| Abstract | 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<C5.1<C13.2<C11.2 whereas in HepG2 cells, C11.2 was significantly more cytotoxic than C12.2; suggesting that the proteins exhibit cell type-specific cytotoxicity. Moreover, C11.2 and C12.2 altered HepG2 cell morphology in the presence of siRNA, compared to C5.1 and C13.2. In a HEK293-dEGFP reporter cell line, a complex of C12.2 and siRNA induced a significant decrease in dEGFP expression, which was not observed with C5.1-siRNA; however, unexpectedly similar effects were observed with C12.2-scrambled siRNA suggesting non-specific siRNA effects. Effects on PTP1B expression were also examined with all purified proteins complexed with the optimum molar concentration of PTPN1 siRNA; none were able to exhibit PTP1B expression knockdown at the protein level.
In this thesis, a comprehensive strategy for the design, purification and testing of novel siRNA carriers has been developed. A number of recombinant siRNA carriers have been successfully produced and characterised. The results highlight common issues encountered with the development CPP-based siRNA delivery vectors; nonetheless, the ability of C12.2 to mediate RNAi-mediated knockdown demonstrates the potential of the development of CPPs as non-covalent siRNA delivery vectors. |
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