Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or... – Recovery or purification
Patent
1998-06-29
2000-11-07
Salimi, Ali
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
Recovery or purification
4352351, 435803, 530412, C12N 702, A23J 100
Patent
active
061435481
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to the purification of large scale quantities of active (infectious) adenovirus and AAV, especially for use in therapeutic applications. In particular, the invention provides improved methods for contacting such viruses with suitable chromatographic materials in a fashion such that any damage to the virus, particularly to surface components thereof, resulting from contact with such chromatographic materials is minimized or eliminated. The result is the ability to rapidly and efficiently purify commercial level quantities of active (infectious) virus suitable for use in therapeutic applications, e.g. gene transfer/therapy procedures.
BACKGROUND OF THE INVENTION
Molecular therapy of disease often involves the administration of nucleic acid to the cells of interest in order to confer a therapeutic benefit. Most commonly, recombinant viruses are engineered which take advantage of the natural infectivity of viruses and their ability to transport heterologous nucleic acid (transgene) to a cell. Widespread use of such recombinant viral vectors depends on strategies for the design and production of such viruses.
Most attempts to use viral vectors for gene therapy have relied on retrovirus vectors, chiefly because of their ability to integrate into the cellular genome. However, the disadvantages of retroviral vectors are becoming increasingly clear, including their tropism for dividing cells only, the possibility of insertional mutagenesis upon integration into the cell genome, decreased expression of the transgene over time, rapid inactivation by serum complement, and the possibility of generation of replication-competent retroviruses (Jolly, D., Cancer Gene Therapy 1:51-64, 1994; Hodgson, C. P., Bio Technology 13:222-225, 1995).
Adenovirus is a nuclear DNA virus with a genome of about 36 kb, which has been well-characterized through studies in classical genetics and molecular biology (Horwitz, M. S., "Adenoviridae and Their Replication," in Virology, 2nd edition, Fields, B. N., et al., eds., Raven Press, New York, 1990). Adenovirus-based vectors offer several unique advantages for delivering a therapeutic transgene to a cell, including, inter alia, tropism for both dividing and non-dividing cells, minimal pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large inserts (Berkner, K. L., Curr. Top. Micro. Immunol. 158:39-66, 1992; Jolly, D., Cancer Gene Therapy 1:51-64, 1994).
Adeno-associated virus (AAV) is a single-stranded non-pathogenic DNA virus which is capable of integrating into the genome of an infected cell. This feature of the virus life cycle has focused attention on the use of AAV as a gene therapy vehicle (creating a recombinant adeno-associated vector, rAAV) to deliver a gene of interest for gene therapy. The ability of AAV to insert a therapeutic gene into the cell genome facilitates persistent expression of the gene of interest and reduces the need for repeated dosing of a gene therapy vector.
Current methods for the purification of adenovirus and adeno-associated virus (AAV) involve the use of density gradient centrifugation, which does not easily allow for large scale production of virus stocks for therapeutic use. A further limitation to widespread use of AAV vectors is the general lack of any adequate purification methods which yield high titers of AAV, while removing contaminating adenovirus required for the propagation of AAV vector stocks.
Ion-exchange, affinity chromatography and gel filtration are widely used column chromatography tools in protein purification. Until recently, however, these methods have been inapplicable to purification of adenoviruses. Such techniques have resulted in damage to the viruses, thereby reducing their ability to bind and infect a target cell. Provisional U.S. patent application Serial No. 60/002,967, filed Aug. 30, 1995, set forth parameters for purifying infectious adenovirus utilizing chromatographic fractionation techniques as describ
REFERENCES:
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Erickson Amy E.
O'Riordan Catherine E.
Smith Alan E.
Genzyme Corporation
Lazar Steven R.
Salimi Ali
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