RP1 Dual-AAV Gene Therapy Preserves Retinal Structure and Ameliorates Photoreceptor Degeneration in a Murine Model of Retinitis Pigmentosa.

Purpose: This study aimed to develop and compare dual-adeno-associated virus (AAV) gene therapy utilizing protein trans-splicing versus mRNA trans-splicing to restore RP1 expression, and to evaluate their safety and efficacy in rescuing photoreceptor degeneration in a mouse model of RP1-associated retinitis pigmentosa (RP).

Methods: By screening various split sites within the RP1 sequence in combination with different inteins or adopting mRNA trans-splicing binding sequences, we generated several plasmid pairs capable of reconstituting RP1 in vitro. Next, we conducted dual-AAV gene therapy via subretinal injection in RP1-/- mice. We validated the RP1 protein reconstitution in vivo by Western blotting. To assess the safety and efficacy of dual-AAV systems in RP1-/- mice, retinal structure was evaluated by histological analysis and transmission electron microscopy (TEM), and visual function was assessed by electroretinography (ERG) and visually guided behavioral tests at 1 and 6 months post-treatment.

Results: The protein trans-splicing dual-AAV system achieved 72.35% of wild-type RP1 expression levels, significantly outperforming the mRNA trans-splicing approach (37.84%) at 1 month post-treatment. Both dual-AAV gene therapies preserved photoreceptor morphology and restored retinal function. The protein trans-splicing dual-AAV system exhibited higher ERG amplitudes and better performance in the water maze than the mRNA trans-splicing system.

Conclusions: Our study demonstrates protein trans-splicing mediated dual-AAV gene therapy as a promising strategy for RP1-associated RP, offering potential therapeutic avenue for patients with RP.