Herpes simplex virus strains deficient for the essential immediate early genes ICP4 and ICP27 and methods for their production, growth and use
AssigneeUniversity of Pittsburgh—Of the Commonwealth System of Higher Education
InventorNeal A. DeLuca
Life of this patent
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Abstract
Novel cell lines that express complementing levels of herpes simplex virus (HSV) essential immediate early proteins ICP4 and ICP27 and a method of producing the novel cell lines. These novel cell lines are utilized to provide novel HSV strains deficient for both ICP4 and ICP27, and their generation, and novel HSV strains deficient for ICP4 and ICP27 and one or more additional genes, and their generation. Vectors are provided from these methods of using these vectors for gene transfer and for producing site-specific homologous recombinations with cellular DNA.
Claims60
A cell line containing DNA encoding both the HSV proteins ICP4 and ICP27.
A method of producing a cell line containing DNA encoding the HSV proteins ICP4 and ICP27, said method comprising:
- cotransfecting cells capable of hosting HSV with pieces of DNA encoding the HSV protein ICP4 and pieces of DNA encoding the HSV protein ICP27;
- incubating said cells;
- and selecting cells harboring both ICP4 and ICP27 encoding pieces of DNA.
An HSV strain whose genome is deficient for the HSV genes encoding ICP4 and ICP27.
A method of producing an HSV strain whose genome is deficient for the HSV genes encoding ICP4 and ICP27, said method comprising:
- coinfecting ICP4ICP27 complementing cells containing DNA encoding the HSV proteins ICP4 and ICP27 with an HSV strain deficient for the ICP4 gene and an HSV strain deficient for the ICP27 gene;
- incubating said cells;
- plating virus progeny from said incubated cells on ICP4ICP27 complementing cells, ICP27 complementing cells, ICP4 complementing cells, and ICP4.sup.- ICP27.sup.- cells;
- picking plaques;
- and identifying virus that plaque on ICP4ICP27 complementing cells and do not plaque on ICP27 complementing cells, ICP4 complementing cells, or ICP4.sup.- ICP27.sup.- cells.
A method of growing an HSV strain whose genome is deficient for the HSV genes encoding ICP4 and ICP27, said method comprising:
- infecting ICP4 ICP27 complementing cells containing DNA encoding the HSV proteins ICP4 and ICP27 with said HSV strain whose genome is deficient for the HSV genes encoding ICP4 and ICP27;
- incubating said infected ICP4 ICP27 complementing cells;
- and collecting virus progeny produced by said incubated cells.
A vector comprising:
- an HSV strain whose genome is deficient for the HSV genes encoding the proteins ICP4 and ICP27, at least one exogenous gene to be transferred to a cell, and an appropriate promoter sequence, wherein said exogenous gene and said promoter sequence are contained within one or more nonessential regions of said HSV genome.
A method of using an ICP4.sup.- ICP27.sup.- HSV strain as a vector, said method comprising:
- inserting at least one exogenous gene and an appropriate promoter sequence into said ICP4.sup.- ICP27.sup.- HSV strain's genome;
- and then infecting cells with said ICP4.sup.- ICP27.sup.- HSV strain.
A method of producing an HSV strain whose genome is deficient for the HSV genes encoding ICP4 and ICP27, and at least one additional HSV gene, said method comprising:
- coinfecting ICP4ICP27 complementing cells containing DNA encoding the HSV proteins ICP4 and ICP27 with an ICP4.sup.- ICP27.sup.- HSV strain and an HSV strain deficient for at least one additional HSV gene;
- incubating said cells;
- plating virus progeny from said incubated cells on said ICP4ICP27 complementing cells;
- and selecting a resultant ICP4.sup.- ICP27.sup.- HSV strain whose genome is further deficient for at least one additional HSV gene.
A method of producing an HSV strain whose genome is deficient for the HSV genes encoding ICP4 and ICP27, and at least one additional HSV gene, said method comprising:
- cotransfecting ICP4ICP27 complementing cells containing DNA encoding the HSV proteins ICP4 and ICP27 with an ICP4.sup.- ICP27.sup.- HSV strain and pieces of DNA encoding an inactivating mutation of at least one additional HSV gene;
- incubating said cells;
- plating virus progeny from said incubated cells on ICP4ICP27 complementing cells;
- and selecting a resultant ICP4.sup.- ICP27.sup.- HSV strain whose genome is further deficient for at least one additional HSV gene.
A method of generating a HSV mutant virus dependent upon complementing functions of ICP4 and ICP27 for growth, which comprises:
- infecting an ICP4.sup.(+) /ICP27.sup.(+) cell line expressing complementing levels of ICP4 and ICP27 with said HSV mutant virus;
- incubating said cell line in the appropriate medium;
- and, collecting said HSV mutant virus produced by said cell line.
Dependencies are inferred from the claim text.