ABE8e Corrects Pax6-Aniridic Variant in Humanized Mouse ESCs and via LNPs in Ex Vivo Cortical Neurons.

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Aniridia is a rare congenital vision-loss disease caused by heterozygous variants in the PAX6 gene. There is no vision-saving therapy, but one exciting approach is to use CRISPR/Cas9 to permanently correct the causal genomic variants. Preclinical studies to develop such a therapy in animal models face the challenge of showing efficacy when binding human DNA. Thus, we hypothesized that a CRISPR gene therapy can be developed and optimized in humanized mouse embryonic stem cells (ESCs) that will be able to distinguish between an aniridia patient variant and nonvariant chromosome and lay the foundation for human therapy. To answer the challenge of binding human DNA, we proposed the "CRISPR Humanized Minimally Mouse Models" (CHuMMMs) strategy. Thus, we minimally humanized Pax6 exon 9, the location of the most common aniridia variant c.718C > T. We generated and characterized a nonvariant CHuMMMs mouse, and a CHuMMMs cell-based disease model, in which we tested five CRISPR enzymes for therapeutic efficacy. We then delivered the therapy via lipid nanoparticles (LNPs) to alter a second variant in exvivo cortical primary neurons. We successfully established a nonvariant CHuMMMs mouse and three novel CHuMMMs aniridia cell lines. We showed that humanization did not disrupt Pax6 function invivo, as the mouse showed no ocular phenotype. We developed and optimized a CRISPR therapeutic strategy for aniridia in the invitro system, and found that the base editor, ABE8e, had the highest correction of the patient variant at 76.8%. In the exvivo system, the LNP-encapsulated ABE8e ribonucleoprotein (RNP) complex altered the second patient variant and rescued 24.8% Pax6 protein expression. We demonstrated the usefulness of the CHuMMMs approach, and showed the first genomic editing by ABE8e encapsulated as an LNP-RNP. Furthermore, we laid the foundation for translation of the proposed CRISPR therapy to preclinical mouse studies and eventually patients with aniridia.