Targeted gene therapies for spinal cord regeneration.

Spinal cord injury presents a debilitating and often insurmountable medical condition characterised by a disruption to axons running through the spinal cord. Due to the poor regenerative capacity of the CNS, this leads frequently leads to loss of motor and sensory function below the level of injury which are never regained. Promoting axon regeneration for functional recovery after spinal cord injury demands innovative solutions.

Even moderate effects could bring about a dramatic improvement. Gaining a deeper understanding of the factors influencing regeneration is crucial, and focusing this research on identifying potential treatments brings us closer to making a tangible difference to patients' lives. Studies targeting the intrinsic growth capacity of CNS neurons have demonstrated that injured axons can be encouraged to grow into and beyond the injury site, but this does not necessarily lead to a recovery of function.

Recent studies have shown that directing regenerating axons to their correct targets using a gradient of chemoattractant can lead to a striking recovery of function.

One molecule, Protrudin (along with its molecular partners), has been identified by Dr. Eva's lab for its role in promoting axon regeneration. Previous studies have identified signalling pathways, genetic factors, the cytoskeleton, synapse maturation events, and deficits in axonal transport as intrinsic mechanisms that can be targeted to promote regeneration.

Protrudin is an integral ER molecule, which causes an increase in ER in the distal part of the axon when overexpressed, crucial for regeneration. The published evidence indicates that Protrudin is a potent driver of axon regeneration, functioning from the axonal ER though mechanisms that have not been completely characterised. Further work with Protrudin has identified other ER-related genes that also drive regeneration, and Golgi-satellites as the organelles contacted by Protrudin in regenerating axons.

Crucially, although potent, protrudin is very small in size (~1.3kb, or ~50kDa) , meaning that in can be packaged into size limited adeno-associated virus (AAV) particles together with other proteins, linked by a self-cleaving peptide. Ongoing research in the Eva lab focuses on developing dual-delivery vectors to optimize the delivery of Protrudin and other therapeutic proteins, thus advancing the prospects of effective treatment strategies.

Concurrently, the Bradbury lab is exploring the therapeutic potential of chondroitinase, a molecule capable of degrading chondroitin sulfate proteoglycans (CSPGs) present in the glial scar post-injury. Glial scars form a physical and biochemical barrier, which can prevent axonal outgrowth and cause aberrant function or death of neurons, interrupting the progression of the inflammatory response from pro-inflammatory to pro-reparative.

By disrupting this inhibitory environment, chondroitinase promotes axonal sprouting and functional recovery in animal models of SCI, offering a complementary approach to Protrudin-based therapies.

Building on these promising studies, we will aim to develop optimised Protrudin-related AAVs, using in vitro regeneration models. Additionally we will aim to produce combined gene therapies for stimulating functional regeneration in the injured spinal cord by combining intrinsic growth stimulators with the extrinsic modifier chondroitinase. It will uncover new axon biology whilst identifying clinically relevant therapies for spinal cord repair.

Aim of the investigation (up to 150 words)

Aim 1: Generate novel dual-delivery Protrudin vectors (Protrudin-Plus AAvs), measure their effects on axon regeneration in vitro, and examine regenerative mechanisms. Aim 2: Validate Protrudin-Plus AAVs in vivo using the optic nerve crush model.

Aim 3: Test Protrudin-Plus, either alone or with Chondroitinase, in a rat model of spinal cord injury, measuring effects on axon regeneration and recov of paw reaching and walking.