Design and evaluation of mRNA accessible siRNA to silence the epidermal growth factor receptor (EGFR)

Fox, Stephen Peter 2007. Design and evaluation of mRNA accessible siRNA to silence the epidermal growth factor receptor (EGFR). PhD Thesis, Cardiff University.

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Abstract

Small interfering RNA siRNA promise to be highly effective molecules but susceptible to some of the limitations experienced with antisense oligonucleotide ASO technology biological instability, design of target mRNA accessible sequences and efficient delivery to subcellular sites of action. This thesis has aim to address each of these limitations for siRNA. The biological stability of 5'-end 32P radiolabeled siRNA and ASO were tested in FBS, FBS-containing cell culture media and in A431 cell lysates. The biological degradation profiles of ASO and siRNA in serum were comparable ASO t1/2 19 minutes and siRNA t1/2 19 minutes. The stability of siRNA was higher than ASO in A431 cell lysates ASO tU2 37 minutes and siRNA t1/2 43 minutes. siRNA were designed using innovative scanning ASO array technologies to EGFR mRNA accessible sites. Initially seven siRNAs were designed against different mRNA accessible sites. Based on above a further five siRNAs were designed incorporating termini modifications to alter the thermodynamic stability of the siRNA duplex. These sequences were evaluated for biological activity following optimisation of a delivery strategy. siRNA delivery to A431 cells was optimised using a FACs assay with Oligofectamine and fluorescendy labelled siRNA. Oligofectamine delivery showed significant cell fluorescence associated with labelled siRNA compared with untreated controls P 0.05. The activity, cell proliferation and EGFR protein and mRNA knockdown, of the designed siRNA was investigated in A431 and TamR cells. RT-PCR, Western blot and immunofluorescent microscopy showed inhibitory trends, but ultimately lacked sufficient resolution and reproducibility A431 growth was not inhibited. A proprietary siRNA showed knockdown of EGFR mRNA in TamR cells, but all the custom designed siRNA produced highly variable effects, failing to show significant knockdown, even when various designed siRNA were pooled. ASO and siRNA share common pharmaceutical developmental problems, although siRNAs can elicit the powerful innate RNAi mechanism. Further understanding of RNAi will allow siRNA to fulfill their enormous therapeutic potential.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Pharmacy
Subjects:	R Medicine > RM Therapeutics. Pharmacology
ISBN:	9781303182112
Date of First Compliant Deposit:	30 March 2016
Last Modified:	12 Feb 2016 23:14
URI:	https://orca.cardiff.ac.uk/id/eprint/55684

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