodified virus, in Dulbecco's modified Eagle's medium were added to the culture medium and exposed to cells for 5 days at 37°C under 5% CO2. Cells were assayed for β-galactosidase activity as a measure of foreign gene expression according to the method of Gorman (Gorman, C. DNA Cloning, vol. 2 eds, Glover, D.M. IRL Press, Washington D.C. pp 157-158 (1986)). In brief, cell monolayers (approximately lxlO6 cells/60 mm dish) were washed with phosphate buffered saline, then lysed. The lysate, 0.1 ml, was reacted with o-nitrophenyl-galactopyranoside (ONPG, Sigma) and β-galactosidase activity quantitated by absorbance at 420 nm after addition of a Cθ3 to terminate the reaction. Results were expressed in U/mg of cellular protein according to the method by Norton, P.A. and Coffin, J.M. Mol. Cell. Biol. 1:281-290 (1985), using purified E___ coli β-galactosidase (Sigma) activity as a standard. Protein concentrations of the cellular samples were determined using a Bio-Rad Protein Assay Kit (Bio-Rad) following the manufacturer's instructions. For competition experiments, virus was added to the cell media together with a 100-fold molar excess of a natural asialoglycoprotein, asialoorosomucoid, prepared by desialylation (Oka, J.A., and Weigel, P.H. J. Biol. Chem. 258: 10253-10262 (1983)) of orosomucoid as previously described by Whitehead., D.H., and Sam ons, H.G. Biochim. Biophys. Acta 124:209-211 (1966). Background enzyme activity was determined in corresponding untreated cells and subtracted from the values of viral-treated samples. All assays were performed in triplicate and the results expressed as means + S.E. Table 1 shows that unmodified virus did not produce enzymatic activity in human HepG2 or SK Hepl cells as expected from the ecotropism of the virus. Also, modified virus did not produce β-galactosidase activity in SK Hepl, asialoglycoprotein receptor (-) cells. However, modified virus did produce high β-galactosidase activity, 71.2 ± 4.8U/mg of cellular protein, in human HepG2, asialoglycoprotein receptor (+) cells. Furthermore, this enzymatic activity was completely suppressed by addition of a large molar excess of asialoorosomucoid, supporting the notion that the transfection by modified virus was, in fact, mediated by asialoglycoprotein receptors. As expected from the ecotropism, β-galactosidase activity was high, 50.6 ± 5.2. in Morris 7777 rat cells after exposure to unmodified virus. Interestingly, β-galactosidase activity in these same cells was significantly lower when exposed to the same amount of modified virus. The same tendency was seen in originally susceptible murine NIH 3T3 cell as enzymatic activity after exposure to unmodified virus, 56.7 + 1.8, was more than double that following exposure to modified virus 27.0 ± 0.9. The coupling reaction linking lactose to protein has been shown to be enhanced under alkaline conditions. Schwartz, B.A. and Gray, G.R. Arch. Biochem. Biophys. 181:542-549 (1977). However, such conditions could be detrimental to the virus. To determine the optimal pH that results in modified, yet functional vectors, virus modified at different pHs were administered to HeρG2 cells, and β-galactosidase activity measured. Table 2 shows that enzymatic activity rose from 50.3 + 1.2, for virus modified at pH 7.4; to 71.2 + 4.8, for virus modified at pH 8.0. However, activity was significantly lower, 25.1 + 2.4, in cells treated with virus modified at pH 8.4.
Table 1
Cellular β-Galactosidase Activity Following Exposure to Viral Preparations-i-
β-Galactosidase Activity* Mean ± S.E. (U/mg)
AsG Unmodified Modified Modified Receptor Virus Virus Virus + Status ASOR**
Cell Line (Source) HepG2
(human) (+) 1.8 + 1.9 71.2 ± 4.8 2.9 + 1.1
SK Hepl
(human) (-) 1.7 ± 3.4 0.8 ± 4.6 1.6 ± 2.5
Morris 7777 (rat) (-) 50.6 ± 5.2 16.3 ± 4.4 15.7 ± 4.7
NIH 3T3
(mouse) (-) 52.1 + 4.9 15.4 + 1.1 16.3 + 3.9
+ virus was modified at pH 8.0 then incubated with cells for 5 days. * calculated as the difference in activity between treated and untreated cells.
** asialoorosomucoid (ASOR) in 100-fold molar excess.
AsG, Asialoglycoprotein Table 2
Effect of the pH During Modification of Viral Transfection in HepG2 Cells
Specific β-Galactosidase Activity (Mean ± S.E. U/mg protein)*
Modified Virus Modified Virus
+ Asialoorosomucoid** S ■
7.0 50.3 ± 1.2 6.4 ± 1.9
8.0 71.8 ± 4.1 4.9 ± 0.4 8.4 25.1 ± 2.4 0.0 ± 1.6
* after 5 days of exposure to modified virus.
** β-galactosidase activity of samples treated with modified virus plus a 100-fold molar excess of asialoorosomucoid.
Histochemical Staining to Demonstrate β-Galactosidase Activity
To confirm the colorimetric results, and to determine the fraction of cells that expressed the β-galactosidase gene after exposure to viral samples, histochemical staining of in situ β-galactosidase activity was performed according to the method of Sanes e_t al. EMBO J. 1:3133-3142 (1986). In brief, cultured cells in 35 mm dishes containing 0.5 -1 x 10*^ cells treated for 5 days with equal amounts, 8.4 μg of viral RNA (0.3 mg viral protein), of modified or unmodified virus. Cells were fixed in 0.5% glutaraldehyde (Sigma), phosphate buffered saline, then incubated with 1 mM MgCl2/ phosphate buffered saline, and overlaid with lmg/ml 4-Cl-5-Br-3-indoylyl-β-galactosidase (X-gal) (BRL, Washington, D.C.), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 2 mM MgCl2 in phosphate buffered saline. After incubation at 37°C for 1 hour, the dishes were washed in phosphate buffered saline to quench the reaction and evaluated by counting positive (blue) cells under a light microscope and the results expressed as the percent of positive/10 high power fields.
In situ staining for β-galactosidase activity in cells treated with various viral preparations is shown in Figure 1. In rodent NIH 3T3 cells treated with unmodified virus, panel B, 12.6% were positive for β-galactosidase activity using the X-gal stain. Background staining in untreated NIH 3T3 cells was not detectable, panel A. After exposure to modified virus, only 3.6% were positive under otherwise identical conditions, panel C. Human SK Hepl cells that were untreated, panel H, or exposed to either unmodified virus, panel I, or modified virus, panel J, failed to develop detectable staining. Similarly, HepG2, asialoglycoprotein receptor (+) cells treated with unmodified virus, panel E, did not develop evidence of significant β-galactosidase activity. However, HepG2, receptor (+) cells treated with modified virus, panel F, did develop substantial staining. Microscopic counting revealed that 36.4 % of the HepG2, cells possessed detectable marker enzyme. The observed color development was completely suppressed by addition of a 100-fold molar excess asialoorosomucoid to compete for uptake by asialoglycoprotein receptors, panel G, indicating involvement of asialoglycoprotein receptors in the transfection process.
Assays for Cellular Uptake of Virus
To determine whether the modified virus was actually taken up by cells and, if so, whether asialoglycoprotein receptors were involved, HepG2, SK Hepl and Morris 7777 cells, 5.0x10s cells/35 mm dish, were incubated at 37°C in serum-free Dulbecco's modified Eagle's medium containing 35s-biolabeled, modified virus, 3.3 μg viral RNA (98 μg viral protein) (Watanabe, N., e£ al. Cancer Immunol. Immunother. 28:157-163 (1989)) with a specific activity of 6.1x10^ cpm/mg viral RNA. At various times, medium was removed, and cells were chilled to 4°C, washed with ice-cold minimum essential medium containing lmg/ml bovine serum albumin. Surface-bound radioactivity was stripped with cold 0.5 ml phosphate buffered saline, pH 7.2 containing 0.4% trypsin, 0.02% EDTA and separated from cells by centrifugation. The cell pellet was solubilized in 0.2 N NaOH and Poly-Fluor (Packard, Chicago, IL) , and trypsin-EDTA resistant
(internalized) radioactivity was measured by scintillation counting (TRI-CARB 4530, Packard). Schwartz, A.L., ej al. J. Biol. Chem. 256:8878-8881 (1981). Non-specific uptake was measured in the presence of a 100-fold molar excess of asialoorosomucoid, and specific uptake calculated as the difference between total and non-specific measurements. All assays were performed in triplicate and the results expressed as means ± S.E. in terms of ng viral RNA/10^ cells as a function of time.
Figure 2 shows that, of the two human and one rodent cell lines, only the human HepG2 asialoglycoprotein receptor (+) cells demonstrated significant specific uptake of labeled virus.
Counts resistant to EDTA and trypsin, increased as a function of time and continued to rise linearly through 120 minutes of incubation at a rate of approximately 800 ng viral protein/hr/105 cells. These data further support the notion that the observed expression of the galactosidase gene by modified virus was in fact due to internalization of the virus by asialoglycoprotein receptors.
Stability of Modified Virus To assess the stability of modified virus, samples of freshly prepared sterile, modified virus were incubated in serum-free Dulbecco's modified Eagle's medium at 4°C and 25°C. At various times, samples were added to the medium of HepG2 cells and incubated for 5 days. Cells were then assayed for β-galactosidase activity by colorimetric assay as described above. All assays were performed in triplicate and the results expressed as means ± S.E, in terms of U/mg cell protein normalized for the amount of virus added as a function of time of incubation. Table 3 shows that enzymatic activity at both 4 °C and 25°C, decreased with time to approximately 50% of original activity after 48 hours.
Table 3
S abilit of Mo ifi Vir
Figure imgf000022_0001
* after 5 days of exposure to modified virus
Specific β-galactosidase activity was calculated as the difference between samples treated with virus alone, and samples treated with modified virus plus a 100-fold molar excess of asialoorosomucoid. The coupling of lactose to proteins to target artificial asialoglycoproteins is based on the specificity of sodium cyanoborohydride to reduce Schiff's bases formed between aldehyde and amino groups to render the bonds irreversible. Treatment of viruses with aldehydes is not always similarly benign. For example formaldehyde has been used to inactivate viruses in the production of vaccines. Buynak, E.B., et al. J. Am. Med. Assoc. 235: 2832-2834 (1976). The data presented here indicate that under the conditions described, the modification process results not only in altered specificity of infection, but also results in preservation of viral gene expression. Furthermore, the data indicate that the production of modified yet functional virus increased with increasing pH of the modification reaction up to a limit of approximately 8.0, beyond which the function of the virus became compromised. Many retroviruses have been shown to enter cells normally via endocytosis and are thought to introduce their genetic material during an acidification step in the pathway. Andersen, K.B. and Nexo, P.A. Virology 125:85-98 (1983). Although the asialoglycoprotein endocytotic pathway is ultimately degradative with delivery of ligands to lysosomes (Tolleshaug, H., ei al. Biochim. Biophvs. Acta 585:71-84 (1979)), early in the internalization process, endosomal endocytotic compartments are acidified prior to fusion with lysosomes. Tycko, B. and Maxfield, R.F. Cell 28:643-651 (1982). This period of acid exposure may be analogous to the natural route of entry for some viruses (Nussbaum, 0., and Loyter, A. FEBS Lett. 221:61-67 (1987)) and may provide the requisite conditions for acid- mediated fusion of the viral envelope of endosomal membrane prior to destruction of the virus.
Helenius, A. Biol. Cell 51:181-186 (1984). The fact that modified virus is still able to introduce its genome into target cells suggests that the process of chemical modification did not abolish the function of those elements of the virus.
B. VIRUS-ASIALOGLYCOPROTEIN CONJUGATES
Crude preparations of virus obtained by low-speed centrifugation of medium from producer BAG cells followed by high-speed centrifugation through a discontinuous sucrose gradient as described previously was dialyzed against 0.9% saline, pH 7.5, at 4°C. After dialysis, NHS-LC-biotin (Pierce Chemical Co., Rockville, IL) was reacted with the virus (0.1 mg/ml of virus) at room temperature for four hours. The sample was then dialyzed against 0.9% saline, pH 7.5 at 4°C. Asialoorosomucoid (AsOR) was obtained by desialylation of serum orosomucoid originally derived from pooled human serum. Whitehead, D.H. and Sammons, H.P. Biochem. Biophvs. Acta 124:209 (1966). AsOR 0.1 mg was added to 1.0 mg of virus, thoroughly mixed and then 1.0 mg of avidin per mg of virus was added and allowed to incubate at room temperature for four hours. The complex was then dialyzed against Modified Eagle's Medium. Complexed virus was purified on a Sephadex G150 molecular sieve column. To determine conditions for purification, a viral complex was prepared in which asialoorosomucoid was labeled with 125I. Figure 3 shows that asialoorosomucoid alone was eluted from the column beginning at fraction number 32. Avidin, as detected by its optical density at 280 nm, eluted slightly later beginning at tube 33. However, unlabeled virus alone was much larger than either of the other two proteins and was eluted earlier with a peak at tube 29. The column was able to completely resolve virus from asialoorosomucoid and avidin. Figure 3 also shows that this virus complexed with l2^I-labeled AsOR mediated by biotin-avidin bonds, the radioactivity from the AsOR moved to the same position as expected for the intact virus, namely with a peak at tube 29. These data indicate that some labeled asialoorosomucoid was bound by the virus and migrated with it through the column.
In order to determine whether this complex could be used to target gene expression specifically to asialoglycoprotein receptor (+) cells, conjugated virus was incubated for 10 days with each of five cell lines: Hep G2, receptor (+); Huh-7, receptor (+); SK Hepl, human hepatoma receptor (-); Mahlavu, receptor (-) and Morris 7777, rat hepatoma receptor (-) cells. Figure 4, lane 1 shows that Hep G2 receptor (+) cells treated with conjugate had beta-galactosidase activity at a level of 2.3 units/mg of cell protein which is approximately 50% of the activity of the producer cell line, BAG shown in lane 11. Hep G2 cells without treatment were at a level of 1.81 units/mg. Huh-7 receptor (+) cells treated with conjugate had higher levels of beta-galactosidase, 3.8 units/mg as shown in lane 3 compared to those cells treated with biotinylated virus without asialoorosomucoid present in a complex shown in lane 4. This was similar to the levels obtained from these cells that were not treated at all as seen in lane 5. Lane 6 shows that Mahlavu receptor (-) cells treated with conjugate did not have any significant beta-galactosidase activity compared to those same cells that were untreated shown in lane 7. Similarly lanes 8 and 9 show that Morris 7777 cells treated with other conjugate or biotinylated virus without asialoorosomucoid, lanes 8 and 9 respectively, showed no significant beta-galactosidase activity compared to those same cells that were untreated shown in lane 10. SK HEPL cells responded similarly to the receptor (-) Morris 7777 cells.
In the staining procedure described in Example 1, Hep G2 cells treated with the conjugated virus produced a bluish coloration as did the Huh-7 cells treated similarly. However, cells that did not receive treatment had no staining.
EXAMPLE 2
Chemical Modification and Alteration of Host Cell Specificity of Hepatitis B Virus (HBV) Hepatitis B virus is a human pathogen that possesses very narrow host (species) and organ (liver) specificities, in vitro, the virus is also very fastidious as evidenced by the fact that human hepatocytes or hepatoma cells in culture cannot be infected by HBV without unusual and highly artificial conditions such as high concentrations of corticosteroids. Cells and Cell Culture
Hepatitis B virus (HBV) was obtained from Hep G2 producer cells chronically infected with HBV as described by Sells et. ai. Proc. Natl. Acad. Sci. :1005-1009 (1987), and maintained in Dulbecco's modified Eagle's medium (MEM) containing G418 as 380 mg/ml, supplemented with 10% heat inactivated fetal bovine serum. To test the infectivity and specificity of unmodified and modified HBV, two human cell lines were cultured. Huh7 human hepatoma cell line which possesses asialoglycoprotein receptors and IMR-90 fibroblasts which do not possess asialoglycoprotein receptors were maintained in Dulbecco's modified Eagle's minimum essential medium supplemented with 10% fetal bovine serum (FBS) .
Isolation of HBV
HeρG2 cells were cultured in serum free media for three days. The medium was centrifuged at 2000 rpm to remove debris and the supernatant applied on 10-20% lactose gradient, pH 7.4, 8.0 or 8.4, and ultracentrifuged at 40000 rpm in VTi55 rotor at 4°C for 16 hours to pellet and isolate the virus.
Chemical Modification of HBV HBV obtained (3.0 mg of protein) was lactosaminated in a similar fashion to that described in Example 1 using 10 mg of sodium cyanoborohydride for 3 hours at 25°C. The modified virus was sterilized by filtration through 0.45 μm membranes and then dialyzed against MEM through membranes with a 12-14000 molecular weight exclusion limit followed by dialysis against MEM plus 10% FBS. Infection of Cells with Unmodified and Modified HBV
Huh7 and IMR-90 cells were plated at 25-50% confluence in 35 or 100 mm diameter plastic dishes. Cell medium was removed and replaced with medium containing modified or unmodified virus and incubated at 37°C. Cells were washed and changed to fresh medium every three days and at regular intervals cells were studied for the presence of HBV DNA and medium analyzed for the presence of hepatitis B surface antigen (HBsAg) .
Detection of Targeted HBV DNA in Huh7 Cells Treated with Modified and Unmodified HBV
DNA was extracted from cells according to the method by Blin, N. and Stafford, D.W. Nucleic Acid Res. 2:2303-2312 (1976), in which the cells were washed twice with 10 ml of cold Tris-buffered saline (TBS), scraped off into TBS and centrifuged at 200 rpm. The cell pellet was resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, was added to the same buffer containing 20 mg/ml RNase, 0.5% SDS, and then treated with proteinase K. Cellular DNA was isolated by ethanol precipitation after phenol extraction. The DNA was analyzed by Southern blot using a γ32P-ATP labeled cDNA probe specific for HBV sequences (a Bam HI restriction fragment of plasmid adw HTD carrying the HBV genome, obtained from Dr. Jake Liang, Massachusetts General Hospital).
The Southern blot showed no hybridizable sequences when probed with our cDNA probe specific for HBV. This confirms the previous finding that Huh7 cells, even though of human origin, cannot be infected by unmodified HBV under the conditions of routine cell culture. In addition, the data indicate that the washing procedures eliminate any detectable non-specifically bound HBV DNA on these cells. However, treatment of the Huh7 cells with modified HBV for as little as one day of incubation resulted in a strong signal of hybridizable bands on the Southern blot corresponding to those expected for the plasmid sequences. IMR-90, asialoglycoprotein (-) cells did not produce hybridizable sequences under any conditions.
Detection of HBsAg in the Supernatant of Huh7 and IMR-90 Cells Exposed to Unmodified or Modified HBV
Medium from Huh7 and IMR-90 cells was incubated with modified or unmodified HBV as described above and at various intervals was assayed for HBsAg by an enzyme i munoassay kit (Auszyme Monoclonal) . The conditions were those recommended by the manufacturer, Abbott Labs.
As shown in Table 4, the background color absorbance was approximately 0.121 in untreated Huh7 cells and there was no significant difference between day 1 and day 7. Unmodified HBV did not result in significant production of HBsAg. Absorbance here was approximately 0.180. Similarly, the color absorbance reflecting HBV levels in IMR-90 cells did not exceed 0.110. However, Huh7 cells treated with modified HBV released HBsAg into their supernatants, with absorbance ranging from 0.760 to 0.865. Table 4
Levels of Hepatitis B Surface Antigen (HBsAg) in
Culture Medium as Determined by Auszyme Assay
(Absorbance Units)
Cells
IMR-90 Huh7
Day Modified Untreated Unmodified Modified HBV HBV HBV
1 .121 ± .054 .135 ± .017 .850 ± .010 3 .186 + .036 .700 ± .012 5 .171 ± .010 .865 ± .053 7 .110 + .023 .764 + .067
Eguivalents
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims

