Safety and Efficacy Demonstrated in Chemogenetic Application of CODA Viral Transgene in Modulating the Motor Activity of Rats Restricted; Files Only

Abstract

Spinal cord injuries impact several thousand people every year in the United States. Nearly 70% of people who suffer spinal injuries will also develop spasticity, which results from the overstimulation of motor neurons in the lower central nervous system. Spasticity causes significant amounts of pain, hampers the ability to function independently, and may lead to other injuries. Currently available treatments are often ineffective, necessitating invasive and costly surgery. This project explores a new and alternative form of treatment for spasticity though chemogenetic gene therapy. The adeno-associated virus serotype 9 (AAV9) vector was used to deliver a transgene through an intraparenchymal injection into the lumbar spinal cord, which the body could then uptake. This new genetic material was used by the body’s machinery to create a modified ɑ7 ligand-binding domain for the nicotinic acetylcholine receptor that was fused with a a splice variant chloride-permeable glycine receptor ion pore domain, called PSAM-GlyR. The PSAM-GlyR was designed to bind only to the partial agonist bradanilcine. The addition of bradanilcine could then force a conformational change of PSAM-GlyR, allowing chloride ions into the motor neuron and hyperpolarizing the neuron to reduce its rate of activation. This project used a rat model, giving 30 rats the transgene one of three different drug doses of either 109, 1010, or  1011 vector genomes per animal to measure the safety of the treatment. The motor function of the animals was measured over eight weeks with the lowest and medium doses showing no significant health issues, while the highest dosage showed significant safety concerns included weakened limbs and severe inflammation of the spinal cord. This indicated that the highest dosage was unsafe for continued use in rats. The medium dosage was selected for additional study. Using the selected dosage, a new cohort of 20 rats received varying doses of either Bradanicline or a placebo following injection of the medium dosage of AAV9. A behavioral test was conducted after introduction of either the drug or a placebo and motor function was evaluated and was repeated with increasing dosages for five weeks. The rats showed a significant decrease in motor activity at dosages of 30 mg/kg and 100 mg/kg, a promising indication that spasticity can be regulated through neuromodulation. In the future, this project will continue to explore the current dosages in use through larger sample sizes or use different dosages to finetune the optimal dosage for maximizing the reduction in motor movement while also minimizing safety risks.

Table of Contents

Introduction…..................................................................................................................................1

1.1 Mechanism of Spasticity….............................................................................................1

1.2 Current Treatments….....................................................................................................4

1.2.1 Physiotherapy/Physical Therapy….................................................................4

1.2.2 Electrical Stimulation…..................................................................................5

1.2.3 Pharmacology….............................................................................................6

1.2.4 Surgical Treatment…......................................................................................7

1.3 Chemogenetics…...........................................................................................................7

1.3.1 Principles of Chemogenetics…......................................................................8

1.3.2 Viability of nAChR as Receptor Target…......................................................9

1.3.3. Bradanicline as ɑ7 AChR Agonist Candidate….........................................10

1.3.4 Transgene Delivery System…......................................................................11

1.4 Implementation….......................................................................................................11

Methods…....................................................................................................................................13

2.1 Motor Function Behavioral Assay….........................................................................13

2.1.1 Grip Strength Test…...................................................................................13

2.1.2 Basso, Beattie and Bresnahan Locomotor Test ….....................................14

2.2 Phase I: Safety Test…...............................................................................................14

2.2.1 Motor Neuron Count…..............................................................................16 

2.2.1 Histological Analysis….............................................................................16

2.3 Phase II: Chemogenetic Ligand Effectivity Test…..................................................17

Results…...................................................................................................................................19

3.1 Phase I: Safety Test Results…................................................................................19

3.1.1 Phase I Grip Test Results…....................................................................19

3.1.2 Phase I Basso, Beattie and Bresnahan Locomotor Test Results…........20

3.1.3 Phase I Motor Neuron Count Results…................................................21 

3.1.4 Phase I Iba1 Inflammation Marker Staining Results…........................22

3.1.5 Grip Strength and BBB Scores following Ligand Dosage…........................23

3.2 Phase II: Chemogenetic Ligand Effectivity Results….................................................26

3.2.1 Phase II Initial Safety Screening Results.....................................................26

3.2.2 Phase II Grip Strength Test Results.............................................................27

3.2.3 Phase II Basso, Beattie and Bresnahan (BBB) Locomotor Test Results.....28

Discussion....................................................................................................................................30

References....................................................................................................................................34

Supplemental Information...........................................................................................................41

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry
Keyword	Chemistry Spinal Cord
Committee Chair / Thesis Advisor	Nicholas Boulis, Emory University
Committee Members	Tracy McGill, Emory University Richard Himes, Emory University

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