Recombinant LuIII Autonomous Parvovirus as a Transient Transducing Vector for Human Cells

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Abstract
Recombinants based on the genome of the autonomous parvovirus, LuIII, were constructed by replacing the viral coding sequences in an infectious clone (pGLu883) by a luciferase or β-galactosidase reporter, which was linked to the viral P4 promoter. In cells cotransfected with either of these constructs, together with a plasmid supplying LuIII nonstructural and capsid proteins, excision and replication of the recombinant 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 homology existed between the recombinant genome and sequences flanking the viral genes in the helper construct, concomitant production of replication-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 free from replication-competent virus. With such preparations, we observed luciferase expression (declining after 3 days) for up to 7 days in recipient HeLa cells. Hybridization of the recombinant viral DNA with strand-specific luciferase probes indicated packaging of both strands (as reported for LuIII), but with a several-fold excess of the (–) strand. We suggest that transducing-autonomous parvoviruses will be useful in gene transfer applications, possibly including gene therapy when only transient expression is desired. Previously, the development of potential vectors for gene therapy has focused on retroviruses and adeno-associated virus (AAV, a defective parvovirus) because of their efficient integration capability. For some applications, such as toxin gene targeting, transient expression without integration would be preferable. Autonomous parvoviruses, such as LuIII, contain a small (5 kb) single-stranded DNA genome and infect human cell lines lytically with no evidence of integration into cellular DNA. Maxwell et al. show that the viral coding sequences of LuIII can be replaced, generating recombinant virions capable of transducing and expressing reporter genes. This system appears promising as a transient expression vector.