Ανάπτυξη ρετροϊικών φορέων τύπου spuma FV για τη γονιδιακή διόρθωση της χρόνιας κοκκιωματώδους νόσου (chronic granulomatous disease)

Abstract

Objective: The X-linked form of chronic granulomatous disease (X-CGD) results from mutations in the CYBB gene encoding gp91phox, the larger subunit of the oxidase flavocytochrome b558. Affected individuals suffer from recurrent life-threatening infections due to impaired superoxide production by the NADPH oxidase in phagocytes. Novel foamy virus (FV) vectors expressing the human codon-optimized gp91phox were evaluated for the genetic correction of the disease in the X-CGD cell line and in X-CGD mouse model.Materials and Methods: The vectors were evaluated in vitro, in the human X-CGD PLB-985 cell line and in the X-CGD BM Lin-cells. Transplantation of transduced Lin-cells was performed in X-CGD mice after busulfan conditioning. Real time PCR was used for chimerism and vector copy number determination. Restoration of NADPH oxidase production was assessed by the NBT and the DHR assays.Results: High and stable gp91phox expression, as well as reconstitution of NADPH activity, was achieved in the human X-CGD PLB-985 cell line and in primary murine X-CGD hematopoietic stem cells (HSCs) ex vivo. Transplantation of transduced bone marrow HSCs in the murine model of X-CGD, even with low MOI, reconstituted the levels of oxidase-producing neutrophils and provided enzymatic activity that reached 70% of normal.Conclusion: FV vectors expressing the human gp91phox transgene constitute potential candidates for the gene therapy of CGD since they combine lack of pathogenicity with efficacy even at low MOI.