# KCNV2-Deficient Retinal Organoid Model of Cone Dystrophy—In Vitro Screening for AAV Gene Replacement Therapy

## TL;DR

Researchers created a human retinal organoid model to test gene therapy for a rare eye disease, showing how in vitro models can reduce animal use in preclinical studies.

## Contribution

A novel KCNV2-deficient retinal organoid model was developed for in vitro AAV gene therapy screening with transgene codon optimization and promoter specificity improvements.

## Key findings

- KCNV2-deficient photoreceptors showed upregulated apoptosis and stress pathways, partially restored by AAV-KCNV2 therapy.
- Codon optimization and the RK promoter improved vector potency and specificity in retinal organoids.
- Single-cell RNA sequencing revealed gene expression changes in transduced retinal organoids.

## Abstract

KCNV2 encodes Kv8.2, an electrically silent voltage-gated potassium channel subunit that is expressed in photoreceptors. Disease-causing variants in KCNV2 cause a monogenic disorder which is classified clinically as cone dystrophy with supernormal rod response (CDSRR). Here, we generated KCNV2-deficient human retinal organoids as a tool for gene therapy vector potency assessment. The organoids were derived from two separate sources: by generating IPSCs from patient blood and by gene editing of a control cell line. Eight KCNV2 gene therapy vectors were assessed in retinal organoids; Kv8.2 protein levels and its in situ interactions with potassium channel binding partners were quantitatively assessed. We show significant enhancements in vector potency and specificity by transgene codon optimisation and the use of the photoreceptor-specific rhodopsin kinase (RK) promoter, respectively. Single-cell RNA sequencing was performed in transduced retinal organoids to assess the performance of the AAV vectors at single-cell resolution. KCNV2-deficient photoreceptors had an upregulation in genes associated with apoptosis, oxidative stress, and hypoxia pathways which were partially restored in AAV-KCNV2 transduced photoreceptors. These data show how human retinal organoids can be used to evaluate AAV gene therapy vector potency in vitro in a physiologically relevant model for the selection of lead therapeutic candidates and to help minimise the use of animals in preclinical development.