# US 5,700,470 A · Recombinant adenovirus with removed EZA gene and method of preparation > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,700,470 A - Document ID: US5700470A - Status: Granted - Kind: A - Application number: 08/615,048 - Filed: 1996-03-12 - Granted: 1997-12-23 - Earliest claimed: 1995-03-15 - Assignees: Sumitomo Pharmaceuticals Company, Limited - Inventors: Izumu Saito; Yumi Kanegae; Michio Nakai - Web page: https://patentgrep.com/patent/US5700470A ## CPC - C12N 15/86: Viral vectors - C12N 2710/10343: viral genome or elements thereof as genetic vector - C12N 2800/30: Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract A recombinant DNA virus for transfecting an animal cell and bearing a foreign gene and a promoter capable of regulating expression of the foreign gene is completely deleted of the function of E2A gene. The recombinant DNA virus can thus stably transduce the foreign gene into various animal cells, which leads to continuous expression of the foreign gene in the animal cells. The continuous expression of the foreign gene can provide an effective treatment of hereditary disease. ## Claims ### Claim 1 A recombinant adenovirus comprising an adenovirus genome having a foreign gene and a promoter for expressing said foreign gene, wherein the function of an E2A gene is completely deleted by removing a part or all of said E2A gene. ### Claim 2 (depends on 1) The recombinant adenovirus of claim 1, wherein said foreign gene and said promoter for expressing said foreign gene are inserted in an orientation opposite to the natural transcription orientation of E1A and E1B genes. ### Claim 3 (depends on 1) The recombinant adenovirus of claim 1, wherein said adenovirus genome has a deletion of an E1A gene. ### Claim 4 (depends on 1, 8) The recombinant adenovirus of claim 1 or 8 wherein said foreign gene and said promoter for expressing said foreign gene are inserted in a deletion. ### Claim 5 (depends on 4) The recombinant adenovirus or claim 4, wherein said adenovirus genome has an E3 gene region deletion of genome map units 79.6 to 84.8%. ### Claim 6 (depends on 1) The recombinant adenovirus of claim 1, wherein said promoter for expressing said foreign gene is a hybrid CAG promoter comprising a cytomegalovirus enhancer, a chicken .beta.-actin promoter, a rabbit .beta.-globin splice acceptor and a poly(A) sequence. ### Claim 7 A recombinant adenovirus comprising an adenovirus genome having two recombinase-recognition sequences in the same orientation located on both sides of an E2A gene. ### Claim 8 (depends on 7) The recombinant adenovirus of claim 7, wherein one of said two recombinase-recognition sequences is located between the termination codons of an E2A gene and an L3 gene, without preventing proliferation of said recombinant adenovirus. ### Claim 9 (depends on 8) The recombinant adenovirus of claim 8, wherein the other of said two recombinase-recognition sequences is located in the segment from 79.6 to 84.8% genome map units of said adenovirus genome. ### Claim 10 (depends on 7) The recombinant adenovirus of claim 7, wherein said recombinase is a Cre recombinase derived from Eschericha coli P1 phage. ### Claim 11 (depends on 7) The recombinant adenovirus of claim 7, wherein each of said recombinase-recognition sequences is representing by SEQ ID NO:1 of loxP which is a substrate for Cre recombinase. ### Claim 12 (depends on 7) The recombinant adenovirus of claim 7, further comprising a foreign gene. ### Claim 13 (depends on 12) The recombinant adenovirus of claim 12, further comprising a promoter for expressing said foreign gene. ### Claim 14 (depends on 13) The recombinant adenovirus of claim 13, wherein said foreign gene and said promoter for expressing said foreign gene are inserted in an orientation opposite to the natural transcription orientation of E1A and E1B genes. ### Claim 15 (depends on 7) The recombinant adenovirus of claim 7, wherein said adenovirus genome has a deletion of an E1A gene. ### Claim 16 (depends on 15) The recombinant adenovirus of claim 15, wherein said foreign gene and said promoter for expressing said foreign gene are inserted in a deletion. ### Claim 17 (depends on 16) The recombinant adenovirus or claim 16, wherein said adenovirus genome has an E3 gene region deletion of genome map units 79.6 to 84.8%. ### Claim 18 (depends on 13) The recombinant adenovirus of claim 13, wherein said promoter for expressing said foreign gene is a hybrid CAG promoter comprising a cytomegalovirus enhancer, a chicken .beta.-actin promoter, a rabbit .beta.-globin splice acceptor and a poly(A) sequence. ### Claim 19 A method for constructing a recombinant adenovirus without a functional E2A gene, which comprises the steps of: transducing an animal cell line with an adenovirus vector (a) having inserted therein a promoter, a recombinase gene and a poly(A) sequence, and a recombinant adenovirus (b) comprising an adenovirus genome having two recombinase-recognition sequences located in the same orientation on both sides of an E2A gene region and excising the E2A gene region located between said two recombinase-recognition sequences. ### Claim 20 (depends on 19) The method for constructing a recombinant adenovirus of claim 19, wherein one of said two recombinase-recognition sequences is located between the termination codons of an E2A gene and an L3 gene, without preventing proliferation of said recombinant adenovirus(b). ### Claim 21 (depends on 19) The method for constructing a recombinant adenovirus of claim 19, wherein said recombinase is a Cre recombinase derived from Escherichia coli P1 phage. ### Claim 22 (depends on 19) The method for constructing a recombinant adenovirus of claim 19, wherein each said recombinase-recognition sequences is represented by SEQ ID NO:1 of loxP which is a substrate for a Cre recombinase. ### Claim 23 (depends on 19) The method for constructing a recombinant adenovirus of claim 19, wherein said recombinant adenovirus (b) further comprises a foreign gene. ### Claim 24 (depends on 19) The method for constructing a recombinant adenovirus of claim 19, wherein said recombinant adenovirus (b) further comprises a promoter for regulating expressing said foreign gene. ### Claim 25 (depends on 24) The recombinant adenovirus of claim 24, wherein said foreign gene and said promoter for expressing said foreign gene are inserted in an orientation opposite to the natural transcription orientation of E 1A and E 1 B genes. ### Claim 26 (depends on 19) The method for constructing a recombinant adenovirus of claim 19, wherein each of said adenovirus vector (a) and said recombinant adenovirus (b) has a deletion of an E1A gene. ### Claim 27 (depends on 26) The method for constructing a recombinant adenovirus of claim 26, wherein said foreign gene and said promoter for expressing said foreign gene of said recombinant adenovirus (b) are inserted in a deletion. ### Claim 28 (depends on 27) The method for constructing a recombinant adenovirus of claim 27, wherein said adenovirus genome of said recombinant adenovirus (b) has an E3 gene region deletion of genome map units 79.6 to 84.8%. ### Claim 29 (depends on 24) The method for constructing a recombinant adenovirus of claim 24, wherein said promoter for expressing said foreign gene is a hybrid CAG promoter comprising a cytomegalovirus enhancer, a chicken .beta.-actin promoter, a rabbit .beta.-globin splice acceptor and a poly(A) sequence. ### Claim 30 (depends on 19) The method for constructing a recombinant adenovirus of claim 19, wherein the function of E1A genes in said adenovirus vector (a) and said recombinant adenovirus (b) is deleted, and said animal cell line expresses an E1A gene. ### Claim 31 A recombinant adenovirus comprising an adenovirus genome having a foreign gene inserted between the termination codons of an E2A gene and an L3 gene.