Human iPSC modeling elucidates mutation-specific responses to gene therapy in a genotypically diverse dominant maculopathy

Dominantly inherited disorders are not typically considered therapeutic candidates for gene augmentation. Here, we utilized patient-specific induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) to test the potential of gene augmentation to treat Best disease, a dominant macular dystrophy caused by over 200 missense mutations in BEST1. Gene augmentation in iPSC-RPE fully restored BEST1 calcium-activated chloride channel activity and improved rhodopsin degradation in iPSC-RPE models of recessive bestrophinopathy and dominant Best disease caused by two different ion binding domain mutations. A dominant Best disease iPSC-RPE model that did not respond to gene augmentation showed normalization of BEST1 channel activity following CRISPR-Cas9 editing of the mutant allele. We then tested gene editing in all three dominant Best disease iPSC-RPE models, which produced premature stop codons exclusively within the mutant BEST1 alleles. Single-cell profiling demonstrated no adverse perturbation of RPE transcriptional programs in any model, although off-target analysis detected a silent genomic alteration in one model. These results suggest that gene augmentation is a viable first-line approach for some dominant Best disease patients and that non-responders are candidates for alternate approaches such as genome editing. However, testing genome editing strategies for on-target efficiency and off-target events using patient-matched iPSC-RPE model systems is warranted. In summary, personalized iPSC-RPE models can be used to select among a growing list of gene therapy options to maximize safety and efficacy while minimizing time and cost. Similar scenarios likely exist for other genotypically diverse channelopathies, expanding the therapeutic landscape for affected patients. SignificanceDominantly inherited disorders pose distinct challenges for gene therapies, particularly in the face of extreme mutational diversity. We tested whether a broad gene replacement strategy could reverse the cellular phenotype of Best disease, a dominant blinding condition that targets retinal pigment epithelium (RPE). Using RPE generated from patient-specific induced pluripotent stem cells (iPSCs), we show that gene replacement functionally overcomes some, but not all, of the tested mutations. In comparison, all dominant Best disease models tested were phenotypically corrected after mutation-specific genome editing, although one off-target genomic alteration was discovered. Our results support a two-tiered approach to gene therapy for Best disease, guided by safety and efficacy testing in iPSC-RPE models to maximize personal and public health value.