Comparing AAV Vector Serotypes for Gene Delivery to the Dorsal Root Ganglion for Pain Relief Open Access

Kim, Nawoo (2015)

Permanent URL: https://etd.library.emory.edu/concern/etds/wd375w937?locale=en
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Abstract

Chronic peripheral neuropathy is a debilitating condition characterized by distal tingling, numbness, and pain. The current treatments only try to control the pain or treat the underlying cause, but there is a surprisingly low success rate. The dorsal root ganglion (DRG) has been a potential target for administering therapeutics, as spontaneous firing from this cluster of sensory neurons and pain fibers is a characteristic of peripheral neuropathy. Viral vectors, such as adeno-associated virus (AAV), can be used to deliver therapeutic genes to dampen the pain sensations. This study focuses on comparing the transduction efficacy of different AAV serotypes, 1, 2, 5, 6, and 8 expressing green fluorescent protein (GFP) administered to embryonic rat DRGs in culture. The cells were also stained with β-tubulin for co-localization with sensory neurons, TRPM8 (transient receptor potential cation channel, subfamily M, member 8) for co-localization with C and δ pain fibers, and IB4 (isolectin-b4) for co-localization with C fibers exclusively. Although significance could not be established for one serotype, all vectors were successful in transduction and co-localization with each type of stain. From an observational standpoint, AAV8 and AAV5 expressed the highest percentage of GFP-positive cells co-localizing with β-tubulin, TRPM8, and IB4. These findings are consistent with previous studies comparing different AAV serotypes in vivo. The determination of the most effective AAV serotype will prompt further studies involving gene therapy to the DRG using different delivery methods and therapeutic genes to provide pain relief.

Table of Contents

Introduction....................................................................................................................................... 1

Materials and Methods....................................................................................................................10

DRG Harvesting and Extraction......................................................................................................10

Preparation of Plates........................................................................................................................10

Dissociation and Plating DRG ......................................................................................................... 11

Transduction.....................................................................................................................................11

Immunohistochemistry.....................................................................................................................12

Cell Counting....................................................................................................................................13

Data Analysis....................................................................................................................................13

Results..................................................................................................................................................14

MOI Comparisons.............................................................................................................................14

AAV Serotype Comparisons.............................................................................................................14

Percentage Co-localization................................................................................................................15

Discussion.............................................................................................................................................16

Shortcomings......................................................................................................................................17

Future Directions.................................................................................................................................. 19

References..............................................................................................................................................21

Figure 1....................................................................................................................................... 25

Figure 2....................................................................................................................................... 26

Figure 3....................................................................................................................................... 27

Figure 4....................................................................................................................................... 28

Figure 5....................................................................................................................................... 29

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