DEVELOPMENT OF A GMP MANUFACTURING PROCESS FOR BASE EDITED HEMATOPOIETIC STEM AND PROGENITOR CELLS (BE-HSPCS) FOR X-LINKED CHRONIC GRANULOMATOUS DISEASE (X-CGD)

Background & AimMutations in CYBB gene disrupt gp91phox expression causing X-CGD, an inherited immune deficiency disorder of granulocytes with impaired NADPH oxidase activity for pathogen killing. We address a common missense mutation that accounts for ∼6% of our clinical X-CGD patient cohort. We developed a process to precisely correct the mutation at genome level in autologous HSPCs using pioneering CRISPR base-editing technology for ex vivo gene therapy.Methods, Results & ConclusionA total of 3 performance qualifications were completed using the post-thawed CD34+ cells enriched from mobilized apheresis products from 2 healthy donors (to knock down gp91phox expression) and 1 patient (to restore gp91phox expression). Thawed CD34+ cells were pre-stimulated for up to 48 hours before electroporation with Adenosine base editor (ABE) and single guide RNA using MaxCyte GT device. Final products (∼48hr post-EP) were cryopreserved after submitting FN assays (cell count, viability, purity, sterility, mycoplasma, endotoxin, base editing rate by deep sequencing, gp91 expression and DHR function in granulocytes differentiated in vitro from final product by flow cytometry). Cells electroporated at varying concentrations, 50-200x106/mL exhibited similar viability, purity and fold expansion in healthy donor final products. We observed slightly lower viability and purity in the patient sample which remained well within the range of acceptability (≥70%). Base editing data from HD cells showed high performance consistency when using 50-200x106cells/mL for electroporation (31.3-34.49%), and robust base editing rate with the larger CL1.1 processing assembly (52.42%). Base editing rate using the mutation-specific base editor/sgRNA was 64.97% correction, which demonstrated the process feasibility. Functionally, we observed in myeloid-differentiated CD34+ successful disruption of gp91 expression (74.8% to 32.7%) and DHR function (74.6% to 28.1%) in HD naïve compared to BE-products. In patient cells, restoration of gp91 expression (0.47% to 61.9%) and DHR function (from 1.6% to 49.3%) was observed in naïve compared with BE-CGD products. Furthermore, cryopreservation did not demonstrate any detrimental effects on final product viability, purity and potency. In summary, the process for GMP manufacturing of base-editing autologous HSPC was successfully validated and generated products that passed all release criteria. We anticipate the start of 1st approved base editing clinical trial (IND 28925) for XCGD by Mid-2024.