Packaging systems for human recombinant adenovirus to be used in gene therapy
First Claim
1. A method for producing a replication defective adenovirus comprising a gene of interest, without the concomitant production of replication competent adenovirus through homologous recombination, said method comprising:
- a) providing a cell, said cell harboring a first adenoviral nucleic acid encoding adenoviral E1A and E1B gene products, and wherein said first adenoviral nucleic acid further comprises a deletion starting directley after a nucleotide after the furthest downstream E1B stop codon;
b) transferring a recombinant nucleic acid into said cell, said recombinant nucleic acid comprising;
a second adenoviral nucleic acid, and comprising at least one functional encapsidating signal, and at least two functional Inverted Terminal Repeats, said recombinant nucleic acid lacking overlapping sequences with the first adenoviral nucleic acid leading to replication competent adenovirus;
c) culturing said cell; and
d) harvesting replication defective adenovirus produced from said cell.
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Accused Products
Abstract
Presented are ways to address the problem of replication competent adenovirus in adenoviral production for use with, for example, gene therapy. Packaging cells having no overlapping sequences with a selected vector and are suited for large scale production of recombinant adenoviruses. A method of the invention produces adenovirus incapable of replicating. The method includes a primary cell containing a nucleic acid based on or derived from adenovirus and an isolated recombinant nucleic acid molecule for transfer into the primary cell. The isolated recombinant nucleic acid molecule is based on or derived from an adenovirus, and further has at least one functional encapsidating signal, and at least one functional Inverted Terminal Repeat. The isolated recombinant nucleic acid molecule lacks overlapping sequences with the nucleic acid of the cell. Otherwise, the overlapping sequences would enable homologous recombination leading to replication competent adenovirus in the primary cell into which the isolated recombinant nucleic acid molecule is to be transferred.
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Citations
14 Claims
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1. A method for producing a replication defective adenovirus comprising a gene of interest, without the concomitant production of replication competent adenovirus through homologous recombination, said method comprising:
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a) providing a cell, said cell harboring a first adenoviral nucleic acid encoding adenoviral E1A and E1B gene products, and wherein said first adenoviral nucleic acid further comprises a deletion starting directley after a nucleotide after the furthest downstream E1B stop codon;
b) transferring a recombinant nucleic acid into said cell, said recombinant nucleic acid comprising;
a second adenoviral nucleic acid, and comprising at least one functional encapsidating signal, and at least two functional Inverted Terminal Repeats, said recombinant nucleic acid lacking overlapping sequences with the first adenoviral nucleic acid leading to replication competent adenovirus;
c) culturing said cell; and
d) harvesting replication defective adenovirus produced from said cell. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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Specification