Combining new gene therapy with non-invasive spinal roots stimulation to improve synaptic plasticity at spino-muscular circuitry after spinal cord injury

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Impaired ability of voluntary walking and bladder dysfunction is an acute problem among veterans with spinal cord injuries (SCI). Results of recent studies, including our own, revealed that there are at least three major factors known to limit recovery from SCI: (1) decreased neuronal excitability, (2) presence of axonal growth/regeneration inhibitors, and (3) lack of neurotrophin support. Using animal models, we have recently demonstrated that degradation of scar-related inhibitory Chondroitin Sulfate Proteoglycans (CSPGs) with the enzyme Chondroitinase-ABC (ChABC), combined with AAV-based delivery of neurotrophin NT3, induced partial improvements following mild contusion SCI. A potential disadvantage to the use of ChABC is that it is not specific, i.e. degrades all CSPGs, including those that are important components of the extracellular matrix. In our search for more specific targets, we have recently demonstrated that one CSPG molecule, NG2, known as a major obstacle to axonal regeneration following brain and spinal cord injury, blocks axonal conduction, but other CSPGs tested did not. Acute administration of monoclonal NG2 function neutralizing antibody (NG2-Ab; designed initially to prevent inhibitory effects of NG2 on axonal growth) prevents the conduction block induced by acute injections of NG2 into the spinal cord. Intrathecal infusion of NG2-Ab, via osmotic mini-pump for 2 weeks, however, induced only limited and transient improvements of motor function following SCI. In an attempt to design an approach for safe, prolonged and clinically feasible delivery of NG2- Ab, we have successfully created a new AAV-10 vector-based gene therapy tool for prolonged and clinically- relevant delivery of a recombinant single chain variable fragment (scFv) anti-NG2 antibody: AAV-NG2Ab. Results of preliminary experiments revealed that combined administration of AAV-NG2Ab and AAV-NT3 induced greater improvements, compared to ChABC/AAV-NT3, following mild (150 kDyn) contusions. Effects of this novel gene therapy (AAV-NG2Ab/AAV-NT3) tool on motor recovery were, however, still limited in rats with mild contusion and less obvious in rats with severe contusion SCI. In attempts to further improve the beneficial effects of AAV-NG2Ab/AAV-NT3 and expand improvements to severe SCI models, we now propose to add a third treatment component, i.e. non-invasive repetitive electro- magnetic stimulation over spinal vertebrae (rSEMS). We recently found that rSEMS strengthens transmission and improves function of NMDA receptor at motoneuron synaptic inputs, which is required to initiate effects of NT-3 at these inputs. Thus, in the proposed project we have designed a new additive treatment comprised of AAV10-NG2Ab, AAV10-NT3 and rSEMS. In addition to a mild contusion model of injury, we propose to use severe mid-thoracic contusions which are known to induce major deficits of motor function and bladder activity in rat and human SCI. An important and novel aspect of this research is evaluation of the proposed novel gene therapy (AAV-NG2Ab/AAV-NT3) combined with rSEMS on bladder function. To evaluate the efficacy of these treatments, we will conduct a multidisciplinary examination, including in vivo physiology, anatomy, immunochemistry and behavior. We will examine the effects (additive or synergistic) of the new therapeutic treatment on (1) strengthening synaptic connections through the injury epicenter to lumbar motoneurons, and then to hindlimb muscles (using in-vivo electrophysiology); (2) anatomical plasticity of fibers accounting for the persistence of the synaptic response after exposure to this novel treatment (using anatomical tracing and confocal microscopy); (3) recovery of locomotor performance (using automated Catwalk gait analyses); (4) recovery of bladder activity (using metabolic chamber and cystometry/ electrophysiology). To better understand the effects of treatment at the cellular level, we will study axon remyelination (using Electron Microscopy) and NG2- positive processes contacting nodes of Ranvier. Preliminary results of on-going experiments show improvements of motor function in rats that have received this novel additive treatment after severe mid-thoracic contusive SCI.