Reconstitution of Neutrophil NADPH Oxidase Activity in Murine X-CGD Following Transplantation of Retrovirus-Transduced Marrow: Potential Impact of Submyeloablative Conditioning.
Authors: Sadat Mohammed , Barese Cecilia , Pech Nancy , Goebel W. Scott , Yoder Mervin C. , Mooney Sean , Cornetta Kenneth , Grez Manuel , Dinauer Mary C. 
Published: April 2020

Abstract
X-linked chronic granulomatous disease (X-CGD) is an inherited disorder of innate immunity in which neutrophils lack the superoxide-generating NADPH oxidase, resulting in recurrent pyogenic infections. Genetic blood diseases can potentially be treated by transplantation of autologous hematopoietic stem cells (HSC) transduced with the functional gene. However, in the absence of a selective advantage for HSC following genetic correction, marrow conditioning is required to enhance engraftment. The current study examined the efficacy of 300 cGy as submyeloablative conditioning using a murine model of X-CGD. We compared reconstitution of peripheral blood neutrophil NADPH oxidase activity in female recipients conditioned with either 300 cGy or ablative irradiation prior to transplantation of male bone marrow transduced with a monocistronic retroviral vector for expression of gp91phox, the product of the X-CGD gene, in the SFFV (spleen focus forming virus) backbone. Transduced cells were transplanted into each of 6 mice receiving 300 cGy (8 x 106 cells per recipient) and also into each of five 1100 cGy-irradiated mice (2 x 106 cells per recipient). Chimerism for donor cells in the 300 cGy group ranged from 40-75 % at 6 months (mean ± SD 63 ± 16%), as monitored by FISH for the Y chromosome. The fraction of oxidase-positive cells in the 1100 cGy group at 6 months was 41 ± 11%, and was stable in this range after 2 months post-transplant. Unexpectedly, the fraction of oxidase-positive donor neutrophils was higher in recipients conditioned with 300 cGy, which increased from 44 ± 17% at four months (N=6) to 100% (N=4) at six months, and was 61, 63, and 100% in 3 mice available for study at nine months. Two mice in each group were sacrificed at five months for analysis of vector copy number in spleen DNA, which was 2–3 in both the 300 and 1100 cGy-irradiated groups. In a second experiment, in 1 of 8 mice conditioned with 300 cGy prior to transplantation of 5 x 106 SFFV-91 transduced cells, the frequency of oxidase positive donor neutrophils was 75% at 5 months compared to 19 ± 6% oxidase-positive donor neutrophils in the other 7 mice and in the group conditioned with 950 cGy (18 ± 5%). Ongoing studies include analysis of provirus marking and insertion sites in secondary and tertiary CFU-S. There has been no evidence of abnormal hematopoiesis in primary and secondary transplant recipients. In summary, 300 cGy is effective conditioning for engraftment of transduced marrow in a murine model of X-CGD, and some recipients had an unexpectedly higher fraction of oxidase-corrected donor neutrophils compared to cohorts conditioned with ablative irradiation. Our previous studies suggest that recipients conditioned with submyeloablative irradiation provide a more competitive environment for engraftment of donor cells undergoing the retroviral transduction procedure (Goebel, et al Exp Hem30, 1432, 2002). We thus postulate that a higher frequency of gene-corrected donor neutrophils in 300 cGy-conditioned murine recipients might reflect a selective advantage for proviral integration at loci that are active in myeloid cells.