Fig. 1: A combined in vitro and in silico strategy to evaluate the efficacy of different gene correction therapeutic strategies.

a Schematic indicating the modeling approach in which samples from patients are collected ex vivo are then genome-edited in vitro. Genotypes and phenotype outcomes from the in vitro studies are inputs for an in silico model that simulates the delivery of the therapeutic in vivo as well as tissue morphogenesis (GETEM model, Fig. 5). The results of the in silico model can ultimately guide dosing and formulation decisions. b Editing strategy for gene correction of Pompe-diseased induced pluripotent stem cells (iPSCs). Pompe disease is caused by two defective copies of the acid-α-glucosidase (GAA) gene. This enzyme is responsible for breakdown of glycogen within lysosomes inside cells. Without GAA, glycogen build up can cause downstream health issues. After correction, GAA expresses a functional protein leading to a reduction in glycogen. The schematic indicates the editing locations within GAA locus and CRISPR gene correction strategy. In the Pompe patient-derived line, cells harbor compound heterozygous mutations in GAA. Allele one, a1, contains a point mutation that causes a premature stop codon (GAA:c.[1441=2237G>A]) while allele two, a2, carries a one base pair deletion (GAA:c.[1441delT;2237=]). For the CRISPR gene correction strategy, single guide RNAs (sgRNAs; the predicted DNA double-strand break by SpyCas9 is denoted by the arrowhead) were designed to be specific to only the diseased allele by containing the mutant bases (red) within the seed region. Single-stranded oligonucleotides (ssODNs) used for genomic repair contained the wildtype sequence at the mutation site (blue) as well as a silent mutation “wobble” to remove the PAM site (green) to prevent re-cutting of the corrected allele while preserving the amino acid sequence of GAA.