Comparative analysis of cell-specific promoters in AAV9-mediated gene therapy targeting the central nervous system.

We present a comprehensive toolkit of ubiquitous and cell-specific promoters for potential use in adeno-associated virus (AAV)9-mediated gene therapy for central nervous system (CNS) disorders, systematically evaluating biodistribution, cellular specificity, and peripheral targeting. While ubiquitous promoters, such as the cytomegalovirus early enhancer/chicken beta-actin (CAG) promoter, drive widespread expression, cell-specific promoters offer greater targeting precision. We introduce a novel, astrocyte-specific, truncated glial fibrillary acidic protein (GFAP) promoter, named gfaABCD1405 (gfa1405), which enhances astrocyte specificity while reducing size, improving utility for gene therapies requiring larger transgenes. gfa1405 demonstrated broader CNS expression than gfaABC(1)D, addressing previous limitations in AAV-mediated applications. The methyl CpG binding protein 2 promoter (p546) effectively targeted neurons, with strong expression in the neocortex and hippocampus, making it a promising candidate for neuronal disorders. Oligodendrocyte-specific promoters exhibited distinct patterns, with the myelin-associated glycoprotein (MAG) promoter driving expression in the corpus callosum, while the 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) promoter showed broader transduction, suggesting wider therapeutic applicability. This study establishes a promoter selection framework for AAV9-based gene therapy, enabling variable CNS cell-type targeting and optimizing therapeutic efficacy. By integrating comparative promoter analysis with gfa1405 development, we provide a practical collection for CNS-directed gene therapy, minimizing off-target effects and advancing treatment strategies for neurological disorders.