Titer- and Intervention Timing-Dependent Functional Effects of AAV9-NeuroD1 Gene Therapy on Spinal Cord Injury

Spinal cord injury (SCI) often results in varying degrees of motor and sensory dysfunction with limited potential for recovery. Research into in vivo astrocyte-to-neuron reprogramming has led to promising results that, if translated to SCI, could offer substantial therapeutic benefit by replenishing lost populations of neurons for functional restoration. Previous studies have shown that AAV9-mediated delivery of NeuroD1 is capable of reprogramming astrocytes into neurons in vivo after chronic SCI in rats. Here we evaluate the dose-dependent functional and neuroprotective potential of the NeuroD1 gene therapy platform for acute and subacute SCI in rats. The Cre-dependent, DIO-based AAV9-NeuroD1 gene therapy platform was administered directly into the spinal cord of female Long-Evans rats after moderate, thoracic level 8/9 contusion SCI at one of two intervention timepoints: immediately after injury (acute) or 1 week post-contusion (subacute). The viruses were administered at a titer of 10¹¹ or 10¹³ GC/mL. We demonstrate that the AAV9-NeuroD1 reprogramming platform successfully, albeit variably, transduced spinal cells that persisted up to 6 weeks post-injury. However, histological analysis revealed substantial neuronal off-targeting, suggesting a lack of astrocyte-specific targeting from the AAV9 DIO-based delivery platform. Surprisingly, we also found titer- and intervention timing-dependent effects on motor function and tissue preservation-as demonstrated by 1-2 point decrease in BBB scoring and near 50% increase in peak lesion cavitation area-when AAV9-NeuroD1 was administered immediately after SCI. While did not find any effects of the AAV9-NeuroD1 platform on sensory function or neuroinflammatory cell density, a subset of NeuroD1 signal reflected phagocytic uptake by Iba1 and CD68-expressing microglia/macrophages. These results indicate that the NeuroD1 reprogramming platform can exacerbate injury-related functional deficits when administered in the acute stage of injury, but has neuroprotective benefit on tissue preservation when administered in the subacute stage of injury. Our study demonstrates that several factors must be considered, including viral titer and intervention timing, to assess the therapeutic potential of AAV9-NeuroD1-mediated reprogramming for SCI.