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

<i>KCNV2</i> encodes Kv8.2, an electrically silent voltage-gated potassium channel subunit that is expressed in photoreceptors. Disease-causing variants in <i>KCNV2</i> cause a monogenic disorder which is classified clinically as cone dystrophy with supernormal rod response (CDSRR). Here, we generated <i>KCNV2</i>-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 <i>KCNV2</i> 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. <i>KCNV2</i>-deficient photoreceptors had an upregulation in genes associated with apoptosis, oxidative stress, and hypoxia pathways which were partially restored in AAV-<i>KCNV2</i> 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.