1. A method of targeting a virus or a cell for internalization into a target cell, comprising introducing onto the surface of the virus, or cell, a molecule which binds to a surface receptor of the target cell to produce a modified virus or cell which binds to the receptor, is internalized selectively by the cell ijri vivo and expresses the delivered nucleic acid.
2. The method of claim 1, wherein the virus or cell is a bacterium, a protozoan or a mammalian cell.
3. The method of claim 1, wherein the virus or cell, in unmodified form, is not normally internalized by the target cell.
4. The method of claim 3, wherein the virus or cell is a human pathogen and the target cell is a nonhuman cell.
5. A method of targeting the internalization of a virus or viral component into a target cell, comprising introducing onto the surface of the virus or viral component a molecule which binds to a receptor of the target cell to produce a modified virus or viral component which binds to the receptor and is internalized selectively by the cell.
6. The method of claim 5, wherein the virus is infective.
7. The method of claim 5, wherein the virus or viral component is replication defective.
8. The method of claim 5, wherein the virus is a retrovirus.
9. The method of claim 5, wherein the virus, or viral component, in unmodified form, does not infect the cell.
10. The method of claim 9, wherein the virus is a human pathogen and the target cell is a nonhuman cell.
11. The method of claim 5, wherein the virus is a pathogen for hepatocytes.
12. The method of claim 10, wherein the virus is a hepatitis virus.
13. The method of claim 12, wherein the receptor mediates endocytosis of the molecule by the cell.
14. The method of claim 13, wherein the receptor is an asialoglycoprotein receptor, the molecule introduced onto the surface of the virus, or viral component, is a ligand for the asialoglycoprotein receptor and the targeted cell bears an asialoglycoprotein receptor.
15. The method of claim 13, wherein the ligand for the asialoglycoprotein receptor is galactose or N-acetyl galactosamine and the target cell bearing an asialoglycoprotein receptor is an hepatocyte.
16. The method of claim 5, wherein the molecule is introduced onto the surface of the virus or viral component by chemical coupling.
17. A method of targeting the infectivity of a virus to a cell bearing an asialoglycoprotein receptor, comprising introducing onto the surface of the virus a ligand for the asialoglycoprotein receptor to produce a modified virus which infects a cell bearing asialoglycoprotein receptor.
18. The method of claim 17, wherein the ligand for the asialoglycoprotein receptor is lactose or galactose.
19. The method of claim 17, wherein the cell bearing the asialoglycoprotein receptor is an hepatocyte.
20. The method of claim 17, wherein the virus is a human pathogen and the cell is a non-human cell
21. The method of claim 20, wherein the virus is hepatitis virus.
22. A modified virus, or component thereof, having on its surface a molecule which binds to a surface component of a cell which is not normally infectable by the virus in its unmodified form, the modified virus, or component thereof, being capable of binding to and being internalized by the cell.
23. The modified virus of claim 22, wherein the cellular surface component of the cell is a receptor which mediates endocytosis by the cell.
24. The modified virus of claim 23, wherein the receptor is an asialoglycoprotein receptor and the molecule is a ligand for the asialoglyco¬ protein receptor.
25. The modified virus of claim 22, which is a human pathogen.
26. The modified virus of claim 25, which is a hepatitis virus.
27. Modified hepatitis virus containing lactose or galactose terminal carbohydrates on its surface.
28. A method of introducing nucleic acid into a cell, comprising: a) incorporating the nucleic acid into a viral vector comprising a modified virus, or viral component, containing a molecule on its surface which binds to a surface component of the cell; and b) contacting the viral vector and the cell under conditions which allow the vector to become internalized by the cell and expresses the introduced nucleic acid.
29. The method of claim 28, wherein the virus, or component thereof, in unmodified form, does not ordinarily infect the cell.
30. The method of claim 28, wherein the nucleic acid is an expressible gene.
31. The method of claim 28, wherein the virus is a retrovirus.
32. The method of claim 28, wherein the molecule introduced onto the surface of a virus or viral component is a galactose derivative, the cellular surface component is a ligand for the asialoglycoprotein receptor and the cell bears an asialoglycoprotein receptor.
33. The method of claim 32, wherein the cell bearing an asialoglycoprotein receptor is an hepatocyte.
34. A method of infecting an animal cell with a human virus that, in unmodified form, does not normally infect the animal cell, comprising providing a modified human virus having on its surface a molecule which binds to a surface component of the animal cell, the modified human virus being capable of binding to and infecting the animal cell and contacting the modified virus and the cell under conditions which allow the modified virus to bind to and infect the cell.
35. The method of claim 34, wherein the human virus is a human pathogen.
36. The method of claim 34, wherein the animal cell and the modified virus are contacted iri vivo.
37. The method of claim 34, wherein the animal cell and the modified virus are contacted in vitro.
38. An animal cell infected with a modified human virus, the cell being uninfectable by the virus in unmodified form.
39. The animal cell of claim 38, comprising an hepatocyte infected with hepatitis virus.
40. An animal infected with a modified human virus, the animal being uninfectable by the virus in unmodified form.
PCT/US1991/007103 1990-10-01 1991-09-27 Targeting viruses and cells for selective internalization by cells WO1992006180A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU88603/91A AU660629B2 (en) 1990-10-01 1991-09-27 Targeting viruses and cells for selective internalization by cells
JP3517570A JPH07500961A (en) 1990-10-01 1991-09-27 Targeting viruses and cells for selective internalization by cells

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US59095690A 1990-10-01 1990-10-01
US590,956 1990-10-01
US72270091A 1991-06-28 1991-06-28
US722,700 1991-06-28

