Genomic and functional assays demonstrate reduced gammaretroviral vector genotoxicity associated with use of the cHS4 chromatin insulator.
Interest in the use of recombinant retroviral vectors for clinical gene therapy has been tempered by evidence of vector-mediated genotoxicity involving the activation of cellular oncogenes flanking sites of vector integration. We report here that the rate of gammaretroviral vector genotoxicity can be significantly reduced by addition of the cHS4 chromatin insulator, based on two complementary approaches for assessing vector-mediated genotoxicity. One approach involves the direct, genomewide assessment of cellular gene dysregulation using panels of transduced cell clones and genomic microarrays, whereas the other involves the functional assessment of malignant transformation using a factor-dependent cell line. Both assays are robust and quantitative, and indicate the cHS4 chromatin insulator can reduce vector-mediated genotoxicity approximately sixfold (ranged three to eight fold). These approaches also provide a means for assessing various aspects of vector-mediated genotoxicity, including the overall rate of cellular gene dysregulation, the potential influence of vector provirus over large genomic distances, and the involvement of oncogenic pathways in vector-mediated malignant transformation.
Li
Chang Long
CL
Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, Washington 98195-7720, USA.
Xiong
Ding
D
Stamatoyannopoulos
George
G
Emery
David W
DW
D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D002843:Chromatin / Q000235:genetics*; D005260:Female; D005434:Flow Cytometry; D017988:Gammaretrovirus / Q000235:genetics*; D005822:Genetic Vectors / Q000009:adverse effects*; D006801:Humans; D038101:Insulator Elements; D051379:Mice; D008809:Mice, Inbred C3H; D011995:Recombination, Genetic
D002843:Chromatin