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Doxycycline-regulated GDNF Expression Promotes Axonal Regeneration and Functional Recovery in Transected Peripheral Nerve
Rajiv Midha, MD, MSc, FRCS(C)1, Antos Shakhbazau, PhD2 and Chandan Mohanty1
1Department of Clinical Neuroscience and Hotchkiss Brain Institute, University of Calgary, Calgary, AB, Canada, 2Department of Clinical Neuroscience and Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, Calgary, AB, Canada
Background and hypothesis: Increased production of neurotrophic factors (NTFs) is one of the key responses seen following peripheral nerve injury, making them an attractive choice for pro-regenerative gene therapies. However, the downside of over-expression of certain NTFs, including glial cell line-derived neurotrophic factor (GDNF), was earlier found to be the trapping and misdirection of regenerating axons, the so-called 'candy-store' effect. We hypothesized that cell-based GDNF delivery could be regulated using gene therapy approaches and this would result in improve behavioural outcomes for nerve injury repair. Methods: Schwann cells (SCs) were transduced with a lentivirus vector system, which could regulate GDNF expression by external administration of doxycycline. A total of 66 adult male Lewis rats were studied, 22 for early assessment (1 and 3 weeks), while 44 adult male Lewis rats were trained to perform a validated skilled locomotion task (horizontal ladder-rung) before undergoing a right sciatic nerve transection and repair. The rats were allocated to the following groups: sham, non-repair, or injections distal to repair with one of, medium, control SCs or SCs with continuous GDNF stimulation and controlled GDNF stimulation based on the type of surgical repair, cell injection and doxycycline dosing. Rats were serially assessed using functional outcome parameters consisting of ladder-rung, mechanical sensitivity with modified von Frey filament test, thermal sensitivity with Hargreaves plantar test and electrophysiological tests, and at study termination at 11 weeks using immunohistochemistry, GDNF protein assays and target organ muscle weights. Results: Injection of these modified SCs distal to nerve injury repair followed by time-restricted administration of doxycycline demonstrated that GDNF expression in SCs can be controlled locally in the peripheral nerves in the early phase termination studies at 1 and 3 weeks. Continuous GDNF over-production in our model caused regenerating axons to enter and be misdirected within the local clusters of DiI-positive GDNF-producing cells. Cell-based GDNF therapy was shown to increase the overall extent of axonal regeneration, while controlled deactivation of GDNF effectively prevented trapping of regenerating axons in GDNF-enriched areas and was associated with significantly improved sensitivity and locomotor performance. Conclusions: This proof-of-principle experiment demonstrates the successful application of a conditional GDNF expression system in injured peripheral nerve, with local control of cell based GDNF delivery and resulting biological and functional effects.
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