The development of bacterial toxin and plant sugar-binding proteins for neuronal tracing and delivery

The blood-brain barrier, while protective, presents a major obstacle for drug delivery to the central nervous system, thus ways to circumvent it for the treatment of neurological disorders are highly desired. Many pathogens and their toxins have evolved to hijack cell entry systems, initiating endocytosis following the binding of sugars on the plasma membrane. This project aims to repurpose these mechanisms to facilitate the delivery of therapeutic proteins to neurones. Viruses with the ability to express sugar-binding proteins were assessed for their ability to target neurones for transduction following retrograde transport. This would allow for the tracing of neuronal circuits as well as potential delivery of therapeutic molecules to higher order neurones after peripheral administration. Canine adenovirus 2 and adeno-associated virus serotypes 2, 5, and 8 did not successfully transduce neurones in rats or mice; future work may pseudotype the viruses for re-targeting. The sub-populations of neurones labelled by B subunits of cholera toxin (CTB) and the related heat-labile enterotoxin (LTB) following various routes of injection in mice were studied. In general, motor neurones, autonomic preganglionic neurones and primary afferents could be targeted following peripheral administration. A fusion protein of CTB and the calcium buffering protein parvalbumin was designed, expressed and tested both in vitro and in vivo; CTB successfully delivered its cargo to neurones. CTB was also engineered for alternative detection and novel conjugation methods; following administration in vivo all engineered CTB proteins could be detected. This study provides a basis for the development of further CTB fusion proteins; the toxin represents a versatile delivery vehicle, both as an experimental tool and as a potential therapeutic in the treatment of neurodegenerative diseases.