AbstractRecent advances in biotechnology have enabled the development and large scale production of protein therapeutics, thereby providing new treatments for numerous diseases. However, oral delivery of biologics remains a challenge as macromolecules are poorly absorbed and rapidly degraded in the gastrointestinal tract. Several strategies have been studied to improve bioavailability, but to date, no safe, efficient and clinically relevant delivery system has been discovered. Here, the reprogramming of bacterial toxins for therapeutic purposes was investigated. Following cellular uptake, Pseudomonas aeruginosa exotoxin A (PE) and Vibrio cholerae cholix toxin (Cho) escape lysosomal degradation and are capable of both trafficking to the cytosol of non-polarised (NP) cells and undergoing transcytosis across polarised cells. This project aimed to investigate the trafficking routes exploited by these toxins to determine how they avoid degradation, and define their potential as vehicles for oral protein delivery.The nature of the conformational change experienced by PE in acidic conditions was first investigated. It was shown that this transition consists of a subtle alteration in the protein’s structure which does not affect its overall size, but results in the exposure of additional trypsin cleavage sites and hydrophobic residues. It was concluded that the transition is involved in the protein’s escape from lysosomal degradation and was most likely caused by the protonation of two histidine residues when the pH was lowered, resulting in the formation of additional salt bridges and thus different structural constraints.The interaction between PE and monosialoganglioside GM1 was also examined to determine whether it could act as a secondary receptor for PE. High-affinity binding was established in both acidic and physiological conditions, and interaction with GM1 was shown to be required for efficient protein internalisation by NP cells. These findings were concluded to be in agreement with GM1 acting as a secondary receptor for PE, leading the toxin away from lysosomal degradation following conformational change.The potential of PE as a vehicle for delivery of biologics was studied using several versions of the toxin connected to a cargo protein. Results showed that a truncated version of PE is capable of carrying a cargo protein (green fluorescent protein, human growth hormone) inside NP cells in vitro and across polarised epithelium in vivo. These data strongly support the idea that PE has the potential to be used as a vehicle for oral delivery of biologics.Similarly, these studies were also carried out on cholix to assess its ability to act as a drug carrier. Cholix was shown to undergo a conformational change similar to that experienced by PE. It was also demonstrated that an interaction with GM1 occurred, although in this case, increased binding appeared to result in decreased internalisation by NP cells. Finally, full-length and truncated cholix could transport a payload inside NP cells in vitro and across polarised epithelial cells in vivo. These results confirm that cholix could also represent a powerful tool for oral administration of macromolecular drugs.
|Date of Award||12 Feb 2016|
|Supervisor||Randy Mrsny (Supervisor)|
Characterization of the trafficking pathway used by <i>Pseudomonas aeruginosa </i>Exotoxin A and application to oral drug delivery
Laurent, F. (Author). 12 Feb 2016
Student thesis: Doctoral Thesis › PhD