|Publicatiedatum||16 april 1992|
|Aanvraagdatum||27 sept 1991|
|Prioriteitsdatum||1 okt 1990|
|Ook gepubliceerd als||CA2092323A1, EP0553235A1, EP0553235A4|
|Publicatienummer||PCT/1991/7103, PCT/US/1991/007103, PCT/US/1991/07103, PCT/US/91/007103, PCT/US/91/07103, PCT/US1991/007103, PCT/US1991/07103, PCT/US1991007103, PCT/US199107103, PCT/US91/007103, PCT/US91/07103, PCT/US91007103, PCT/US9107103, WO 1992/006180 A1, WO 1992006180 A1, WO 1992006180A1, WO 9206180 A1, WO 9206180A1, WO-A1-1992006180, WO-A1-9206180, WO1992/006180A1, WO1992006180 A1, WO1992006180A1, WO9206180 A1, WO9206180A1|
|Uitvinders||George Y. Wu, Catherine H. Wu|
|Aanvrager||University Of Connecticut|
|Citatie exporteren||BiBTeX, EndNote, RefMan|
|Niet-patentcitaties (6), Verwijzingen naar dit patent (258), Classificaties (23), Juridische gebeurtenissen (7)|
|Externe links: Patentscope, Espacenet|
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.
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.
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
(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
(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.
S abilit of Mo ifi Vir
* 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.
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
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
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.
|1||*||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.|
|2||*||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|
|3||*||See also references of EP0553235A4|
|4||*||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.|
|5||*||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.|
|6||*||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.|
|WO1993009221A1 *||28 okt 1992||13 mei 1993||Thera Gene Hb||Targeted delivery of virus vector to mammalian cells|
|WO1993022433A2 *||28 april 1993||11 nov 1993||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 *||28 april 1993||7 juli 1994||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 *||25 juni 1993||6 jan 1994||British Technology Group Ltd.||Protein based delivery system|
|WO1994006923A1 *||23 sept 1993||31 maart 1994||The University Of Connecticut||Modification of a virus to redirect infectivity and enhance targeted delivery of polynucleotides to cells|
|WO1994010323A1 *||4 nov 1993||11 mei 1994||Imperial Cancer Research Technology Limited||Virus with modified binding moiety specific for the target cells|
|WO1994024299A1 *||6 april 1994||27 okt 1994||Boehringer Ingelheim International Gmbh||Adenovirus for the transfer of foreign dna into higher eucaryotic cells|
|WO1995031566A1 *||15 mei 1995||23 nov 1995||Chiron Viagene, Inc.||Compositions and methods for targeting gene delivery vehicles|
|WO1999051748A2||7 april 1999||14 okt 1999||Corixa Corporation||Fusion proteins of mycobacterium tuberculosis antigens and their uses|
|WO2000029600A1||19 nov 1999||25 mei 2000||Georgetown University||Systemic viral/ligand gene delivery system and gene therapy|
|WO2002002641A1||15 juni 2001||10 jan 2002||Human Genome Sciences, Inc.||Antibodies that immunospecifically bind to blys|
|WO2002079447A2||29 maart 2002||10 okt 2002||Avigenics, Inc.||Avian lysozyme promoter|
|WO2002097033A2||7 mei 2002||5 dec 2002||Human Genome Sciences, Inc.||Antibodies that immunospecifically bind to trail receptors|
|WO2003084470A2 *||2 april 2003||16 okt 2003||Arizeke Pharmaceuticals, Inc.||Compositions and methods for targeted biological delivery of molecular carriers|
|WO2003084470A3 *||2 april 2003||12 jan 2006||Arizeke Pharmaceuticals Inc||Compositions and methods for targeted biological delivery of molecular carriers|
|WO2003086458A1||11 april 2003||23 okt 2003||Medimmune, Inc.||Recombinant anti-interleukin-9 antibodies|
|WO2005001038A2||28 mei 2004||6 jan 2005||Seattle Genetics, Inc.||Recombinant anti-cd30 antibodies and uses thereof|
|WO2005032572A2||4 okt 2004||14 april 2005||Vib Vzw||Means and methods for the recruitment and identification of stem cells|
|WO2005042708A2||27 okt 2004||12 mei 2005||Rosetta Inpharmatics Llc||METHOD OF DESIGNING siRNAS FOR GENE SILENCING|
|WO2006001888A2||18 april 2005||5 jan 2006||Acuity Pharmaceuticals Inc||Compositions and methods for inhibiting angiogenesis|
|WO2006046994A2||1 aug 2005||4 mei 2006||Mount Sinai School Of Medicine Of New York University||Klf6 alternative splice forms and a germline klf6 dna polymorphism associated with increased cancer risk|
