Development of a gmp manufacturing process for nadph oxidase correction in mRNA transfected granulocytes and monocytes for patients with chronic granulomatous disease

Background & Aim Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency caused by functional impairment of any one of five subunits of the NADPH oxidase complex in granulocytes and monocytes. GP91 and p47 deficiency account for the majority of CGD cases, with ∼70% attributable to defects in gp91 and ∼30% to p47. Patients with such impairments harbor cells that are incapable of producing reactive oxygen species (ROS) necessary for normal killing of bacteria and fungi and therefore exhibit an increase in severe infections. Allogeneic HSC transplantation has been a successful treatment option for some patients, with an overall survival rate of >80%, however, patients with the worst outcomes also had active infections upon receiving the transplant. We developed a process that would allow for treatment of active infections using autologous corrected gran/mono transfusions with the hope that this will lead to an improvement in survival post-transplant. Methods, Results & Conclusion A GMP-compliant manufacturing process was established for mRNA transfection of gran/mono by electroporation using the MaxCyte GT system. A total of 5 performance qualifications were completed using the gran/mono enriched RO fraction from elutriated apheresis products from 3 healthy donors and 2 GCSF mobilized CGD patients (gp91- or p47-deficiency). Overnight stored RO fraction cells from healthy donors were transfected with GFP mRNA to validate the performance consistency of the MaxCyte system. GFP expression can be detected at 1hr post-EP. Viability and GFP transfection efficiency were high in final product (∼ 4 hours post transfection)(99±0.6% and 91.9±3.6%, respectively). Patient stored RO fraction cells were successfully transfected with gp91 or p47 mRNA to confirm performance feasibility (74.7% gp91; 92.4±2.4% p47). The purity of gran/mono, and the presence of other cell types (T/B/NK cells) were assessed by flow cytometry and were within acceptable ranges. Gp91/p47 transfected cells were harvested at 3hr-post-EP and placed in functional assays to determine their capability of producing superoxide anion and H2O2. In all cases tested, transfected cells had significantly more DHR activity than untransfected cells, indicating that they regained NADPH oxidase functionality. In summary, the qualification of this process to be used for GMP manufacturing of autologous cells was successful and generated products that passed all release criteria. We anticipate the start of this clinical trial by mid-2020.