# US 8,465,732 B2 · Endothelial cells expressing adenovirus E4ORF1 and methods of use thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,465,732 B2 - Document ID: US8465732B2 - Status: Granted - Kind: B2 - Application number: 12/523,372 - Filed: 2008-01-18 - Published: 2010-04-15 - Granted: 2013-06-18 - Earliest claimed: 2007-01-19 - Assignees: Cornell Research Foundation, Inc.; Sloan-Kettering Institute for Cancer Research - Applicants: Shahin Rafii; Fan Zhang; Marco Seandel - Inventors: Shahin Rafii; Fan Zhang; Marco Seandel - Web page: https://patentgrep.com/patent/US8465732B2 ## CPC - C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)} - C07K 14/005: from viruses - C12N 5/0647: {Haematopoietic stem cells; Uncommitted or multipotent progenitors} - C12N 5/069: {Vascular Endothelial cells} - C12N 5/0693: {Tumour cells; Cancer cells} - C12N 15/1131: {against viruses} - C12N 15/1136: {against growth factors, growth regulators, cytokines, lymphokines or hormones} - C12N 2310/14: interfering nucleic acids [NA] - C12N 2501/998: Proteins not provided for elsewhere - C12N 2710/10322: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - C12N 2740/10043: viral genome or elements thereof as genetic vector - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract The present invention relates to adenovirus E4ORF 1 gene and to endothelial cells engineered to express the E40RF 1 gene. The present invention also relates to uses of the E40RF 1 gene, and cells expressing the E40RF1 gene, and to compositions comprising the E4ORF 1 gene, or comprising cells expressing the E4ORF 1 gene. ## Claims ### Claim 1 A method for maintaining or expanding endothelial cells in culture, comprising: (a) introducing a nucleic acid sequence encoding adenovirus E4ORF1 under the control of a promoter into isolated endothelial cells, wherein the nucleic acid sequence does not encode adenovirus E4ORF2, E4ORF3, E4ORF4, E4ORF5, or E4ORF6, such that the endothelial cells express E4ORF1 to a level sufficient to maintain or expand the isolated endothelial cells in culture, and (b) culturing the isolated endothelial cells expressing E4ORF1. ### Claim 2 (depends on 1) The method of claim 1, wherein the step of introducing is performed by transfection. ### Claim 3 (depends on 2) The method of claim 2, wherein the transfection is performed using a method selected from the group consisting of liposome-mediated transfection, polybrene-mediated transfection, DEAE dextran-mediated transfection, electroporation, calcium phosphate precipitation, microinjection, or micro-particle bombardment. ### Claim 4 (depends on 1) The method of claim 1, wherein the step of introducing is performed by viral-mediated transduction. ### Claim 5 (depends on 4) The method of claim 4, wherein the viral-mediated transduction is selected from the group consisting of lentivirus-mediated transduction, adenovirus-mediated transduction, retrovirus-mediated transduction, adeno-associated virus-mediated transduction and herpesvirus-mediated transduction. ### Claim 6 (depends on 1) The method of claim 1 wherein the step of culturing is performed in the absence of serum. ### Claim 7 (depends on 1) The method of claim 1 wherein the step of culturing is performed in the absence of exogenous growth factors. ### Claim 8 (depends on 1) The method of claim 1, wherein the endothelial cells are primary endothelial cells. ### Claim 9 A population of isolated endothelial cells expressing adenovirus E4ORF1 but not expressing adenovirus E4ORF2, E4ORF3, E4ORF4, E4ORF5, or E4ORF6. ### Claim 10 (depends on 9) A composition comprising a population of endothelial cells according to claim 9. ### Claim 11 (depends on 10) The composition of claim 10 further comprising a carrier solution. ### Claim 12 (depends on 11) The composition of claim 11, wherein the carrier solution is a physiological saline solution. ### Claim 13 (depends on 9) A therapeutic composition comprising a population of endothelial cells according to claim 9 and a carrier solution suitable for administration to a subject, wherein the endothelial cells are human umbilical vein endothelial cells. ### Claim 14 (depends on 13) The therapeutic composition of claim 13, wherein the carrier solution is a physiological saline solution. ### Claim 15 A method of culturing primary cancer cells, stem cells or progenitor cells, comprising: (a) obtaining or generating a population of isolated E4ORF1-expressing human umbilical vein endothelial cells, wherein the E4ORF1-expressing human umbilical vein endothelial cells do not express E4ORF2, E4ORF3, E4ORF4, E4ORF5, or E4ORF6, and (b) culturing the E4ORF1-expressing human umbilical vein endothelial cells in the same culture vessel with primary cancer cells, stem cells or progenitor cells. ### Claim 16 (depends on 15) The method of claim 15, wherein the E4ORF1-expressing human umbilical vein endothelial cells form a feeder cell layer on the surface of the culture vessel, and wherein the primary cancer cells, stem cells or progenitor cells are placed on top of the feeder cell layer. ### Claim 17 (depends on 15) The method of claim 15, wherein the stem cells are hematopoietic stem cells or embryonic stem cells. ### Claim 18 (depends on 15) The method of claim 15, wherein the stem cells are hematopoietic stem cells and wherein the human umbilical vein endothelial cells and the hematopoietic stem cells are grown in the absence of serum. ### Claim 19 (depends on 15) The method of claim 15, wherein the stem cells are embryonic stem cells and wherein the human umbilical vein endothelial cells and the embryonic stem cells are grown in the absence of serum. ### Claim 20 A method of culturing primary cancer cells, stem cells or progenitor cells, comprising (a) obtaining or generating a population of isolated E4ORF1-expressing human umbilical vein endothelial cells, wherein the E4ORF1-expressing human umbilical vein endothelial cells do not express E4ORF2, E4ORF3, E4ORF4, E4ORF5, or E4ORF6, (b) culturing the E4ORF 1-expressing human umbilical vein endothelial cells in a culture vessel, (c) collecting conditioned medium from the culture vessel, and (d) adding the conditioned medium to a culture of primary cancer cells, stem cells or progenitor cells. ### Claim 21 A method for maintaining or expanding endothelial cells in culture, the method comprising: (a) obtaining a population of isolated endothelial cells expressing adenovirus E4ORF1 to a level sufficient to maintain or expand the isolated endothelial cells in culture, but not expressing adenovirus E4ORF2, E4ORF3, E4ORF4, E4ORF5, or E4ORF6, and (b) culturing the population of isolated endothelial cells. ### Claim 22 (depends on 21) The method of claim 21 wherein the step of culturing is performed in the absence of serum. ### Claim 23 (depends on 21) The method of claim 21 wherein the step of culturing is performed in the absence of exogenous growth factors.