Project Summary: 30 lines of text (Currently 28): Spinal cord injuries (SCI) disrupt the communication pathways in the spinal cord, leading to paralysis, primarily affecting the corticospinal tract (CST) responsible for voluntary movements. Unfortunately, damaged CST axons have limited regrowth capacity due to intrinsic and extrinsic factors. Efforts to address intrinsic factors have focused on targeting proteins like PTEN, which inhibits neuronal growth. In addition, there has been a focus on targeting extrinsic molecules such as chondroitin sulfate proteoglycans (CSPGs) through chondroitiase ABC (chABC) digestion, which hinder axon growth. Despite progress in individually targeting these factors, overall growth remains limited, posing a significant challenge. Adeno-associated virus (AAV)- based delivery of growth-promoting molecules has shown promise, but permanent expression can have off- target effects. Our recent findings demonstrated that permanent AAV-driven PTEN deletion resulted in significant functional recovery after SCI, albeit with pathophysiology at 2 months post-SCI. The long-term goal of this project is to develop effective therapeutic strategies for SCI by evaluating controlled PTEN deletion and CSPGs degradation in promoting axon regeneration and functional recovery. The following three specific aims are proposed: 1) Temporal evaluation of PTEN knockdown after SCI without development of pathophysiologies; 2) Determine the role of CST in PTEN-deletion induced recovery of function; 3) Examine the role of CSPGs in growth impairment of PTEN deleted axons. For the first aim, our approach involves regulating PTEN deletion using a regulatory system within our AAVs. This system enables the activation of PTEN deletion in the presence of an inducer drug, enabling us to pinpoint the crucial temporal window for CST regeneration while avoiding the development of pathophysiologies. In the second aim, our approach is to interrupt PTEN-deleted CSTs to assess the extent to which CST regeneration contributes to the observed recovery of function in PTEN-deleted mice after SCI. Lastly, for the third aim, our approach entails testing whether CSPGs are the main impediment for the growth of PTEN-deleted axons by temporally regulating the digestion of CSPGs using chondroitinase ABC (chABC). The proposed studies are innovative as they aim to determine the optimal timing for PTEN deletion to maximize regeneration while avoiding adverse effects, investigate the mechanistic link between PTEN deletion in the CST and functional recovery, and examine the role of CSPGs in inhibiting axonal growth of PTEN-deleted axons. This project is significant as it addresses the critical challenge of limited axon regeneration following spinal cord injury and seeks to identify effective strategies for promoting functional recovery. The completion of the proposed research will establish new knowledge and approaches to enhance axon regeneration and improve outcomes for indivi...