Single Cell Profiling To Define Biomarkers Of Photoreceptor Dysfunction After Gene Editing Within PSC-Derived Organoids

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PROJECT SUMMARY Genome editors make targeted changes in the genome and hold great promise in both basic and translational research. Unfortunately, they often produce unwanted adverse effects, including genotoxicity, immune response, and reductions in cellular function. Therefore, screening for adverse events is essential for the development of safe genome editing therapies. Here we propose to develop a generalizable and scalable approach to define biomarkers for adverse events after delivery of a genome editor. Our strategy combines state-of-the-art, label-free optical metabolic imaging (OMI) to measure the physiological, functional, and high-content morphological status, with single cell transcriptomic profiling (scRNA-seq) and regulatory network-based methods to analyze single cell data. The inferred gene regulatory networks can be used to develop a small (~50) set of biomarkers for adverse events within functional cells. Proof-of-concept studies will focus on the retina, specifically on rod and cone photoreceptors (PR) within 3D optic vesicle (OV) organoids derived from human pluripotent stem cells (PSCs). Creation of this dataset and validation of this approach will leverage these bioengineering technologies toward the development of safer genome editing therapeutics. In Aim 1, we will adapt an existing imaging and culture platform to administer Cas9 genome editors into OVs. Cells will be edited with important PR master regulators and challenged with light and chemical perturbations to test functional phototransduction post genome editing. In Aim 2, we will discover gene regulatory networks and biomarkers associated with abnormal metabolism within normal and dysfunctional gene-edited OVs. We will perform scRNA-seq and OMI on metabolically-distinct, gene-edited OVs, and then map the gene regulatory network associated with adverse events within PRs. We plan to validate the biomarker panel with qPCR/immunocytochemistry (ICC) and electrophysiology. In Aim 3, we will test and refine the platform with novel sgRNAs and genome editors within the SCGE toolkit. And finally, in Aim 4, we will expand the platform to detect adverse events that occur only in cone PRs, which constitute a minority of PRs within the retina, yet are critical for human vision. By tackling a 3D, heterogeneous organoid culture, our approach will extend to more complex cultures. Thus, the impact of this work could be broad, with the potential to advance the development of genome editors administered to any tissue.