|WO2006069253A2||22 dec 2005||29 juni 2006||Auckland Uniservices Limited||Trefoil factors and methods of treating proliferation disorders using same|
|WO2006081331A2||25 jan 2006||3 aug 2006||Prolexys Pharmaceuticals, Inc.||Quinoxaline derivatives as antitumor agents|
|WO2006091871A1||23 feb 2006||31 aug 2006||Halozyme Therapeutics, Inc.||Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases|
|WO2006102095A2||17 maart 2006||28 sept 2006||Medimmune, Inc.||Framework-shuffling of antibodies|
|WO2008105797A2||29 juni 2007||4 sept 2008||Bristol-Myers Squibb Company||Polynucleotides encoding novel pcsk9 variants|
|WO2008157379A2||13 juni 2008||24 dec 2008||Macrogenics, Inc.||Covalent diabodies and uses thereof|
|WO2009002193A1||25 juni 2008||31 dec 2008||Auckland Uniservices Limited||Polypeptides and polynucleotides for artemin and related ligands, and methods of use thereof|
|WO2009123894A2||25 maart 2009||8 okt 2009||Macrogenics, Inc.||Her2/neu-specific antibodies and methods of using same|
|WO2009151717A2||25 maart 2009||17 dec 2009||Macrogenics, Inc.||Bcr-complex-specific antibodies and methods of using same|
|WO2010027364A1||7 sept 2008||11 maart 2010||Glyconex Inc.||Anti-extended type i glycosphingolipid antibody, derivatives thereof and use|
|WO2010033279A2||4 juni 2009||25 maart 2010||Macrogenics, Inc.||Antibodies with altered binding to fcrn and methods of using same|
|WO2010072684A1||18 dec 2009||1 juli 2010||Universität Regensburg||Norrin in the treatment of diseases associated with an increased tgf-beta activity|
|WO2010080538A1||17 dec 2009||15 juli 2010||Macrogenics, Inc.||Covalent diabodies and uses thereof|
|WO2010096388A2||16 feb 2010||26 aug 2010||Carnegie Mellon University||Quenched dendrimeric dyes for bright detection|
|WO2010124365A1||27 april 2010||4 nov 2010||Ottawa Hospital Research Institute||Compositions and methods for modulating stem cells and uses thereof|
|WO2010141329A1||28 mei 2010||9 dec 2010||Medimmune, Llc||Molecules with extended half-lives and uses thereof|
|WO2011020079A1||13 aug 2010||17 feb 2011||Calmune Corporation||Antibodies against human respiratory syncytial virus (rsv) and methods of use|
|WO2011035205A2||17 sept 2010||24 maart 2011||Calmune Corporation||Antibodies against candida, collections thereof and methods of use|
|WO2011046457A1||15 okt 2010||21 april 2011||Auckland Uniservices Limited||Anti-neoplastic uses of artemin antagonists|
|WO2011057188A1||8 nov 2010||12 mei 2011||Idexx Laboratories, Inc.||Canine anti-cd20 antibodies|
|WO2011150079A1||25 mei 2011||1 dec 2011||Carnegie Mellon University||Targeted probes of cellular physiology|
|WO2012006596A2||8 juli 2011||12 jan 2012||Calmune Corporation||Anti-human respiratory syncytial virus (rsv) antibodies and methods of use|
|WO2012018687A1||29 juli 2011||9 feb 2012||Macrogenics, Inc.||Covalent diabodies and uses thereof|
|WO2012162068A2||16 mei 2012||29 nov 2012||Macrogenics, Inc.||Deimmunized serum-binding domains and their use for extending serum half-life|
|WO2013040341A2||14 sept 2012||21 maart 2013||Ottawa Hospital Research Institute||Wnt7a compositions and methods of using the same|
|WO2014006063A2||2 juli 2013||9 jan 2014||Medizinische Universität Wien||Complement split product c4d for the treatment of inflammatory conditions|
|EP0672129A1 *||18 nov 1993||20 sept 1995||University Of Medicine And Dentistry Of New Jersey||Cell-type specific gene transfer using retroviral vectors containing antibody-envelope fusion proteins|
|EP0672129A4 *||18 nov 1993||11 juni 1997||Univ New Jersey Med||Cell-type specific gene transfer using retroviral vectors containing antibody-envelope fusion proteins.|
|EP0746625A1 *||3 nov 1993||11 dec 1996||UNITED STATES GOVERNMENT as represented by THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES||Targetable vector particles|
|EP0746625A4 *||3 nov 1993||2 mei 1997||Us Health||Targetable vector particles|
|EP1038967A2 *||4 nov 1993||27 sept 2000||Transgene S.A.||Virus with modified binding moiety specific for the target cells|
|EP1038967A3 *||4 nov 1993||1 feb 2006||Transgene S.A.||Virus with modified binding moiety specific for the target cells|
|EP1146125A1 *||12 april 2001||17 okt 2001||Transgene S.A.||Poxvirus with targeted infection specificity|
|EP1516932A1 *||12 april 2001||23 maart 2005||Transgene S.A.||Poxvirus with targeted infection specificity|
