Gene expression profiling of CD4+ and CD8+ T-cells from gene therapy treated ADA patients and from healthy controls

Gene transfer into HSCs by gammaretroviral vectors (RV) is an effective treatment for inherited blood disorders, although potentially limited by the risk of insertional mutagenesis. We evaluated the genomic impact of RV integration in T-lymphocytes from adenosine deaminase (ADA)-Severe combined immunodeficiency (SCID) patients 10 to 30 months after infusion of autologous, genetically-corrected CD34+ cells. Expression profiling on ex vivo T-cell bulk population revealed no difference with respect to healthy controls. To assess the effect of vector integration on gene expression at the single cell level, primary T-cell clones were isolated from two patients. T-cell clones harboured either one or two vector copies per cell and displayed partial to full correction of ADA expression, purine metabolism and TCR-driven functions. Analysis of retroviral integration sites (RIS) indicated a high diversity in T-cell origin, consistent with the polyclonal TCR-Vbeta repertoire. Quantitative transcript analysis of 120 genes within a 200kb-window around RIS showed modest (2.8- to 5.2-fold) disregulation of 5.8% genes in 18.6% of the T-cell clones compared to controls. Nonetheless, affected clones maintained a stable phenotype and normal functions in vitro. These results confirm that RV-mediated gene transfer for ADA-SCID is safe, and provide crucial information for the development of future gene therapy protocols.

13

Cassani B, Montini E, Maruggi G, Ambrosi A, Mirolo M, Selleri S, Biral E, Frugnoli I, Hernandez-Trujillo V, Di Serio C, Roncarolo MG, Naldini L, Mavilio F, Aiuti A