292. Lentivirus Vector Mediated Gene Correction in Artemis-Deficient Severe Combined Immunodeficiency

Mutations in DCLRE1C/Artemis, a DNA repair gene, cause T-B-NK+ SCID by preventing V(D)J recombination in T and B cell progenitors and also confer heightened sensitivity to irradiation and alkylator chemotherapy. SCID newborn screening identifies Artemis-deficient SCID (ART-SCID) early in life and while allogeneic hematopoietic cell transplantation can cure ART-SCID, preparative regimens for conditioning are poorly tolerated. Without alkylating chemotherapy patients may have graft failure while toxic effects of chemotherapy include increased mortality, short stature and abnormal dental development. Thus, building on experience with X-linked and ADA deficient SCID, we found addition of a normal Artemis gene to hematopoietic stem cells (HSC) an attractive strategy to treat ART-SCID.Since overexpression of Artemis protein causes cellular cytotoxicity, a lentivirus vector with human Artemis cDNA and its endogenous promoter (Apro-hART) was produced and used to transduce fibroblasts from ART-SCID patients and controls. Apro-hART transduced ART-SCID fibroblasts showed correction of radiosensitivity by enumeration of foci of DNA damage and proliferation assays. Radiosensitivity of cells lacking the DNA repair enzyme Ligase-4 was not corrected. View Large Image | Download PowerPoint SlideMobilized peripheral blood CD34+ cells from an ART-SCID patient, incapable of differentiation into T and B cells, were transduced with Apro-hART or GFP lentivirus and cultured on OP9 cells or injected into irradiated newborn NSG mice. OP9 cocultures and blood and spleen cells from NSG mice showed that Apro-hART-corrected, but not GFP-transduced, ART-SCID CD34+ cells differentiated into B cells and T and B cells, respectively, as did GFP-transduced control CD34+ cells. Lymphocyte maturation was proven by lineage specific surface markers and measures of V(D)J diversity and recombination, T cell receptor Vbeta spectratyping and Kappa chain receptor excision circles (KRECs), respectively. View Large Image | Download PowerPoint SlideColony forming assays with transduced cells revealed transduction efficiency of 28.6%, a mean vector copy number of 3/cell and a diverse profile of lentivirus integration sites.This successful gene correction and restoration of Artemis function in fibroblasts and HSCs from ART-SCID patients supports institution of a clinical trial of gene addition therapy for ART-SCID.