Treatment of Infections in Patients with X-Linked Chronic Granulomatous Disease (XCGD) with Gene Therapy.
Authors: Kang Elizabeth M. , Linton Gilda , Theobald Narda , O’Brien Sandra Anaya , Hilligoss Dianne , Malech Harry L. 
Published: October 2019

CGD is a monogenetic disorder that results in significant morbidity and mortality and is therefore a good candidate for gene therapy techniques. Genetic modification has been used successfully in mouse models of the disease and further, allogeneic bone marrow or peripheral blood transplantation has been shown to be curative in humans. The first clinical trial for CGD performed by Malech et al., (PNAS 1997) done without conditioning, resulted in marking levels of 0.1 to 0.01%. More recently with the use of busulfan 8mg/kg and an SFFV retrovirus, Ott et al., (Nature, 2006) achieved much higher (>20%) peripheral blood levels of corrected cells. Subsequently there was an oligoclonal expansion of those cells in which probable activation of myeloid specific genes EVI1-MDS, SetBP1 or PRDM16 occurred due to vector insertion. While there was low production of superoxide on a per cell basis, there was a clinical benefit to these patients as their underlying infections resolved with the therapy. As such, we have initiated a clinical trial with the primary goal to treat infections in X-CGD patients with genetically modified cells. We used busulfan 10mg/kg over two days as a conditioning agent and a standard ex vivo transduction method using an amphotropic pseudotyped MFGS vector encoding only the gp91phox transgene, culturing the cells for 96 hours in retronectin coated bags and media supplemented with, SCF, Flt-3, IL-3, and MDGF. To date we have treated two patients. The first is a 28 yo male who presented with multiple staph aureus positive liver abscesses not amenable to resection or radio frequency ablation. He received 40x10e6 CD34+cells/kg after the busulfan and the transduction efficiency was 73% as measured from Day 4 of the culture. At day 13 post transplant, the patient had a marking level of 24% that subsequently decreased over time, but now remains at approximately 0.7–1% more than 9 months post treatment. The patient tolerated the treatment well and 6 months after his treatment, his liver abscesses were completely resolved. He remains on routine antibiotic prophylaxis. LAM PCR analysis in this patient has not shown any clonal dominance. We have found one insert in MDS-EVI1 out of 60 insertions found and sequenced, but none in SetBP1 or PRDM16. The second patient, a 31 year old male, was enrolled due to a fungal chest wall infection unresolved despite two years of combination antifungal treatment. The patient’s cells were transduced with the same vector as the first, but using a different production lot with a lower titre. The transduction efficiency was therefore lower with 41% gp91 positive cells at end of transduction. The patient received a total of 71x10e6 CD34+cells/kg and the initial marking was 5% at two weeks post infusion while the neutrophil count was still low but recovering. By day 21 however, the marking had dropped to less than 1%. Interestingly, there was a small positive fraction in the unstimulated control of the DHR, suggesting auto-activation of the cells. Hence given the timing of the drop and the presence of these autostimulated cells, we hypothesize that the patient has had an immune mediated reaction to the gp91phox expressing cells. It does not appear to be silencing as the realtime PCR data also suggests clearance. Studies are in progress to determine if this is a T or more likely, B cell mediated rejection. In the meantime, we will add an immunosuppressant peritransplant to avoid any possible immune mediated rejections in future patients.