Suppression of clonal dominance in cultured human lymphoid cells by addition of the cHS4 insulator to a lentiviral vector

Lentiviral vectors efficiently transduce quiescent stem cells and are being evaluated for gene therapy of blood disorders. The risk of genotoxicity as a result of insertional mutagenesis is an important safety consideration. The hypersensitive site 4 insulator from the chicken beta-globin locus (cHS4) possesses chromatin barrier and enhancer-blocking functions. A control lentiviral vector encoding green fluorescent protein was compared with a vector in which the cHS4 insulator element flanked the green fluorescent protein expression cassette in single cell isolates of transduced human T cells (Jurkat) after 9 days in culture. The insulator had minimal effect on mean fluorescent intensity and only modestly reduced the variability of green fluorescent protein expression among individual single cell isolates. Most unique integration sites were within genes, but the insulator-containing vector had a moderate predilection to integrate near the transcriptional start site compared with the control vector. Clonal dominance developed in cultures of cells containing the integrated vector genomes, as reflected by the recovery of multiple single cell isolates containing the same integration site. We infer that certain integrations conferred a proliferative or survival advantage by affecting gene expression through insertional mutagenesis, leading to this clonal dominance. This effect was diminished by including the insulator element in the vector genome.