Suppression of Clonal Dominance in Cultured Human Lymphoid Cells by Addition of the cHS4 Insulator to a Lentiviral Vector

Abstract

Lentiviral vectors efficiently transduce quiescent stem cells and are being evaluated for gene therapy of blood dis-orders. The risk of genotoxicity as a result of insertional mutagenesis is an important safety consideration. The hy-persensitive site 4 insulator from the chicken beta-globin locus (cHS4) possesses chromatin bar-rier 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 indi-vidual 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 domi-nance developed in cultures of cells containing the integrated vector genomes, as reflected by the recovery of mul-tiple single cell isolates containing the same integration site. We infer that certain integrations conferred a prolifera-tive 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.

Leukemia induction after a single retroviral vector insertion in Evi1 or Prdm16

“…In our cell-based immortalization assay, we recently obtained evidence for a reduced genotoxic potential of g-retroviral and lentiviral vectors with more physiological cellular promoters (and Modlich U, unpublished data). The present study thus supports the emerging concept 17,19,54 that the type of the vector backbone and the nature of its cis-regulatory sequences are crucial variables to improve the therapeutic index of integrating vectors. The use of retroviral vectors containing strong, uninsulated enhancers should rather be restricted for the discovery of cancer-initiating genes in model systems (as shown here for Evi1 and Prdm16), or to clinical situations that offer no alternative.…”

CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors

“…Here, our results introduce novel insulator elements suitable for implementation in viral vectors, endorsing previous reports on insulator elements able to reduce insertional genotoxicity. 9,26,44,45 The barrier activity of the novel insulating elements was probed in the context of random transgene chromosomal integration upon stable transfection. Flanking the transgene with CTCF binding sites led to a decrease in its expression, stably propagated over cell division.…”

Physiological and tissue-specific vectors for treatment of inherited diseases

“…New cellular 140 and animal 13 models have been developed to study genotoxicity potential of integrative vectors. Using these models, several groups have demonstrated the increase in safety of SIN vectors 12,141 and better performance of lentiviral over murine leukemia-based vectors. 13 In addition, although it was largely hypothesized that regulated expression should also reduce genotoxicity, Baum's group 113 recently demonstrated that tissue-specific and physiologically regulated vectors are indeed safer with no detectable transforming potential.…”