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Evaluation of AAV8 as a gene therapy vector to deliver NT-3 and shRNA\(_{RhoA}\) to injured dorsal root ganglion neurones

Jacques, Steven John (2012)
Ph.D. thesis, University of Birmingham.

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Abstract

Two major reasons for the failure of central nervous system axon regeneration are (i) lack of neurotrophic factors available to CNS neurones and (ii) the presence of molecules that inhibit the growth of axons. In this study a gene therapy approach using adeno-associated virus 8 (AAV8) was used to manipulate these two factors. The following major aims were addressed: (i) confirm the bioactivity of transgenes that would be packaged into the AAV8 vector; (ii) assess the cellular tropism of AAV8 in the dorsal root ganglion (DRG); (iii) evaluate the inflammatory responses of the nervous system to AAV8 after intra-DRG and intrathecal injection; (iv) determine the axon regenerative effect of AAV8-mediated delivery of nt-3 (a neurotrophic factor) and shRNA\(_{RhoA}\) (a disinhibitory therapy) to dorsal root ganglion neurones after spinal cord injury in the rat. Delivery of the nt-3 transgene in vitro resulted in production of high levels of NT-3 protein. Transfection of shRNA\(_{RhoA}\)-containing plasmids into cell lines resulted in a marked decrease in the amount of RhoA detectable in cell lysates. AAV8 was found to preferentially transduce large diameter, proprioceptive DRG neurones (DRGN) but in the context of a significant inflammatory response after intra-DRG injection 28d following intra-DRG injection. Axon regenerative effects of AAV8-mediated transgene delivery before lesioning were ambiguous and further work need to be undertaken to clarify this matter.

Type of Work:Ph.D. thesis.
Supervisor(s):Logan, Ann
School/Faculty:Colleges (2008 onwards) > College of Medical & Dental Sciences
Department:Molecular Neuroscience Group, School of Clinical and Experimental Medicine
Subjects:QH301 Biology
QH426 Genetics
RZ Other systems of medicine
Institution:University of Birmingham
ID Code:3461
This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
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