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Title: Novel cell-penetrating peptide-based vectors for siRNA delivery
Author: Tsimon, Myrsini
ISNI:       0000 0004 5354 014X
Awarding Body: University of Westminster
Current Institution: University of Westminster
Date of Award: 2014
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Harnessing the RNAi pathway with synthetic siRNA as a potent and selective mode of post-transcriptional gene silencing and has therapeutic potential in personalised medicine; however, the large size and negative charge of siRNA creates a hurdle for intracellular delivery that has thus far limited its development as a therapeutic. Cell penetrating peptides (CPPs), such as the Antennapedia homeodomain (AntpHD) and its third helix, Penetratin, are well characterised cationic motifs used previously to deliver covalently linked nucleic acid cargo, such as siRNA in vitro and in vivo, may offer a strategy to address this challenge. This thesis aimed to design, purify and characterize novel recombinant fusion proteins, that would have broad applicability as carrier molecules for non-covalent siRNA delivery. The fusion proteins were comprised of a cell penetrating peptide sequence (either AntpHD, Penetratin, HIV-Tat or EB1) fused to either the first dsRNA binding motif 1 (DRBM) or the tandem motifs (DRBMx2) from human PKR, which bind dsRNA with high avidity in a sequence independent manner. A panel of constructs were cloned, expressed in a bacterial cell system, and purified by affinity chromatography under both native and denaturing conditions. Several of the constructs were either poorly expressed, insoluble or prone to precipitation during purification or dialysis; however, construct C5.1 was successfully purified and its identity confirmed by mass spectrometry. Construct C5.1 bound siRNA only at a high ratio of protein to siRNA due to the presence of co-purifying nucleic acid, whereas constructs C12.2 and C13.2 bound siRNA at low molar ratios. Both C12.2 and C5.1 were efficiently internalized in either live or fixed HEK293 and HepG2 cells; however, the proteins appeared to be sequestered in endosomes whether in the presence or absence of cargo. Cytotoxicity of the fusion proteins in HEK293 cells increased in the order of C12.2
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available