AAV-delivered multicopy suppressor tRNA restores visual function in a Leber Congenital Amaurosis mouse model

Abstract: Nonsense mutations that introduce premature termination codons (PTCs) are a major cause of inherited retinal diseases (IRDs), as they prematurely halt protein synthesis and alter gene function. However, therapeutic strategies capable of directly overcoming PTC-induced translational arrest in vivo remain limited. Here, we develop an adeno-associated virus (AAV)-delivered suppressor tRNA (sup-tRNA) therapy to overcome PTC-mediated translational arrest in the rd12 mouse model of Leber Congenital Amaurosis (LCA), which harbors a nonsense mutation in the Rpe65 gene. We first conducted systematic in vitro screening to identify a sup-tRNA variant with high fidelity and efficiency in decoding a UGA PTC, then significantly enhanced the therapeutic efficacy by constructing an expression cassette with four tandem copies (4×) of the sup-tRNA. In the rd12 mouse model, subretinal injection of AAV2/8-4×sup-tRNA successfully restored the full-length expression of the RPE65 protein, validating the copy number-dependent mechanism of the therapy in vivo. The treatment led to significant functional and structural improvements, including the preservation of photoreceptor cells, increased outer nuclear layer thickness, and rescued visual function as demonstrated by ERG and behavioral tests. Furthermore, the safety of this therapeutic approach was confirmed in wild-type mice. This study supports multicopy sup-tRNA delivery as an efficient and potentially broadly applicable strategy for treating IRDs caused by nonsense mutations.