Retroviral vector integration deregulates gene expression but has no consequence on the biology and function of transplanted T cells

doi:10.1073/pnas.0507496103

Retroviral vector integration deregulates gene expression but has no consequence on the biology and function of transplanted T cells

^*Cancer Immunotherapy and Gene Therapy Program, and ^‡Bone Marrow Transplantation Unit, Istituto Scientifico H. San Raffaele, Via Olgettina 58, 20132 Milan, Italy; ^†Department of Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41100 Modena, Italy; ^§MolMed S.p.A., Via Olgettina 58, 20132 Milan, Italy; and ^¶San Raffaele “Vita e Salute” University, Via Olgettina 58, 20132 Milan, Italy

Edited by Malcolm A. Martin, National Institutes of Health, Bethesda, MD (received for review September 1, 2005)

Abstract

The use of retroviral vectors in gene therapy has raised safety concerns for the genotoxic risk associated with their uncontrolled insertion into the human genome. We have analyzed the consequences of retroviral transduction in T cells from leukemic patients treated with allogeneic stem cell transplantation and donor lymphocytes genetically modified with a suicide gene (HSV-TK). Retroviral vectors integrate preferentially within or near transcribed regions of the genome, with a preference for sequences around promoters and for genes active in T cells at the time of transduction. Quantitative transcript analysis shows 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 9 yr after administration. Analysis of integrated proviruses in 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 in vivo. Despite the potentially dangerous interactions with the T cell genome, retroviral integration has therefore little consequence on the safety and efficacy of T cell transplantation.

Footnotes

↵ ∥ To whom correspondence should be addressed. E-mail: f.mavilio{at}hsr.it.
Author contributions: C. Bonini, C. Bordignon, and F.M. designed research; A.R., C. Bonini, Z.M., F.U., D.S., S.M., A.B., M.T.L.S., M.B., A.P., and F.C. performed research; E.T. contributed new reagents/analytic tools; A.R., C. Bonini, F.U., E.T., and F.M. analyzed data; and C. Bordignon and F.M. wrote the paper.
Conflict of interest statement: C. Bordignon and F.M. are paid consultants of MolMed S.p.A., an Italian biotechnology company based in Milan, which sponsors one of the two clinical trials described in this paper.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: HSC, hematopoietic stem cell; DLI, donor lymphocyte infusion; GvHD, graft-versus-host disease; TK, timidine kinase; TCR, T cell receptor; CT, cycle of threshold.