X-linked SCID (SCID-X1) is amenable to correction by gene therapy using conventional gammaretroviral vectors. Here, we describe the occurrence of clonal T cell acute lymphoblastic leukemia (T-ALL) promoted by insertional mutagenesis in a completed gene therapy trial of 10 SCID-X1 patients. Integration of the vector in an antisense orientation 35 kb upstream of the protooncogene LIM domain only 2 (LMO2) caused overexpression of LMO2 in the leukemic clone. However, leukemogenesis was likely precipitated by the acquisition of other genetic abnormalities unrelated to vector insertion, including a gain-of-function mutation in NOTCH1, deletion of the tumor suppressor gene locus cyclin-dependent kinase 2A (CDKN2A), and translocation of the TCR-β region to the STIL-TAL1 locus. These findings highlight a general toxicity of endogenous gammaretroviral enhancer elements and also identify a combinatorial process during leukemic evolution that will be important for risk stratification and for future protocol design.

X-linked agammaglobulinaemia (XLA) is a human immunodeficiency caused by failure of pre-B cells in the bone marrow to develop into circulating mature B cells. A novel gene has been isolated which maps to the XLA locus, is expressed in B cells, and shows mutations in families with the disorder. The gene is a member of the src family of proto-oncogenes which encode protein-tyrosine kinases. This is, to our knowledge, the first evidence that mutations in a src-related gene are involved in human genetic disease.

Prolonged exposure of human hematopoietic stem cells (HSC) to growth factors for efficient transduction by murine oncoretroviral vectors has major detrimental effects on repopulating activity. In this study, we have used a vesicular stomatitis virus G envelope protein (VSV-G)-pseudotyped human immunodeficiency virus type 1 (HIV-1) lentiviral-based vector system to transduce cord blood (CB) CD34+ cells over a limited time period (< or =24 hours). Under these conditions, significant gene marking was observed in engrafted human lymphoid, myeloid, and progenitor cells in all transplanted Severe Combined Immunodeficient (SCID) mice. To enhance the level of gene expression in hematopoietic cells, we also generated a series of lentiviral vectors incorporating the spleen focus forming virus (SFFV) long terminal repeat (LTR) sequences, and the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). By including the central polypurine tract (cPPT) sequence of HIV-1 we were then able to achieve high levels of transduction (over 80%) and gene expression in vivo after a single exposure to viral supernatant. These results demonstrate that lentiviral vectors are highly effective for gene transfer to human HSC, and that SFFV regulatory sequences can be successfully incorporated to enhance the long-term expression of a transgene in primary human hematopoietic cells in vivo.

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Retroviral and lentiviral vector integration into host-cell chromosomes carries with it a finite chance of causing insertional mutagenesis. This risk has been highlighted by the induction of malignancy in mouse models, and development of lymphoproliferative disease in three individuals with severe combined immunodeficiency-X1 (refs. 2,3). Therefore, a key challenge for clinical therapies based on retroviral vectors is to achieve stable transgene expression while minimizing insertional mutagenesis. Recent in vitro studies have shown that integration-deficient lentiviral vectors can mediate stable transduction. With similar vectors, we now show efficient and sustained transgene expression in vivo in rodent ocular and brain tissues. We also show substantial rescue of clinically relevant rodent models of retinal degeneration. Therefore, the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration. For therapeutic application to postmitotic tissues, this system substantially reduces the risk of insertional mutagenesis.