Design of efficacious somatic cell genome editing strategies for recessive and polygenic diseases

Abstract

Gene correction of multiple alleles for compound heterozygous recessive or polygenic diseases is a promising therapeutic strategy. However, the targeting of multiple alleles using genome editors in a single cell could lead to mixed genotypes and adverse events that amplify during tissue morphogenesis. Here we demonstrate that SpyCas9-based S1mplex genome editors can be designed and developed to correct two distinct mutant alleles within a single human cell precisely. Gene-corrected cells in a patient-derived, induced pluripotent stem cell (iPSC) model of Pompe disease robustly expressed the corrected transcript from both corrected alleles. The translated protein from the gene-corrected cells was properly processed after translation and was able to enzymatically cross-correct diseased cells at levels equivalent to standard-of-care, enzyme replacement therapy (ERT). Using a novel in silico model for the in vivo delivery of these and many other genome editors into a developing liver of a human infant, we identify progenitor cell targeting, delivery efficiencies, and suppression of imprecise editing outcomes at the on-target site as key design parameters controlling the potency and efficacy of in vivo somatic cell genome editing. Both single and double gene correction are efficacious for in vivo somatic cell editing strategies, while double gene correction is more effective than single-gene correction for autologous cell therapy with ex vivo gene-corrected cells. This work establishes that precise gene correction using genome editors to correct multiple distinct gene variants could be efficacious in the treatment of recessive and polygenic disorders.