Publications (1)

Publication Number Publication Date
WO1992006180A1 true WO1992006180A1 (en) 1992-04-16

Family

ID=27081003

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/007103 WO1992006180A1 (en) 1990-10-01 1991-09-27 Targeting viruses and cells for selective internalization by cells

Country Status (5)

Country Link
EP (1) EP0553235A1 (en)
JP (1) JPH07500961A (en)
AU (1) AU660629B2 (en)
CA (1) CA2092323A1 (en)
WO (1) WO1992006180A1 (en)

Cited By (181)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993009221A1 (en) * 1991-10-30 1993-05-13 Thera Gene Hb Targeted delivery of virus vector to mammalian cells
WO1993022433A2 (en) * 1992-04-28 1993-11-11 Frank Andreas Harald Meyer Medicament for the gene-therapeutic treatment of human beings, animals and plants, especially to block virus multiplication and carcinogenes and process for producing the medicament
WO1994000588A1 (en) * 1992-06-26 1994-01-06 British Technology Group Ltd. Protein based delivery system
WO1994006923A1 (en) * 1992-09-24 1994-03-31 The University Of Connecticut Modification of a virus to redirect infectivity and enhance targeted delivery of polynucleotides to cells
WO1994010323A1 (en) * 1992-11-04 1994-05-11 Imperial Cancer Research Technology Limited Virus with modified binding moiety specific for the target cells
WO1994024299A1 (en) * 1993-04-08 1994-10-27 Boehringer Ingelheim International Gmbh Adenovirus for the transfer of foreign dna into higher eucaryotic cells
EP0672129A1 (en) * 1992-11-20 1995-09-20 University Of Medicine & Dentistry Of New Jersey Cell-type specific gene transfer using retroviral vectors containing antibody-envelope fusion proteins
WO1995031566A1 (en) * 1994-05-13 1995-11-23 Chiron Viagene, Inc. Compositions and methods for targeting gene delivery vehicles
US5521291A (en) * 1991-09-30 1996-05-28 Boehringer Ingelheim International, Gmbh Conjugates for introducing nucleic acid into higher eucaryotic cells
US5547932A (en) * 1991-09-30 1996-08-20 Boehringer Ingelheim International Gmbh Composition for introducing nucleic acid complexes into higher eucaryotic cells
EP0746625A1 (en) * 1992-11-09 1996-12-11 UNITED STATES GOVERNMENT as represented by THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES Targetable vector particles
US5645829A (en) * 1993-06-18 1997-07-08 Beth Israel Hospital Association Mesothelial cell gene therapy
US5728399A (en) * 1994-06-29 1998-03-17 University Of Conn. Use of a bacterial component to enhance targeted delivery of polynucleotides to cells
US5837533A (en) * 1994-09-28 1998-11-17 American Home Products Corporation Complexes comprising a nucleic acid bound to a cationic polyamine having an endosome disruption agent
US5869331A (en) * 1992-11-20 1999-02-09 University Of Medicine & Dentistry Of New Jersey Cell type specific gene transfer using retroviral vectors containing antibody-envelope fusion proteins and wild-type envelope fusion proteins
US5889169A (en) * 1991-05-16 1999-03-30 Cold Spring Harbor Laboratory Cell cycle regulatory protein p16 gene
US5922859A (en) * 1992-02-01 1999-07-13 Boehringer Ingelheim International Gmbh Complexes containing nucleic acid which can be taken-up by endocytosis into higher eukaryotic cells
US5962316A (en) * 1992-10-16 1999-10-05 Cold Spring Harbor Laboratory Cell-cycle regulatory proteins, and uses related thereto
WO1999051748A2 (en) 1998-04-07 1999-10-14 Corixa Corporation Fusion proteins of mycobacterium tuberculosis antigens and their uses
US5981273A (en) * 1991-09-30 1999-11-09 Boehringer Ingelheim Int'l. Gmbh Composition comprising an endosomolytic agent for introducing nucleic acid complexes into higher eucaryotic cells
US6004798A (en) * 1997-05-14 1999-12-21 University Of Southern California Retroviral envelopes having modified hypervariable polyproline regions
US6043030A (en) * 1992-12-17 2000-03-28 Cold Spring Harbor Laboratory Cell-cycle regulatory proteins, and uses related thereto
US6057155A (en) * 1995-11-28 2000-05-02 Genvec, Inc. Targeting adenovirus with use of constrained peptide motifs
US6057299A (en) * 1994-01-13 2000-05-02 Calydon, Inc. Tissue-specific enhancer active in prostate
WO2000029600A1 (en) 1998-11-19 2000-05-25 Georgetown University Systemic viral/ligand gene delivery system and gene therapy
US6074850A (en) * 1996-11-15 2000-06-13 Canji, Inc. Retinoblastoma fusion polypeptides
US6136792A (en) * 1994-01-13 2000-10-24 Calydon, Inc. Prostate specific enhancer polynucleotides and methods of use thereof
US6153435A (en) * 1995-02-21 2000-11-28 Cornell Research Foundation, Inc. Nucleic acid that encodes a chimeric adenoviral coat protein
US6162641A (en) * 1997-06-06 2000-12-19 The Regents Of The University Of Michigan Neuregulin response element and uses therefor
US6211334B1 (en) 1992-10-16 2001-04-03 Cold Spring Harbor Cell-cycle regulatory proteins, and uses related thereto
EP1146125A1 (en) * 2000-04-14 2001-10-17 Transgene S.A. Poxvirus with targeted infection specificity
US6331390B1 (en) 1992-12-17 2001-12-18 Cold Spring Harbor Laboratory Cell-cycle regulatory proteins, and uses related thereto
WO2002002641A1 (en) 2000-06-16 2002-01-10 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to blys
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
WO2002079447A2 (en) 2001-03-30 2002-10-10 Avigenics, Inc. Avian lysozyme promoter
US6465253B1 (en) 1994-09-08 2002-10-15 Genvec, Inc. Vectors and methods for gene transfer to cells
WO2002097033A2 (en) 2001-05-25 2002-12-05 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to trail receptors
US6534051B1 (en) 1992-11-20 2003-03-18 University Of Medicine And Dentistry Of New Jersey Cell type specific gene transfer using retroviral vectors containing antibody-envelope fusion proteins and wild-type envelope fusion proteins
US6576456B2 (en) 1995-02-21 2003-06-10 Cornell Research Foundation, Inc. Chimeric adenovirus fiber protein
WO2003084470A2 (en) * 2002-04-02 2003-10-16 Arizeke Pharmaceuticals, Inc. Compositions and methods for targeted biological delivery of molecular carriers
WO2003086458A1 (en) 2002-04-12 2003-10-23 Medimmune, Inc. Recombinant anti-interleukin-9 antibodies
US6660264B1 (en) 1999-04-09 2003-12-09 Health Protection Agency Treatment of intracellular infection
US6699656B2 (en) 1996-06-24 2004-03-02 University Of Maryland Biotechnology Institute Treatment and prevention of HIV infection by administration of derivatives of human chorionic gonadotropin
WO2005001038A2 (en) 2003-05-28 2005-01-06 Seattle Genetics, Inc. Recombinant anti-cd30 antibodies and uses thereof
US6849399B1 (en) 1996-05-23 2005-02-01 Bio-Rad Laboratories, Inc. Methods and compositions for diagnosis and treatment of iron misregulation diseases
US6864082B2 (en) 1997-04-10 2005-03-08 University Of Southern California Modified viral surface proteins for binding to extracellular matrix components
US6875588B2 (en) 2001-11-30 2005-04-05 Avigenics, Inc. Ovomucoid promoter and methods of use
WO2005032572A2 (en) 2003-10-03 2005-04-14 Vib Vzw Means and methods for the recruitment and identification of stem cells
WO2005042708A2 (en) 2003-10-27 2005-05-12 Rosetta Inpharmatics Llc METHOD OF DESIGNING siRNAS FOR GENE SILENCING
US6916918B2 (en) 1997-08-04 2005-07-12 Cell Genesys, Inc. Human glandular kallikrein enhancer, vectors comprising the enhancer and methods of use thereof
WO2006001888A2 (en) 2004-04-16 2006-01-05 Acuity Pharmaceuticals Inc Compositions and methods for inhibiting angiogenesis
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
WO2006046994A2 (en) 2004-07-30 2006-05-04 Mount Sinai School Of Medicine Of New York University Klf6 alternative splice forms and a germline klf6 dna polymorphism associated with increased cancer risk
US7067255B2 (en) 1996-04-04 2006-06-27 Bio-Rad Laboratories, Inc. Hereditary hemochromatosis gene
WO2006069253A2 (en) 2004-12-22 2006-06-29 Auckland Uniservices Limited Trefoil factors and methods of treating proliferation disorders using same
US7078483B2 (en) 1998-04-29 2006-07-18 University Of Southern California Retroviral vectors including modified envelope escort proteins
WO2006081331A2 (en) 2005-01-25 2006-08-03 Prolexys Pharmaceuticals, Inc. Quinoxaline derivatives as antitumor agents
WO2006091871A1 (en) 2005-02-23 2006-08-31 Halozyme Therapeutics, Inc. Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases
WO2006102095A2 (en) 2005-03-18 2006-09-28 Medimmune, Inc. Framework-shuffling of antibodies
US7135562B2 (en) 2002-03-14 2006-11-14 University Of Cincinnati Avian iFABP gene expression controlling region
US7176300B2 (en) 2001-03-30 2007-02-13 Avigenics, Inc. Avian lysozyme promoter
US7276364B1 (en) 1999-11-18 2007-10-02 Dendreon Corporation Nucleic acids encoding endotheliases, endotheliases and uses thereof
US7294507B2 (en) 2001-11-30 2007-11-13 Avigenics, Inc. Ovomucoid promoters and methods of use
US7335761B2 (en) 2001-11-30 2008-02-26 Avigenics, Inc. Avian gene expression controlling regions
US7348014B2 (en) 2000-04-14 2008-03-25 Transgene, S.A. Poxvirus with targeted infection specificity
US20080103108A1 (en) * 1999-08-19 2008-05-01 Yanina Rozenberg Targeted artificial gene delivery
US7393534B2 (en) 2003-07-15 2008-07-01 Barros Research Institute Compositions and methods for immunotherapy of cancer and infectious diseases
WO2008105797A2 (en) 2006-06-30 2008-09-04 Bristol-Myers Squibb Company Polynucleotides encoding novel pcsk9 variants
EP1967205A2 (en) 2000-08-30 2008-09-10 Pfizer Products Inc. Anti-IgE vaccines
EP1978098A2 (en) 1999-12-10 2008-10-08 Invitrogen Corporation Use of multiple recombination sites with unique specificity in recombinational cloning
US7449562B2 (en) 2001-06-29 2008-11-11 Novartis Ag PERV screening method and use thereof
WO2008157379A2 (en) 2007-06-21 2008-12-24 Macrogenics, Inc. Covalent diabodies and uses thereof
WO2009002193A1 (en) 2007-06-27 2008-12-31 Auckland Uniservices Limited Polypeptides and polynucleotides for artemin and related ligands, and methods of use thereof
EP2014674A1 (en) 2001-11-26 2009-01-14 Cellvir Protein-protein interactions in human immunodeficiency virus
EP2027874A2 (en) 2000-11-28 2009-02-25 Medimmune, Inc. Methods of administering/dosing anti-rsv antibodies for prophylaxis and treatment
US7498314B2 (en) 2001-05-03 2009-03-03 Fit Biotech Oyj Plc Expression vectors and uses thereof
US7541512B2 (en) 2001-03-30 2009-06-02 Synageva Biopharma Corp. Avians containing a lysozyme promoter transgene
US7550561B1 (en) 1991-05-16 2009-06-23 Cold Spring Harbor Laboratory p16INK4 polypeptides
US7550650B2 (en) 2001-09-18 2009-06-23 Synageva Biopharma Corp. Production of a transgenic avian by cytoplasmic injection
US7566452B1 (en) 1999-05-04 2009-07-28 New York University Cancer treatment with endothelin receptor antagonists
WO2009123894A2 (en) 2008-04-02 2009-10-08 Macrogenics, Inc. Her2/neu-specific antibodies and methods of using same
EP2128270A1 (en) 2003-08-08 2009-12-02 Genenews Inc. Osteoarthritis biomarkers and uses thereof
WO2009151717A2 (en) 2008-04-02 2009-12-17 Macrogenics, Inc. Bcr-complex-specific antibodies and methods of using same
WO2010027364A1 (en) 2008-09-07 2010-03-11 Glyconex Inc. Anti-extended type i glycosphingolipid antibody, derivatives thereof and use
EP2163643A1 (en) 2003-03-05 2010-03-17 Halozyme, Inc. Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof
WO2010033279A2 (en) 2008-06-04 2010-03-25 Macrogenics, Inc. Antibodies with altered binding to fcrn and methods of using same
US7691632B2 (en) 1993-11-18 2010-04-06 Cold Spring Harbor Laboratory Kit for detecting the level of cyclin-dependent kinase inhibitor P16 gene expression
US7700341B2 (en) 2000-02-03 2010-04-20 Dendreon Corporation Nucleic acid molecules encoding transmembrane serine proteases, the encoded proteins and methods based thereon
WO2010072684A1 (en) 2008-12-22 2010-07-01 Universität Regensburg Norrin in the treatment of diseases associated with an increased tgf-beta activity
WO2010080538A1 (en) 2008-12-19 2010-07-15 Macrogenics, Inc. Covalent diabodies and uses thereof
EP2218779A1 (en) 2002-12-16 2010-08-18 Halozyme, Inc. Human chondroitinase glycoprotein (chasegp), process for preparing the same, and pharmaceutical compositions comprising thereof
WO2010096388A2 (en) 2009-02-18 2010-08-26 Carnegie Mellon University Quenched dendrimeric dyes for bright detection
EP2228389A2 (en) 2001-04-13 2010-09-15 Human Genome Sciences, Inc. Antibodies against vascular endothelial growth factor 2
WO2010124365A1 (en) 2009-04-27 2010-11-04 Ottawa Hospital Research Institute Compositions and methods for modulating stem cells and uses thereof
WO2010141329A1 (en) 2009-06-01 2010-12-09 Medimmune, Llc Molecules with extended half-lives and uses thereof
EP2272566A2 (en) 2003-08-18 2011-01-12 MedImmune, LLC Humanisation of antibodies
WO2011020079A1 (en) 2009-08-13 2011-02-17 Calmune Corporation Antibodies against human respiratory syncytial virus (rsv) and methods of use
EP2292663A2 (en) 2006-08-28 2011-03-09 Kyowa Hakko Kirin Co., Ltd. Antagonistic human light-specific human monoclonal antibodies
EP2298869A1 (en) 2003-06-13 2011-03-23 University Of Medicine And Dentistry Of New Jersey Recombinant protein production in the presence of mRNA interferase
WO2011035205A2 (en) 2009-09-18 2011-03-24 Calmune Corporation Antibodies against candida, collections thereof and methods of use
EP2302385A1 (en) 2002-02-13 2011-03-30 American Diagnostica Inc. Methods for selecting treatment regimens and predicting outcomes in cancer patients
EP2308996A1 (en) 1998-03-30 2011-04-13 NorthWest Biotherapeutics, Inc. Therapeutic and diagnostic applications based on the role of the CXCR-4 and SDF-1 genes in tumourigenesis
US7928189B2 (en) 2008-05-05 2011-04-19 Ottawa Health Research Institute PCSK9 polypeptide fragment
WO2011046457A1 (en) 2009-10-16 2011-04-21 Auckland Uniservices Limited Anti-neoplastic uses of artemin antagonists
EP2316487A1 (en) 2003-04-11 2011-05-04 MedImmune, LLC Recombinant IL-9 antibodies & uses thereof
EP2319941A2 (en) 2005-10-21 2011-05-11 GeneNews Inc. Method and apparatus for correlating levels of biomarker products with disease
WO2011057188A1 (en) 2009-11-06 2011-05-12 Idexx Laboratories, Inc. Canine anti-cd20 antibodies
US7964708B2 (en) 2006-11-15 2011-06-21 Limin Li Anti-TSG101 antibodies and their uses for treatment of viral infections
US7973139B2 (en) 2004-03-26 2011-07-05 Human Genome Sciences, Inc. Antibodies against nogo receptor
EP2341060A1 (en) 2000-12-12 2011-07-06 MedImmune, LLC Molecules with extended half-lives, compositions and uses thereof
EP2351584A1 (en) 2003-12-23 2011-08-03 Genentech, Inc. Novel anti-IL 13 antibodies and uses thereof
EP2357192A1 (en) 1999-02-26 2011-08-17 Human Genome Sciences, Inc. Human endokine alpha and methods of use
US8022189B2 (en) 1998-08-21 2011-09-20 Albany Medical College Isolated antibodies against biologically active leptin-related peptides
US8021833B2 (en) 2003-02-12 2011-09-20 Functional Genetics, Inc. Method for reducing HIV viral budding by administering a VPS28-specfic antibody that disrupts Gag-TSG101-VPS28 binding interactions
EP2368578A1 (en) 2003-01-09 2011-09-28 Macrogenics, Inc. Identification and engineering of antibodies with variant Fc regions and methods of using same
EP2371389A2 (en) 2002-08-14 2011-10-05 MacroGenics, Inc. FcgammaRIIB-specific antibodies and methods of use thereof
EP2383350A1 (en) 2004-05-07 2011-11-02 Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. Methods of diagnosing or treating prostate cancer using the erg gene, alone or in combination with other over or under expressed genes in prostate cancer
EP2385124A2 (en) 1999-05-14 2011-11-09 Arbor Vita Corporation Peptides or peptide analogues for modulating the binding of a PDZ protein and a PL protein
EP2387995A1 (en) 2006-03-30 2011-11-23 PTC Therapeutics, Inc. Methods for the production of functional protein from DNA having a nonsense mutation and the treatment of disorders associated therewith
WO2011150079A1 (en) 2010-05-25 2011-12-01 Carnegie Mellon University Targeted probes of cellular physiology
US8088382B2 (en) 2005-07-05 2012-01-03 Cornell Research Foundation, Inc. Methods of inhibiting transendothelial migration of neutrophils and monocytes with anti-CD99L2 antibodies
WO2012006596A2 (en) 2010-07-09 2012-01-12 Calmune Corporation Anti-human respiratory syncytial virus (rsv) antibodies and methods of use
EP2407548A1 (en) 2006-10-16 2012-01-18 MedImmune, LLC Molecules with reduced half-lives, compositions and uses thereof
WO2012018687A1 (en) 2010-08-02 2012-02-09 Macrogenics, Inc. Covalent diabodies and uses thereof
EP2422811A2 (en) 2004-10-27 2012-02-29 MedImmune, LLC Modulation of antibody specificity by tailoring the affinity to cognate antigens
EP2431054A2 (en) 2000-06-15 2012-03-21 Human Genome Sciences, Inc. Human tumor necrosis factor delta and epsilon
EP2453024A2 (en) 2004-06-21 2012-05-16 The Board of Trustees of The Leland Stanford Junior University Genes and pathways differentially expressed in bipolar disorder and/or major depressive disorder
US8211858B2 (en) 2007-04-27 2012-07-03 The University Of Toledo Modified plasminogen activator inhibitor type-1 molecule and methods based thereon
US8231878B2 (en) 2001-03-20 2012-07-31 Cosmo Research & Development S.P.A. Receptor trem (triggering receptor expressed on myeloid cells) and uses thereof
EP2500030A2 (en) 2005-11-04 2012-09-19 Genentech, Inc. Use of complement pathway inhibitors to treat ocular diseases
EP2505209A1 (en) 2006-06-26 2012-10-03 MacroGenics, Inc. Fcgamma-RIIB-specific antibodies and methods of the use thereof
EP2518163A2 (en) 2006-10-10 2012-10-31 The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. Prostate cancer specific alterations in erg gene expression and detection and treatment methods based on those alterations
EP2520669A2 (en) 2005-02-07 2012-11-07 GeneNews Inc. Mild osteoathritis biomarkers and uses thereof
WO2012162068A2 (en) 2011-05-21 2012-11-29 Macrogenics, Inc. Deimmunized serum-binding domains and their use for extending serum half-life
EP2564864A2 (en) 2005-11-12 2013-03-06 The Board of Trustees of the Leland FGF2-related methods for diagnosing and treating depression
WO2013040341A2 (en) 2011-09-16 2013-03-21 Ottawa Hospital Research Institute Wnt7a compositions and methods of using the same
EP2573114A1 (en) 2005-08-10 2013-03-27 MacroGenics, Inc. Identification and engineering of antibodies with variant Fc regions and methods of using same
EP2610267A1 (en) 2006-12-18 2013-07-03 Genentech, Inc. Antagonist anti-Notch3 antibodies and their use in the prevention and treatment of Notch3-related diseases
EP2629094A1 (en) 2007-01-24 2013-08-21 Carnegie Mellon University Optical biosensors
WO2014006063A2 (en) 2012-07-02 2014-01-09 Medizinische Universität Wien Complement split product c4d for the treatment of inflammatory conditions
US8647622B2 (en) 2007-08-29 2014-02-11 Sanofi Humanized anti-CXCR5 antibodies, derivatives thereof and their use
US8809287B2 (en) 2004-11-15 2014-08-19 Icahn School Of Medicine At Mount Sinai Compositions and methods for altering Wnt autocrine signaling
US8916517B2 (en) 2009-11-02 2014-12-23 The Administrators Of The Tulane Educational Fund Analogs of pituitary adenylate cyclase-activating polypeptide (PACAP) and methods for their use
US8937169B2 (en) 1996-01-11 2015-01-20 Human Genome Sciences, Inc. Human G-protein chemokine receptor HSATU68
US8981061B2 (en) 2001-03-20 2015-03-17 Novo Nordisk A/S Receptor TREM (triggering receptor expressed on myeloid cells) and uses thereof
US9000127B2 (en) 2012-02-15 2015-04-07 Novo Nordisk A/S Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1)
US9006181B2 (en) 2004-07-21 2015-04-14 The Administrators Of The Tulane Educational Fund Treatment of renal dysfunction and multiple myeloma using PACAP compounds
EP2932982A1 (en) 2005-05-17 2015-10-21 Amicus Therapeutics, Inc. A method for the treatment of pompe disease using 1-deoxynojirimycin and derivatives
US9211315B2 (en) 2004-03-05 2015-12-15 Halozyme, Inc. Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases
US9273111B2 (en) 2004-11-29 2016-03-01 Universite De Lorraine Therapeutic TREM-1 peptides
EP3026432A2 (en) 2010-12-27 2016-06-01 Brown University Method for predicting patient's response to biglycan treatment
US9447454B2 (en) 2003-10-23 2016-09-20 The Rockefeller University Method of purifying RNA binding protein-RNA complexes
US9458464B2 (en) 2014-06-23 2016-10-04 The Johns Hopkins University Treatment of neuropathic pain
US9550830B2 (en) 2012-02-15 2017-01-24 Novo Nordisk A/S Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1)
US9592289B2 (en) 2012-03-26 2017-03-14 Sanofi Stable IgG4 based binding agent formulations
US9597346B2 (en) 2010-01-15 2017-03-21 Cornell University Methods for reducing protein levels in a cell
US9663568B2 (en) 2012-02-15 2017-05-30 Novo Nordisk A/S Antibodies that bind peptidoglycan recognition protein 1
WO2017182981A1 (en) 2016-04-20 2017-10-26 Washington University Ppar agonist or lxr agonist for use in the treatment of systemic lupus erythematosus by modulation of lap activity
US9873722B2 (en) 2011-09-16 2018-01-23 Fate Therapeutics, Inc. Wnt compositions and therapeutic uses of such compositions
US9920100B2 (en) 2015-06-05 2018-03-20 The Chinese University Of Hong Kong Mimotopes of tropomyosin for use in immunotherapy for shellfish and/or arthropod allergy
EP3320906A1 (en) 2012-10-26 2018-05-16 The Chinese University of Hong Kong Treatment of melanoma and lung carcinoma using a smad3 inhibitor
US10130687B2 (en) 2011-01-05 2018-11-20 Rhode Island Hospital Compositions and methods for the treatment of orthopedic disease or injury
US10179814B2 (en) 2014-07-17 2019-01-15 Novo Nordisk A/S Site directed mutagenesis of TREM-1 antibodies for decreasing viscosity
EP3450571A1 (en) 2014-02-24 2019-03-06 Celgene Corporation Methods of using an activator of cereblon for neural cell expansion and the treatment of central nervous system disorders
EP3479844A1 (en) 2005-04-15 2019-05-08 MacroGenics, Inc. Covalent diabodies and uses thereof
US10434177B2 (en) 2014-11-17 2019-10-08 Carnegie Mellon University Activatable two-component photosensitizers
US10570200B2 (en) 2013-02-01 2020-02-25 California Institute Of Technology Antibody-mediated immunocontraception
WO2020097261A1 (en) 2018-11-06 2020-05-14 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services New compositions and methods for treating beta-globinopathies
US10729790B2 (en) 2015-05-26 2020-08-04 Salk Institute For Biological Studies Motor neuron-specific expression vectors
WO2021064421A1 (en) 2019-10-03 2021-04-08 Oxford University Innovation Limited Treatment
EP3888690A2 (en) 2014-05-16 2021-10-06 MedImmune, LLC Molecules with altered neonate fc receptor binding having enhanced therapeutic and diagnostic properties
US11155618B2 (en) 2018-04-02 2021-10-26 Bristol-Myers Squibb Company Anti-TREM-1 antibodies and uses thereof
WO2022157548A1 (en) 2021-01-24 2022-07-28 Forrest Michael David Inhibitors of atp synthase - cosmetic and therapeutic uses
WO2022240824A1 (en) 2021-05-13 2022-11-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Compositions and methods for treating sickle cell diseases
WO2023004332A2 (en) 2021-07-19 2023-01-26 New York University Adeno-associated viral vector compositions and methods of promoting muscle regeneration
WO2023081167A2 (en) 2021-11-02 2023-05-11 The Regents Of The University Of California P-selectin mutants and modulation of integrin-mediated signaling
WO2023131901A1 (en) 2022-01-07 2023-07-13 Johnson & Johnson Enterprise Innovation Inc. Materials and methods of il-1beta binding proteins
WO2023146807A1 (en) 2022-01-25 2023-08-03 The Regents Of The University Of California Vegf mutants and modulation of integrin-mediated signaling
WO2024013727A1 (en) 2022-07-15 2024-01-18 Janssen Biotech, Inc. Material and methods for improved bioengineered pairing of antigen-binding variable regions