|EP1967205A2||24 aug 2001||10 sept 2008||Pfizer Products Inc.||Anti-IgE vaccines|
|EP1967206A2||24 aug 2001||10 sept 2008||Pfizer Products Inc.||Anti-IgE vaccines|
|EP1978098A2||11 dec 2000||8 okt 2008||Invitrogen Corporation||Use of multiple recombination sites with unique specificity in recombinational cloning|
|EP2014674A1||26 nov 2002||14 jan 2009||Cellvir||Protein-protein interactions in human immunodeficiency virus|
|EP2027874A2||28 nov 2001||25 feb 2009||Medimmune, Inc.||Methods of administering/dosing anti-rsv antibodies for prophylaxis and treatment|
|EP2065052A2||24 aug 2001||3 juni 2009||Pfizer Products Inc.||Anti-IgE vaccines|
|EP2106807A1||8 juli 1998||7 okt 2009||CANJI, Inc.||Compositions and kits for enhancing delivery of therapeutic agents to cells|
|EP2128270A1||9 aug 2004||2 dec 2009||Genenews Inc.||Osteoarthritis biomarkers and uses thereof|
|EP2163643A1||5 maart 2004||17 maart 2010||Halozyme, Inc.||Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof|
|EP2177620A1||5 maart 2004||21 april 2010||Halozyme, Inc.||Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof|
|EP2210948A2||11 dec 2000||28 juli 2010||Life Technologies Corporation||Use of multiple recombination sites with unique specificity in recombinational cloning|
|EP2218779A1||15 dec 2003||18 aug 2010||Halozyme, Inc.||Human chondroitinase glycoprotein (chasegp), process for preparing the same, and pharmaceutical compositions comprising thereof|
|EP2228389A2||12 april 2002||15 sept 2010||Human Genome Sciences, Inc.||Antibodies against vascular endothelial growth factor 2|
|EP2270049A2||11 april 2003||5 jan 2011||Medimmune, Inc.||Recombinant anti-interleukin-9-antibody|
|EP2272566A2||18 aug 2004||12 jan 2011||MedImmune, LLC||Humanisation of antibodies|
|EP2275449A1||15 juni 2001||19 jan 2011||Human Genome Sciences, Inc.||Antibodies that immunospecifically bind to blys|
|EP2281842A1||15 juni 2001||9 feb 2011||Human Genome Sciences, Inc.||Antibodies that immunospecifically bind to BLyS|
|EP2281843A1||15 juni 2001||9 feb 2011||Human Genome Sciences, Inc.||Antibodies that immunospecifically bind to blys|
|EP2292663A2||24 aug 2007||9 maart 2011||Kyowa Hakko Kirin Co., Ltd.||Antagonistic human light-specific human monoclonal antibodies|
|EP2298869A1||14 juni 2004||23 maart 2011||University Of Medicine And Dentistry Of New Jersey||Recombinant protein production in the presence of mRNA interferase|
|EP2298874A1||15 dec 2003||23 maart 2011||Halozyme, Inc.||Human chondroitinase glycoprotein (CHASEGP), process for preparing the same, and pharmaceutical compositions comprising thereof|
|EP2302040A1||14 juni 2004||30 maart 2011||University Of Medicine And Dentistry Of New Jersey||Medical use of mRNA interferase|
|EP2302385A1||13 feb 2003||30 maart 2011||American Diagnostica Inc.||Methods for selecting treatment regimens and predicting outcomes in cancer patients|
|EP2308996A1||29 maart 1999||13 april 2011||NorthWest Biotherapeutics, Inc.||Therapeutic and diagnostic applications based on the role of the CXCR-4 and SDF-1 genes in tumourigenesis|
|EP2311973A1||5 maart 2004||20 april 2011||Halozyme, Inc.||Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof|
|EP2316487A1||12 april 2004||4 mei 2011||MedImmune, LLC||Recombinant IL-9 antibodies & uses thereof|
|EP2319941A2||23 okt 2006||11 mei 2011||GeneNews Inc.||Method and apparatus for correlating levels of biomarker products with disease|
|EP2330213A1||5 maart 2004||8 juni 2011||Halozyme, Inc.|
|EP2338512A1||28 nov 2001||29 juni 2011||MedImmune, LLC||Methods of administering/dosing anti-RSV antibodies for prophylaxis and treatment|
|EP2341060A1||12 dec 2001||6 juli 2011||MedImmune, LLC||Molecules with extended half-lives, compositions and uses thereof|
|EP2351584A1||23 dec 2004||3 aug 2011||Genentech, Inc.||Novel anti-IL 13 antibodies and uses thereof|
|EP2354149A1||12 dec 2001||10 aug 2011||MedImmune, LLC||Molecules with extended half-lives, compositions and uses thereof|
|EP2357187A1||12 dec 2001||17 aug 2011||MedImmune, LLC||Molecules with extended half-lives, compositions and uses thereof|
|EP2357192A1||25 feb 2000||17 aug 2011||Human Genome Sciences, Inc.||Human endokine alpha and methods of use|
|EP2360476A1||13 feb 2003||24 aug 2011||American Diagnostica Inc.||Methods for selecting treatment regimens and predicting outcomes in cancer patients|
|EP2361635A2||24 aug 2001||31 aug 2011||Pfizer Products Inc.||Anti IgE vaccines|
