Point mutations in the KCNJ13 gene cause autosomal recessive childhood blindness, Leber congenital amaurosis (LCA16), by disrupting Kir7.1 channel function.We describe the etiology of the LCA16 retinopathy phenotype in three patients from two unrelated families harboring a homozygous KCNJ13 missense mutation (c.431T>C, p.Leu144Pro).Our in silico prediction and in vitro validation using a human iPSC-derived retinal pigmented epithelium (RPE) model created via lipid nanoparticle-mediated delivery of the adenine base editor (ABE8e) demonstrated that the L144P mutation impairs Kir7.1 channel function and confirmed that non-viral biologic delivery is clinically translatable.We used two cytosine base editors (CBEs, BE4max-WTCas9 and evoCDA-SpCas9-NG) to correct this mutation in an L144P HEK293 stable cell model, achieving high on-target editing efficiency.However, our electrophysiological measurements showed minimal functional rescue of the channel in CBE-edited cells due to bystander nucleotide editing.Editing with evoCDA introduced a bystander missense mutation (L143F), whereas BE4max primarily generated silent mutations.Extended characterization of BE4max-edited cells revealed a distorted mRNA structure, altered half-life, and reduced abundance of cognate tRNA, all associated with these silent changes.In contrast, prime editing successfully restored channel function.Prime editors targeting the L144P locus achieved approximately 20% on-target editing without introducing bystander nucleotide editing or synonymous changes.Functional assessment demonstrated a strong genotype-phenotype correlation, with restored Kir7.1 channel activity observed in 28% of edited cells (12/43).Overall, these results highlight the importance of comprehensive functional validation of genome editing outcomes and emphasize the need for rigorous preclinical studies to translate therapeutic genome editing into first-in-human trials for genetically diverse diseases.
Kabra et al. (Thu,) studied this question.