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

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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:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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