RAAV-MEDIATED GENE SUPPLEMENTATION APPROACH ADDRESSING A <EM >CACNA1F</EM> CSNB2 TRUNCATION VARIANT IN MICE

The L-type calcium channel truncation mutation Ca_V1.4-R1827X (RX) disrupts the distal C-terminal regulatory domain, altering channel function and expected to compromise sustained calcium entry at rod ribbon synapses. This variant causes incomplete congenital stationary night blindness type 2 (CSNB2) in humans. Prior work in a heterologous expression system showed that co-expression of the missing C-terminal peptide rescued truncated channels to wild-type function. Here, we establish preclinical proof-of-concept for gene supplementation therapy by delivering a complementary peptide to rod photoreceptors. We generated a rhodopsin promoter-driven tdTomato-peptide construct and investigated photoreceptor targeting and subcellular localization immunohistochemically following subretinal delivery in C57BL/6J and RX mice. The construct drove robust rod-restricted expression confined to the photoreceptor containing layers. Importantly, tdTomato-peptide displayed enrichment beneath the ribbon in the outer plexiform layer in RX retinas, whereas wildtype retinas lacked this enrichment, suggesting specific localization to synaptic sites where the therapeutic peptide is needed.
We also compared delivery routes in wild-type mice: subretinal injection achieved high transduction confined to the injection bleb, while intravitreal injection produced retina-wide distribution but lower transduction efficiency.
These findings demonstrate rod-specific expression and appropriate subcellular localization of the therapeutic peptide at ribbon synapses in mutant photoreceptors, supporting the feasibility of gene supplementation therapy for this truncation mutation. Ongoing work focuses on optimizing the transduction efficiency to enable functional and behavioral rescue studies.