P754: prime editing as a novel tool for precise correction of causal mutations in fanconi anaemia group a patient-derived cells

Topic: 11. Bone marrow failure syndromes incl. PNH - Biology & Translational Research Background: Fanconi anaemia (FA) is a rare genetic disorder caused by mutations in genes involved in the repair of DNA inter-strand crosslinks. The most common of FA complementation groups is FANC-A in which the FANCA gene is mutated. Most FA patients develop bone marrow failure already during childhood and require allogeneic haematopoietic stem and progenitor cell (HSPC) transplantation. This treatment necessitates an available donor and can be associated with severe side effects. Correction of patient’s own HSPCs ex vivo with the use of genome-editing technologies followed by infusion of the corrected cells into patient holds great potential for the treatment of FA patients, as neither allogeneic HSPCs donor nor intensive conditioning treatment is needed. Prime editing is an advanced genome-editing technology capable of precise correction of mutations even in the absence of homology-directed repair (HDR) and thus could potentially be used in FA patient cells in which the HDR activity is considerably reduced. Aims: We set out to utilize prime editing for the correction of causal mutations found in FA patients in the Czech Republic. We focused on two compound heterozygote FANC-A patients (patient 1: FANCA c.1A>G and c.4010 + 1-c.4010 + 18del; patient 2: FANCA c.1A>G and c. 3788_3790delTCT), specifically on their shared c.1A>G mutation. Methods: We designed and prepared the pegRNAs carrying the targeting sequence and also a correcting template specific for c.1A>G and introduced them along with prime editor and nicking sgRNAs into primary fibroblasts derived from both FA patients by nucleofection. Different forms of prime editor and pegRNA (plasmid or mRNA delivery, engineered pegRNAs) were tested. Efficiency of prime editing was analysed by next generation sequencing, recovery of production of full-length FANCA protein was detected by immunoblotting. Results: We obtained on average 15% of correct editing in fibroblasts from patient 1 and 11.5% in fibroblasts from patient 2 with imprecise editing/indels under 3%. Restoration of full-length FANCA protein expression was detected in edited samples. We also observed an increase in the amount of corrected DNA over time in culture in agreement with the hypothesized functional restoration of FA pathway and associated proliferation advantage of corrected cells. Summary/Conclusion: Our results show that prime editing can be successfully used for the correction of FANCA mutations in FA patient-derived cells. The correction was reproducible and efficient in cells obtained from two unrelated FA patients. Experiments aiming at further increase of prime editing efficiency in FA cells (e.g. recently published upgrades of prime-editing technology) and also at the transfer of the editing strategy into HSPCs are ongoing. Supported by KOG-202108-0090, LX22NPO5102, and UNCE/MED/015. Keywords: Fanconi anemia, FANCA, Gene therapy