Use of an autonomous parvovirus vector for selective transfer of a foreign gene into transformed human cells of different tissue origins and its expression therein

Abstract

In this work, we report the transduction of a chloramphenicol acetyltransferase (CAT) reporter gene into a variety of normal and transformed human cells of various tissue origins. The vector used was MVM/P38cat, a recombinant of the prototype strain of the autonomous parvovirus minute virus of mice (MVMp). The CAT gene was inserted into the capsid-encoding region of the infectious molecular clone of MVMp genome, under the control of the MVM P38 promoter. When used to transfect permissive cells, the MVM/P38cat DNA was efficiently replicated and expressed the foreign CAT gene at high levels. By cotransfecting with a helper plasmid expressing the capsid proteins, it was possible to produce mixed virus stocks containing MVM/P38cat infectious particles and variable amounts of recombinant MVM. MVM/P38cat viral particles were successfully used to transfer the CAT gene and to express it in a variety of human cells. Both viral DNA replication and P38-driven CAT expression were achieved in fibroblasts, epithelial cells, T lymphocytes, and macrophages in a transformation-dependent way, but with an efficiency depending on the cell type. In transformed B

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“…Our previous data on the selectivity of rMVMpmediated transduction 23 and those presented here indicate a low but significant level of transgene expression in several rMVMp-infected normal cells, particularly normal human fibroblasts and NRA. Whether this could be deleterious to normal cells when the transgene codes for a cytotoxic protein remains to be addressed.…”

“…46 The rMVMp and RCPV titers of the different virus stocks were determined by replication center assay (RCA) on NB324K cells. 23 The rMVMp titers, whatever the recombinant, were always at least 5 × 10 8 IU/ml. RCPV contamination of the recombinant virus stocks was approximately 0.05% for MVM/P38-GFP and less than 0.5% for both MVM/P38-CAT and MVM/P38-TK.…”

“…pMVM/P38-CAT has previously been described. 23 pMVM/P38-TK and pMVM/P38-GFP were constructed from pMVM/P38-CAT by substituting the HSV-TK or GFP gene for the CAT gene. The HSV-1 TK gene was obtained by digesting plasmid pFG5 45 with BglII and AspI.…”

“…various foreign cDNAs/genes under the control of P38, notably human interleukin-2 and murine interleukin-4 cDNAs and the CAT reporter gene. 22,23 Recombinants of MVMp (rMVMp) were produced by transient cotransfection of permissive cells with a vector plasmid containing the rMVMp genome and a complementary helper plasmid carrying the viral capsid genes. These MVMp-based vectors were shown in vitro to promote transgene expression in a variety of murine and human cells, this expression being transformation-dependent.…”

“…These MVMp-based vectors were shown in vitro to promote transgene expression in a variety of murine and human cells, this expression being transformation-dependent. 22,23 The selective gene transfer potential of MVMp-based vectors has so far always been assessed at the level of the whole cell population (eg by CAT ELISA or CAT assay). The efficacy of cancer gene therapy depends primarily on the percentage of cells expressing the transgene in the infected cell population.…”