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4802401B2 (en) * 2000-11-07 2011-10-26 トランスジェン・ソシエテ・アノニム Poxvirus with targeted infection specificity

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0400047B1 (en) * 1988-02-05 1997-04-23 Whitehead Institute For Biomedical Research Modified hepatocytes and uses therefor

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
J. Gen. Virol., Vol. 68, issued 1987, GRUNDY, et al., "B2 Microglobulin Enhances the Infectivity of Cytomegalovirus and when Bound to the Virus Enables Class I HLA Molecules To Be Used as a Virus Receptor", pages 793-803, see figures 1-8, tables 1-2, pages 795-799. *
Proc. Natl. Acad. Sci. USA, Vol. 86, issued December 1989, ROUX et al., "A versatile and potentially general approach to the targeting of specific cell types by retroviruses: Application to the infection of human cells by means of major histocompatibility complex class I and class II antigens by mouse ecotropic murine *
See also references of EP0553235A4 *
The Journal of Biological Chemistry, Vol. 263, No. 29, issued 15 October 1988, WU et al., "Receptor - mediated Gene Delivery and Expression in Vivo", pages 14621-14624, see the entire document. *
Virology, Vol. 163, issued 1988, KOMAI et al., "The Vero Cell Receptor for the Hepatitis B Virus Small S Protein is a Sialoglycoprotein", pages 629-634, see figures 1-2, tables 1-3, pages 630-631 and 633. *
Virology, Vol. 172, issued 1989, PUGH et al., "Infection and Uptake of Duck Hepatitis B Virus by Duck Hepatocytes Maintained in the Presence of Dimethyl Sulfoxide", pages 564-572, see figures 1-8, pages 565-571. *