|EP2368578A1||9 jan 2004||28 sept 2011||Macrogenics, Inc.||Identification and engineering of antibodies with variant Fc regions and methods of using same|
|EP2371389A2||14 aug 2003||5 okt 2011||MacroGenics, Inc.||FcgammaRIIB-specific antibodies and methods of use thereof|
|EP2383350A1||6 mei 2005||2 nov 2011||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||12 mei 2000||9 nov 2011||Arbor Vita Corporation||Peptides or peptide analogues for modulating the binding of a PDZ protein and a PL protein|
|EP2387995A1||29 maart 2007||23 nov 2011||PTC Therapeutics, Inc.||Methods for the production of functional protein from DNA having a nonsense mutation and the treatment of disorders associated therewith|
|EP2405015A2||5 maart 2004||11 jan 2012||Halozyme, Inc.|
|EP2407548A1||16 okt 2007||18 jan 2012||MedImmune, LLC||Molecules with reduced half-lives, compositions and uses thereof|
|EP2412384A1||28 nov 2001||1 feb 2012||MedImmune, LLC||Methods of administering/dosing anti-RSV antibodies for prophylaxis and treatment|
|EP2422811A2||27 okt 2005||29 feb 2012||MedImmune, LLC||Modulation of antibody specificity by tailoring the affinity to cognate antigens|
|EP2431054A2||14 juni 2001||21 maart 2012||Human Genome Sciences, Inc.||Human tumor necrosis factor delta and epsilon|
|EP2453024A2||21 juni 2005||16 mei 2012||The Board of Trustees of The Leland Stanford Junior University||Genes and pathways differentially expressed in bipolar disorder and/or major depressive disorder|
|EP2484696A1||24 aug 2007||8 aug 2012||Kyowa Hakko Kirin Co., Ltd.||Antagonistic human light-specific human monoclonal antibodies|
|EP2500030A2||4 nov 2006||19 sept 2012||Genentech, Inc.||Use of complement pathway inhibitors to treat ocular diseases|
|EP2505209A1||26 juni 2007||3 okt 2012||MacroGenics, Inc.||Fcgamma-RIIB-specific antibodies and methods of the use thereof|
|EP2514439A1||15 nov 2007||24 okt 2012||Functional Genetics, Inc.||Anti-TSG101antibodies and their uses for treatment of viral infections|
|EP2518163A2||9 okt 2007||31 okt 2012||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||6 feb 2006||7 nov 2012||GeneNews Inc.||Mild osteoathritis biomarkers and uses thereof|
|EP2564864A2||13 nov 2006||6 maart 2013||The Board of Trustees of the Leland||FGF2-related methods for diagnosing and treating depression|
|EP2573114A1||10 aug 2006||27 maart 2013||MacroGenics, Inc.||Identification and engineering of antibodies with variant Fc regions and methods of using same|
|EP2610267A1||17 dec 2007||3 juli 2013||Genentech, Inc.||Antagonist anti-Notch3 antibodies and their use in the prevention and treatment of Notch3-related diseases|
|EP2629094A1||24 jan 2008||21 aug 2013||Carnegie Mellon University||Optical biosensors|
|EP2639301A2||29 juni 2007||18 sept 2013||Bristol-Myers Squibb Company||Polynucleotides encoding novel PCSK9 variants|
|EP2671946A1||29 juni 2007||11 dec 2013||Bristol-Myers Squibb Company||Polynucleotides encoding novel PCSK9 variants|
|EP2786762A2||17 dec 2009||8 okt 2014||MacroGenics, Inc.||Covalent diabodies and uses thereof|
|EP2805728A1||23 dec 2004||26 nov 2014||Genentech, Inc.||Novel anti-IL 13 antibodies and uses thereof|
|EP2837697A2||9 okt 2007||18 feb 2015||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|
|EP2932982A1||17 mei 2006||21 okt 2015||Amicus Therapeutics, Inc.||A method for the treatment of pompe disease using 1-deoxynojirimycin and derivatives|
|EP2998318A1||4 nov 2006||23 maart 2016||Genentech, Inc.||Use of complement pathway inhibitors to treat ocular diseases|
|EP3009517A1||5 maart 2004||20 april 2016||Halozyme, Inc.||Soluble hyaluronidase glycoprotein (shasegp), process for preparing the same, uses and pharmaceutical compositions comprising thereof|
|EP3026432A2||27 dec 2011||1 juni 2016||Brown University||Method for predicting patient's response to biglycan treatment|
|EP3045472A1||23 feb 2006||20 juli 2016||Halozyme, Inc.||Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases|
|EP3045475A1||25 maart 2009||20 juli 2016||MacroGenics, Inc.||Bcr-complex-specific antibodies and methods of using same|
|EP3067063A1||25 maart 2009||14 sept 2016||MacroGenics, Inc.||Her2/neu-specific antibodies and methods of using same|
|US5521291 *||15 dec 1993||28 mei 1996||Boehringer Ingelheim International, Gmbh||Conjugates for introducing nucleic acid into higher eucaryotic cells|
|US5547932 *||23 sept 1992||20 aug 1996||Boehringer Ingelheim International Gmbh||Composition for introducing nucleic acid complexes into higher eucaryotic cells|
