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 described more fully herein. Recent studies have shown that column chromatography may be used in the purification of recombinant adenovirus (Huyghe et al., Human Gene Therapy 6:1403-1416, 1995).
Column chromatography, using other systems such as so-called "macroporous" resins, which comprise beads having pores therein, the average diameter of which is approximately the same as the diameter of adenovirus (diameter=about 80 nm, excluding the fibres and about 140 nm with the fibre molecules), have not resulted in the recovery of infectious adenovirus. The most likely reason for this is that the passage of adenovirus through such resins shears the fibres from the viral surface through intimate contact of the virus with the pores in the beads. The adenovirus fibre molecules, inter alia, are believed to be involved in the virus ability to bind to and infect target cells. Thus, damage or loss of the fibre molecules (as well as other surface molecules) by such prior art column methods results in the recovery of inactive (non-infectious) virus.
As is well known in the art, AAV propagation requires the use of helper virus, such as adenovirus. The requirement for helper virus complicates purification of AAV. Current approaches to AAV purification involve lysing of AAV infected cells using repeated cycles of freeze-thawing followed by the use of density gradient centrifugation to fractionate the cell lysate in order to obtain infectious AAV, free of cellular contaminants and substantially free of helper virus (such as adenovirus) required for AAV propagation. (Flotte et al., Gene Therapy 2:29-37, 1995; Chiorini et al., Human Gene Therapy 6:1531-1541, 1995; Fisher et al., J. Virol. 70: 520-532, 1996). Standard purification techniques generally result in very low yields (0.3-5%) of active (infectious) virus. Moreover, because of the helper-virus requirement, it has been difficult to obtain AAV that is totally free of the helper (e.g. adenovirus).