Cited By (311)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5889169A (en) * 1991-05-16 1999-03-30 Cold Spring Harbor Laboratory Cell cycle regulatory protein p16 gene
US7550561B1 (en) 1991-05-16 2009-06-23 Cold Spring Harbor Laboratory p16INK4 polypeptides
US5521291A (en) * 1991-09-30 1996-05-28 Boehringer Ingelheim International, Gmbh Conjugates for introducing nucleic acid into higher eucaryotic cells
US6274322B1 (en) * 1991-09-30 2001-08-14 Boehringer Ingelheim International Gmbh Composition for introducing nucleic acid complexes into higher eucaryotic cells
US6022735A (en) * 1991-09-30 2000-02-08 Boehringer Ingelheim International Gmbh Composition for introducing nucleic acid complexes into higher eucaryotic cells
US5981273A (en) * 1991-09-30 1999-11-09 Boehringer Ingelheim Int'l. Gmbh Composition comprising an endosomolytic agent for introducing nucleic acid complexes into higher eucaryotic cells
US5547932A (en) * 1991-09-30 1996-08-20 Boehringer Ingelheim International Gmbh Composition for introducing nucleic acid complexes into higher eucaryotic cells
WO1993009221A1 (en) * 1991-10-30 1993-05-13 Thera Gene Hb Targeted delivery of virus vector to mammalian cells
US5695991A (en) * 1991-10-30 1997-12-09 Got-A-Gene Ab Targeted delivery of virus vector to mammalian cells
US5922859A (en) * 1992-02-01 1999-07-13 Boehringer Ingelheim International Gmbh Complexes containing nucleic acid which can be taken-up by endocytosis into higher eukaryotic cells
WO1993022433A2 (en) * 1992-04-28 1993-11-11 Frank Andreas Harald Meyer Medicament for the gene-therapeutic treatment of human beings, animals and plants, especially to block virus multiplication and carcinogenes and process for producing the medicament
WO1993022433A3 (en) * 1992-04-28 1994-07-07 Frank Andreas Harald Meyer Medicament for the gene-therapeutic treatment of human beings, animals and plants, especially to block virus multiplication and carcinogenes and process for producing the medicament
US6159728A (en) * 1992-06-26 2000-12-12 Btg International Limited RNA bacteriophage-based delivery system
WO1994000588A1 (en) * 1992-06-26 1994-01-06 British Technology Group Ltd. Protein based delivery system
WO1994006923A1 (en) * 1992-09-24 1994-03-31 The University Of Connecticut Modification of a virus to redirect infectivity and enhance targeted delivery of polynucleotides to cells
US7425617B2 (en) 1992-10-16 2008-09-16 Cold Spring Harbor Laboratory Antibodies to the cell cycle regulatory protein p16
US6211334B1 (en) 1992-10-16 2001-04-03 Cold Spring Harbor Cell-cycle regulatory proteins, and uses related thereto
US6486131B2 (en) 1992-10-16 2002-11-26 Cold Spring Harbor Laboratory Cell-cycle regulatory proteins, and uses related thereto
US5968821A (en) * 1992-10-16 1999-10-19 Cold Spring Harbor Laboratories, Inc. Cell-cycle regulatory proteins, and uses related thereto
US5962316A (en) * 1992-10-16 1999-10-05 Cold Spring Harbor Laboratory Cell-cycle regulatory proteins, and uses related thereto
EP1038967A2 (en) * 1992-11-04 2000-09-27 Transgene S.A. Virus with modified binding moiety specific for the target cells
GB2286593A (en) * 1992-11-04 1995-08-23 Imp Cancer Res Tech Virus with modified binding moiety specific for the target cells
EP1038967A3 (en) * 1992-11-04 2006-02-01 Transgene S.A. Virus with modified binding moiety specific for the target cells
WO1994010323A1 (en) * 1992-11-04 1994-05-11 Imperial Cancer Research Technology Limited Virus with modified binding moiety specific for the target cells
EP0746625A1 (en) * 1992-11-09 1996-12-11 UNITED STATES GOVERNMENT as represented by THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES Targetable vector particles
US6503501B1 (en) 1992-11-09 2003-01-07 W. French Anderson Targetable vector particles
EP0746625A4 (en) * 1992-11-09 1997-05-02 Us Health Targetable vector particles
US5985655A (en) * 1992-11-09 1999-11-16 The United States Of America As Represented By The Department Of Health And Human Sevices Targetable vector particles
EP0672129A4 (en) * 1992-11-20 1997-06-11 Univ New Jersey Med Cell-type specific gene transfer using retroviral vectors containing antibody-envelope fusion proteins.
US5869331A (en) * 1992-11-20 1999-02-09 University Of Medicine & Dentistry Of New Jersey Cell type specific gene transfer using retroviral vectors containing antibody-envelope fusion proteins and wild-type envelope fusion proteins
EP0672129A1 (en) * 1992-11-20 1995-09-20 University Of Medicine & Dentistry Of New Jersey Cell-type specific gene transfer using retroviral vectors containing antibody-envelope fusion proteins
US6534051B1 (en) 1992-11-20 2003-03-18 University Of Medicine And Dentistry Of New Jersey Cell type specific gene transfer using retroviral vectors containing antibody-envelope fusion proteins and wild-type envelope fusion proteins
US6146885A (en) * 1992-11-20 2000-11-14 University Of Medicine And Dentistry Of New Jersey Cell-type specific gene transfer using retroviral vectors containing antibody-envelope fusion proteins
US6043030A (en) * 1992-12-17 2000-03-28 Cold Spring Harbor Laboratory Cell-cycle regulatory proteins, and uses related thereto
US6331390B1 (en) 1992-12-17 2001-12-18 Cold Spring Harbor Laboratory Cell-cycle regulatory proteins, and uses related thereto
WO1994024299A1 (en) * 1993-04-08 1994-10-27 Boehringer Ingelheim International Gmbh Adenovirus for the transfer of foreign dna into higher eucaryotic cells
US5693509A (en) * 1993-04-08 1997-12-02 Boehringer Ingelheim International Gmbh Adenovirus for delivering foreign DNA into higher eukaryotic cells
US6068837A (en) * 1993-06-18 2000-05-30 Beth Israel Hospital Association Mesothelial cell gene therapy
US5645829A (en) * 1993-06-18 1997-07-08 Beth Israel Hospital Association Mesothelial cell gene therapy
US7691632B2 (en) 1993-11-18 2010-04-06 Cold Spring Harbor Laboratory Kit for detecting the level of cyclin-dependent kinase inhibitor P16 gene expression
US6057299A (en) * 1994-01-13 2000-05-02 Calydon, Inc. Tissue-specific enhancer active in prostate
US6136792A (en) * 1994-01-13 2000-10-24 Calydon, Inc. Prostate specific enhancer polynucleotides and methods of use thereof
WO1995031566A1 (en) * 1994-05-13 1995-11-23 Chiron Viagene, Inc. Compositions and methods for targeting gene delivery vehicles
US5728399A (en) * 1994-06-29 1998-03-17 University Of Conn. Use of a bacterial component to enhance targeted delivery of polynucleotides to cells
US6465253B1 (en) 1994-09-08 2002-10-15 Genvec, Inc. Vectors and methods for gene transfer to cells
US6951755B2 (en) 1994-09-08 2005-10-04 Genvec, Inc. Vectors and methods for gene transfer
US6379965B1 (en) 1994-09-28 2002-04-30 American Home Products Corporation Multifunctional complexes for gene transfer into cells comprising a nucleic acid bound to a polyamine and having an endosome disruption agent
US7202227B2 (en) 1994-09-28 2007-04-10 Wyeth Multifunctional molecular complexes for gene transfer to cells
US5837533A (en) * 1994-09-28 1998-11-17 American Home Products Corporation Complexes comprising a nucleic acid bound to a cationic polyamine having an endosome disruption agent
US6127170A (en) * 1994-09-28 2000-10-03 American Home Products Corporation Multifunctional complexes for gene transfer into cells comprising a nucleic acid bound to a polyamine and having a endosome disruption agent
US6576456B2 (en) 1995-02-21 2003-06-10 Cornell Research Foundation, Inc. Chimeric adenovirus fiber protein
US6153435A (en) * 1995-02-21 2000-11-28 Cornell Research Foundation, Inc. Nucleic acid that encodes a chimeric adenoviral coat protein
US6649407B2 (en) 1995-11-28 2003-11-18 Genvec, Inc. Targeting adenovirus with use of constrained peptide motifs
US6329190B1 (en) 1995-11-28 2001-12-11 Genvec, Inc. Targetting adenovirus with use of constrained peptide motifs
US6057155A (en) * 1995-11-28 2000-05-02 Genvec, Inc. Targeting adenovirus with use of constrained peptide motifs
US6392069B2 (en) 1996-01-08 2002-05-21 Canji, Inc. Compositions for enhancing delivery of nucleic acids to cells
US7534769B2 (en) 1996-01-08 2009-05-19 Canji, Inc. Compositions and methods for enhancing delivery of therapeutic agents to cells
US7002027B1 (en) 1996-01-08 2006-02-21 Canji, Inc. Compositions and methods for therapeutic use
US8937169B2 (en) 1996-01-11 2015-01-20 Human Genome Sciences, Inc. Human G-protein chemokine receptor HSATU68
US7026116B1 (en) 1996-04-04 2006-04-11 Bio-Rad Laboratories, Inc. Polymorphisms in the region of the human hemochromatosis gene
US7595385B2 (en) 1996-04-04 2009-09-29 Bio-Rad Laboratories, Inc. Polymorphisms in the region of the human hemochromatosis gene
US7579169B2 (en) 1996-04-04 2009-08-25 Bio-Rad Laboratories, Inc. Hereditary hemochromatosis gene
US8257927B2 (en) 1996-04-04 2012-09-04 Bio-Rad Laboratories, Inc. Hereditary hemochromatosis gene
US7067255B2 (en) 1996-04-04 2006-06-27 Bio-Rad Laboratories, Inc. Hereditary hemochromatosis gene
US7052845B2 (en) 1996-04-04 2006-05-30 Bio-Rad Laboratories, Inc. Polymorphisms in the region of the human hemochromatosis gene
US7998680B2 (en) 1996-04-04 2011-08-16 Bio-Rad Laboratories, Inc. Determining genotype of a polymorphic site in the hereditary hemochromatosis gene
US6849399B1 (en) 1996-05-23 2005-02-01 Bio-Rad Laboratories, Inc. Methods and compositions for diagnosis and treatment of iron misregulation diseases
US6699656B2 (en) 1996-06-24 2004-03-02 University Of Maryland Biotechnology Institute Treatment and prevention of HIV infection by administration of derivatives of human chorionic gonadotropin
US6074850A (en) * 1996-11-15 2000-06-13 Canji, Inc. Retinoblastoma fusion polypeptides
US6902731B1 (en) 1996-11-15 2005-06-07 Canji, Inc. Methods of treating hyperproliferative disorders using retinoblastoma fusion proteins
US6379927B1 (en) 1996-11-15 2002-04-30 Canji, Inc. Retinoblastoma fusion proteins
US6864082B2 (en) 1997-04-10 2005-03-08 University Of Southern California Modified viral surface proteins for binding to extracellular matrix components
US7347998B2 (en) 1997-04-10 2008-03-25 University Of Southern California Method of delivering therapeutic agents to site of tissue injury
US8871734B2 (en) 1997-04-10 2014-10-28 The University Of Southern California Transgene delivering retrovirus targeting collagen exposed at site of tissue injury
US8148509B2 (en) 1997-04-10 2012-04-03 University Of Southern California Transgene delivering retrovirus targeting collagen exposed at site of tissue injury
US8530441B2 (en) 1997-04-10 2013-09-10 University Of Southern California Transgene delivering retrovirus targeting collagen exposed at site of tissue injury
US7820157B2 (en) 1997-04-10 2010-10-26 University Of Southern California Transgene delivering retrovirus targeting collagen exposed at site of tissue injury
US6004798A (en) * 1997-05-14 1999-12-21 University Of Southern California Retroviral envelopes having modified hypervariable polyproline regions
US6162641A (en) * 1997-06-06 2000-12-19 The Regents Of The University Of Michigan Neuregulin response element and uses therefor
EP2106807A1 (en) 1997-07-08 2009-10-07 CANJI, Inc. Compositions and kits for enhancing delivery of therapeutic agents to cells
US6916918B2 (en) 1997-08-04 2005-07-12 Cell Genesys, Inc. Human glandular kallikrein enhancer, vectors comprising the enhancer and methods of use thereof
EP2308996A1 (en) 1998-03-30 2011-04-13 NorthWest Biotherapeutics, Inc. Therapeutic and diagnostic applications based on the role of the CXCR-4 and SDF-1 genes in tumourigenesis
WO1999051748A2 (en) 1998-04-07 1999-10-14 Corixa Corporation Fusion proteins of mycobacterium tuberculosis antigens and their uses
US7078483B2 (en) 1998-04-29 2006-07-18 University Of Southern California Retroviral vectors including modified envelope escort proteins
US8022189B2 (en) 1998-08-21 2011-09-20 Albany Medical College Isolated antibodies against biologically active leptin-related peptides
US8067545B2 (en) 1998-08-21 2011-11-29 Albany Medical College Isolated antibodies against biologically active leptin-related peptides
WO2000029600A1 (en) 1998-11-19 2000-05-25 Georgetown University Systemic viral/ligand gene delivery system and gene therapy
EP2357192A1 (en) 1999-02-26 2011-08-17 Human Genome Sciences, Inc. Human endokine alpha and methods of use
US6660264B1 (en) 1999-04-09 2003-12-09 Health Protection Agency Treatment of intracellular infection
US8597645B2 (en) 1999-05-04 2013-12-03 New York University Cancer treatment with endothelin receptor antagonists
US7566452B1 (en) 1999-05-04 2009-07-28 New York University Cancer treatment with endothelin receptor antagonists
US9125897B2 (en) 1999-05-04 2015-09-08 New York University Cancer treatment with endothelin receptor antagonists
US8067000B2 (en) 1999-05-04 2011-11-29 New York University Cancer treatment with endothelin receptor antagonists
EP2385124A2 (en) 1999-05-14 2011-11-09 Arbor Vita Corporation Peptides or peptide analogues for modulating the binding of a PDZ protein and a PL protein
US20080103108A1 (en) * 1999-08-19 2008-05-01 Yanina Rozenberg Targeted artificial gene delivery
US7276364B1 (en) 1999-11-18 2007-10-02 Dendreon Corporation Nucleic acids encoding endotheliases, endotheliases and uses thereof
EP1978098A2 (en) 1999-12-10 2008-10-08 Invitrogen Corporation Use of multiple recombination sites with unique specificity in recombinational cloning
