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US 2002/0160516 A1Published application
Episomal expression vector for human gene therapy and expression system for production of proteins
OwnerMark J. Cooperinventor
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Abstract
A method is described for expressing biologically functional proteins without host cell toxicity. This method takes advantage of the surprising replication activating ability of the 107/402-T antigen. The invention also provides expression vectors, human cells, and fusion proteins for practicing the method. The invention thus provides the art with an expression system for proteins which is useful to the pharmaceutical and biotechnology industries
Claims200
A kit, comprising:
- (a) a DNA molecule comprising:
- (1) a coding sequence for a mutant form of a papovavirus large T antigen which contains a replication-competent binding site for a papovavirus origin of replication and which is negative for binding to a wild-type p53 gene product due to a mutation in a p53 binding domain of the large T antigen and which is negative for binding to a wild-type retinoblastoma tumor suppressor gene product due to a mutation in an RB binding domain of the large T antigen;
- and
- (2) a first promoter which controls expression of the mutant form of the papovavirus large T antigen;
- and
- (b) a first episome comprising:
- (1) the papovavirus origin of replication;
- and
- (2) a coding sequence for a protein or a site for inserting the coding sequence for the protein.
6 more elements
A kit, comprising:
- (a) a mammalian cell;
- (b) a DNA molecule comprising:
- (1) a coding sequence for a mutant form of a papovavirus large T antigen which contains a replication-competent binding site for a papovavirus origin of replication and which is negative for binding to a wild-type p53 gene product due to a mutation in a p53 binding domain of the large T antigen and which is negative for binding to a wild-type retinoblastoma tumor suppressor gene product due to a mutation in an RB binding domain of the large T antigen, wherein the DNA molecule is integrated into the genome of the mammalian cell;
- and
- (2) a first promoter which controls expression of the mutant form of the papovavirus large T antigen;
- and
- (c) a first episome comprising:
- (1) the papovavirus origin of replication;
- and
- (2) a coding sequence for a protein or a site for inserting the coding sequence for the protein;
- and
- (3) a second promoter for controlling expression of the coding sequence for the protein.
9 more elements
A kit, comprising:
- (a) a mammalian cell;
- and
- (b) an episome comprising:
- (1) a coding sequence for a mutant form of a papovavirus large T antigen which contains a replication-competent binding site for a papovavirus origin of replication and which is negative for binding to a wild-type p53 gene product due to a mutation in a p53 binding domain of the large T antigen and which is negative for binding to a wild-type retinoblastoma tumor suppressor gene product due to a mutation in an RB binding domain of the large T antigen;
- (2) a promoter which controls expression of the mutant form of the papovavirus large T antigen;
- (3) the papovavirus origin of replication;
- and
- (4) a coding sequence for a protein or a site for inserting the coding sequence for the protein.
5 more elements
A mammalian cell comprising:
- (a) a DNA molecule comprising:
- (1) a coding sequence for a mutant form of a papovavirus large T antigen which contains a replication-competent binding site for a papovavirus origin of replication and which is negative for binding to a wild-type p53 gene product due to a mutation in a p53 binding domain of the large T antigen and which is negative for binding to a wild-type retinoblastoma tumor suppressor gene product due to a mutation in an RB binding domain of the large T antigen;
- and
- (2) a first promoter which controls expression of the mutant form of the papovavirus large T antigen;
- and
- (b) a first episome comprising:
- (1) a coding sequence for a protein or a site for inserting the coding sequence for the protein;
- and
- (2) the papovavirus origin of replication.
6 more elements
A mammalian cell, comprising:
- a DNA molecule which comprises:
- (a) a coding sequence for a mutant form of a papovavirus large T antigen which contains a replication-competent binding site for a papovavirus origin of replication and which is negative for binding to a wild-type p53 gene product due to a mutation in a p53 binding domain of the large T antigen and which is negative for binding to a wild-type retinoblastoma tumor suppressor gene product due to a mutation in an RB binding domain of the large T antigen;
- and
- (b) a promoter which controls expression of the mutant form of the papovavirus large T antigen.
1 more element
A method, comprising the step of:
- culturing a mammalian cell which comprises:
- (a) a DNA molecule comprising:
- (1) a coding sequence for a mutant form of a papovavirus large T antigen which contains a replication-competent binding site for a papovavirus origin of replication and which is negative for binding to a wild-type p53 gene product due to a mutation in a p53 binding domain of the large T antigen and which is negative for binding to a wild-type retinoblastoma tumor suppressor gene product due to a mutation in an RB binding domain of the large T antigen;
- and
- (2) a first promoter which controls expression of the mutant form of the papovavirus large T antigen;
- and
- (b) a first episome comprising:
- (1) a coding sequence for a protein;
- and
- (2) the papovavirus origin of replication, wherein the step of culturing is carried out under conditions suitable for expressing the protein.
7 more elements
A method comprising the step of:
- transfecting a mammalian cell with a first episome comprising (a) a coding sequence for a protein and (b) a papovavirus origin of replication, wherein the mammalian cell comprises a DNA molecule which comprises:
- (a) a coding sequence for a mutant form of a papovavirus large T antigen which contains a replication-competent binding site for the papovavirus origin of replication and which is negative for binding to a wild-type p53 gene product due to a mutation in a p53 binding domain of the large T antigen and which is negative for binding to a wild-type retinoblastoma tumor suppressor gene product due to a mutation in an RB binding domain of the large T antigen;
- and
- (b) a first promoter which controls expression of the mutant form of the papovavirus large T antigen.
1 more element
A kit, comprising:
- (a) a mammalian cell;
- (b) a first episome comprising:
- (1) a coding sequence for a mutant form of a papovavirus large T antigen which contains a replication-competent binding site for a papovavirus origin of replication and which is negative for binding to a wild-type p53 gene product due to a mutation in a p53 binding domain of the large T antigen and which is negative for binding to a wild-type retinoblastoma tumor suppressor gene product due to a mutation in an RB binding domain of the large T antigen;
- and
- (2) a first promoter which controls expression of the mutant form of the papovavirus large T antigen;
- and
- (c) a second episome comprising:
- (1) the papovavirus origin of replication;
- and
- (2) a coding sequence for a protein or a site for inserting the coding sequence for the protein;
- and
- (3) a second promoter which controls expression of the coding sequence for the protein.
9 more elements
A method comprising the step of:
- transfecting a mammalian cell with an episome comprising:
- (a) a coding sequence for a protein;
- (b) a papovavirus origin of replication;
- (c) a coding sequence for a mutant form of a papovavirus large T antigen which contains a replication-competent binding site for the papovavirus origin of replication and which is negative for binding to a wild-type p53 gene product due to a mutation in a p53 binding domain of the large T antigen and which is negative for binding to a wild-type retinoblastoma tumor suppressor gene product due to a mutation in an RB binding domain of the large T antigen;
- and
- (d) a promoter which controls expression of the mutant form of the papovavirus large T antigen.
3 more elements
Dependencies are inferred from the claim text.