Biased Engraftment of Retrovirus-Transduced Marrow in Murine X-CGD Following Submyeloablative Conditioning.

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 gp91^phox 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, SF71gp91^phox, in which human gp91^phox 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.