41. Therapeutic Level CRISPR-Oligomer-Mediated Correction of X-CGD Patient Hematopoietic Stem Cells Using Non-Viral, cGMP Compliant, Scalable, and Closed System

Gene therapy using integrating viral vectors in hematopoietic stem cells (HSC) has shown clinical benefit in genetic diseases. However, there remain safety concerns associated with random integration and the lack of regulation of gene expression. Efficient and site-specific correction of mutation(s) in HSC using non-viral methods may improve safety and regulation of gene expression. Chronic granulomatous disease (CGD) due to defective phagocyte NADPH oxidase complex and lack of bactericidal superoxide and other reactive oxidative species (ROS) is characterized by severe infections and hyperinflammation. Although the X-linked form of CGD with gp91phox deficiency results from mutations that span the CYBB gene, we identified a ‘hotspot’ mutation at Exon 7 c. 676C>T, causing a premature stop codon in 17 out of 285 patients with X-linked CGD at the NIH. Here we report the result of the efficient correction of the hotspot CYBB mutation using highly efficient CRISPR (Cas9 and sgRNA) system with an oligomer as donor repair template, using MaxCyte's commercially/clinically validated cGMP/regulatory compliant and closed platform technology. Plasmids encoding Cas9 and gRNA were purchased from the Genomic Engineering Center at Washington University (St. Louis, MO). The mRNA encoding Cas9 and gRNA were in vitro transcribed at MaxCyte using mMESSAGE mMACHINE® T7 Ultra kit, (Ambion, Austin, TX). We screened and selected best gRNA from four gRNA candidates for correction and then optimized transfection conditions with EBV-transformed B cell line (B-LCL) derived from an adult patient (P1) with the hotspot CYBB mutation. Transfected B-LCL exhibit 80±6% viability, minimal detectable toxicity as determined by cell proliferation rate referenced to control cells, and efficient site-specific gene correction with 20-50% WT gp91 expression. These developed protocols were used to treat G-CSF and pleraxifor mobilized CD34+ HSC from P1. Following optimization, in vitro treated HSC from P1 achieved 20-30% WT gp91 expression, with >50% viability, and minimal loss of cell proliferation capacity compared with control cells. CD34+ HSCs undergo myeloid differentiation in DMEM supplemented with G-CSF prior to functional evaluation using flow cytometric dihydrorhodamine (DHR) assay, demonstrating ~20% ROS+ cells in treated samples compared to ~80% in normal controls. P1's HSC treated the same way were transplanted into immunodeficient mice, and analyzed 8 weeks later. Bone marrow from mice transplanted with P1 treated cells showed CD45+ human cell engraftment rates at 50-80%, and of the forward/side scatter-gated granulocytes, 11-26% express gp91phox relative to 68% in normal control. Peripheral blood from mice demonstrated 11-23% human CD45+ cells, of which 9-21% expressed gp91phox, compared to 79% in normal controls. Deep sequencing of human CD45+ cells sorted from mouse bone marrow confirmed high rates (up to 21%) of genetic correction from the ‘T’ mutation to the wildtype ‘C’. Since female carriers of X-CGD with ~10-15% normal functioning neutrophils appear to have normal resistance to infections, this level of correction at 10-20% in human CD45+ cells from transplanted mice suggest CRISPR/oligo approach a feasible therapeutic option for treatment of CGD patients with the Ex7, c. 676C>T mutation.