# DEVELOPMENT OF AAV-CRISPR/CAS9-BASED THERAPIES FOR CONE ROD DYSTROPHY

> **NIH NIH R01** · UNIVERSITY OF FLORIDA · 2022 · $521,101

## Abstract

ABSTRACT
Mutations in GUCY2D, the gene encoding retinal guanylate cyclase-1 (retGC1), are the leading cause of
autosomal dominant cone-rod dystrophy. GUCY2D-CORD6 patients present with loss of visual acuity, abnormal
color vision, photophobia, visual field loss and macular atrophy within the first decade. Rod degeneration and
peripheral visual field loss follow. Significant progress towards clinical application of gene replacement therapy
for LCA due to recessive mutations in GUCY2D (LCA1) has been made, but a different approach is needed to
treat CORD6 where gain of function mutations cause dysfunction and dystrophy. Our preliminary data show that
1) selective and efficient somatic knock-out of GUCY2D and Gucy2e (murine homologue) with AAV-
CRISPR/Cas9 results in a subsequent loss of retinal structure/function that manifests from reduced retGC1
expression in macaque and mouse, respectively, 2) a ‘knock-out + complementation in trans’ approach (wherein
complementation is performed with ‘hardened’ Gucy2e not recognized by Gucy2e gRNA) preserves retinal
function in mice, 3) AAV-CRISPR/Cas9- based editing of GUCY2D is therapeutic in a R838S transgenic (Tg)
mouse model of CORD6, and 4) Cas9 variants identified by directed evolution exhibit allele specificity for
GUCY2D(R838S). We will build upon these results in the following Aims. Aim 1 will establish the optimal
parameters for AAV-CRISPR/Cas9-based gene editing in two R838S CORD6 Tg mouse lines. We will establish
the optimal AAV capsid/dose, durability of therapy, treatment window, and feasibility of transient Cas9 expression
systems. Aim 2 will evaluate safety/efficacy of AAV-CRISPR/Cas9-based gene editing in macaque by looking
for off-target editing and assessing the potential impact of AAV vector insertions and long-term Cas9 expression.
We will also evaluate regional differences in editing efficiency and conduct dose-ranging studies. Aim 3 will
compare ‘knock out + complementation in trans’ vs. ‘allele-targeted’ approaches for treating CORD6. The optimal
AAV capsids and Cas9 expression system from Aim 1 will be used to test KO + complementation in GC2-/- mice.
Allele-targeted editing will be performed in humanized R838S CORD6 mice that carry both the wt and R838S-
containing exon 13 of GUCY2D. Approaches will include a novel, engineered Cas9 that specifically edits the
R838S mutant allele, gRNAs containing mismatches to mutant but not wt allele, or utilization of an alternative
PAM site found in the mutant, but not wt allele. Successful approaches will be tested in macaque for
efficiency/safety. Our findings will identify the optimal capsid/dose, and treatment age for therapeutic AAV-
CRISPR/Cas9-based disruption of R838S GUCY2D in vivo. We will establish the safety profile, and regional
efficiencies of gene editing by AAV-CRISPR/Cas9 in a species with both genomic and clinical relevance. In
addition, we will identify materials and approaches that will allow clinical application of AAV-CRISPR/C...

## Key facts

- **NIH application ID:** 10412033
- **Project number:** 5R01EY028968-04
- **Recipient organization:** UNIVERSITY OF FLORIDA
- **Principal Investigator:** Shannon Elizabeth Boye
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $521,101
- **Award type:** 5
- **Project period:** 2019-06-01 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10412033

## Citation

> US National Institutes of Health, RePORTER application 10412033, DEVELOPMENT OF AAV-CRISPR/CAS9-BASED THERAPIES FOR CONE ROD DYSTROPHY (5R01EY028968-04). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10412033. Licensed CC0.

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