The corticospinal tract (CST), a crucial motor pathway, suffers from limited intrinsic axon regeneration capacity after injury, leading to paralysis. Gene therapy, using AAV vectors, offers a potential solution by enhancing regeneration-associated gene expression. AAV serotype and promoter choice are critical factors influencing transgene expression. Previous research showed AAV1's efficacy in transducing layer V corticospinal neurons, but the optimal promoter remained undetermined. This study aims to identify a promoter for strong and selective transgene expression in layer V corticospinal neurons by directly comparing four commonly used promoters: sCAG, hCMV, mPGK, and hSYN. These promoters were chosen for their relatively small size, previous use in nervous system transduction, and the need for strong expression in the large CST. The study contributes to the optimization of AAV-mediated gene transfer to the CST and cortex.

Recombinant AAVs are favored for CNS neuron targeting and have been employed in clinical trials for neurological disorders, showcasing their increasing acceptance as a clinical gene delivery vector. Different AAV serotypes exhibit varying cellular tropism, with transduction efficiency depending on capsid-receptor interactions, cellular uptake, endosomal release, nuclear trafficking, and transgene transcription/translation. AAV1, followed by AAV5, demonstrated the highest efficiency in transducing layer V cortical neurons in rats after direct injection in previous studies. However, while the promoter is a key determinant of transgene expression levels and cellular specificity, a direct comparison of promoters for optimal layer V corticospinal neuron transduction was lacking.

Four AAV1 vectors were created, each expressing enhanced green fluorescent protein (eGFP) under the control of a different promoter (sCAG, hCMV, mPGK, hSYN). The vectors were characterized using quantitative PCR to determine their titres. In vitro, primary cortical neuron cultures from embryonic day 18 Sprague Dawley rats were transfected using magnetofection at 10 days in vitro (DIV) and analyzed at 14 DIV using fluorescence microscopy and SynD software for fluorescence intensity, soma area, and dendritic analysis. In vivo, the titre-matched AAVs (1.42 x 10^12 gc/ml) were stereotactically injected into the sensorimotor cortex of 20 adult female Lister Hooded rats and 20 adult female C5BL/6 mice. After 6 weeks, animals were perfused, and brain tissue was processed for immunohistochemistry using antibodies against NeuN (neuronal marker), GFAP (astrocyte marker), Iba1 (microglia marker), O4 (oligodendrocyte lineage marker), and WFA (perineuronal net marker). Image analysis using ImageJ and custom macros was employed to quantify eGFP expression, cell type specificity (e.g., number of eGFP+ NeuN+ cells), and axon transduction in the dorsolateral corticospinal tract (dCST).

The AAV1 vectors harboring the hCMV and sCAG promoters resulted in transgene expression across neurons, astrocytes, and oligodendrocytes. In contrast, the mPGK and hSYN promoters drove stronger, more cell-type-specific transgene expression. The mPGK promoter directed expression primarily in cortical neurons and oligodendrocytes. Remarkably, the hSYN promoter resulted in neuron-specific expression, including in perineuronal net-expressing interneurons and layer V corticospinal neurons. This suggests that hSYN might be the optimal promoter for targeting specific neuronal populations within the corticospinal tract for gene therapy applications.

The study demonstrates that promoter selection significantly impacts the efficacy and cell-type specificity of AAV-mediated gene delivery to the corticospinal tract. The hSYN promoter's ability to drive strong and neuron-specific expression in layer V corticospinal neurons is particularly valuable for gene therapy strategies aimed at promoting axon regeneration after spinal cord injury. This finding addresses the need for targeted gene delivery in this specific neuronal population. The findings highlight the importance of careful promoter selection for optimizing transgene expression and cell-type specificity in gene therapy applications targeting the CNS.

This study provides a direct comparison of four promoters for AAV1-mediated gene delivery to the corticospinal tract, identifying the hSYN promoter as a promising candidate for achieving neuron-specific transgene expression. Future research could focus on optimizing the hSYN promoter further or exploring other promoters for enhanced specificity and transduction efficiency in different neuronal subtypes within the CST. Investigating the long-term effects of hSYN-driven transgene expression in animal models of spinal cord injury could also provide valuable insights for potential therapeutic applications.

The study focused on a 6-week time point, and longer-term studies are needed to assess the duration of transgene expression. The study primarily used female animals, limiting the generalizability of the findings to male animals. Additional investigations are needed to determine the optimal promoter in other animal models or human cells.