EP2210948A2 (en) 1999-12-10 2010-07-28 Life Technologies Corporation Use of multiple recombination sites with unique specificity in recombinational cloning
US7700341B2 (en) 2000-02-03 2010-04-20 Dendreon Corporation Nucleic acid molecules encoding transmembrane serine proteases, the encoded proteins and methods based thereon
US7348014B2 (en) 2000-04-14 2008-03-25 Transgene, S.A. Poxvirus with targeted infection specificity
US7354591B2 (en) 2000-04-14 2008-04-08 Transgene S.A. Poxvirus with targeted infection specificity
EP1516932A1 (en) * 2000-04-14 2005-03-23 Transgene S.A. Poxvirus with targeted infection specificity
EP1146125A1 (en) * 2000-04-14 2001-10-17 Transgene S.A. Poxvirus with targeted infection specificity
EP2431054A2 (en) 2000-06-15 2012-03-21 Human Genome Sciences, Inc. Human tumor necrosis factor delta and epsilon
EP2281843A1 (en) 2000-06-16 2011-02-09 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to blys
EP2281842A1 (en) 2000-06-16 2011-02-09 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to BLyS
EP2275449A1 (en) 2000-06-16 2011-01-19 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to blys
WO2002002641A1 (en) 2000-06-16 2002-01-10 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to blys
US8273356B2 (en) 2000-08-30 2012-09-25 Pfizer Inc. Anti-IgE vaccines
US7897151B2 (en) 2000-08-30 2011-03-01 Pharmacia & Upjohn Company, Llc Anti-IgE vaccines
EP1967205A2 (en) 2000-08-30 2008-09-10 Pfizer Products Inc. Anti-IgE vaccines
EP1967206A2 (en) 2000-08-30 2008-09-10 Pfizer Products Inc. Anti-IgE vaccines
EP2361635A2 (en) 2000-08-30 2011-08-31 Pfizer Products Inc. Anti IgE vaccines
EP2065052A2 (en) 2000-08-30 2009-06-03 Pfizer Products Inc. Anti-IgE vaccines
EP2338512A1 (en) 2000-11-28 2011-06-29 MedImmune, LLC Methods of administering/dosing anti-RSV antibodies for prophylaxis and treatment
EP2412384A1 (en) 2000-11-28 2012-02-01 MedImmune, LLC Methods of administering/dosing anti-RSV antibodies for prophylaxis and treatment
EP2027874A2 (en) 2000-11-28 2009-02-25 Medimmune, Inc. Methods of administering/dosing anti-rsv antibodies for prophylaxis and treatment
EP3569610A2 (en) 2000-12-12 2019-11-20 Medlmmune, LLC Molecules with extended half lives, compositions and uses thereof
EP2357187A1 (en) 2000-12-12 2011-08-17 MedImmune, LLC Molecules with extended half-lives, compositions and uses thereof
EP2354149A1 (en) 2000-12-12 2011-08-10 MedImmune, LLC Molecules with extended half-lives, compositions and uses thereof
EP2341060A1 (en) 2000-12-12 2011-07-06 MedImmune, LLC Molecules with extended half-lives, compositions and uses thereof
US8981061B2 (en) 2001-03-20 2015-03-17 Novo Nordisk A/S Receptor TREM (triggering receptor expressed on myeloid cells) and uses thereof
US8231878B2 (en) 2001-03-20 2012-07-31 Cosmo Research & Development S.P.A. Receptor trem (triggering receptor expressed on myeloid cells) and uses thereof
WO2002079447A2 (en) 2001-03-30 2002-10-10 Avigenics, Inc. Avian lysozyme promoter
US7199279B2 (en) 2001-03-30 2007-04-03 Avigenics, Inc. Recombinant promoters in avian cells
US7176300B2 (en) 2001-03-30 2007-02-13 Avigenics, Inc. Avian lysozyme promoter
US7541512B2 (en) 2001-03-30 2009-06-02 Synageva Biopharma Corp. Avians containing a lysozyme promoter transgene
EP2228389A2 (en) 2001-04-13 2010-09-15 Human Genome Sciences, Inc. Antibodies against vascular endothelial growth factor 2
US7510718B2 (en) 2001-05-03 2009-03-31 Fit Biotech Oyj Plc Expression vectors and uses thereof
US7498314B2 (en) 2001-05-03 2009-03-03 Fit Biotech Oyj Plc Expression vectors and uses thereof
US9725486B2 (en) 2001-05-03 2017-08-08 Fit Biotech Oy Methods of treating HIV diseases using novel expression vectors
WO2002097033A2 (en) 2001-05-25 2002-12-05 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to trail receptors
US7449562B2 (en) 2001-06-29 2008-11-11 Novartis Ag PERV screening method and use thereof
US7550650B2 (en) 2001-09-18 2009-06-23 Synageva Biopharma Corp. Production of a transgenic avian by cytoplasmic injection
EP2014674A1 (en) 2001-11-26 2009-01-14 Cellvir Protein-protein interactions in human immunodeficiency virus
US7335761B2 (en) 2001-11-30 2008-02-26 Avigenics, Inc. Avian gene expression controlling regions
US7294507B2 (en) 2001-11-30 2007-11-13 Avigenics, Inc. Ovomucoid promoters and methods of use
US7812215B2 (en) 2001-11-30 2010-10-12 Synageva Biopharma Corp. Methods and protein production using ovomucoid promoters
US6875588B2 (en) 2001-11-30 2005-04-05 Avigenics, Inc. Ovomucoid promoter and methods of use
US7507873B2 (en) 2001-11-30 2009-03-24 Avigenics, Inc. Transgenic avians containing recombinant ovomucoid promoters
US7375258B2 (en) 2001-11-30 2008-05-20 Avigenics, Inc. Transgenic avians with an ovomucoid gene expression control region linked to a nucleotide sequence encoding a heterologous polypeptide
EP2360476A1 (en) 2002-02-13 2011-08-24 American Diagnostica Inc. Methods for selecting treatment regimens and predicting outcomes in cancer patients
EP2302385A1 (en) 2002-02-13 2011-03-30 American Diagnostica Inc. Methods for selecting treatment regimens and predicting outcomes in cancer patients
US7135562B2 (en) 2002-03-14 2006-11-14 University Of Cincinnati Avian iFABP gene expression controlling region
WO2003084470A3 (en) * 2002-04-02 2006-01-12 Arizeke Pharmaceuticals Inc Compositions and methods for targeted biological delivery of molecular carriers
WO2003084470A2 (en) * 2002-04-02 2003-10-16 Arizeke Pharmaceuticals, Inc. Compositions and methods for targeted biological delivery of molecular carriers
EP2270049A2 (en) 2002-04-12 2011-01-05 Medimmune, Inc. Recombinant anti-interleukin-9-antibody
WO2003086458A1 (en) 2002-04-12 2003-10-23 Medimmune, Inc. Recombinant anti-interleukin-9 antibodies
EP2371389A2 (en) 2002-08-14 2011-10-05 MacroGenics, Inc. FcgammaRIIB-specific antibodies and methods of use thereof
EP2298874A1 (en) 2002-12-16 2011-03-23 Halozyme, Inc. Human chondroitinase glycoprotein (CHASEGP), process for preparing the same, and pharmaceutical compositions comprising thereof
EP2218779A1 (en) 2002-12-16 2010-08-18 Halozyme, Inc. Human chondroitinase glycoprotein (chasegp), process for preparing the same, and pharmaceutical compositions comprising thereof
US8815558B2 (en) 2002-12-16 2014-08-26 Halozyme, Inc. Human chondroitinase glycoprotein (CHASEGP), process for preparing the same, and pharmaceutical compositions comprising thereof
EP2368578A1 (en) 2003-01-09 2011-09-28 Macrogenics, Inc. Identification and engineering of antibodies with variant Fc regions and methods of using same
US8021833B2 (en) 2003-02-12 2011-09-20 Functional Genetics, Inc. Method for reducing HIV viral budding by administering a VPS28-specfic antibody that disrupts Gag-TSG101-VPS28 binding interactions
US9562223B2 (en) 2003-03-05 2017-02-07 Halozyme, Inc. Methods for reducing intraocular pressure by administering a soluble hyaluronidase glycoprotein (sHASEGP)
EP2405015A2 (en) 2003-03-05 2012-01-11 Halozyme, Inc. Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof
EP2311973A1 (en) 2003-03-05 2011-04-20 Halozyme, Inc. Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof
EP2177620A1 (en) 2003-03-05 2010-04-21 Halozyme, Inc. Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof
US10286044B2 (en) 2003-03-05 2019-05-14 Halozyme, Inc. Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof
US11723959B2 (en) 2003-03-05 2023-08-15 Halozyme, Inc. Preparation of mammalian oocyte for fertilization via a soluble human PH20 hyaluronidase polypeptide
EP2163643A1 (en) 2003-03-05 2010-03-17 Halozyme, Inc. Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof
EP2330213A1 (en) 2003-03-05 2011-06-08 Halozyme, Inc. Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof
US10898551B2 (en) 2003-03-05 2021-01-26 Halozyme, Inc. Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof
US9677062B2 (en) 2003-03-05 2017-06-13 Halozyme, Inc. Hyaluronidase and factor VIII compositions
EP3009517A1 (en) 2003-03-05 2016-04-20 Halozyme, Inc. Soluble hyaluronidase glycoprotein (shasegp), process for preparing the same, uses and pharmaceutical compositions comprising thereof
US9677061B2 (en) 2003-03-05 2017-06-13 Halozyme, Inc. Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof
EP2316487A1 (en) 2003-04-11 2011-05-04 MedImmune, LLC Recombinant IL-9 antibodies & uses thereof
WO2005001038A2 (en) 2003-05-28 2005-01-06 Seattle Genetics, Inc. Recombinant anti-cd30 antibodies and uses thereof
EP2302040A1 (en) 2003-06-13 2011-03-30 University Of Medicine And Dentistry Of New Jersey Medical use of mRNA interferase
EP2298869A1 (en) 2003-06-13 2011-03-23 University Of Medicine And Dentistry Of New Jersey Recombinant protein production in the presence of mRNA interferase
US8257714B2 (en) 2003-07-15 2012-09-04 Michigan State University Compositions and methods for immunotherapy of cancer and infectious diseases
US7393534B2 (en) 2003-07-15 2008-07-01 Barros Research Institute Compositions and methods for immunotherapy of cancer and infectious diseases
EP2128270A1 (en) 2003-08-08 2009-12-02 Genenews Inc. Osteoarthritis biomarkers and uses thereof
EP2272566A2 (en) 2003-08-18 2011-01-12 MedImmune, LLC Humanisation of antibodies
US8003096B2 (en) 2003-10-03 2011-08-23 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Means and methods for the recruitment and identification of stem cells
WO2005032572A2 (en) 2003-10-03 2005-04-14 Vib Vzw Means and methods for the recruitment and identification of stem cells
US9447454B2 (en) 2003-10-23 2016-09-20 The Rockefeller University Method of purifying RNA binding protein-RNA complexes
WO2005042708A2 (en) 2003-10-27 2005-05-12 Rosetta Inpharmatics Llc METHOD OF DESIGNING siRNAS FOR GENE SILENCING
EP3718564A1 (en) 2003-12-23 2020-10-07 Genentech, Inc. Novel anti-il 13 antibodies and uses thereof
EP2351584A1 (en) 2003-12-23 2011-08-03 Genentech, Inc. Novel anti-IL 13 antibodies and uses thereof
EP2805728A1 (en) 2003-12-23 2014-11-26 Genentech, Inc. Novel anti-IL 13 antibodies and uses thereof
US9211315B2 (en) 2004-03-05 2015-12-15 Halozyme, Inc. Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases
US10016491B2 (en) 2004-03-05 2018-07-10 Halozyme, Inc. Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases
US7973139B2 (en) 2004-03-26 2011-07-05 Human Genome Sciences, Inc. Antibodies against nogo receptor
WO2006001888A2 (en) 2004-04-16 2006-01-05 Acuity Pharmaceuticals Inc Compositions and methods for inhibiting angiogenesis
EP2383350A1 (en) 2004-05-07 2011-11-02 Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. Methods of diagnosing or treating prostate cancer using the erg gene, alone or in combination with other over or under expressed genes in prostate cancer
EP2453024A2 (en) 2004-06-21 2012-05-16 The Board of Trustees of The Leland Stanford Junior University Genes and pathways differentially expressed in bipolar disorder and/or major depressive disorder
US9006181B2 (en) 2004-07-21 2015-04-14 The Administrators Of The Tulane Educational Fund Treatment of renal dysfunction and multiple myeloma using PACAP compounds
WO2006046994A2 (en) 2004-07-30 2006-05-04 Mount Sinai School Of Medicine Of New York University Klf6 alternative splice forms and a germline klf6 dna polymorphism associated with increased cancer risk
EP2422811A2 (en) 2004-10-27 2012-02-29 MedImmune, LLC Modulation of antibody specificity by tailoring the affinity to cognate antigens
US8809287B2 (en) 2004-11-15 2014-08-19 Icahn School Of Medicine At Mount Sinai Compositions and methods for altering Wnt autocrine signaling
US10603357B2 (en) 2004-11-29 2020-03-31 Bristol-Myers Squibb Company Therapeutic TREM-1 peptides
US9273111B2 (en) 2004-11-29 2016-03-01 Universite De Lorraine Therapeutic TREM-1 peptides
WO2006069253A2 (en) 2004-12-22 2006-06-29 Auckland Uniservices Limited Trefoil factors and methods of treating proliferation disorders using same
WO2006081331A2 (en) 2005-01-25 2006-08-03 Prolexys Pharmaceuticals, Inc. Quinoxaline derivatives as antitumor agents
EP2520669A2 (en) 2005-02-07 2012-11-07 GeneNews Inc. Mild osteoathritis biomarkers and uses thereof
EP3045472A1 (en) 2005-02-23 2016-07-20 Halozyme, Inc. Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases
WO2006091871A1 (en) 2005-02-23 2006-08-31 Halozyme Therapeutics, Inc. Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases
EP3943501A1 (en) 2005-02-23 2022-01-26 Halozyme, Inc. Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases
US10588983B2 (en) 2005-02-23 2020-03-17 Halozyme, Inc. Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases
WO2006102095A2 (en) 2005-03-18 2006-09-28 Medimmune, Inc. Framework-shuffling of antibodies
EP3479844A1 (en) 2005-04-15 2019-05-08 MacroGenics, Inc. Covalent diabodies and uses thereof
EP3782655A1 (en) 2005-05-17 2021-02-24 Amicus Therapeutics, Inc. A method for the treatment of pompe disease using 1-deoxynojirimycin and derivatives
EP3441090A1 (en) 2005-05-17 2019-02-13 Amicus Therapeutics, Inc. A method for the treatment of pompe disease using 1-deoxynojirimycin and derivatives
EP2932982A1 (en) 2005-05-17 2015-10-21 Amicus Therapeutics, Inc. A method for the treatment of pompe disease using 1-deoxynojirimycin and derivatives
US8088382B2 (en) 2005-07-05 2012-01-03 Cornell Research Foundation, Inc. Methods of inhibiting transendothelial migration of neutrophils and monocytes with anti-CD99L2 antibodies
EP2573114A1 (en) 2005-08-10 2013-03-27 MacroGenics, Inc. Identification and engineering of antibodies with variant Fc regions and methods of using same
