a. RATIONALE. Certain parvoviruses were found to become localized in tumours. This oncotropism results from the greater capacity of neoplastic cells for amplifying and expressing parvoviral DNA. Parvovirus-based vectors may thus be used for the transfer and targeted expression of therapeutic genes in cancer cells.
b. STATE OF THE ART AND MAIN OBJECTIVES. Parvoviral genomes harbouring heterologous genes can be constructed and packaged into recombinant particles which are able to transduce these genes in target cells. Recombinants retain the oncotropic properties of wild-type parvoviruses in-vitro. Therefore,it is now important to determine whether they can target a toxic phenotype in tumour cells in vivo. The main restriction to this assessment lies in the low yields of recombinant parvoviruses produced by present packaging systems. Our purpose is to optimize, by combining different expertises in heterologous gene expression systems, the production and purification procedures so as to prepare stocks of recombinants which express cytokine genes and can be tested for their antineoplastic activity against model tumours in animals.
c. EXPERIMENTAL APPROACH. The rodent parvoviruses MVM and H-l will be used, given their ability to grow in a wide variety of cells of both human and animal origin, and their apparent innocuousness for humans. Genomic infectious clones of these viruses proved to be suitable for the insertion, transduction and expression of foreign genes.
- Construction of chimeric and second generation parvoviral vectors. Three approaches will be followed. (i) To boost the production of recombinants, known replication/ excision-promoting elements of parvoviral DNA will be incorporated into the parvoviral vectors. (ii) Chimeric adenovirus and retrovirus-based vectors harbouring a parvovirus expression cassette will be constructed in order to combine the efficient packaging of the former vectors with the oncotropism of the latter viruses. (iii) Surface targeting of tumour cells will be investigated by generating parvoviruses with modified capsids that display ligands to tumour-specific markers.
- Development of efficient packaging systems. Supply of wild-type capsid proteins (VP) may constitute a limitation to the assembly of recombinant parvoviruses. This will be tested by expressing the parvoviral VP genes either from stably transfected packaging cell lines or from adeno- and alphaviruses which allow their amplification in the form of episomes.
- Transduction of cytokine genes. The local secretion of cytokines in a tumour stimulates host defence mechanisms against neoplastic cells, while avoiding the deleterious side effects of systemic cytokine administration. To achieve this local secretion, we will construct and use parvovirus-based vectors carrying various cytokine genes. An important advantage of this approach is that only a fraction of a tumour cell population needs to express and secrete the recombinant cytokine to achieve a therapeutic effect.
- Cytokine gene therapy of model tumours. The cytokine-carrying vectors will be tested for their oncosuppressive potential in vivo, using a unique animal skin carcinoma model and various human and murine tumour cell implants. - Establishment of methods for parvovirus purification. Successful outcome of animal experiments might open the door to human applications. However, a prerequisite is to achieve the purification of clinical grade quality parvoviruses, which will be attempted hv means of monoclonal antibodies or tagging and affinitY chromatography.
Funding SchemeCSC - Cost-sharing contracts
2300 AH Leiden