This subproject will test the feasibility and efficacy of gene therapy for promoting neuronal survival and regeneration in the injured adult rat spinal cord either alone or in combination with a cellular transplantation intervention. Recombinant adeno-associated virus (rAAV) constructs will be employed to determine whether: (i) the efficacy of in vivo gene delivery to neurons can vary relative to post-injury times and the probability of either retrograde cell death or atrophy; (ii) in vivo gene delivery can promote rescue of embryonic and adult neuronal populations susceptible to post-axotomy cell death; and (iii) an effective delivery and expression of transgenes can be used to enhance axonal outgrowth from intraspinal grafts of fetal raphe tissue. Thus, the central hypothesis to be addressed is that either ex vivo and/or in vivo gene delivery can foster the synthesis and release of neurotrophic actors that in turn can serve to promote sparing of neuronal populations and stimulate or enhance axonal regeneration /sprouting following spinal cord injury (SCI) or peripheral nerve damage. Using sciatic nerve lumbar neuron pools (L4-L6) as a model, the efficacy of gene delivery will be compared at acute and chronic post- injury periods in Aim I. The selected post-injury times will relate to different epochs associated with initial retrograde (e.g., chromatolytic) responses and subsequent induced atrophy. Likewise, other experiments will examine the efficacy of gene delivery in two other intrinsic neuronal populations, rubrospinal and dorsal nucleus of Clarke, with contrasting cell survivals after SCI.
Aim II will determine whether a model population of neurons (i.e., motoneurons) can be rescued in both the adult spinal cord and grafts of fetal spinal cord (FSC) tissue by in vivo delivery of rAAVs expressing GDNF. The ability of transgene expression to rescue DNC neurons also will be examined. Lastly, Aim III will test the hypothesis that in vivo gene delivery, involving the expression of neurotrophins in conjunction with intraspinal grafts of fetal CNS tissue, can be used to augment host-graft connectivity by enhancing donor neuron-derived axonal sprouting and/or elongation. These experiments will investigate (i) whether the pattern of outgrowth exhibits any preferential disbursement relative to the distribution of transduced cells and (ii) to what extent the presence of NT-3 can promote growth of axons through a cellular milieu (e.g., astrogliotic scar) that can be inhibitory or non permissive to axonal regrowth. This subproject builds on a base of established expertise in SCI neurobiology/neuropathology, neurotransplantation, and AAV development to investigate fundamental issues related to gene transfer and repair of the damaged spinal cord.
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