EP2319941A2 (en) 2005-10-21 2011-05-11 GeneNews Inc. Method and apparatus for correlating levels of biomarker products with disease
EP3299027A1 (en) 2005-11-04 2018-03-28 Genentech, Inc. Use of complement pathway inhibitors to treat ocular diseases
EP2998318A1 (en) 2005-11-04 2016-03-23 Genentech, Inc. Use of complement pathway inhibitors to treat ocular diseases
EP2500030A2 (en) 2005-11-04 2012-09-19 Genentech, Inc. Use of complement pathway inhibitors to treat ocular diseases
EP2564864A2 (en) 2005-11-12 2013-03-06 The Board of Trustees of the Leland FGF2-related methods for diagnosing and treating depression
EP2387995A1 (en) 2006-03-30 2011-11-23 PTC Therapeutics, Inc. Methods for the production of functional protein from DNA having a nonsense mutation and the treatment of disorders associated therewith
EP2505209A1 (en) 2006-06-26 2012-10-03 MacroGenics, Inc. Fcgamma-RIIB-specific antibodies and methods of the use thereof
EP2639301A2 (en) 2006-06-30 2013-09-18 Bristol-Myers Squibb Company Polynucleotides encoding novel PCSK9 variants
WO2008105797A2 (en) 2006-06-30 2008-09-04 Bristol-Myers Squibb Company Polynucleotides encoding novel pcsk9 variants
EP2671946A1 (en) 2006-06-30 2013-12-11 Bristol-Myers Squibb Company Polynucleotides encoding novel PCSK9 variants
EP2292663A2 (en) 2006-08-28 2011-03-09 Kyowa Hakko Kirin Co., Ltd. Antagonistic human light-specific human monoclonal antibodies
EP2484696A1 (en) 2006-08-28 2012-08-08 Kyowa Hakko Kirin Co., Ltd. Antagonistic human light-specific human monoclonal antibodies
EP2518163A2 (en) 2006-10-10 2012-10-31 The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. Prostate cancer specific alterations in erg gene expression and detection and treatment methods based on those alterations
EP2837697A2 (en) 2006-10-10 2015-02-18 The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. Prostate cancer-specific alterations in ERG gene expression and detection and treatment methods based on those alternations
EP2407548A1 (en) 2006-10-16 2012-01-18 MedImmune, LLC Molecules with reduced half-lives, compositions and uses thereof
EP2514439A1 (en) 2006-11-15 2012-10-24 Functional Genetics, Inc. Anti-TSG101antibodies and their uses for treatment of viral infections
US8796423B2 (en) 2006-11-15 2014-08-05 Eli Lilly And Company Anti-TSG101 antibodies and their uses for treatment of viral infections
US7964708B2 (en) 2006-11-15 2011-06-21 Limin Li Anti-TSG101 antibodies and their uses for treatment of viral infections
EP2610267A1 (en) 2006-12-18 2013-07-03 Genentech, Inc. Antagonist anti-Notch3 antibodies and their use in the prevention and treatment of Notch3-related diseases
EP2629094A1 (en) 2007-01-24 2013-08-21 Carnegie Mellon University Optical biosensors
US8211858B2 (en) 2007-04-27 2012-07-03 The University Of Toledo Modified plasminogen activator inhibitor type-1 molecule and methods based thereon
EP3424951A1 (en) 2007-06-21 2019-01-09 MacroGenics, Inc. Covalent diabodies and uses thereof
WO2008157379A2 (en) 2007-06-21 2008-12-24 Macrogenics, Inc. Covalent diabodies and uses thereof
WO2009002193A1 (en) 2007-06-27 2008-12-31 Auckland Uniservices Limited Polypeptides and polynucleotides for artemin and related ligands, and methods of use thereof
US9815902B2 (en) 2007-08-29 2017-11-14 Sanofi Humanized anti-CXCR5 antibodies, derivatives thereof and their uses
US8647622B2 (en) 2007-08-29 2014-02-11 Sanofi Humanized anti-CXCR5 antibodies, derivatives thereof and their use
US9228019B2 (en) 2007-08-29 2016-01-05 Sanofi Humanized anti-CXCR5 antibodies, derivatives thereof and their use
US9243067B2 (en) 2007-08-29 2016-01-26 Sanofi Humanized anti-CXCR5 antibodies, derivatives thereof and their use
US9175087B2 (en) 2007-08-29 2015-11-03 Sanofi Humanized anti-CXCR5 antibodies, derivatives thereof and their use
US8980262B2 (en) 2007-08-29 2015-03-17 Sanofi Humanized anti-CXCR5 antibodies, derivatives thereof and their use
WO2009151717A2 (en) 2008-04-02 2009-12-17 Macrogenics, Inc. Bcr-complex-specific antibodies and methods of using same
EP3045475A1 (en) 2008-04-02 2016-07-20 MacroGenics, Inc. Bcr-complex-specific antibodies and methods of using same
EP3067063A1 (en) 2008-04-02 2016-09-14 MacroGenics, Inc. Her2/neu-specific antibodies and methods of using same
WO2009123894A2 (en) 2008-04-02 2009-10-08 Macrogenics, Inc. Her2/neu-specific antibodies and methods of using same
US7928189B2 (en) 2008-05-05 2011-04-19 Ottawa Health Research Institute PCSK9 polypeptide fragment
WO2010033279A2 (en) 2008-06-04 2010-03-25 Macrogenics, Inc. Antibodies with altered binding to fcrn and methods of using same
WO2010027364A1 (en) 2008-09-07 2010-03-11 Glyconex Inc. Anti-extended type i glycosphingolipid antibody, derivatives thereof and use
WO2010080538A1 (en) 2008-12-19 2010-07-15 Macrogenics, Inc. Covalent diabodies and uses thereof
EP2786762A2 (en) 2008-12-19 2014-10-08 MacroGenics, Inc. Covalent diabodies and uses thereof
EP3482769A1 (en) 2008-12-19 2019-05-15 MacroGenics, Inc. Covalent diabodies and uses thereof
WO2010072684A1 (en) 2008-12-22 2010-07-01 Universität Regensburg Norrin in the treatment of diseases associated with an increased tgf-beta activity
US9249306B2 (en) 2009-02-18 2016-02-02 Carnegie Mellon University Quenched dendrimeric dyes for florescence detection
WO2010096388A2 (en) 2009-02-18 2010-08-26 Carnegie Mellon University Quenched dendrimeric dyes for bright detection
US10071138B2 (en) 2009-04-27 2018-09-11 Ottawa Hospital Research Institute Compositions and methods for modulating stem cells and uses thereof
WO2010124365A1 (en) 2009-04-27 2010-11-04 Ottawa Hospital Research Institute Compositions and methods for modulating stem cells and uses thereof
US10828346B2 (en) 2009-04-27 2020-11-10 Ottawa Hospital Research Institute Compositions and methods for modulating stem cells and uses thereof
WO2010141329A1 (en) 2009-06-01 2010-12-09 Medimmune, Llc Molecules with extended half-lives and uses thereof
US8568719B2 (en) 2009-08-13 2013-10-29 Crucell Holland B.V. Antibodies against human respiratory syncytial virus (RSV) and methods of use
US9403900B2 (en) 2009-08-13 2016-08-02 Crucell Holland B.V. Anti-human respiratory syncytial virus (RSV) antibodies and methods of use
WO2011020079A1 (en) 2009-08-13 2011-02-17 Calmune Corporation Antibodies against human respiratory syncytial virus (rsv) and methods of use
US9365638B2 (en) 2009-08-13 2016-06-14 Crucell Holland B. V. Antibodies against human respiratory syncytial virus (RSV) and methods of use
US9988437B2 (en) 2009-08-13 2018-06-05 Janssen Vaccines & Prevention B.V. Anti-human Respiratory Syncytial Virus (RSV) antibodies and methods of use
WO2011035205A2 (en) 2009-09-18 2011-03-24 Calmune Corporation Antibodies against candida, collections thereof and methods of use
WO2011046457A1 (en) 2009-10-16 2011-04-21 Auckland Uniservices Limited Anti-neoplastic uses of artemin antagonists
US8916517B2 (en) 2009-11-02 2014-12-23 The Administrators Of The Tulane Educational Fund Analogs of pituitary adenylate cyclase-activating polypeptide (PACAP) and methods for their use
WO2011057188A1 (en) 2009-11-06 2011-05-12 Idexx Laboratories, Inc. Canine anti-cd20 antibodies
US9597346B2 (en) 2010-01-15 2017-03-21 Cornell University Methods for reducing protein levels in a cell
US9995679B2 (en) 2010-05-25 2018-06-12 Carnegie Mellon University Targeted probes of cellular physiology
WO2011150079A1 (en) 2010-05-25 2011-12-01 Carnegie Mellon University Targeted probes of cellular physiology
WO2012006596A2 (en) 2010-07-09 2012-01-12 Calmune Corporation Anti-human respiratory syncytial virus (rsv) antibodies and methods of use
US9139642B2 (en) 2010-07-09 2015-09-22 Crucell Holland B.V. Anti-human respiratory syncytial virus (RSV) antibodies and methods of use
WO2012018687A1 (en) 2010-08-02 2012-02-09 Macrogenics, Inc. Covalent diabodies and uses thereof
EP3026432A2 (en) 2010-12-27 2016-06-01 Brown University Method for predicting patient's response to biglycan treatment
US10130687B2 (en) 2011-01-05 2018-11-20 Rhode Island Hospital Compositions and methods for the treatment of orthopedic disease or injury
WO2012162068A2 (en) 2011-05-21 2012-11-29 Macrogenics, Inc. Deimmunized serum-binding domains and their use for extending serum half-life
US9873722B2 (en) 2011-09-16 2018-01-23 Fate Therapeutics, Inc. Wnt compositions and therapeutic uses of such compositions
WO2013040341A2 (en) 2011-09-16 2013-03-21 Ottawa Hospital Research Institute Wnt7a compositions and methods of using the same
US9732130B2 (en) 2011-09-16 2017-08-15 Ottawa Hospital Research Institute WNT7A compositions and method of using the same
US9663568B2 (en) 2012-02-15 2017-05-30 Novo Nordisk A/S Antibodies that bind peptidoglycan recognition protein 1
US10906975B2 (en) 2012-02-15 2021-02-02 Novo Nordisk A/S Methods of treating autoimmune disease or chronic inflammation with antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1)
US10189904B2 (en) 2012-02-15 2019-01-29 Novo Nordisk A/S Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1)
US10906965B2 (en) 2012-02-15 2021-02-02 Novo Nordisk A/S Methods of treating autoimmune disease or chronic inflammation wtih antibodies that bind peptidoglycan recognition protein 1
US9000127B2 (en) 2012-02-15 2015-04-07 Novo Nordisk A/S Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1)
US9550830B2 (en) 2012-02-15 2017-01-24 Novo Nordisk A/S Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1)
US10150809B2 (en) 2012-02-15 2018-12-11 Bristol-Myers Squibb Company Antibodies that bind peptidoglycan recognition protein 1
US9592289B2 (en) 2012-03-26 2017-03-14 Sanofi Stable IgG4 based binding agent formulations
US10525130B2 (en) 2012-03-26 2020-01-07 Sanofi Stable IGG4 based binding agent formulations
WO2014006063A2 (en) 2012-07-02 2014-01-09 Medizinische Universität Wien Complement split product c4d for the treatment of inflammatory conditions
EP3527213A1 (en) 2012-10-26 2019-08-21 The Chinese University Of Hong Kong Treatment of cancer using a smad3 inhibitor
EP3320906A1 (en) 2012-10-26 2018-05-16 The Chinese University of Hong Kong Treatment of melanoma and lung carcinoma using a smad3 inhibitor
US10570200B2 (en) 2013-02-01 2020-02-25 California Institute Of Technology Antibody-mediated immunocontraception
EP3450571A1 (en) 2014-02-24 2019-03-06 Celgene Corporation Methods of using an activator of cereblon for neural cell expansion and the treatment of central nervous system disorders
EP3888690A2 (en) 2014-05-16 2021-10-06 MedImmune, LLC Molecules with altered neonate fc receptor binding having enhanced therapeutic and diagnostic properties
US9458464B2 (en) 2014-06-23 2016-10-04 The Johns Hopkins University Treatment of neuropathic pain
US11072654B2 (en) 2014-07-17 2021-07-27 Novo Nordisk A/S Site directed mutagenesis of TREM-1 antibodies for decreasing viscosity
US10179814B2 (en) 2014-07-17 2019-01-15 Novo Nordisk A/S Site directed mutagenesis of TREM-1 antibodies for decreasing viscosity
US10434177B2 (en) 2014-11-17 2019-10-08 Carnegie Mellon University Activatable two-component photosensitizers
US10946098B2 (en) 2014-11-17 2021-03-16 Carnegie Mellon University Activatable two-component photosensitizers
US10729790B2 (en) 2015-05-26 2020-08-04 Salk Institute For Biological Studies Motor neuron-specific expression vectors
US11642423B2 (en) 2015-05-26 2023-05-09 Salk Institute For Biological Studies Motor neuron-specific expression vectors
US9920100B2 (en) 2015-06-05 2018-03-20 The Chinese University Of Hong Kong Mimotopes of tropomyosin for use in immunotherapy for shellfish and/or arthropod allergy
WO2017182981A1 (en) 2016-04-20 2017-10-26 Washington University Ppar agonist or lxr agonist for use in the treatment of systemic lupus erythematosus by modulation of lap activity
US11155618B2 (en) 2018-04-02 2021-10-26 Bristol-Myers Squibb Company Anti-TREM-1 antibodies and uses thereof
US11919954B2 (en) 2018-04-02 2024-03-05 Bristol-Myers Squibb Company Anti-TREM-1 antibodies and uses thereof
WO2020097261A1 (en) 2018-11-06 2020-05-14 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services New compositions and methods for treating beta-globinopathies
US11325978B2 (en) 2018-11-06 2022-05-10 The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Compositions and methods for treating beta-globinopathies
WO2021064421A1 (en) 2019-10-03 2021-04-08 Oxford University Innovation Limited Treatment
WO2022157548A1 (en) 2021-01-24 2022-07-28 Forrest Michael David Inhibitors of atp synthase - cosmetic and therapeutic uses
WO2022240824A1 (en) 2021-05-13 2022-11-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Compositions and methods for treating sickle cell diseases
WO2023004332A2 (en) 2021-07-19 2023-01-26 New York University Adeno-associated viral vector compositions and methods of promoting muscle regeneration
WO2023081167A2 (en) 2021-11-02 2023-05-11 The Regents Of The University Of California P-selectin mutants and modulation of integrin-mediated signaling
WO2023131901A1 (en) 2022-01-07 2023-07-13 Johnson & Johnson Enterprise Innovation Inc. Materials and methods of il-1beta binding proteins
WO2023146807A1 (en) 2022-01-25 2023-08-03 The Regents Of The University Of California Vegf mutants and modulation of integrin-mediated signaling
WO2024013727A1 (en) 2022-07-15 2024-01-18 Janssen Biotech, Inc. Material and methods for improved bioengineered pairing of antigen-binding variable regions

