Adeno-associated Viral Vector Delivery of Clostridial Tetanus Toxin Light Chain for Clinical Use Open Access

Huang, Jeremiah W. (2012)

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Clostridial bacterial toxins are gaining greater significance as therapeutic agents in the
field of neuromodulation. The success of botulinum neurotoxin has encouraged further
investigation into other Clostridial toxins, such as tetanus neurotoxin, as possible means of gene
therapy. The inhibitory function of tetanus light chain can provide an avenue for
neuromodulation for conditions characterized by aberrant neural firing. Viral vectors can be
employed to induce stable LC expression as opposed to the commonly used botulinum treatment
method, which requires repeated injections. Earlier studies have achieved effective synaptic
inhibition using adenoviral (Ad) directed-LC expression. However, the desired effect was only
temporary because of adenovirus's transient gene expression in the spinal cord. To overcome
the short-lived adenoviral gene expression, adeno-associated viral (AAV) vector was evaluated
for its ability to provide persistent LC expression following ipsilateral spinal cord injection of
viral vector. As exemplified in previous studies, administration of Ad.LC resulted in an initial
inhibition of motor function followed by recovery while AAV.LC induced sustained locomotor
inhibition that lasted for thirty days post treatment. AAV.LC specifically inhibited sensorimotor
activity to the ipsilateral hind limbs. Expression of LC selectively decreased expression of
vesicle associated membrane protein 1 (VAMP1) compared to that of other proteins found in the
soluble NSF attachment protein receptor (SNARE) complex. The control vectors, Ad.GFP and
AAV.GFP, revealed no changes in motor function, which suggests inhibition was due to LC
expression in motor neurons. Furthermore, no gross changes were observed in motor neuron
density or structure in the spinal cord following administration of any of the four vectors.
Results from the study prompt continued investigations into the therapeutic possibilities of
delivering tetanus toxin LC to treat conditions involving atypical spinal neural transmission.

Table of Contents

1 Introduction 1

2 Materials and Methods 8

2.1 Vector Construction 8

2.2 Animal Use 9

2.3 Behavioral Testing 9

2.4 Gene Delivery 10

2.5 Perfusion 11

2.6 Immunohistochemistry 12

2.7 Motor Neuron Density Measurements 12

2.8 Western Blotting 13

2.9 Statistical Analysis 14

3 Results 15

3.1 BBB 15

3.2 Tarlov Motor Score 15

3.3 Grip Strength 16

3.4 Rotarod 17

3.5 Immunohistochemistry 18

3.6 Western Blotting 18

4 Discussion 20

5 Conclusion 24

6 References 25

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