Polyclonal distribution of lentiviral vector integration sites in gene therapy for x-adrenoleukodystrophy

Retrovirus gene therapy has been successful in inherited monogenetic disorders, but insertion of functional retrovirus LTRs can cause severe side effects. Self-inactivating (SIN) lentiviral vectors have the potential to avoid many of these risks. Here we show that stable and sustained restoration of the genetic defect in cerebral adrenoleukodystrophy (X-ALD) in the first HIV-1 lentiviral SIN-vector gene therapy trial in humans is not accompanied by signs of clonal dominance or even premalignant disproportional distribution of cellular contributions. Integration site (IS) distribution (P1: 2217 IS; P2:1380 IS) was analyzed in ex vivo transduced cells prior to reinfusion and on engrafted cells (P1: 6-24 months (M); P2: 6-20 M) by LAM-PCR and pyrosequencing, revealing gene coding regions as preferred targets for lentiviral vector integration (P1: 72%; P2: 76%). Interestingly, we found common IS (CIS) that affected the same genes in samples of both patients, likely reflecting a preference of lentiviral vector insertion for particular genes. Identical IS were identified in myeloid and lymphoid lineages, indicating successful ex vivo transduction of early hematopoietic progenitors. Our molecular follow-up presents the first molecular data on lentiviral clinical gene therapy in humans. High throughput distribution analysis of the IS repertoire indicates that lentivirus vectors offer great promise for safe and effective correction of human stem and progenitor cells.