Adenovector complementing cells

Details Adenovector complementing cells Adenovector complementing cells

2001-07-23

2004-01-27

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Primate cell, per se

C435S320100, C435S325000, C435S366000

Reexamination Certificate

active

06682929

ABSTRACT:

FIELD OF THE INVENTION
This invention pertains to cells for the propagation of adenoviral vectors.
BACKGROUND OF THE INVENTION
Recombinant eukaryotic viral vectors have become a preferred means of gene transfer for many researchers and clinicians. The human adenovirus is one of the most widely used recombinant viral vectors in current gene therapy protocols. As the use of adenoviral vectors becomes more prevalent, the need for systems that efficiently produce adenoviral vectors suitable for administration is increasingly important.
A concern associated with recombinant adenoviral vectors is uncontrolled propagation of the vector upon administration. To address this concern, replication-deficient adenoviral vectors, typically lacking the essential E1 region of the adenoviral genome, have been developed. The relatively small foreign gene insert capacity of E1-deleted adenoviral vectors has led to the development of adenoviral vectors with additional early region gene deletions, particularly deletions in the E4 region (see, e.g., WO 96/18418 and U.S. Pat. No. 6,127,175). Such vectors are propagated in complementing cell lines expressing adenoviral E1 and E4 gene products, such as those described by Wang et al.,
Gene Ther.,
2, 775-783 (1995), and Yeh et al.,
J. Virol.,
70, 559-565 (1996).
Adenoviral vector technology is also limited by the difficulties associated with large-scale propagation of adenoviral vectors using currently available complementing cell lines. For example, while the A549 cell line supports sufficient propagation of wild-type adenovirus, adenoviral propagation is significantly reduced or nonexistent when A549 cells are engineered to constitutively express E1 gene products for complementation (see, e.g., Imler et al.,
Gene Ther.,
1, 75-84 (1996), and Gao et al.,
Human Gene Ther.,
11, 213-219 (2000)). Moreover, propagation of wild-type adenovirus on the widely used HEK 293 cell line (Graham et al.,
J. Gen. Virol.,
36, 59-72 (1977)) results in approximately 50-75% of the yield of wild-type adenovirus on A549 cells.
Accordingly, there remains a need for alternative cells for propagating replication-deficient adenoviral vectors. The invention provides such cells. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
The invention provides a cell having a cellular genome comprising at least one adenoviral nucleic acid sequence, which upon expression produces a gene product that complements in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome so as to propagate a replication-deficient adenoviral vector comprising an adenoviral genome deficient in the at least one essential gene function of the one or more regions when present in the cell. The cell (i) is a pleural effusion, large cell lung carcinoma, (ii) is epithelial, and (iii) comprises a wild-type p53 gene. Alternatively, the cell (i) is a lung carcinoma, (ii) comprises a homozygous deletion of the p53 gene, and (iii) is heterozygous for a K-ras codon 12 mutation. The inventive cell preferably is an NCI-H460 cell or a Calu-1 cell.
The invention also provides a system comprising the inventive cell and a replication-defective adenoviral vector comprising an adenoviral genome deficient in the at least one essential gene function of the one or more regions. The invention further provides a method of propagating a replication-deficient adenoviral vector, wherein the method comprises providing the inventive cell, introducing a replication-deficient adenoviral vector into the inventive cell, and maintaining the cell to propagate the replication-deficient adenoviral vector.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a cell having a cellular genome comprising at least one adenoviral nucleic acid sequence, which upon expression produces a gene product that complements in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome of a replication-deficient adenoviral vector so as to propagate (i.e., replicate the entire life cycle of, or replicate to any stage of the life cycle of) the replication-deficient adenoviral vector when present in the cell.
The cell (i) is a large cell lung carcinoma derived from a pleural effusion (i.e., a pleural effusion, large cell lung carcinoma), (ii) is epithelial, and (iii) comprises a wild-type p53 gene. By “derived” from a pleural effusion is meant that the cell is isolated from a large cell lung carcinoma that originated from an effusion of the lung pleura. By “epithelial” is meant that the cell participates in lining the inner and outer surfaces of the organism from which it is isolated. The cell has a wild-typep53 gene in that the nucleic acid sequence encoding the p53 gene does not comprise any alterations that change the normal function of the p53 gene product in the inventive cell. Advantageously, the cell comprises a homozygous K-ras codon 12 mutation. The cell comprises a homozygous K-ras codon 12 mutation in that both alleles of the K-ras gene locus are mutated in the inventive cell. Moreover, the cell does not express the p16INK4a protein. The cell also desirably exhibits adherent growth in culture, and comprises two X chromosomes and two Y chromosomes.
The cell alternatively (i) is a lung carcinoma, (ii) comprises a homozygous deletion of the p53 gene, and (iii) is heterozygous for a K-ras codon 12 mutation. The cell comprises a homozygous deletion of the p53 gene in that both alleles of the p53 gene locus comprise deletions which, for example, prevent expression of the p53 gene product or render the p53 gene product non-functional. The cell is heterozygous for a K-ras codon 12 mutation in that the cell comprises a K-ras gene locus comprising a wild-type allele and a codon 12 mutation in the other allele. Advantageously, the cell does not express the p16INK4a protein. The cell also desirably exhibits adherent growth in culture. Desirably, the antigen expression profile of the cell comprises (i) blood type A, (ii) Rh positive, and (iii) HLA antigens A10, A11, B15, and Bw35. By “antigen expression profile” is meant the collection of antigens that are expressed on the surface of the inventive cell.
The cell can be any suitable such cell into which can be incorporated and preferably retained the adenoviral nucleic acid encoding at least one gene product which complements in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome. The cell desirably can propagate adenoviral vectors and/or adeno-associated viral (AAV) vectors when infected with such vectors or with nucleic acid sequences encoding the adenoviral or AAV genome. Most preferably, the cell can propagate a suitable replication-deficient adenoviral vector upon infection with an appropriate replication-deficient adenoviral vector or transfection with an appropriate replication-deficient viral genome. The cell preferably is an NCI-H460 cell or a Calu-1 cell having a cellular genome comprising at least one adenoviral nucleic acid sequence, which upon expression produces a gene product that complements in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome of a replication-deficient adenoviral vector so as to propagate the replication-deficient adenoviral vector when present in the cell.
Particularly desirable cell types are those that support high levels of wild-type adenovirus propagation. The cell desirably produces at least about 100% more wild-type adenovirus, preferably at least about 200% more wild-type adenovirus, and most preferably at least about 300% more wild-type adenovirus, than a 293 cell. The cell also desirably produces at least about 90% more wild-type adenovirus, more preferably at least about 100% more wild-type adenovirus, and most preferably at least about 130% more wild-type adenovirus, than an A549 cell. The cell preferably

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