In Vivo Gene Therapy For Sciatic Nerve Injury Of Adult Rats By Adenovirus-mediated Gene Transfer Of BDNF

Despite recent advances in microsurgical techniques and the understanding of nerve regeneration, functional recovery following repair of transected peripheral nerves often remains poor. The clinical results of peripheral nerve repair remain disappointing, with loss of muscle function, unpaired sensation and/or painful neuropathies. The poor functional outcome after peripheral nerve repair has motivated a better understanding of the molecular and cellular events surrounding nerve regenerationAfter axotomy and during peripheral nerve regeneration, the neurotrophins show a well beneficial effect on the survival and phenotypic expression of primary sensory neurons in dorsal root ganglia and of motoneurons in spinal cord. The role of neurotrophic factors in the maintenance and survival of peripheral neuronal cells and in promotion of nerve regeneration has been the subject of numerous studies. The field of neurotrophic factor pharmacology emerged during the past decade with the discovery that these proteins can counteract neuronal atrophy and death in the adult nervous system. These concepts are being tested in clinical trials. Therapeutic use of neurotrophic proteins seems practical for diseases of the peripheral nervous system (PNS), where they can be given by systemic administration. Administration of exogenous neurotrophic factors after nerve injury has been shown to mimic the effect of target organ-derived trophic factors on neuronal cells.Neurotrophic factors have been demonstrated to prevent the development of peripheral neuropathy in animal models, but the therapeutic use of these factors in human disease has been limited by the short serum half-life and dose-limitinginBDNFside effects of these potent peptides. Effective gene transfer into regenerating sciatic nerves by adenoviral vectors is the potentials for gene therapy of peripheral nerve injury. Replication defective adenoviral vectors have been demonstrated as an effective method for delivering genes into a variety of cell types and tissues both in vivo and in vitro. Transfecting genes into neuronal cells has proven to be difficult because of their lack of cell division. Since the major problem in sciatic nerve injury is the degeneration of the terminally differentiated neuronal cells, the adenoviral vector's ability to transfer genes into differentiated post-mitotic cells makes them advantageous for a gene delivery system for the nervous system. Here we showed that a replication defective recombinant adenovirus carrying the brain-derived neurotrophic factor (BDNF) gene could infect Schwann cells in injured sciatic nerves of rat in vivo with microinjection. With the methods of in situ hybridization, immunocytochemistry and unage analysis, BDNF gene delivered into the Schwann cells was expressed efficiently. The expression of the BDNF gene lasted for 1 months, within which nerve regeneration is accomplished in the rat. With traditional methods of assessing nerve recovery following peripheral nerve injury and repair, such as histomorphometry, retrograde axonal transport of HRP tracer, electrophysiology and sciatic function index (SFI), the conclusion is the adenoviral vectors might thus be used to modulate Schwann cell gene expression for treating peripheral nerve injury. The main results and conclusions of our experiments are showed as follows:1. 293 cell, a human embryonic kidney cell line as a packaging cell was used for culture of recombinant adenovirus vectors containing BDNF gene. We reproduced and purified the helper-free replication-defective recombined adenovirus vector containing the BDNF cDNA expression cassette , (AxCA-BDNF, a gift from Dr. Wang GQ, constructed by Kojima H, Japan). After two rounds of CsCl centrifugation, biological activity identification wasIVcarried out using plaques test. The purified recombined adenovirus AxC A-BDNF were obtained with total volume of 50ul, with liter of 2.2 108~1.8 1010pru/ml.2. An animal model was prepared in order to study peripheral nerve regeneration: Based on...