Self-Inactivating gp91 Lentivector Corrects the Oxidase Defect in NOD/SCID Mouse Repopulating Peripheral Blood Mobilized CD34 Cells from Patients with X-linked Chronic Granulomatous Disease Running head: Lentivector correction of X-linked CGD Scientific section heading: Gene Therapy

HIV-1 derived lentivectors are promising for gene transfer into hematopoietic stem cells, but require pre-clinical in vivo evaluation relevant to specific human diseases. Non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice accept human hematopoietic stem cell grafts providing a unique opportunity for in vivo evaluation of therapies targeting human hematopoietic diseases. We demonstrate for the first time that hematopoietic stem cells from X-linked chronic granulomatous disease (X-CGD) patients give rise to X-CGD phenotype neutrophils in the NOD/SCID model that can be corrected using VSV-G pseudotyped 3 generation self-inactivating (SIN) lentivector encoding gp91. We transduced X-CGD patient mobilized CD34 peripheral blood stem cells (CD34PBSC) with lentivector-gp91 or amphotropic onco-retrovirus MFGS-gp91 and evaluated correction ex vivo and in vivo in NOD/SCID mice. Only lentivector transduced CD34PBSC under ex vivo conditions non-permissive for cell division, but both vectors performed best under conditions permissive for proliferation (multiple growth factors). Under the latter conditions both lentivector and MFGS achieved significant ex vivo correction of X-CGD CD34PBSC (18% and 54% of cells expressing gp91, associated with 53% and 163% of normal superoxide production, respectively). However, lentivector, but not MFGS, achieved significant correction of human X-CGD neutrophils arising in vivo in transplanted NOD/SCID mice (20% and 2.4%, respectively). Thus, 3 generation SIN lentivector-gp91 performs well as assessed in human X-CGD neutrophils differentiating in vivo, and our studies suggest that the NOD/SCID model is generally applicable for in vivo study of therapies evaluated in human blood cells expressing a specific disease phenotype. email: [email protected] only. For personal use at PENN STATE UNIVERSITY on February 23, 2013. bloodjournal.hematologylibrary.org From