Packaging systems for human recombinant adenovirus to be used in gene therapy
First Claim
1. A system for producing adenovirus incapable of replicating, said system comprising:
- a primary cell containing a nucleic acid encoding adenoviral E1A and E1B gene products, wherein aid nucleic acid lacks a gene coding for active or functional pIX; and
an isolated recombinant nucleic acid molecule for transfer into said primary cell, said isolated recombinant nucleic acid molecule based on or derived from an adenovirus of the family Adenoviridae, and further havingat least a functional encapsidating signal, andat least one functional Inverted Terminal Repeat,said isolated recombinant nucleic acid molecule lacking overlapping sequences with the nucleic acid of the cell, the overlapping sequences otherwise enabling homologous recombination leading to replication competent adenovirus in said primary cell into which said isolated recombinant nucleic acid molecule is to be transferred.
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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 system for use with the invention produces adenovirus incapable of replicating. The system 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
20 Claims
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1. A system for producing adenovirus incapable of replicating, said system comprising:
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a primary cell containing a nucleic acid encoding adenoviral E1A and E1B gene products, wherein aid nucleic acid lacks a gene coding for active or functional pIX; and
an isolated recombinant nucleic acid molecule for transfer into said primary cell, said isolated recombinant nucleic acid molecule based on or derived from an adenovirus of the family Adenoviridae, and further havingat least a functional encapsidating signal, and at least one functional Inverted Terminal Repeat, said isolated recombinant nucleic acid molecule lacking overlapping sequences with the nucleic acid of the cell, the overlapping sequences otherwise enabling homologous recombination leading to replication competent adenovirus in said primary cell into which said isolated recombinant nucleic acid molecule is to be transferred. - View Dependent Claims (2, 3, 11, 12, 20)
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4. An adenoviral producer cell that does not produce replication competent adenovirus, said adenoviral producer cell comprising:
one or more recombinant nucleic acid molecules having no overlapping sequences with respect to one another which would otherwise allow for homologous recombination leading to replication competent adenovirus in said adenoviral producer cell, and wherein said adenoviral producer cell comprises a nucleic acid encoding adenoviral E1A and E1B gene products, said nucleic acid lacking a gene coding for functional or active pIX. - View Dependent Claims (7, 8, 9, 10, 13, 14, 15)
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5. An adenoviral producer cell that does not produce replication competent adenovirus, said adenoviral producer cell comprising:
one or more recombinant nucleic acid molecules having no overlapping sequences with respect to one another which would otherwise allow for homologous recombination leading to replication competent adenovirus in said adenoviral producer cell, and wherein said adenoviral producer cell comprises a nucleic acid encoding adenoviral E1 and E1B gene products, said nucleic acid lacking a gene coding for functional or active pIX, and E2A gene product under the control of an inducible promoter.
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6. An adenoviral producer cell that does not produce replication competent adenovirus, said adenoviral producer cell comprising:
one or more recombinant nucleic acid molecules having no overlapping sequences with respect to one another which would otherwise allow for homologous recombination leading to replication competent adenovirus in said adenoviral producer cell, said adenoviral producer cell comprises a nucleic acid encoding adenoviral E1 and E2A gene products, said nucleic acid lacking a gene coding for functional or active pIX, and wherein said E2A gene product encodes a temperature sensitive mutation. - View Dependent Claims (19)
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16. An established adenoviral producer cell that lacks a gene encoding for active or functional pIX and does not produce replication competent adenovirus, said established adenoviral producer cell derived from a primary cell, said established adenoviral producer cell comprising:
one or more recombinant nucleic acid molecules having no overlapping sequences within each of the recombinant nucleic acid molecules, wherein said adenoviral producer cell comprises DNA sequences encoding functional adenoviral E1A and E1B gene products. - View Dependent Claims (17, 18)
Specification