Correction of Human X-Linked Chronic Granulomatous Disease (X-CGD) Following Transduction of X-CGD CD34+ Cells with a Modified RD114-Pseudotyped Simian Immunodeficiency Virus Lentiviral Vector in a NOD/SCID Mouse Xenograft Model.
Authors: Naumann Nora , De Ravin Suk See , Choi Uimook , Moayeri Morvarid , Ikeda Yasuhiro , Malech Harry L. 
Published: October 2019

Abstract
X-linked chronic granulomatous disease (X-CGD) is an immune deficiency that results from mutations in the gp91phox subunit of the phagocyte NADPH-oxidase. We have developed a modified RD114-pseudotyped simian immunodeficiency virus (SIVmac) vector encoding human gp91phox and report here that this vector efficiently targets peripheral blood-mobilized CD34+ hematopoietic stem cells (PBSCs) from X-CGD patients, correcting the oxidase deficient phenotype. The majority of lentiviral gene transfer studies have relied on human immunodeficiency virus (HIV) 1-derived vectors pseudotyped with the vesicular stomatitis virus G protein (VSV-G) envelope. Because HIV is a human pathogen, there are theoretical benefits to considering other types of lentivectors engineered from viruses less pathogenic for humans such as SIV-based vectors. In addition, the VSV-G envelope has some toxicity for cells, limiting the titers of vector that can be used. RD114/TR-pseudotyped SIV gp91phox encoding vector particles were transiently produced following transfection of 293T cells and concentrated by high-speed centrifugation achieving stock titers of 1.5 × 10E7 infectious units/ml. PBSCs from two patients with X-CGD (P1 and P2) were cultured and transduced (multiplicity of infection 2–3) overnight four times before transplantation into sublethally irradiated NOD/SCID mice on day 5 of culture. Ex vivo transduction efficiency was 40.5% and 46%, respectively. Cells maintained in culture showed a progressive decrease in marking over the first 9 days, after which marking stabilized at 22.7% (P1) and 27.1% (P2) out to day 26 of culture with an averaged copy number of 2.2–2.4 per transduced cell at that time. Compared to healthy control, 18–19% of myeloid colonies derived from patient CD34+ cells stained positive in the nitroblue tetrazolium (NBT) test. Liquid cell cultures at 20 days were analyzed for production of reactive oxidative species using the flow cytometry dihydrorhodamine 123 (DHR) assay. Cells from P1 demonstrated 15.3% and from P2 9.1% of the activity of healthy cells. At 6 weeks after transplant, analysis of mouse bone marrow (mBM) samples revealed human cell engraftment levels of 36.8–74% (CD45+ cells). Compared to analysis of mice transplanted with healthy human PBSCs, 10.5% (P1) and 7.3% (P2) of the myeloid cells (CD13+) developing from X-CGD CD34+ cells in the chimeric mBM expressed gp91phox transgene. Human CD34+ cells were isolated from mBM and grown in liquid culture or plated for colony assays. 2.1% (P1) and 1.2% (P2) of cultured cells were gp91phox positive at day 14 with an averaged copy number of 2.3–5.2 per marked cell. 4.8% and 3.6% of colonies derived from the same cells were positive in the NBT test. DHR assay at day 21 of culture demonstrated 2.7% (P1) and 2.0% (P2) of the activity of healthy cells. Linear amplification-mediated PCR retrieved multiple unique vector insertion sites in CD34+ cells from both patients isolated from chimeric mBM, without predominance of any one clone suggesting polyclonal marking. In conclusion, these data suggest that modified RD114-pseudotyped SIVmac-based vectors may be suitable for hematopoietic gene therapy for CGD and represent an alternative to HIV-1 based vectors. Studies are ongoing to further improve vector production titers to achieve higher transduction levels of PBSCs.