Synonymous editing alters ion channel function, favoring prime editing for retinal disease correction

Area of Science:

• Genetics and Molecular Biology
• Ophthalmology
• Gene Therapy

Background:

• Point mutations in the KCNJ13 gene lead to Leber congenital amaurosis 16 (LCA16), a form of childhood blindness, by impairing Kir7.1 channel function.
• Three patients from two families presented with LCA16 due to a homozygous KCNJ13 missense mutation (c.431T>C, p.Leu144Pro).

Purpose of the Study:

• To investigate the functional consequences of the L144P mutation in KCNJ13 and evaluate different genome editing strategies for potential therapeutic intervention.
• To assess the clinical translatability of non-viral delivery methods for gene editing in retinal diseases.

Main Methods:

• Utilized in silico prediction and in vitro validation with human induced pluripotent stem cell (iPSC)-derived retinal pigmented epithelium (RPE) models.
• Employed adenine base editor (ABE8e) for non-viral delivery and cytosine base editors (CBEs: BE4max, evoCDA) and prime editors for mutation correction in cell models.
• Performed electrophysiological measurements and comprehensive molecular characterization to assess on-target editing efficiency, bystander effects, and functional rescue.

Main Results:

• The L144P mutation was confirmed to impair Kir7.1 channel function, with non-viral delivery proving clinically translatable.
• Cytosine base editors achieved high on-target editing but showed minimal functional rescue due to bystander editing, leading to mRNA structural changes and reduced tRNA abundance.
• Prime editing successfully restored Kir7.1 channel function with approximately 20% on-target editing, avoiding bystander effects and synonymous mutations, and demonstrating a strong genotype-phenotype correlation.

Conclusions:

• Comprehensive functional validation is crucial for assessing genome editing outcomes, as off-target or bystander edits can significantly impact therapeutic efficacy.
• Prime editing shows promise for correcting the KCNJ13 mutation and restoring Kir7.1 channel function, offering a potential therapeutic strategy for LCA16.
• Rigorous preclinical studies are essential for translating genome editing technologies into safe and effective first-in-human trials for genetic disorders.