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.

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 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.