Efficient introduction of genetic information into cells is the major physical challenge for the successful development of gene transfer as a therapeutic mode. We are developing recombinant adeno-associated virus vectors to exploit the natural ability of the virus to infect cells and deliver the encapsidated DNA into the cell nucleus. Once the vector DNA is nuclearly localized, cellular processes result in synthesis of the vector encoded gene product. Recombinant AAV is produced in cells which transiently express the viral gene products necessary for the replication of the recombinant vector and production of the viral capsids. The rAAV contains no virus open reading frames and thus the transduced cell is incapable of producing any viral specific proteins. This is important for long-term transgene expression and avoidance of a cellular immune response to the virus proteins. Human coagulation factor IX (rAAV/FIX) is used as a model for a secreted therapeutic gene product. We produced rAAV/FIX and characterized the expression in tissue culture. Our findings indicate that transiently, there are high levels of secreted FIX. This amount of secreted FIX dimishes within two weeks to less than 10% of the highest levels. Analysis of clonally derived cell lines indicated that FIX was being secreted from about 1% of the cells. This result suggests that in dividing cells, rAAV integrates inefficiently. However, in non-mitotically active cells, the vector DNA remains episomal for prolonged periods. We are evaluating the human insulin promoter for regulated gene expression. Many polypeptides that may be useful for gene therapy are potent cytokines or hormones that have to be strictly regulated. Insulin is one example where too much insulin produces insulin shock and can be fatal. Therefore, the proper amount of insulin produced in response to extracellular stimulus, e.g. glucose, is essential. Purification of rAAV from cellular lysate, tissue culture medium, and from the helper adenovirus is a tedious and inefficient process. We are developing methods that exploit the physico-chemical differences between rAAV and the other elements in the system to achieve both purification and concentration of vector. We currently produce rAAV derived from two serotypes: AAV type 2 and AAV type 4. We have recently cloned and sequenced the genome for AAV 4 and demonstrated that the infection with both serotypes is non-competitive, therefore, it is most likely that type 2 and type 4 utilize distinct receptors. This is an important development for several reasons. First, although the receptors for AAV types 2 and 4 are unknown, different receptors imply that the cell tropism may be different as well. Second, neutralizing antibodies to type 2 should not neutralize type 4, thereby permitting re-administration of recombinant AAV to a sensitized individual.
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