Method for concentrating and purifying recombinant autonomous parvovirus vectors designed for tumour-cell-targeted gene therapy

Avalosse, Bernard; Dupont, Francis; Spegelaere, Pierre; Mine, Natacha; Burny, Arsène

doi:10.1016/s0166-0934(96)02105-2

Cited by 14 publications

(12 citation statements)

References 9 publications

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“…Recombinant rAAV-EGFP viral particles obtained from 30 plates were concentrated and purified by two rounds of CsCl gradients in a Beckmann SW41 rotos (Beckmann, Fullerton, CA, USA) at 154 000 g for 48 h. Positive fractions were dialyzed in microcollodion tubes (Sartorius, Goettingen, Germany) and further concentrated in 50-300 l PBS on Amicon Centricon 30 membranes (Millipore, Bedford, MA, USA) as described by Avalosse et al 43 for the purification of MVMp-based parvoviral vectors. Adenovirus was inactivated by heating at 56°C for 30 min.…”

Section: Purified Preparationsmentioning

confidence: 99%

Cellular contaminants of adeno-associated virus vector stocks can enhance transduction

Tenenbaum¹,

Hamdane²,

Pouzet³

et al. 1999

Gene Ther

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Section: Purified Preparationsmentioning

confidence: 99%

Cellular contaminants of adeno-associated virus vector stocks can enhance transduction

Tenenbaum¹,

Hamdane²,

Pouzet³

et al. 1999

Gene Ther

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“…20,21 These various observations led to the idea that one might achieve tumorselective expression of a foreign gene by means of a parvoviral vector obtained by replacing the capsid protein genes with the foreign gene. 10 Vectors based on MVMp were constructed, expressing 46 The resulting recombinant virus stocks were purified and concentrated as described. 46 The rMVMp and RCPV titers of the different virus stocks were determined by replication center assay (RCA) on NB324K cells.…”

mentioning

confidence: 99%

“…10 Vectors based on MVMp were constructed, expressing 46 The resulting recombinant virus stocks were purified and concentrated as described. 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 …”

mentioning

confidence: 99%

Tumor-selective gene transduction and cell killing with an oncotropic autonomous parvovirus-based vector

Dupont¹,

Avalosse²,

Karim³

et al. 2000

Gene Ther

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A recombinant MVMp of the fibrotropic strain of minute virus of mice (MVMp) expressing the chloramphenicol acetyltransferase reporter gene was used to infect a series of biologically relevant cultured cells, normal or tumor-derived, including normal melanocytes versus melanoma cells, normal mammary epithelial cells versus breast adenocarcinoma cells, and normal neurons or astrocytes versus glioma cells. As a reference cell system we used normal human fibroblasts versus the SV40-transformed fibroblast cell line NB324K. After infection, we observed good expression of the reporter gene in the different tumor cell types, but only poor expression if any in the corresponding normal cells. We also constructed a recombinant MVMp expressing the green fluorescent protein reporter gene and assessed by flow cytometry the efficiency of gene transduction into the different target cells. At a multiplicity of infection of 30, we observed

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“…COS-7 cells were cotransfected with the new helper pHVR5 and each of the different vectors. Two days after transfection, the cells were harvested, the viruses were purified and concentrated as previously described 6 and the yield of recombinant vectors was assessed by RCA, using a CAT-specific 32 P-lab- eled probe. Figure 4 (hatched box) shows that the packaging ability of all vector genomes was essentially similar.…”

Section: Functional Analysis Of the New Helper And Vector Genomesmentioning

confidence: 99%

“…(ෂ10 9 infectious units (IU)/ml) 6 are currently produced by transiently co-transfecting permissive COS-7 cells, a SV40-transformed simian cell line, with a plasmid carrying the vector genome and a helper plasmid expressing the capsid genes (pSP116, Figure 1b).…”

Section: Introductionmentioning

confidence: 99%

A novel MVMp-based vector system specifically designed to reduce the risk of replication-competent virus generation by homologous recombination

Dupont

Karim

et al. 2001

Gene Ther

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Recent work highlights the potential usefulness of MVMbased vectors as selective vehicles for cancer gene therapy (Dupont et al, Gene Therapy, 2000; 7: 790-796). To implement this strategy, however, it is necessary to develop optimized methods for producing high-titer, helper-free parvovirus stocks. Recombinants of MVMp (rMVMp) are currently generated by transiently co-transfecting permissive cell lines with a plasmid carrying the vector genome and a helper plasmid expressing the capsid genes (replaced with a foreign gene in the vector genome). The resulting stocks, however, are always heavily contaminated with replicationcompetent viruses (RCV), which precludes their use in vivo and particularly in gene therapy. In the present work we have developed a second-generation MVMp-based vector system specifically designed to reduce the probability of RCV generation by homologous recombination. We have constructed a new MVMp-based vector and a new helper genome with minimal sequence overlap and have used the degeneracy of the genetic code to further decrease vector-

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Method for concentrating and purifying recombinant autonomous parvovirus vectors designed for tumour-cell-targeted gene therapy

Cited by 14 publications

References 9 publications

Cellular contaminants of adeno-associated virus vector stocks can enhance transduction

Cellular contaminants of adeno-associated virus vector stocks can enhance transduction

Tumor-selective gene transduction and cell killing with an oncotropic autonomous parvovirus-based vector

A novel MVMp-based vector system specifically designed to reduce the risk of replication-competent virus generation by homologous recombination

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