Compound heterozygous recessive or polygenic diseases could be addressed through gene correction of multiple alleles. However, targeting of multiple alleles using genome editors could lead to mixed genotypes and adverse events that amplify during tissue morphogenesis. Here we demonstrate that Cas9-ribonucleoprotein-based genome editors can correct two distinct mutant alleles within a single human cell precisely. Gene-corrected cells in an induced pluripotent stem cell model of Pompe disease expressed the corrected transcript from both corrected alleles, leading to enzymatic cross-correction of diseased cells. Using a quantitative in silico model for the in vivo delivery of genome editors into the developing human infant liver, we identify progenitor targeting, delivery efficiencies, and suppression of imprecise editing outcomes at the on-target site as key design parameters that control the efficacy of various therapeutic strategies. This work establishes that precise gene editing to correct multiple distinct gene variants could be highly efficacious if designed appropriately.

Introduction: Human-induced pluripotent stem cells (iPSCs) represent a promising cell source for the construction of organotypic culture models for chemical toxicity screening and characterization. Materials and Methods: To characterize the effects of chemical exposure on the human neurovasculature, we constructed neurovascular unit (NVU) models consisting of endothelial cells (ECs) and astrocytes (ACs) derived from human-iPSCs, as well as human brain-derived pericytes (PCs). The cells were cocultured on synthetic poly(ethylene glycol) (PEG) hydrogels that guided the self-assembly of capillary-like vascular networks. High-content epifluorescence microscopy evaluated dose-dependent changes to multiple aspects of NVU morphology. Results: Cultured vascular networks underwent quantifiable morphological changes when incubated with vascular disrupting chemicals. The activity of predicted vascular disrupting chemicals from a panel of 38 compounds (U.S. Environmental Protection Agency) was ranked based on morphological features detected in the NVU model. In addition, unique morphological neurovascular disruption signatures were detected per chemical. A comparison of PEG-based NVU and Matrigel Ô-based NVU models found greater sensitivity and consistency in chemical detection by the PEG-based NVU models. Discussion: We suspect that specific morphological changes may be used for discerning adverse outcome pathways initiated by chemical exposure and rapid mechanistic characterization of chemical exposure to neurovascular function. Conclusion: The use of human stem cell-derived vascular tissue and PEG hydrogels in the construction of NVU models leads to rapid detection of adverse chemical effects on neurovascular stability. The use of multiple cell types in coculture elucidates potential mechanisms of action by chemicals applied to the model.

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.

No abstract