Human Cell Assay Core

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PROJECT SUMMARY/ABSTRACT – HUMAN CELL ASSAYS CORE The overarching goal of this Core is to support the preclinical and IND-enabling studies in human cells with the therapeutic leads generated in Projects 1-3. Human pluripotent stem cells (hPSCs) are a unique cell source to generate retinal cells, tissue, and organoids. The unique attributes of hPSC model systems are particularly critical for diseases that have high genotypic diversity, like channelopathies, as generating an animal model with a knocked-in human allele for every patient mutation would be time and cost prohibitive and – even if accomplished – would not provide a platform to assess potential off-target effects. Assays using hPSC-derived cells, organoids, and tissues containing pathogenic mutations can provide patient-relevant information regarding (1) the efficiency of intended on-target editing of mutant alleles, (2) the frequency of unintended on-target genomic editing outcomes (e.g., large deletions, translocations) and off-target genomic editing (in both the target cell type and other cell types exposed to the therapeutic product), and (3) the strength of functional rescue and adverse responses. Because our proposed subretinal route of nonviral editor administration will likely deliver payload to photoreceptors (PRs) as a cellular off-target, we developed an imaging and single cell transcriptional pipeline to identify biomarkers of adverse events in PR-containing hPSC-derived retinal organoids (ROs), pioneered by our team. We will apply these tools to profile potential adverse events in PRs, including changes in phototransduction, p53 response, and innate immune response. We plan to produce iPSC-RPE suitable for on- and off-target analysis of therapeutic editors, perform electrophysiology assays for functional analysis of iPSC-RPE treated with therapeutic candidates, and produce hPSC-ROs for profiling potential adverse events in human photoreceptors exposed to therapeutic candidates. The expected outcomes of HCA Core activities include continuous provision or execution of disease-relevant, rigorous, and reproducible human cell products and ion channel assays. We expect to leverage a world-class stem cell biology and bioengineering community at the University of Wisconsin-Madison to pave the way to use hPSCs in IND-enabling studies in the genome editing field. If successful, this effort could reduce the need for preclinical animal models and provide relevant safety and efficacy information to accelerate the translation of genome editing into first-in- human trials.