Production Of Recombinant Parvoviruses

Despite the antioncogenic properties of parvoviruses and absence of disease pathogen-esis with the exception of parvovirus B19, utilization of wild-type parvoviruses in cancer gene therapy is highly unlikely. Thus, major efforts centered on the advancements in the production of recombinant parvoviral vectors for their potential gene therapy applications in vivo (5,44,45). Elucidation of the genomic organization, sequence determination of par-voviruses and cloning of parvovirus genome in plasmid vectors has led to the development of methods to produce recombinant parvoviral vectors (44,45). In a typical methodology, a gene of interest is cloned between the terminal repeat sequences of parvoviral genome in a plasmid. Genes encoding NS/Rep or VP of the wild type virus and that of required helper viral proteins are provided in trans, also from nonreplicating helper plasmids. Whereas the helper-dependent parvoviruses require the functions of helper virus proteins for a productive life cycle, the APV do not require helper functions from other viruses. However, both dependoviruses and APV require host cell machinery for the replication of viral genome. In the production of recombinant parvoviruses, a combination of the packaging and helper plasmid DNA is transfected into packaging cells (45-48). Approximately 48 to 72 h after the transfection, the cells are lysed and extract containing recombinant parvoviruses subjected to gradient centrifugation or affinity/ion exchange chromatography to purify the virions. Methods such as infectious center assay, genomic slot blot, and real-time PCR are routinely used to determine the titer of the purified recombinant virus.

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