Method for production of high titer virus and high efficiency retroviral mediated transduction of mammalian cells

A retroviral packaging plasmid for the production of high titers of recombinant retrovirus in human cells comprising two retroviral helper DNA sequences derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector and for producing virion proteins capable of packaging said replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral DNA sequence lacking the region encoding the native enhancer and/or promoter of the viral 5′ LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3′LTR, and encoding a foreign enhancer and/or promoter functional in a selected mammalian cell, and a foreign polyadenylation site,

• wherein a first retroviral helper sequence comprises a cDNA sequence encoding the gag and pol proteins of ectropic MMLV or GALV and a second retroviral helper sequence comprises a cDNA encoding the envelope protein, and
• wherein said second retroviral helper DNA sequence codes for an envelope protein from the group consisting of 10A1 murine leukemia virus envelope protein, gibbon ape leukemia virus envelope protein, HIV envelope protein, vesicular stomatitus virus G protein, human T cell leukemia type I envelope protein and human T cell leukemia type II envelope protein.

A retroviral packaging plasmid for the production of high titers of recombinant retrovirus in human cells comprising two retroviral helper DNA sequences derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector and for producing virion proteins capable of packaging said replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral DNA sequences lacking the region encoding the native enhancer and/or promoter of the viral 5′ LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3′LTR, and encoding a foreign enhancer and/or promoter functional in a selected mammalian cell, and a foreign polyadenylation site,

• wherein a first retroviral helper sequence comprises a cDNA sequence encoding the gag and pol proteins of ectropic MMLV or GALV and a second retroviral helper sequence comprises a cDNA encoding the envelope protein,
• wherein said second retroviral helper DNA sequence codes for an envelope protein selected from a xenotropic, amphotropic, ecotropic or polytropic envelope protein;
• and
• wherein said foreign enhancer is the RSV enhancer and promoter.

A retroviral packaging plasmid for the production of high titers of recombinant retrovirus in human cells comprising two retroviral helper DNA sequences derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector and for producing virion proteins capable of packaging said replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral DNA sequence lacking the region encoding the native enhancer and/or promoter of the viral 5′ LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3′LTR, and encoding a foreign enhancer and/or promoter functional in a selected mammalian cell, and a foreign polyadenylation site,

• wherein a first retroviral helper sequence comprises a cDNA sequence encoding the gag and pol proteins of ectropic MMLV or GALV and a second retroviral helper sequence comprises a CDNA encoding the envelope protein,
• wherein said second retroviral helper DNA sequence codes for an envelope protein from the group consisting of 10 A1 murine leukemia virus envelope protein, gibbon ape leukemia virus envelope protein, HIV envelope protein, vesicular stomatitus virus G protein, human T cell leukemia type I envelope protein and human T cell leukemia type II envelope protein, and
• wherein said foreign enhancer is the RSV enhancer and promoter.

A high efficiency method to transduce mammalian hematopoietic stem cells with retroviral supernatants produced by transient transfection comprising the steps of

• A) transient cotransfection of a first population of mammalian cells that can produce virus with:
• (i) one retroviral helper DNA sequence derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector and for producing virion proteins capable of packaging said replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral DNA sequence lacking the region encoding the native enhancer and/or promoter of the viral 5′ LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3′LTR, and encoding a foreign enhancer and/or promoter functional in a selected mammalian cell, and a foreign polyadenylation site;
• and
• (ii) a retroviral vector encoding a foreign gene to produce replication-defective recombinant retroviral vectors carrying said foreign gene in said first population of mammalian cells;
• B) separation of said first population of mammalian cells from cell supernatant;
• C) adding adhesion molecules or antibodies to adhesion molecules to culture plates;
• D) growing a second population of mammalian hematopoietic stem cells on said culture plates;
• and
• E) incubating said supernatant containing replication-defective recombinant retroviral vectors carrying said foreign gene with said second population of mammalian hematopoietc stem cells, to transduce said second population of cells with said foreign gene,
• whereby target cells efficiently transduced with said foreign gene are obtained.

A high efficiency method to transduce mammalian hematopoietic stem cells with retroviral supernatants produced by transient transfection comprising the steps of

• A) transient cotransfection of a first population of mammalian cells that can produce virus with:
• (i) two retroviral helper DNA sequences derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector and for producing virion proteins capable of packaging said replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral DNA sequences lacking the region encoding the native enhancer and/or promoter of the viral 5′ LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3′LTR, and encoding a foreign enhancer and/or promoter functional in a selected mammalian cell, and a foreign polyadenylation site,
• wherein a first retroviral helper sequence comprises a cDNA sequence encoding the gag and pol proteins of ectropic MMLV or GALV and a second retroviral helper sequence comprises a cDNA encoding the envelope protein, and
• wherein said second retroviral helper DNA sequence codes for an envelope protein;
• and
• (ii) a retroviral vector encoding a foreign gene to produce replication-defective recombinant retroviral vectors carrying said foreign gene in said first population of mammalian cells;
• B) separation of said first population of mammalian cells from cell supernatant;
• C) adding adhesion molecules or antibodies to adhesion molecules to culture plates;
• D) growing a second population of mammalian hematopoietic stem cells on said culture plates;
• and
• E) incubating said supernatant containing replication-defective recombinant retroviral vectors carrying said foreign gene with said second population of mammalian hematopoietc stem cells, to transduce said second population of cells with said foreign gene,
• whereby target cells efficiently transduced with said foreign gene are obtained.