|US5645829 *||18 juni 1993||8 juli 1997||Beth Israel Hospital Association||Mesothelial cell gene therapy|
|US5693509 *||6 april 1994||2 dec 1997||Boehringer Ingelheim International Gmbh||Adenovirus for delivering foreign DNA into higher eukaryotic cells|
|US5695991 *||28 okt 1992||9 dec 1997||Got-A-Gene Ab||Targeted delivery of virus vector to mammalian cells|
|US5728399 *||7 juni 1995||17 maart 1998||University Of Conn.||Use of a bacterial component to enhance targeted delivery of polynucleotides to cells|
|US5837533 *||28 sept 1994||17 nov 1998||American Home Products Corporation||Complexes comprising a nucleic acid bound to a cationic polyamine having an endosome disruption agent|
|US5869331 *||28 aug 1997||9 feb 1999||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|
|US5889169 *||25 mei 1994||30 maart 1999||Cold Spring Harbor Laboratory||Cell cycle regulatory protein p16 gene|
|US5922859 *||1 feb 1992||13 juli 1999||Boehringer Ingelheim International Gmbh||Complexes containing nucleic acid which can be taken-up by endocytosis into higher eukaryotic cells|
|US5962316 *||14 sept 1994||5 okt 1999||Cold Spring Harbor Laboratory||Cell-cycle regulatory proteins, and uses related thereto|
|US5968821 *||15 juli 1997||19 okt 1999||Cold Spring Harbor Laboratories, Inc.||Cell-cycle regulatory proteins, and uses related thereto|
|US5981273 *||25 mei 1995||9 nov 1999||Boehringer Ingelheim Int'l. Gmbh||Composition comprising an endosomolytic agent for introducing nucleic acid complexes into higher eucaryotic cells|
|US5985655 *||7 juni 1995||16 nov 1999||The United States Of America As Represented By The Department Of Health And Human Sevices||Targetable vector particles|
|US6004798 *||14 mei 1997||21 dec 1999||University Of Southern California||Retroviral envelopes having modified hypervariable polyproline regions|
|US6022735 *||25 mei 1995||8 feb 2000||Boehringer Ingelheim International Gmbh||Composition for introducing nucleic acid complexes into higher eucaryotic cells|
|US6043030 *||2 jan 1996||28 maart 2000||Cold Spring Harbor Laboratory||Cell-cycle regulatory proteins, and uses related thereto|
|US6057155 *||6 aug 1998||2 mei 2000||Genvec, Inc.||Targeting adenovirus with use of constrained peptide motifs|
|US6057299 *||27 sept 1996||2 mei 2000||Calydon, Inc.||Tissue-specific enhancer active in prostate|
|US6068837 *||3 dec 1997||30 mei 2000||Beth Israel Hospital Association||Mesothelial cell gene therapy|
|US6074850 *||14 feb 1997||13 juni 2000||Canji, Inc.||Retinoblastoma fusion polypeptides|
|US6127170 *||28 sept 1995||3 okt 2000||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|
|US6136792 *||11 juli 1997||24 okt 2000||Calydon, Inc.||Prostate specific enhancer polynucleotides and methods of use thereof|
|US6146885 *||22 juli 1994||14 nov 2000||University Of Medicine And Dentistry Of New Jersey||Cell-type specific gene transfer using retroviral vectors containing antibody-envelope fusion proteins|
|US6153435 *||17 juni 1999||28 nov 2000||Cornell Research Foundation, Inc.||Nucleic acid that encodes a chimeric adenoviral coat protein|
|US6159728 *||25 juni 1993||12 dec 2000||Btg International Limited||RNA bacteriophage-based delivery system|
|US6162641 *||5 juni 1998||19 dec 2000||The Regents Of The University Of Michigan||Neuregulin response element and uses therefor|
|US6211334||29 nov 1994||3 april 2001||Cold Spring Harbor||Cell-cycle regulatory proteins, and uses related thereto|
|US6274322 *||16 dec 1999||14 aug 2001||Boehringer Ingelheim International Gmbh||Composition for introducing nucleic acid complexes into higher eucaryotic cells|
|US6329190||6 dec 1999||11 dec 2001||Genvec, Inc.||Targetting adenovirus with use of constrained peptide motifs|
|US6331390||30 juni 1995||18 dec 2001||Cold Spring Harbor Laboratory||Cell-cycle regulatory proteins, and uses related thereto|
|US6379927||19 mei 1999||30 april 2002||Canji, Inc.||Retinoblastoma fusion proteins|
|US6379965||22 okt 1999||30 april 2002||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|
|US6392069||8 juli 1998||21 mei 2002||Canji, Inc.||Compositions for enhancing delivery of nucleic acids to cells|
|US6465253||27 nov 1996||15 okt 2002||Genvec, Inc.||Vectors and methods for gene transfer to cells|
|US6486131||30 jan 1998||26 nov 2002||Cold Spring Harbor Laboratory||Cell-cycle regulatory proteins, and uses related thereto|
