Widespread AAV-mediated CSPG digestion impairs functional recovery after cervical spinal cord injury

Spinal cord injury (SCI) disrupts long-distance communication between the brain and spinal circuits, resulting in persistent motor, sensory and autonomic dysfunction¹. These deficits arise from the limited regenerative capacity of adult central nervous system (CNS) neurons and the presence of a growth-inhibitory extracellular environment. Among pathways that control voluntary movement, failure of corticospinal tract (CST) regeneration is a major contributor to impaired motor function. Strategies to promote regeneration have focused on enhancing the intrinsic growth capacity of injured neurons, such as through activation of the mTOR pathway via phosphatase and tensin homolog (PTEN) suppression, as well as reducing extrinsic inhibition through enzymatic digestion of chondroitin sulfate proteoglycans (CSPGs) using chondroitinase ABC (chABC). Because these mechanisms act through distinct but complementary processes, we investigated their combination as a strategy to improve regeneration. Adeno-associated viral (AAV) vectors are widely used to enhance intrinsic growth pathways and represent a clinically relevant platform for gene delivery. In contrast, CSPG digestion has primarily been achieved using lentiviral or focal delivery approaches. We therefore examined whether reducing extrinsic inhibition could be implemented using AAV2-mediated chABC delivery, alone and in combination with AAV2-retro-mediated PTEN knockdown, following cervical SCI. AAV2-mediated delivery of chABC produced robust and persistent CSPG digestion that extended beyond the injection site, and this spatial extent was influenced by viral dose and expression magnitude. Despite effective CSPG degradation, AAV2-chABC treatment did not improve functional outcomes relative to controls and did not enhance the effects of intrinsic growth activation via PTEN knockdown. Instead, AAV2-chABC treatment, alone or in combination with AAV2-retro-mediated PTEN knockdown, was associated with impaired motor performance in behavioral assays. These findings indicate that the extent and persistence of CSPG degradation critically shape functional outcomes after SCI and that simultaneous enhancement of intrinsic growth capacity and extracellular permissiveness does not necessarily translate into improved functional recovery. Together, these results underscore the importance of carefully controlling transgene expression levels and duration in AAV-based gene therapies, where suboptimal delivery parameters may offset the benefits of otherwise promising targets.