AAV9-Mediated Targeting of Defined Neuronal Populations in Spinal Cord Through Intrathecal Injection

Background: Adeno-associated viruses (AAVs) are established vectors for efficient gene delivery to the central nervous system (CNS). Increasingly, strategies aim to restrict transduction to specific neuronal subtypes defined by the associated functional properties, thereby enhancing precision and therapeutic potential. Methods: Recombinant AAV9 vectors carrying fluorescent reporters under the control of cytomegalovirus (CMV), human synapsin (hSyn), or homeobox 9 (Hb9) promoters were delivered intrathecally in Wistar rats. Transgene expression was evaluated 7 days post-injection by confocal microscopy. Neurons in laminae VII–X were quantified across cervical, thoracic, lumbar, and sacral spinal cord levels. Statistical analysis was performed using the Kruskal–Wallis test followed by Mann–Whitney U tests with Bonferroni correction. Results: In lamina VII, consistent neuronal expression was mediated by hSyn across all spinal levels, with significantly higher transduction at cervical compared to thoracic and lumbar regions (p < 0.01). CMV and Hb9 showed no detectable tropism for this lamina. In lamina VIII, CMV drove markedly higher expression than hSyn and Hb9, with a 2.8-fold difference at the lumbar level (p < 0.001). In lamina X, CMV expression exceeded hSyn at the lumbar and sacral levels (p < 0.05), while Hb9 showed no activity. In lamina IX, all promoters mediated motoneuron transduction, but only Hb9 restricted expression specifically to motoneuron somata. Notably, CMV induced off-target expression in glial cells. Conclusions: AAV9-mediated expression patterns in the spinal cord are strongly shaped by promoter choice and segmental level. Hb9 provides high motoneuron specificity, hSyn supports broad neuronal activation across laminae VII–X, whereas CMV drives robust but non-specific expression with significant off-target activity. These findings highlight the importance of rational promoter selection for spinal cord gene therapy and strategies aimed at functional recovery in motor system disorders.