Differential expression of a recombinant adeno-associated virus 2 vector in human CD34+ cells and breast cancer cells

Abstract

The use of autologous hematopoietic stem cell (HSC) grafts after high-dose chemotherapy protocols may be hampered by contamination of the grafts with tumor cells. Because epithelial cells seem to be the natural hosts of adeno-associated virus 2 (AAV-2), we speculated that epithelial tumor cells in HSC grafts might be selective targets for AAV-2-based vectors. To test this hypothesis, the breast cancer cell lines T47D and MCF-7 were infected with a recombinant AAV-2 vector expressing the green fluorescent protein (GFP) gene; in addition, human CD34 ϩ mobilized peripheral progenitor cells were infected with the same vector. At a multiplicity of infection of 100, only 1.39% Ϯ 0.51% CD34 ϩ cells expressed the GFP gene whereas, 36.06% Ϯ 6.53% of the infected T47D cells and 41.52% Ϯ 3.16% of the infected MCF-7 cells expressed the transduced GFP gene. After further optimizing the transduction procedure by using higher multiplicities of infection (100 -500) and preincubation of samples with the tyrosine kinase inhibitor genistein, up to 82.52% and 85.35% GFP ϩ T47D and MCF-7 cells, respectively, were observed. The GFP fluorescence intensity in transduced mammary tumor cells was up to 3 logs higher than that of transduced CD34 ϩ cells. The differential expression of recombinant AAV-2 vectors in hematopoietic and epithelial tumor cells warrants further research with this vector system, including the use of suicide genes for the purging of autologous HSC grafts. Cancer Gene Therapy (2000) 7, 597-604

Suicide gene therapy of sarcoma cell lines using recombinant adeno-associated virus 2 vectors

“…In previous experiments, 15,16 where several tumor cell lines were screened for susceptibility to infection with rAAV-2, we were able to show the highest infection rates and expression of an AAV-2 vector containing the GFP marker gene in soft-tissue sarcoma cells. Therefore, in this study the susceptibility of sarcoma cells was further investigated.…”

“…Previous data suggest that an increase in the MOI will also result in a further increase in the rate of transduction. 15,16 Vector construction strategies for rAAV-2 suicide gene therapy…”

“…7,10 We and others have investigated the infectability of tumor cells by recombinant adeno-associated virus 2 (rAAV-2) vectors. [11][12][13][14][15][16] For further reading on TK-mediated gene therapy, please refer to a recent review by Fillat et al 17 In this study, we investigated the susceptibility of six human sarcoma cell lines to rAAV-2 suicide vectors and the subsequent killing of the infected sarcoma cells following exposure to the prodrug ganciclovir.…”

Gene Therapy for Sarcoma

“…3) containing the hGFP gene ( fig. 4a) [Veldwijk et al, 1999[Veldwijk et al, , 2000. Among a series of primary cells and eight solid tumor cell lines infected with rAAV-2 stocks, we observed the highest infection rates (functional titer: 200 IU/cell) in soft tissue sarcoma cells (HS-1; mean: 96% hGFP+ cells) and in breast cancer cells (T47D; mean: 83%, MCF-7; mean: 85%).…”

Adeno-associated virus vectors: potential applications for cancer gene therapy

“…In vitro experiments have revealed that AAV2 transduces some tumor cells very efficiently but does not transduce human hematopoietic stem/progenitor cells. 156,157 Based on this finding, AAV2 vector-mediated gene transfer may be used to purge contaminating tumor cells from the hematopoietic stem cell population prior to transplantation. Fruehauf et al found that AAV2 vectors efficiently infect human HS-1 and HT 1080 sarcoma cells (more than 95%), while primary human mobilized peripheral blood progenitor cells are more resilient to transduction by AAV2.…”