Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease

Chronic granulomatous disease (CGD) is a severe inborn error of immunity (IEI) caused by defects in the NADPH oxidase. The best current treatment option for patients with CGD, allogeneic hematopoietic stem cell (HSC) transplantation, is associated with severe adverse effects such as graft-versus-host disease, highlighting a need for improved treatment options based on transplantation of autologous ex vivo gene-edited HSCs. Here, we generate CRISPR/Cas9-based gene editing strategies for the correction of two CGD-causing variants; CYBA c.287+1G>T (p22phox) causing autosomal CGD and CYBB c.252G>A (NOX2) causing X-linked CGD. We find that rAAV6 outperforms ssODNs and IDLVs as HDR repair templates in CD34+ hematopoietic stem and progenitor cells (HSPCs), and we optimize gene editing strategies further by including mRNA-encoded inhibitors/effectors. In addition, we develop a near-universal gene editing strategy for X-CGD by targeted integration of a truncated CYBB cDNA, covering 86% of X-CGD patients, and show functional ROS production in edited cells. We find prevalent off-target editing and chromosomal translocations that severely decreased the ability of gene edited HSPCs to engraft in immunodeficient mice, however, by limiting off-target editing through the use of a high-fidelity Cas9, we can show that we can substantially rescue the multilineage engraftment potential of the gene edited HSPCs. To further improve safety, we finally we develop a novel paired D10A Cas9n gene editing approach targeting CYBB. We demonstrate that using this D10A Cas9 approach, we can retain high on-target efficacy while we do not detect any off-target editing or translocations between on- or off-target cleavage sites. Collectively, we bring new insights into to safety and off-target effects of gene editing approaches targeting CYBA and CYBB, highlighting key challenges of these approaches while offering a potentially curative D10A Cas9n-based treatment option for patients with CGD with improved safety. Overall design: DISCOVER-Seq was carried out as previously described. Briefly, 2x107 K562 cells were nucleofected with either mock, sg3/WT-Cas9 RNPs or sg3/HiFi-Cas9. 12h after nucleofection, the cells were crosslinked in 1% formaldehyde for 15 minutes, washed in PBS and pelleted. The pellets were subsequently resuspended in 10 mL LB1 and incubated for 10 minutes on ice before being spun down at 1000xG for 3 minutes at 4°C. The pellets were then resuspended in 10 mL LB2 and incubated on ice for 5 minutes, before being spun down and resuspended in 1 mL LB3. The nuclear extracts were then sonicated using a Q800 sonicator (Qsonica) (Amplitude: 70%, Pulse Rate: 15s on/45s off, Total Sonication On Time: 40 min, Temperatur: 4oC). Hereafter, the samples were spun down at 17.000xG for 15 minutes to pellet debris. The supernatant was mixed with 1.85 mL LB3 and 150 µL 20% Triton X-100 and mixed with anti-MRE11-bound Dynabeads™ Protein A (Thermo Fisher Scientific). The beads were then incubated, rotating at 4°C for 16 hours, after which the beads were washed 5 times in RIPA buffer and once in TBS before resuspending in 200 µL elution buffer. The crosslink was subsequently reversed by 6 hours of incubation at 65°C. Hereafter, 100 µL TE buffer was added to samples before being treated with RNAse A (Thermo Fisher Scientific) and hereafter Proteinase K (Thermo Fisher Scientific). The DNA was then purified from the samples using the MinElute PCR Purification Kit (Qiagen). The samples were library-prepped using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) and sequenced with 50 million 150 bp PE at the AUH NGS core on a NovaSeqTM 6000. Reads were mapped using BWA and analyzed using BLENDER2 with default filtering options.