Also Published As

Publication number Publication date
AU8860391A (en) 1992-04-28
AU660629B2 (en) 1995-07-06
EP0553235A4 (en) 1994-02-09
EP0553235A1 (en) 1993-08-04
JPH07500961A (en) 1995-02-02
CA2092323A1 (en) 1992-04-02

Similar Documents

Publication Publication Date Title
AU660629B2 (en) Targeting viruses and cells for selective internalization by cells
Neda et al. Chemical modification of an ecotropic murine leukemia virus results in redirection of its target cell specificity
US6287857B1 (en) Nucleic acid delivery vehicles
Lu et al. Protease-induced infectivity of hepatitis B virus for a human hepatoblastoma cell line
JP3934005B2 (en) Enhanced virus-mediated DNA transfer
JP3479298B2 (en) Novel conjugates for introducing nucleic acids into higher eukaryotic cells
CA2192687C (en) Use of a bacterial component to enhance targeted delivery of polynucleotides to cells
Hobman et al. Assembly of rubella virus structural proteins into virus-like particles in transfected cells
SK36894A3 (en) Composition of transfection of higher eucaryotic cells
WO1994006923A1 (en) Modification of a virus to redirect infectivity and enhance targeted delivery of polynucleotides to cells
US5843770A (en) Antisense constructs directed against viral post-transcriptional regulatory sequences
Lu et al. Study of the early steps of the Hepatitis B Virus life cycle
AU2662999A (en) Nucleic acid delivery vehicles
Fujisawa et al. Characterization of glycosylated Gag expressed by a neurovirulent murine leukemia virus: identification of differences in processing in vitro and in vivo
Bitzer et al. Sendai virus efficiently infects cells via the asialoglycoprotein receptor and requires the presence of cleaved F0 precursor proteins for this alternative route of cell entry
CA2189582A1 (en) Sv-40 derived dna constructs comprising exogenous dna sequences
US6503499B1 (en) Viral vector complexes having adapters of predefined valence
US6743631B1 (en) Use of human serum resistant vector particles and cell lines for human gene therapy
US6610471B1 (en) Methods and compositions to investigate infection by hepatitis B virus and agents to prevent and treat the infection
CA2216871A1 (en) High efficiency ex vivo transduction of cells by high titer recombinant retroviral preparations
EP0866654B1 (en) Method for treating hepatitis virus infection
RU2138553C1 (en) Transfection composition for higher eucaryotic cells, nucleic acid complex useful as component of transfection composition, conjugate useful as component of transfection composition, endosomolytic peptide useful as component of transfection composition
JP2003532368A (en) Nucleic acid delivery vehicle
Kasahara Cell-specific targeting of retroviral vectors via ligand-receptor interaction
JP2002537809A (en) Methods and reagents for inhibiting smooth muscle cell proliferation

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU NL SE

WWE Wipo information: entry into national phase

Ref document number: 2092323

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 1991919436

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1991919436

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1991919436

Country of ref document: EP