Abstract
Adeno-associated virus (AAV) is a single-stranded DNA virus that can either integrate or replicate in host cells. Production of recombinant viral particles (rAAV) requires expression of the viral structural genes and the viral inverted terminal repeats in cis. By using an SV40 replicon to amplify the structural genes, the yield of recombinant viral particles was increased 60-fold over a nonreplicating helper plasmid. The rAAV particles produced by this system have similar physical properties to wild-type particles, including buoyant density, size, and morphology. This novel rAAV packaging system was used to produce rAAV particles that contain the gene for the T cell co-stimulatory protein B7-2. Transduction of the human nonadherent lymphoid cell line LP-1 with these particles significantly increased the percentage of cells expressing B7-2 from 6.8% to 78.0%. Expression of B7-2 in the human lymphoid cell line RPMI-8226 was also substantially increased. Targeting of tumor cells grown in suspension was hampered by low-efficiency transduction using other viral or nonviral vector systems. Our new packaging system for recombinant AAV should allow generation of sufficient quantities of B7-2 containing particles to develop tumor vaccines for non-Hodgkin's lymphoma.
Overview summary
By using an SV40 replicon to amplify the structural genes of adeno-associated virus (AAV), the yield of recombinant viral particles can be increased 60-fold over a nonreplicating helper plasmid. The rAAV particles have physical properties similar to wild-type particles, including buoyant density, size, and morphology. This novel recombinant (r) AAV packaging system was used to produce rAAV particles that contain the gene for the T cell co-stimulatory protein B7-2. The human nonadherent lymphoid cell line LP-1 and RPMI-8662 were both efficiently transduced by the rAAV particles and expressed high levels of B7-2.
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