1004. Integration Analyses in ADA-Deficient SCID Patients Successfully Treated with Stem Cell Gene Therapy
Authors: A. Aiuti, B. Cassani, F. Cattaneo, G. Andolfi, M. Mirolo, A. Recchia, L. Callegaro, A. Tabucchi, F. Carlucci, S. Slavin, R. Miniero, F. Mavilio, M. G. Roncarolo, C. Bordignon
Journal: Molecular Therapy (2004)

Adenosine deaminase (ADA)-deficiency results in severe combined immunodeficiency (SCID) associated with systemic metabolic toxicity. Gene therapy with hematopoietic stem cells (HSC) may represent a definitive treatment for this disease, considering the current limitations of bone marrow transplantation from non-HLA identical donors or of enzyme replacement therapy (PEG-ADA). We treated four children with autologous bone marrow CD34+ cells engineered with a retroviral vector encoding ADA, combined with low-intensity conditioning with busulfan. Long-term engraftment of gene corrected HSC with differentiation into lymphoid and myeloid lineages, was sustained during the follow-up. All patients showed increased peripheral blood lymphocyte counts, restoration of thymopoiesis, and normalization of proliferative responses to mitogens and antigens. Serum immunoglobulin levels improved, allowing IVIG discontinuation in Pt1 and Pt3, with production of specific antibodies after antigen vaccination. Sustained ADA expression resulted in correction of the systemic toxicity and restoration of normal patients' growth, in the absence of enzyme replacement therapy. All the children are presently healthy, they did not experience any severe infections or adverse effect, with the longest follow up being of 40 months after gene therapy. A systematic analyses to identify and map vector integration sites by inverse-PCR and LM-PCR was undertaken. Cloned integrations were selected for analyses only if contained the correct retroviral and primer sequences and yielded a unique best hit with ≥95% identity to the human genome (NCBI34). Collective data from 160 mapped integrations showed a frequency of insertions inside RefSeq genes of 33.4% (n=53), similar to the one reported in cell lines infected in vitro with retroviral vectors. Genomic regions within 5 Kb upstream from transcription start site were favored sites of integrations (15.1%). There was no preferential target locus, with the exception that two independent integrations were found outside a gene on chromosome 3p13, within a distance of 120 Kb. Integrations in circulating T cells were highly polyclonal (>50 in Pt1 and Pt3), as shown by the random cloning of PCR products from peripheral blood T cells as well as by PCR analyses in T-cell clones generated ex vivo. Fewer integrations (3-15) were detected in myeloid cells from bone marrow and peripheral blood. Common integrants were identified in various lineages during the follow-up of patients, demonstrating the engraftment of transduced HSC with multilineage potential. Collectively, these data show the efficacy of gene therapy in correcting the immune and metabolic defect of ADA-SCID. In addition, these studies are providing new information on the safety and biology of retroviral vectors as well as on the in vivo dynamics of HSC and their progeny.