|US6503501||13 aug 1999||7 jan 2003||W. French Anderson||Targetable vector particles|
|US6534051||17 aug 1998||18 maart 2003||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|
|US6576456||4 juni 1999||10 juni 2003||Cornell Research Foundation, Inc.||Chimeric adenovirus fiber protein|
|US6649407||1 okt 2001||18 nov 2003||Genvec, Inc.||Targeting adenovirus with use of constrained peptide motifs|
|US6660264||10 april 2000||9 dec 2003||Health Protection Agency||Treatment of intracellular infection|
|US6699656||20 nov 2001||2 maart 2004||University Of Maryland Biotechnology Institute||Treatment and prevention of HIV infection by administration of derivatives of human chorionic gonadotropin|
|US6849399||27 aug 1997||1 feb 2005||Bio-Rad Laboratories, Inc.||Methods and compositions for diagnosis and treatment of iron misregulation diseases|
|US6864082||13 juli 2001||8 maart 2005||University Of Southern California||Modified viral surface proteins for binding to extracellular matrix components|
|US6875588||30 nov 2001||5 april 2005||Avigenics, Inc.||Ovomucoid promoter and methods of use|
|US6902731||19 mei 1999||7 juni 2005||Canji, Inc.||Methods of treating hyperproliferative disorders using retinoblastoma fusion proteins|
|US6916918||5 juni 2001||12 juli 2005||Cell Genesys, Inc.||Human glandular kallikrein enhancer, vectors comprising the enhancer and methods of use thereof|
|US6951755||24 okt 2001||4 okt 2005||Genvec, Inc.||Vectors and methods for gene transfer|
|US7002027||8 juli 1997||21 feb 2006||Canji, Inc.||Compositions and methods for therapeutic use|
|US7026116||7 mei 1997||11 april 2006||Bio-Rad Laboratories, Inc.||Polymorphisms in the region of the human hemochromatosis gene|
|US7052845||20 nov 2002||30 mei 2006||Bio-Rad Laboratories, Inc.||Polymorphisms in the region of the human hemochromatosis gene|
|US7067255||2 mei 2002||27 juni 2006||Bio-Rad Laboratories, Inc.||Hereditary hemochromatosis gene|
|US7078483||19 aug 2002||18 juli 2006||University Of Southern California||Retroviral vectors including modified envelope escort proteins|
|US7135562||14 maart 2002||14 nov 2006||University Of Cincinnati||Avian iFABP gene expression controlling region|
|US7176300||3 aug 2001||13 feb 2007||Avigenics, Inc.||Avian lysozyme promoter|
|US7199279||1 april 2002||3 april 2007||Avigenics, Inc.||Recombinant promoters in avian cells|
|US7202227||13 nov 2001||10 april 2007||Wyeth||Multifunctional molecular complexes for gene transfer to cells|
|US7276364||20 nov 2000||2 okt 2007||Dendreon Corporation||Nucleic acids encoding endotheliases, endotheliases and uses thereof|
|US7294507||28 mei 2004||13 nov 2007||Avigenics, Inc.||Ovomucoid promoters and methods of use|
|US7335761||31 jan 2005||26 feb 2008||Avigenics, Inc.||Avian gene expression controlling regions|
|US7347998||15 dec 2004||25 maart 2008||University Of Southern California||Method of delivering therapeutic agents to site of tissue injury|
|US7348014||7 sept 2004||25 maart 2008||Transgene, S.A.||Poxvirus with targeted infection specificity|
|US7354591||12 april 2001||8 april 2008||Transgene S.A.||Poxvirus with targeted infection specificity|
|US7375258||2 dec 2002||20 mei 2008||Avigenics, Inc.||Transgenic avians with an ovomucoid gene expression control region linked to a nucleotide sequence encoding a heterologous polypeptide|
|US7393534||15 juli 2004||1 juli 2008||Barros Research Institute||Compositions and methods for immunotherapy of cancer and infectious diseases|
|US7425617||30 jan 1998||16 sept 2008||Cold Spring Harbor Laboratory||Antibodies to the cell cycle regulatory protein p16|
|US7449562||28 juni 2002||11 nov 2008||Novartis Ag||PERV screening method and use thereof|
|US7498314||3 mei 2002||3 maart 2009||Fit Biotech Oyj Plc||Expression vectors and uses thereof|
|US7507873||4 jan 2007||24 maart 2009||Avigenics, Inc.||Transgenic avians containing recombinant ovomucoid promoters|
|US7510718||3 mei 2002||31 maart 2009||Fit Biotech Oyj Plc||Expression vectors and uses thereof|
|US7534769||22 jan 2002||19 mei 2009||Canji, Inc.||Compositions and methods for enhancing delivery of therapeutic agents to cells|
|US7541512||26 jan 2007||2 juni 2009||Synageva Biopharma Corp.||Avians containing a lysozyme promoter transgene|
|US7550561||14 april 1994||23 juni 2009||Cold Spring Harbor Laboratory||p16INK4 polypeptides|
|US7550650||2 okt 2003||23 juni 2009||Synageva Biopharma Corp.||Production of a transgenic avian by cytoplasmic injection|
