Autonomous parvovirus vectors: preventing the generation of wild-type or replication-competent virus

The preferential expression of autonomous parvoviruses in tumour cells and their oncolytic activity has attracted attention to the potential use of these viruses as vectors for cancer gene therapy. Moreover, they are non-pathogenic in adult animals and they seem to be associated with low or no immunogenicity. Other interesting features are their episomal replication and high stability. Vectors derived from the autonomous parvoviruses MVM(p) or H1 express proteins that can directly or indirectly interfere with tumour development. They retain cis- and trans-acting sequences required for viral DNA amplification; the transgene replaces part of the capsid coding genes. Their development has been hampered by low titres and contamination with replication-competent virus (RCV) that is generated through homologous recombination with helper plasmids. Several approaches have been used to avoid recombination between vectors and helpers. In most instances, reducing the homology up- or downstream of the transgene in either the vector or the helper did not significantly affect RCV production. However, completely eliminating homology downstream of the transgene, splitting VP genes on different helpers or pseudotyping vectors resulted in the production of RCV-free stocks. Although VP-containing particles could sometimes be identified in these stocks by in situ hybridisation, they did not amplify and are therefore not true RCV. The integration of capsid-coding sequences into packaging cells also reduced contamination by RCV and allowed for the amplification of vectors through serial infections. Great progress has been made recently towards the generation of truly RCV-free stocks of vectors derived from autonomous parvoviruses H1 and MVMp. Combining these new vectors with a new packaging cell line should greatly facilitate their development.