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IntroductionPioneering clinical studies have shown that transplantation of genetically modified hematopoietic stem cells may cure severe genetic diseases such as severe combined immunodeficiencies (SCID), 1,2 chronic granulomatous disease (CGD), 3 and lysosomal storage disorders. 4 Unfortunately, some of these studies showed also the limitations of retroviral gene transfer technology, which may cause severe and sometimes fatal adverse effects. In particular, insertional activation of proto-oncogenes by vectors derived from the Moloney murine leukemia virus (MLV) caused T-cell lymphoproliferative disorders in patients with X-linked SCID 5,6 and premalignant expansion of myeloid progenitors in patients with CGD. 3 Preclinical studies showed that HIV-derived lentiviral vectors are less likely to cause insertional gene activation, 7,8 although they can still interfere with normal gene expression at the posttranscriptional level, as observed in a clinical trial of gene therapy for ␤-thalassemia. 9 The molecular bases of vector-induced genotoxicity and the influence of vector design, transduction protocols, and the patient's genetic background in inducing severe adverse effects are still poorly understood. A better understanding of the interactions between retroviral vectors and the human genome may provide new cues to explain these phenomena and a rational basis for predicting genotoxic risks in gene therapy.A large number of studies have focused on the molecular mechanisms by which mammalian retroviruses choose their integration sites in the target cell genome. After entering a cell, retroviral RNA genomes are reverse transcribed into double-stranded DNA and assembled in preintegration complexes (PICs) containing viral and cellular proteins. PICs translocate to the nucleus and associate with the host cell chromatin, where the viral integrase mediates proviral insertion in genomic DNA. Integration is a nonrandom process, whereby PICs of different viruses recognize components or features of the host cell chromatin in a specific fashion. 10 For HIV and other lentiviruses, the LEDGF/p75 protein has been identified as the main factor tethering PICs to active chromatin, 11 whereas mechanisms underlying integration site selection of other retroviruses remain largely unknown. We recently showed that MLV-derived vectors integrate preferentially in hot spots near genes controlling growth and development of hematopoietic cells and flanked by defined subsets of transcription factor binding sites (TFBSs) and suggested that MLV PICs are tethered to active regulatory regions by basal components of the transcriptional machinery. 12,13 The MLV integrase and long terminal repeat enhancer are the main determinants of the selection of TFBS-rich regions of the genome. 13,14 We used ligation-mediated polymerase chain reaction (LM-PCR) and pyrosequencing to build a genomewide, high-definition map of Ͼ 32 000 MLV integration sites in the genome of human CD34 ϩ hematopoietic progenitor cells (HPCs) and used gene expression profiling, chroma...

Gene transfer into HSCs is an effective treatment for SCID, although potentially limited by the risk of insertional mutagenesis. We performed a genome-wide analysis of retroviral vector integrations in genetically corrected HSCs and their multilineage progeny before and up to 47 months after transplantation into 5 patients with adenosine deaminase-deficient SCID. Gene-dense regions, promoters, and transcriptionally active genes were preferred retroviral integrations sites (RISs) both in preinfusion transduced CD34 + cells and in vivo after gene therapy. The occurrence of insertion sites proximal to protooncogenes or genes controlling cell growth and self renewal, including LMO2, was not associated with clonal selection or expansion in vivo. Clonal analysis of long-term repopulating cell progeny in vivo revealed highly polyclonal T cell populations and shared RISs among multiple lineages, demonstrating the engraftment of multipotent HSCs. These data have important implications for the biology of retroviral vectors, the dynamics of genetically modified HSCs, and the safety of gene therapy.

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.donor lymphocyte infusion ͉ gene therapy ͉ graft-versus-host disease ͉ insertional mutagenesis ͉ retroviral integration

Retroviral vectors integrate in genes and