Generation of recombinant toxin molecules from Clostridium tetani and Clostridium botulinum

Obstructive sleep apnoea is a muscular disorder that disrupts breathing during sleep and is caused by a loss of muscle tone in the muscles of the upper airway which become flaccid and physically block the airway. Sleep is disrupted due to the restriction of air intake caused by upper airway resistance and is characterised by pauses in breathing which may last for up to 60 seconds. Individuals are forced to gasp for air in order for breathing to resume, and the disorder is characterised by daytime sleepiness, waking with a headache and disturbances in concentration and memory. If left untreated, obstructive sleep apnoea can lead to increased blood pressure and risk of stroke, brain damage or heart attack. Current treatment options include mechanical and non-mechanical devices to increase upper airway patency, and surgery to remove excess soft tissue which blocks the airway during sleep. These treatments have low adherence rates due to their impact on lifestyle, or are not suitable for all individuals. This project focussed on developing a novel treatment for obstructive sleep apnoea to correct the loss of muscle tone. Tetanus toxin produced by Clostridium tetani, causes spastic paralysis by preventing the release of inhibitory neurotransmitters within the central nervous system which normally signal muscles to relax. The result is an increase in muscle tone leading to rigid paralysis, which can be exploited for the treatment of obstructive sleep apnoea. Localised injection of tetanus toxin has been demonstrated to induce tetany in the upper airway of British Bulldogs, who naturally have sleep apnoea -like events during sleep, without systemic effects. Due to the widespread immunisation against tetanus toxin through vaccination with tetanus toxoid, this project aims to develop a chimeric holotoxin consisting of the heavy chain of tetanus toxin and the botulinum type A light chain to circumvent pre-existing immunity to tetanus toxin. Tetanus and botulinum toxins have structural similarities, and while they elicit opposing effects, their mode of action is similar. The individual domains, which retain function upon separation, have previously been substituted among domains of other Clostridial neurotoxins. In conclusion, individual toxin domains from tetanus and botulinum type A toxin will be developed and recombined to produce a novel chimeric holotoxin for the treatment of obstructive sleep apnoea.