A high efficiency method to transduce mammalian hematopoietic stem cells with retroviral supernatants produced by transient transfection comprising the steps of:

• A) transient cotransfection of a first population of mammalian cells stably transfected with an expression vector encoding the gag and pol proteins and a selectable marker wherein the expression of the gag and pol proteins are stable in the absence of a selective agent with:
• (i) one retroviral helper DNA sequence derived from a replication-incompetent retroviral genome, said retroviral DNA sequence lacking the region encoding the native enhancer and/or promoter of the viral 5′ LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3′LTR, and encoding a foreign enhancer and/or promoter functional in a selected mammalian cell, and a foreign polyadenylation site, and encoding an envelope protein;
• and
• (ii) a retroviral vector encoding a foreign gene to produce replication-defective recombinant retroviral vectors carrying said foreign gene in said first population of mammalian cells;
• B) separation of said first population of mammalian cells from cell supernatant;
• C) adding adhesion molecules or antibodies to adhesion molecules to culture plates;
• D) growing a second population of mammalian hematopoietic stem cells on said culture plates;
• and
• E) incubating said supernatant containing replication-defective recombinant retroviral vectors carrying said foreign gene with said second population of mammalian hematopoietc stem cells, to transduce said second population of cells with said foreign gene,
• whereby target cells efficiently transduced with said foreign gene are obtained.

A high efficiency method to transduce mammalian hematopoietic stem cells with retroviral supernatants produced by transient transfection comprising the steps of:

• A) transient transfection of a first population of mammalian cells stably transfected with at least one expression vector encoding the gag, pol and env proteins and a selectable marker wherein the expression of the gag, pol and env proteins are stable in the absence of a selective agent with a retroviral vector encoding a foreign gene to produce replication-defective recombinant retroviral vectors carrying said foreign gene in said first population of mammalian cells;
• B) separation of said first population of mammalian cells from cell supernatant;
• C) adding adhesion molecules or antibodies to adhesion molecules to culture plates;
• D) growing a second population of mammalian hematopoietic stem cells on said culture plates;
• and
• E) incubating said supernatant containing replication-defective recombinant retroviral vectors carrying said foreign gene with said second population of mammalian hematopoietc stem cells, to transduce said second population of cells with said foreign gene,
• whereby target cells efficiently transduced with said foreign gene are obtained.

A high efficiency method to transduce mammalian hematopoietic stem cells with retroviral supernatants produced by stable mammalian viral producer cells comprising the steps of:

• A) separation of said first population of stable mammalian viral producer cells from cell supernatant;
• B) adding adhesion molecules or antibodies to adhesion molecules to culture plates;
• C) growing a second population of mammalian hematopoietic stem cells on said culture plates;
• and
• D) incubating said supernatant containing replication-defective recombinant retroviral vectors carrying said foreign gene with said second population of mammalian hematopoietc stem cells, to transduce said second population of cells with said foreign gene,
• whereby target cells efficiently transduced with said foreign gene are obtained.

A high efficiency method to transduce mammalian T and B lymphocytes with retroviral supernatants produced by transient transfection comprising the steps of:

• A) transient cotransfection of a first population of mammalian cells that can produce virus with:
• (i) one retroviral helper DNA sequence derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector and for producing virion proteins capable of packaging said replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral DNA sequence lacking the region encoding the native enhancer and/or promoter of the viral 5′ LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3′LTR, and encoding a foreign enhancer and/or promoter functional in a selected mammalian cell, and a foreign polyadenylation site;
• and
• (ii) a retroviral vector encoding a foreign gene to produce replication-defective recombinant retroviral vectors carrying said foreign gene in said first population of mammalian cells;
• B) separation of said first population of mammalian cells from cell supernatant;
• C) adding antibodies to adhesion molecules to culture plates;
• D) growing a second population of mammalian T or B lymphocytes on said culture plates;
• and
• E) incubating said supernatant containing replication-defective recombinant retroviral vectors carrying said foreign gene with said second population of mammalian T or B lymphocytes, to transduce said second population of cells with said foreign gene,
• whereby target cells efficiently transduced with said foreign gene are obtained.

