Biased Engraftment of Retrovirus-Transduced Marrow in Murine X-CGD Following Submyeloablative Conditioning.
Authors: Sadat Mohammed , Dirscherl Sara , Pech Nancy , Getty Robert , Cross Scott , Barese Cecilia , Goebel W. Scott , Yoder Mervin C. , Mooney Sean , Grez Manuel , Cornetta Kenneth , Dinauer Mary C. 
Published: October 2019

Abstract
X-linked chronic granulomatous disease is an inherited disorder of innate immunity in which neutrophils are unable to generate microbicidal oxidants due to mutations in the gene encoding the gp91phox subunit of the NADPH oxidase. Genetic blood diseases can be treated by transplantation of autologous hematopoietic stem cells (HSC) transduced with the functional gene. In the absence of a selective advantage following genetic correction, marrow conditioning is required to facilitate engraftment. Using a murine model of X-CGD, we examined the efficacy of 300 cGy as conditioning prior to transplantation of HSC transduced with a monocistronic gammaretroviral vector, SF71gp91phox, in which human gp91phox expression is driven by the SFFV LTR. Although packaged with an ecotropic envelope for this study, this is the same vector as used in an ongoing clinical trial for X-CGD, where two reported patients showed an unexpected increase in the fraction of oxidase-corrected neutrophils, which appeared to reflect expansion of gene-modified myeloid cells due to insertional activation of adjacent loci (Ott et al, Nat Med, 2006). We compared donor engraftment and reconstitution of NADPH oxidase activity in female X-CGD mice conditioned with either 300 cGy or ablative irradiation (IR) (950–1100 cGy) prior to transplantation of transduced male marrow (5–8 × 106 and 2 × 106 cells, respectively). Donor chimerism in the 300 cGy group was 67 ± 14% by six months (mean ± SD, from two independent experiments), as monitored by FISH for the Y chromosome. The fraction of oxidase-positive cells in mice conditioned with ablative IR was 41 ± 11% and 18 ± 5% at 6 months in the two experiments, and was stable after 2 months post-transplant. In contrast, the fraction of oxidase-positive donor-derived neutrophils was higher in many 300 cGy-conditioned recipients; in the first experiment, 100% of donor neutrophils were oxidase-positive in 4 recipients at six months, and in the second, oxidase positive donor neutrophils increased to 100% in 1 of 8 recipients. The number of vector integrations in primary recipient spleen DNA was similar (≈2) for both conditioning regimens. However, the fraction of vector-marked secondary CFU-S derived from transplanted male cells was higher for mice receiving 300 cGy compared to those receiving ablative IR (vector-positive CFU-S 87 ± 19% vs 36 ± 20%, respectively; from 6–7 primary recipients for each regimen; p < 0.001). There has been no evidence of leukemic transformation in a total of 24 primary, 39 secondary, and 24 tertiary recipients (majority followed for ≥6 months post transplant). Ongoing studies include analysis of proviral insertion sites. In summary, 300 cGy is effective conditioning for engraftment of transduced marrow in a murine model of X-CGD. Our previous studies suggest that submyeloablative IR results in a more competitive environment for engraftment of donor cells cultured ex vivo for retroviral transduction (Goebel, et al Exp Hem, 2002). Analysis of proviral marking frequency in secondary CFU-S in the current study suggests that vector integration may confer a selection advantage for engraftment of long-term repopulating cells in primary recipients conditioned with 300 cGy, associated with a higher frequency of oxidase-corrected neutrophils.