|US7566452||4 mei 1999||28 juli 2009||New York University||Cancer treatment with endothelin receptor antagonists|
|US7579169||5 jan 2006||25 aug 2009||Bio-Rad Laboratories, Inc.||Hereditary hemochromatosis gene|
|US7595385||20 juni 2005||29 sept 2009||Bio-Rad Laboratories, Inc.||Polymorphisms in the region of the human hemochromatosis gene|
|US7691632||20 aug 2008||6 april 2010||Cold Spring Harbor Laboratory||Kit for detecting the level of cyclin-dependent kinase inhibitor P16 gene expression|
|US7700341||2 feb 2001||20 april 2010||Dendreon Corporation||Nucleic acid molecules encoding transmembrane serine proteases, the encoded proteins and methods based thereon|
|US7812215||17 nov 2008||12 okt 2010||Synageva Biopharma Corp.||Methods and protein production using ovomucoid promoters|
|US7820157||18 jan 2008||26 okt 2010||University Of Southern California||Transgene delivering retrovirus targeting collagen exposed at site of tissue injury|
|US7897151||4 aug 2005||1 maart 2011||Pharmacia & Upjohn Company, Llc||Anti-IgE vaccines|
|US7928189||5 mei 2008||19 april 2011||Ottawa Health Research Institute||PCSK9 polypeptide fragment|
|US7964708||15 nov 2007||21 juni 2011||Limin Li||Anti-TSG101 antibodies and their uses for treatment of viral infections|
|US7973139||25 maart 2005||5 juli 2011||Human Genome Sciences, Inc.||Antibodies against nogo receptor|
|US7998680||19 feb 2009||16 aug 2011||Bio-Rad Laboratories, Inc.||Determining genotype of a polymorphic site in the hereditary hemochromatosis gene|
|US8003096||28 maart 2006||23 aug 2011||Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw||Means and methods for the recruitment and identification of stem cells|
|US8021833||12 feb 2004||20 sept 2011||Functional Genetics, Inc.||Method for reducing HIV viral budding by administering a VPS28-specfic antibody that disrupts Gag-TSG101-VPS28 binding interactions|
|US8022189||12 april 2010||20 sept 2011||Albany Medical College||Isolated antibodies against biologically active leptin-related peptides|
|US8067000||22 mei 2009||29 nov 2011||New York University||Cancer treatment with endothelin receptor antagonists|
|US8067545||22 dec 2010||29 nov 2011||Albany Medical College||Isolated antibodies against biologically active leptin-related peptides|
|US8088382||30 juni 2006||3 jan 2012||Cornell Research Foundation, Inc.||Methods of inhibiting transendothelial migration of neutrophils and monocytes with anti-CD99L2 antibodies|
|US8148509||10 sept 2010||3 april 2012||University Of Southern California||Transgene delivering retrovirus targeting collagen exposed at site of tissue injury|
|US8211858||24 april 2008||3 juli 2012||The University Of Toledo||Modified plasminogen activator inhibitor type-1 molecule and methods based thereon|
|US8231878||10 juni 2009||31 juli 2012||Cosmo Research & Development S.P.A.||Receptor trem (triggering receptor expressed on myeloid cells) and uses thereof|
|US8257714||14 april 2008||4 sept 2012||Michigan State University||Compositions and methods for immunotherapy of cancer and infectious diseases|
|US8257927||7 juli 2011||4 sept 2012||Bio-Rad Laboratories, Inc.||Hereditary hemochromatosis gene|
|US8273356||24 nov 2009||25 sept 2012||Pfizer Inc.||Anti-IgE vaccines|
|US8530441||29 maart 2012||10 sept 2013||University Of Southern California||Transgene delivering retrovirus targeting collagen exposed at site of tissue injury|
|US8568719||13 aug 2010||29 okt 2013||Crucell Holland B.V.||Antibodies against human respiratory syncytial virus (RSV) and methods of use|
|US8597645||6 okt 2011||3 dec 2013||New York University||Cancer treatment with endothelin receptor antagonists|
|US8647622||27 aug 2008||11 feb 2014||Sanofi||Humanized anti-CXCR5 antibodies, derivatives thereof and their use|
|US8796423||18 april 2008||5 aug 2014||Eli Lilly And Company||Anti-TSG101 antibodies and their uses for treatment of viral infections|
|US8809287||15 nov 2005||19 aug 2014||Icahn School Of Medicine At Mount Sinai||Compositions and methods for altering Wnt autocrine signaling|
|US8815558||29 mei 2009||26 aug 2014||Halozyme, Inc.||Human chondroitinase glycoprotein (CHASEGP), process for preparing the same, and pharmaceutical compositions comprising thereof|
|US8871734||5 aug 2013||28 okt 2014||The University Of Southern California||Transgene delivering retrovirus targeting collagen exposed at site of tissue injury|