A high efficiency method to transduce mammalian T or B lymphocytes with retroviral supernatants produced by transient transfection comprising the steps of:

• A) transient cotransfection of a first population of mammalian cells that can produce virus with:
• (i) two retroviral helper DNA sequences derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector and for producing virion proteins capable of packaging said replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, said retroviral DNA sequences lacking the region encoding the native enhancer and/or promoter of the viral 5′ LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3′LTR, and encoding a foreign enhancer and/or promoter functional in a selected mammalian cell, and a foreign polyadenylation site,
• wherein a first retroviral helper sequence comprises a cDNA sequence encoding the gag and pol proteins of ectropic MMLV or GALV and a second retroviral helper sequence comprises a cDNA encoding the envelope protein, and
• wherein said second retroviral helper DNA sequence codes for an envelope protein;
• and
• (ii) a retroviral vector encoding a foreign gene to produce replication-defective recombinant retroviral vectors carrying said foreign gene in said first population of mammalian cells;
• B) separation of said first population of mammalian cells from cell supernatant;
• C) adding antibodies to adhesion molecules to culture plates;
• D) growing a second population of mammalian T or B lymphocytes on said culture plates;
• and
• E) incubating said supernatant containing replication-defective recombinant retroviral vectors carrying said foreign gene with said second population of mammalian T or B lymphocytes, to transduce said second population of cells with said foreign gene,
• whereby target cells efficiently transduced with said foreign gene are obtained.

A high efficiency method to transduce mammalian T or B lymphocytes with retroviral supernatants produced by transient transfection comprising the steps of:

• A) transient cotransfection of a first population of mammalian cells stably transfected with an expression vector encoding the gag and pol proteins and a selectable marker wherein the expression of the gag and pol proteins are stable in the absence of a selective agent with:
• (i) one retroviral helper DNA sequence derived from a replication-incompetent retroviral genome, said retroviral DNA sequence lacking the region encoding the native enhancer and/or promoter of the viral 5′ LTR of said virus and lacking both the psi function sequence responsible for packaging helper genome and the 3′LTR, and encoding a foreign enhancer and/or promoter functional in a selected mammalian cell, and a foreign polyadenylation site, and encoding an envelope protein;
• and
• (ii) a retroviral vector encoding a foreign gene to produce replication-defective recombinant retroviral vectors carrying said foreign gene in said first population of mammalian cells;
• B) separation of said first population of mammalian cells from cell supernatant;
• C) adding antibodies to adhesion molecules to culture plates;
• D) growing a second population of mammalian T or B lymphocytes on said culture plates;
• and
• E) incubating said supernatant containing replication-defective recombinant retroviral vectors carrying said foreign gene with said second population of mammalian T or B lymphocytes, to transduce said second population of cells with said foreign gene,
• whereby target cells efficiently transduced with said foreign gene are obtained.

A high efficiency method to transduce mammalian T or B lymphocytes with retroviral supernatants produced by transient transfection comprising the steps of:

• A) transient transfection of a first population of mammalian cells stably transfected with at least one expression vector encoding the gag, pol and env proteins and a selectable marker wherein the expression of the gag, pol and env proteins are stable in the absence of a selective agent with a retroviral vector encoding a foreign gene to produce replication-defective recombinant retroviral vectors carrying said foreign gene in said first population of mammalian cells;
• B) separation of said first population of mammalian cells from cell supernatant;
• C) adding antibodies to adhesion molecules to culture plates;
• D) growing a second population of mammalian T or B lymphocytes on said culture plates;
• and
• E) incubating said supernatant containing replication-defective recombinant retroviral vectors carrying said foreign gene with said second population of mammalian T or B lymphocytes, to transduce said second population of cells with said foreign gene,
• whereby target cells efficiently transduced with said foreign gene are obtained.

A high efficiency method to transduce mammalian T or B lymphocytes with retroviral supernatants produced by stable mammalian viral producer cells comprising the steps of:

• A) separation of said first population of stable mammalian viral producer cells from cell supernatant;
• B) adding antibodies to adhesion molecules to culture plates;
• C) growing a second population of mammalian T or B lymphocytes on said culture plates;
• and
• D) incubating said supernatant containing replication-defective recombinant retroviral vectors carrying said foreign gene with said second population of mammalian T or B lymphocytes, to transduce said second population of cells with said foreign gene,
• whereby target cells efficiently transduced with said foreign gene are obtained.

A improved method to efficiently transduce mammalian cells with a retroviral supernatant, comprising the steps of:

• i) growing said population of mammalian cells on culture plates;
• and
• ii) incubating said supernatant containing replication-defective recombinant retroviral vectors carrying a foreign gene with said population of mammalian cells, to transduce said population of mammalian cells with said foreign gene,
• whereby target cells efficiently transduced with said foreign gene are obtained, wherein the improvement comprises adding antibodies to adhesion molecules present on said population of mammalian cells to culture plates.