Gene-based neuromodulation for sustained botulinum toxin expression in the treatment of muscle spasticity: a proof of principle study Open Access

Di, Long (Spring 2018)

Permanent URL: https://etd.library.emory.edu/concern/etds/08612n52b?locale=en
Published

Abstract

Introduction: Botulinum toxin, often used to treat peripheral nervous system dysfunction, suffers from its transient effects and central nervous system toxicity. Vector-mediated gene transfer has the potential for cell-specific, sustained neural inhibition. Here, we tested the adenoviral vector-mediated expression of select botulinum toxin/A (serotype A) protein domains in focal neural inhibition without cytotoxicity.

 

Methods: Adenoviral vectors encoding different botulinum toxin/A domains were injected into rat spinal cords at 4×104 transforming units to test their neural inhibitory effects: light chain, light chain and receptor binding domain, and light chain and translocation domain. To assess for dosage dependency, vectors were also tested at 4×105 and 4×106 transforming units. Pre-surgical baseline and post-operative motor function was measured by Basso-Beattie-Bresnahan open-field locomotor scoring, grip strength, and rotarod performance. After 3-weeks of postoperative behavioral testing, rats were euthanized, and spinal cords were immunostained and counted for neuronal nuclei to assess cytotoxicity from transgene expression. Prior to injection, vectors were tested in vitro in HEK293 cells to control for differences in transduction efficiency.

 

Results: Animals injected with adenoviral vectors containing genes for the light chain and receptor binding domain displayed the most consistent and sustained deficits in open-field assessment, grip strength, and rotarod performance. Rats injected with adenovirus containing light chain showed spontaneous recovery at 16 days post-injection, coinciding with the cessation of adenoviral gene expression. Spinal cord neuron counts revealed no signs of cytotoxicity between treatment groups and controls at injection titers of 4×104 and 4×105 but not at 4×106 transforming units. In vitro testing showed no difference in transduction efficiency between vectors and there were no differences in neuron density compared to controls.

 

Conclusion: These results suggest that vector-mediated gene transfer of botulinum toxin light chain and receptor-binding domain is most effective at synaptic inhibition that is not cytotoxic. This gene-based approach at focal neuromodulation may hold future applications in the treatment of neurological disease and the study of neural circuitry. Furthermore, these data suggest that the heavy chain may play a role in the characteristic persistence of botulinum neurotoxin/A inactivation of synaptic function that is still unexplained.

Table of Contents

Abstract……………………………………………………………………………………………1

Introduction………………………………………………………………………………………..3

Materials and Methods…………………………….………………………………………………8

Results……………………………………………………………………………………………16

Discussion………………………………………………………………………………………..24

Tables and Figures……………………………………………………………………………….31

            Figure 1: Spinal Cord Surgery…………………………………………………………...31

            Figure 2: Grip Strength and Rotarod…………………………………………………….31

            Figure 3: Automated Cell Count Procedure……………………………………………...32

            Table 1: Viral Vectors……………………………………………………………………32

            Figure 4: Plasmid Map/Vector Confirmation In Vitro…………………………………..33

            Figure 5: Comparison of Gene Combinations…………………………….……………..33

            Table 2: One-Way ANOVA Statistics…………………………………….……………..34

            Figure 6: Dosage Dependence…………………………………………….……………..35

            Figure 7: Vector Confirmation In Vivo……………………………...…….…………….36

            Figure 8: Neurodegeneration at 106 TU………………………………………………….37

Author Disclosure Statement/ References……………………………………………………….39

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