Human iPSC Modeling Reveals Mutation-Specific Responses to Gene Therapy in a Genotypically Diverse Dominant Maculopathy.

Dominantly inherited disorders are not typically considered to be therapeutic candidates for gene augmentation. Here, we utilized 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 an iPSC-RPE model of recessive bestrophinopathy as well as in two models of dominant Best disease caused by different mutations in regions encoding ion-binding domains. A third dominant Best disease iPSC-RPE model did not respond to gene augmentation, but showed normalization of BEST1 channel activity following CRISPR-Cas9 editing of the mutant allele. We then subjected all three dominant Best disease iPSC-RPE models to gene editing, which produced premature stop codons specifically within the mutant BEST1 alleles. Single-cell profiling demonstrated no adverse perturbation of retinal pigment epithelium (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 individuals with dominant Best disease and that non-responders are candidates for alternate approaches such as gene editing. However, testing gene editing strategies for on-target efficiency and off-target events using personalized 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 individuals.

Developing precision medicine using scarless genome editing of human pluripotent stem cells.

Many avenues exist for human pluripotent stem cells (hPSCs) to impact medical care, but they may have their greatest impact on the development of precision medicine. Recent advances in genome editing and stem cell technology have enabled construction of clinically-relevant, genotype-specific "disease-in-a-dish" models. In this review, we outline the use of genome-edited hPSCs in precision disease modeling and drug screening as well as describe methodological advances in scarless genome editing. Scarless genome-editing approaches are attractive for genotype-specific disease modeling as only the intended DNA base-pair edits are incorporated without additional genomic modification. Emerging evidentiary standards for development and approval of precision therapies are likely to increase application of disease models derived from genome-edited hPSCs.

Scarless Genome Editing of Human Pluripotent Stem Cells via Transient Puromycin Selection.

Genome-edited human pluripotent stem cells (hPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. We present and characterize a robust method for rapid, scarless introduction or correction of disease-associated variants in hPSCs using CRISPR/Cas9. Utilizing non-integrated plasmid vectors that express a puromycin N-acetyl-transferase (PAC) gene, whose expression and translation is linked to that of Cas9, we transiently select for cells based on their early levels of Cas9 protein. Under optimized conditions, co-delivery with single-stranded donor DNA enabled isolation of clonal cell populations containing both heterozygous and homozygous precise genome edits in as little as 2 weeks without requiring cell sorting or high-throughput sequencing. Edited cells isolated using this method did not contain any detectable off-target mutations and displayed expected functional phenotypes after directed differentiation. We apply the approach to a variety of genomic loci in five hPSC lines cultured using both feeder and feeder-free conditions.