Preclinical Development of a Lentiviral Vector for Gene Therapy of X-linked Severe Combined Immunodeficiency

X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the interleukin-2 receptor γ chain gene (IL2RG), and is characterized by profound defects in T-, B- and NK-cell functions. Transplantation of hematopoietic stem/progenitor cells (HSPCs) genetically corrected with early MLV-derived retroviral vectors showed restoration of T-cell immunity in patients but resulted in vector-induced insertional oncogenesis. We developed a SIN lentiviral vector carrying a codon-optimized human IL2RG cDNA driven by the EF1αShort promoter (EFS-IL2RG), and tested its efficacy and safety in vivo by transplanting transduced Il2rg-deficient Lin- HSPCs in an Il2rg-/-/Rag2-/- mouse model. The study showed restoration of T, B and NK cell counts in bone marrow and peripheral blood and normalization of thymus and spleen cellularity and architecture. High-definition insertion site analysis defined the EFS-IL2RG genomic integration profile and showed no sign of vector-induced clonal selection or skewing in primarily and secondarily transplanted animals. The study enables a phase-I/II clinical trial aimed at restoring both T- and B-cell immunity in SCID-X1 children upon non-myeloablative conditioning. Interestingly, we found that EFS-IL2RG target a substantially different set of genes in murine vs. human HSPCs, indicating limits in the predictive power of mouse-based genotoxicity studies designed to mimic human intervention