Development of regulated lentiviral vectors for gene therapy of x-linked chronic granulomatous disease

Introduction. Chronic Granulomatous Disease (CGD) is caused by defects of the NADPH oxidase complex, responsible for the production of the oxidative burst in phagocytes. Patients present increased susceptibility to life-threatening fungal and bacterial infections and are treated with a lifelong prophylaxis. Currently, the only curative option is bone marrow transplantation. Gene therapy with hematopoietic stem cells (HSC) may represent a valid alternative to conventional transplant. Clinical trials for X-CGD employing gp91phox-expressing gammaretroviral vectors have been limited by insertional oncogenesis and lack of persistent engraftment. Methods. We developed a novel strategy based on regulated, self-inactivating lentiviral vectors (LVs) that target gp91phox expression to the differentiated myeloid cells while sparing HSC, to reduce the risk of genotoxicity and perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and a posttranscriptional, microRNA mediated regulation. We designed different therapeutic gp91phox-expressing LVs for CGD gene therapy: 1) PGK.gp91, in which gp91phox is driven by an ubiquitous cellular promoter; 2) MSP.gp91, to control the transgene expression at transcriptional level using a myeloid specific promoter; 3) PGK.gp91_126T(2), in which we exploited the miRNA system, incorporating miR-126 target sequences, to prevent the transgene off-target expression in HSC; 4) MSP.gp91_126T(2), combining the posttranscriptional de-targeting with the MSP. Vectors were tested in human cell line, human bone marrow HSC, their progeny differentiated in vitro and in the mouse model of X-CGD. Results. All vectors restored gp91phox expression and NADPH oxidase function in human X-CGD PLB-985 cell line and in myeloid cell lines and in macrophages from peripheral blood monocytes of 3 X-CGD patients (22-48%). While unregulated LVs ectopically expressed gp91phox in CD34+ cells, both transcriptionally and post-transcriptionally regulated LVs substantially reduced this off-target expression. By combining transcriptional and posttranscriptional targeting in the dual regulated vector, we achieved high levels of myeloid-specific transgene expression, entirely sparing the most primitive CD34+CD38-CD90+ HSC compartment (5-fold reduction). XCGD mice transplanted with all vectors engrafted and restored gp91phox expression, with 20-70% of granulocytes and monocytes expressing human gp91phox. MSP-driven vectors were superior in maintaining regulation during BM development as well as in peripheral blood B and T cells. Oxidase activity in corrected granulocytes was superior using MSP-driven vectors (38-59% of WT NADPH oxidase activity) as compared to PGK (17-23%). The MSP transcriptional control combined to miR-126 detargeting represent a promising approach for further clinical development of gp91phox therapeutic vectors.