| Popis: |
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterised pathologically by the accumulation of amyloid-β plaques and neurofibrillary tangles within the brain. Currently, there are no disease-modifying therapeutic interventions available. This thesis describes the development and delivery of novel RNA-based therapeutics for AD. Recently, significant advances have occurred in the field of RNA interference (RNAi)-based therapeutics, allowing targeted silencing of disease-relevant genes through the use of short interfering RNAs (siRNAs) or microRNAs (miRNAs). However, development has been impeded by poor penetrance of the blood-brain-barrier (BBB) in vivo. Exosomes, as secreted extracellular vesicles (EVs), may overcome this problem as they naturally deliver RNA between cells and cross the BBB. Furthermore, recent evidence suggests that they may be loaded with exogenous RNA and preferentially targeted to neuronal tissue. Thus, this study investigated EV-mediated delivery of RNAi-based therapeutics shown to regulate AD-relevant genes. A comparison of EV-loading methods revealed that electroporation was confounded by the formation of large siRNA aggregates and indicated that endogenous loading strategies may be preferable. Endogenous loading of RNAi molecules was shown to be enhanced by co-expression with RNA-binding proteins, particularly following inclusion of an acylation domain. These findings highlight the potential for EV-mediated delivery of RNAi-based therapeutics. A combinatorial therapeutic approach is increasingly recognised to hold the most promise for the future of AD. Thus, this study also focused upon the development of novel multi-gene targeting locked nucleic acid-modified antisense oligonucleotides (LNA-ASOs) as a combinatorial approach towards AD therapy. Two multi-gene LNA-ASOs each produced potent silencing of two separate target genes simultaneously, resulting in a significant reduction in amyloid-β production in vitro. A murine-specific multi-gene LNA-ASO was also identified, paving the way for future in vivo studies. These findings suggest that multi-gene targeting LNA-ASOs may represent a promising novel therapeutic strategy for AD. In summary, this thesis has investigated novel strategies for both RNA delivery and multi-gene targeting, demonstrating the potential of RNA-based therapeutics for the treatment of AD. |
