Retroviral Vector Integration Deregulates Gene Expression but Has No Consequences on the Biology and Function of Transplanted T Cells.

Retroviral vectors have raised safety concerns for the genotoxic risk associated with their uncontrolled insertion into the human genome. The occurrence of T-cell leukemia in patients treated by gene therapy for X-SCID has been correlated with insertional activation of a T-cell proto-oncogene. However, no such event has been reported in other pre-clinical or clinical trials, suggesting the existence of disease- and/or transgene-specific risk factors in the X-SCID case. We have analyzed the integration pattern of a retroviral vector expressing a suicide gene (HSV-TK) and a surface marker (ΔLNGFR) in T-lymphocytes from patients treated with donor lymphocyte infusion (DLI) after hematopoietic stem cell transplantation for therapy of acute and chronic leukemia. The suicide gene allows to control graft-versus-host disease, a major complication of DLI, while preserving anti-tumor and antiviral activities. Proviral integrations were analyzed by sequencing the vector-genome junctions in T-cell DNA obtained from 4 different patients up to 9 years after treatment. Over 85% of proviral integrations occurred within transcription units, with a preference for first introns and upstream promoter regions. Over 75% of intragenic integrations were in genes active in T cells at the time of transduction, and ~4% occurred at recurrent genomic locations (hot spots). Microfluidic real-time PCR in RNA extracted from individual T-cell clones harboring proviral integrations in proximity of transcription start sites showed that one fifth of these integrations affect the expression of nearby genes. However, transduced T-cell populations maintain remarkably stable gene expression profiles, phenotype, biological functions and immune repertoire in vivo, with no evidence of clonal selection up to nine years after administration. Analysis of transduced cells before and after transplantation indicates that integrations interfering with normal T-cell function are more likely to lead to clonal ablation than expansion. Despite the potentially dangerous interactions with the T-cell genome, retroviral integration has therefore little consequences on the safety and efficacy of T-cell transplantation.