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Title: Engineering cholera toxin for the targeted delivery of oligonucleotides
Author: Machin, Darren Christopher
ISNI:       0000 0004 6060 3321
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2016
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Half of the 20 best-selling prescription drugs of 2014 were large biologic therapeutics, however to date there have only been two licensed non-aptamer oligonucleotide drugs. Oligonucleotides have proven highly successful at modification of gene expression in the laboratory, but the lack of a robust delivery vehicle has largely prevented their transferal to clinical use. That role could be filled by cholera toxin, a protein produced by Vibrio cholerae, responsible for the diarrhoeal symptoms of cholera infection. Cholera toxin targets cells presenting GM1 ganglioside on their surface for endocytosis and carries a toxic payload to the endoplasmic reticulum (ER) by retrograde translocation. Several non-toxic variants of cholera toxin have been produced which are capable of carrying a cargo into cells, but to date it has not been tested for oligonucleotide delivery. The non-toxic subunit of cholera toxin responsible for endocytosis, CTB, was expressed in the absence of the toxic A subunit. CTB was site-specifically labelled with fluorescein using sortase A and shown to carry the payload into Vero cells. The endocytic pathway was able to be altered through modification of the protein sequence. Subsequently, a short RNA duplex was delivered to different locations in Vero cells by two subtly different CTB variants. Several strategies were then trialled to promote oligonucleotide release from CTB and export to the cytosol. While oligonucleotides conjugated via a disulfide bond were released in the ER, and a short hydrophobic peptide mediated export of an oligonucleotide-CTB complex from the ER, a combination of both was not observed. This study showed CTB has the potential to function as an effective delivery vehicle for therapeutic oligonucleotides, but also that there remains work to do and are hurdles to overcome to achieve this goal. It provides a platform for the development of CTB, or other similar proteins, for clinical use.
Supervisor: Webb, Michael E. ; Turnbull, W.Bruce ; Macdonald, Andrew ; Andrews, Benjamin I. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available