Streamlined synthetic regulatory cassette for efficient and photoreceptor-enriched retinal gene expression

Inherited retinal diseases (IRDs) are major causes of vision loss and often associated with the degeneration of retinal neurons, such as photoreceptors. Adeno-associated virus (AAV) vectors hold therapeutic potential for IRDs; however, their off-target expression has prompted the development of refined engineering strategies to enhance cell-type preference without compromising transgene expression. Here, we present G5mP, a streamlined 315-bp synthetic promoter-enhancer construct designed for robust retinal gene expression. G5mP integrates three components: G5, a G protein-coupled receptor kinase 1-derived enhancer; mP, a minimal promoter from phosphodiesterase 6B; and a 5'-untranslated region (UTR) derived from retinoschisin 1 (RS1). Compared with the clinically used 742-bp RIR cassette-comprising the RS1 promoter, interphotoreceptor retinoid-binding protein enhancer, and RS1-derived 5'-UTR-G5mP drove stronger overall retinal expression and showed enhanced activity within photoreceptor cells in retinal cell lines, human retinal organoids, and mouse retina without inducing detectable cytotoxicity in vivo. Notably, across retinal cell lines, human retinal organoids, and mouse retina, G5mP induced more robust and distinct photoreceptor-preferential transgene expression than the ubiquitous CAG promoter did. These results highlight its potential as a compact and efficient regulatory element suitable for AAV-mediated gene delivery across retinal cell types, including the effective targeting of photoreceptors.