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

> **NIH NIH U01** · UNIVERSITY OF WISCONSIN-MADISON · 2020 · $611,987

## Abstract

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.

## Key facts

- **NIH application ID:** 9970475
- **Project number:** 8U01EY032333-03
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** David M Gamm
- **Activity code:** U01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $611,987
- **Award type:** 8
- **Project period:** 2018-09-05 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9970475

## Citation

> US National Institutes of Health, RePORTER application 9970475, Single Cell Profiling To Define Biomarkers Of Photoreceptor Dysfunction After Gene Editing Within PSC-Derived Organoids (8U01EY032333-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9970475. Licensed CC0.

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