RECOMBINANT LUIII AUTONOMOUS PARVOVIRUS AS A TRANSIENT TRANSDUCING VECTOR FOR HUMAN-CELLS

Recombinants based on the genome of the autonomous parvovirus, LuIII, were constructed by replacing the viral coding sequences in an infecti ous clone (pGLu883) by a luciferase or beta-galactosidase reporter, wh ich was linked to the viral P4 promoter. In cells cotransfected with e ither of these constructs, together with a plasmid supplying LuIII non structural and capsid proteins, excision and replication of the recomb inant genome occurred. Transducing virions accumulated in the culture medium of the cotransfected cells, as assayed by reporter activity in recipient cells exposed to this medium. Transducing activity could be neutralized by antiserum to LuIII. Production of replicative form DNA and transducing virions were observed following cotransfection of HeLa , 293, or NB324K cells, in increasing order of efficiency. When homolo gy existed between the recombinant genome and sequences flanking the v iral genes in the helper construct, concomitant production of replicat ion-competent, cytopathic virus was sometimes observed. This could be minimized by removal of the left end homology from the helper; by this means, preparations of luciferase transducing virus were obtained fre e from replication-competent virus. With such preparations, we observe d luciferase expression (declining after 3 days) for up to 7 days in r ecipient HeLa cells. Hybridization of the recombinant viral DNA with s trand-specific luciferase probes indicated packaging of both strands ( as reported for LuIII), but with a several-fold excess of the (-) stra nd. We suggest that transducing-autonomous parvoviruses will be useful in gene transfer applications, possibly including gene therapy when o nly transient expression is desired.