684. Expanded Parvoviral Replicons as Novel Nonviral Vectors for Cancer Gene Therapy

Abstract

Gene therapy is an appealing approach to the treatment of human malignancies but significant barriers exist. The use of viral gene therapy vectors is limited due to immunogenicity and toxicity. Nonviral gene transfer systems achieve only low transduction efficiencies. Autonomous parvoviruses are known for their oncotropism and oncosuppressive potential. In addition, recombinant parvoviruses provide high level transgene expression. We therefore developed and optimized parvoviral replicon vectors based on the autonomous parvovirus Minute Virus of Mice (MVMp) and MVMp/EBV (Epstein Barr virus) hybrid vectors. The use of parvoviral replicon vectors will take advantage of the tumor cell-specific cytotoxicity of parvoviruses. Gene delivery of these replicons as SPLP-liposomes will be nontoxic and non-immunogenic allowing repetitive administration. Transgene size, normally limited by inefficient encapsidation of recombinant parvoviruses exceeding a total size of 106% of the wildtype, is not limited in these parvoviral replicon vectors. The parvoviral genome is excised, replicated and expressed in tumor cells resulting in high plasmid copy numbers and high-level transgene expression. Comparison to other highly potent expression vectors, replicative and non-replicative, showed the desirable characteristics of parvoviral replicon vectors. We expect high level cytotoxicity on tumor cells with major bystander effects by using these MVMp-based replicon vectors for expression of the human sodium iodide symporter gene (hNIS) and the Gibbon ape leukaemia virus fusogenic membrane glycoprotein gene (Galv-FMG). Both of these therapeutic genes have proved their strong antitumoral potential in different tumor-models in the past. By packaging these therapeutic MVMp-replicons in SPLP-liposomes we expect a highly efficient vector for cytoreductive cancer gene therapy. Results: MVMp and MVMp/EBV-hybrid replicon vectors containing the marker genes CEA, luciferase and GFP and the therapeutic transgenes Galv-FMG and hNIS have been cloned. Excision and replication of parvoviral DNA has been shown as early as 24 h after transfection. High-level transgene expression in different cell lines was achieved. Excision, replication and transgene expression has been shown up to an increase of the parvoviral genome size of 355% of the wildtype. Comparison to CMV-promoter driven replicative and non-replicative expression vectors showed higher and longer transgene expression levels by the parvoviral replicon vectors. Transgene expression by MVMp and MVMp/EBV-hybrid vectors increased over time in dependence of the susceptibility of the cell line towards MVMp and EBV. Parvoviral replicons containing Galv-FMG were shown to have a very strong cytotoxic potential resulting in an almost complete destruction of susceptible cell layers. Transduction of cells with hNIS expressing parvoviral replicon vectors resulted in high level 125I-uptake.