|US8916517||2 nov 2010||23 dec 2014||The Administrators Of The Tulane Educational Fund||Analogs of pituitary adenylate cyclase-activating polypeptide (PACAP) and methods for their use|
|US8937169||31 jan 2011||20 jan 2015||Human Genome Sciences, Inc.||Human G-protein chemokine receptor HSATU68|
|US8980262||11 maart 2013||17 maart 2015||Sanofi||Humanized anti-CXCR5 antibodies, derivatives thereof and their use|
|US8981061||30 nov 2012||17 maart 2015||Novo Nordisk A/S||Receptor TREM (triggering receptor expressed on myeloid cells) and uses thereof|
|US9000127||30 nov 2012||7 april 2015||Novo Nordisk A/S||Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1)|
|US9006181||21 juli 2005||14 april 2015||The Administrators Of The Tulane Educational Fund||Treatment of renal dysfunction and multiple myeloma using PACAP compounds|
|US9125897||24 juli 2013||8 sept 2015||New York University||Cancer treatment with endothelin receptor antagonists|
|US9139642||8 juli 2011||22 sept 2015||Crucell Holland B.V.||Anti-human respiratory syncytial virus (RSV) antibodies and methods of use|
|US9175087||11 maart 2013||3 nov 2015||Sanofi||Humanized anti-CXCR5 antibodies, derivatives thereof and their use|
|US9211315||7 mei 2014||15 dec 2015||Halozyme, Inc.||Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases|
|US9228019||11 maart 2013||5 jan 2016||Sanofi||Humanized anti-CXCR5 antibodies, derivatives thereof and their use|
|US9243067||11 maart 2013||26 jan 2016||Sanofi||Humanized anti-CXCR5 antibodies, derivatives thereof and their use|
|US9249306||16 feb 2010||2 feb 2016||Carnegie Mellon University||Quenched dendrimeric dyes for florescence detection|
|US9273111||12 juli 2011||1 maart 2016||Universite De Lorraine||Therapeutic TREM-1 peptides|
|US9365638||10 sept 2013||14 juni 2016||Crucell Holland B. V.||Antibodies against human respiratory syncytial virus (RSV) and methods of use|
|US9403900||5 juni 2014||2 aug 2016||Crucell Holland B.V.||Anti-human respiratory syncytial virus (RSV) antibodies and methods of use|
|US9447454||12 dec 2013||20 sept 2016||The Rockefeller University||Method of purifying RNA binding protein-RNA complexes|
|US9458464||23 juni 2014||4 okt 2016||The Johns Hopkins University||Treatment of neuropathic pain|
|US9550830||30 juli 2013||24 jan 2017||Novo Nordisk A/S||Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1)|
|US9562223||26 feb 2014||7 feb 2017||Halozyme, Inc.||Methods for reducing intraocular pressure by administering a soluble hyaluronidase glycoprotein (sHASEGP)|
|US9592289||26 maart 2013||14 maart 2017||Sanofi||Stable IgG4 based binding agent formulations|
|US9597346||17 jan 2011||21 maart 2017||Cornell University||Methods for reducing protein levels in a cell|
|US9663568||30 nov 2012||30 mei 2017||Novo Nordisk A/S||Antibodies that bind peptidoglycan recognition protein 1|
|US9677061||21 dec 2010||13 juni 2017||Halozyme, Inc.|
|US9677062||28 dec 2011||13 juni 2017||Halozyme, Inc.||Hyaluronidase and factor VIII compositions|
|US9725486||13 jan 2014||8 aug 2017||Fit Biotech Oy||Methods of treating HIV diseases using novel expression vectors|
|US9732130||14 sept 2012||15 aug 2017||Ottawa Hospital Research Institute||WNT7A compositions and method of using the same|
|US20080103108 *||2 mei 2007||1 mei 2008||Yanina Rozenberg||Targeted artificial gene delivery|
|Internationale classificatie||A61K31/70, A61K48/00, C12N15/867, A61K35/12, C12N15/00, C12N15/87, A01K67/027, C12N7/00, C12N5/10|
|Coöperatieve classificatie||C12N15/87, A61K48/00, C12N2810/10, C12N2740/13045, C12N15/86, C12N2810/859, C12N2810/80, C12N2740/13043, C12N7/00, C12N2730/10122|
|Europese classificatie||C12N15/86, A61K48/00, C12N7/00, C12N15/87|
|16 april 1992||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
|16 april 1992||AK||Designated states|
Kind code of ref document: A1
Designated state(s): AU CA JP
|11 maart 1993||ENP||Entry into the national phase in:|
Ref country code: CA
Ref document number: 2092323
Kind code of ref document: A
Format of ref document f/p: F
|11 maart 1993||WWE||Wipo information: entry into national phase|
Ref document number: 2092323
Country of ref document: CA
|19 april 1993||WWE||Wipo information: entry into national phase|
Ref document number: 1991919436
Country of ref document: EP
|4 aug 1993||WWP||Wipo information: published in national office|
Ref document number: 1991919436
Country of ref document: EP
|1 april 1999||WWW||Wipo information: withdrawn in national office|
Ref document number: 1991919436
Country of ref document: EP