A peptide-based small interfering RNA delivery system as an RNA interference approach for neurodegenerative disorders

Neurodegenerative disorders (NDDs) have become a major global health burden. Despite persistent advances in understanding the neurodegeneration process, pathogenesis has not been fully clarified and no cures are yet available. As a therapeutic approach for NDDs, RNA interference (RNAi) can be a potent mode of treatment because it can specifically downregulate target genes that are directly or indirectly associated with the onset and progression of neurodegeneration. For example, Keap1, a negative regulator of the antioxidant responsive element (ARE) can be targeted for gene downregulation in order to enhance the endogenous antioxidant capacity and protect brain cells against extensive oxidative stress, a pathological hallmark observed in many NDDs. However, small interfering RNAs (siRNAs) bear limiting factors, including instability in the bloodstream and limited capacity to cross the blood-brain barrier (BBB) mainly due to their bulky size and negatively charged backbone. Thus, there is need for an adequate carrier that can protectively load and deliver bioactive siRNAs to brain cells. In this dissertation we detail the evaluation of a peptide-based siRNA carrier for neurotargeted siRNA delivery and the assessment of Keap1 RNAi therapeutic potential in brain cells. First, we have designed a myristoylated cell-penetrating peptide equipped with a transferrin receptor targeting sequence (myr-TP-Tf) and examined its physicochemical properties and biological functions. The myr-TP-Tf was shown to stably encapsulate siRNAs and deliver them to brain cells, leading to functional silencing of the target gene. Myr-TP-Tf also transported the siRNAs across a brain endothelial cell monolayer in a transwell system. Second, we have assessed the therapeutic potential of Keap1 RNAi against beta amyloid (Aβ) peptide-induced oxidative stress in a human glioma cell culture. It was found that the Keap1 siRNA-peptide complex-pretreated group had better tolerance to the cytotoxicity from the Aβ and displayed lower levels of oxidative stress and autophagic activity compared to the control groups, demonstrating the neuroprotective effect of Keap1 RNAi. Third, we examined the brain-targeting and functional target gene silencing abilities of the siRNA-peptide carrier complex in vivo. Although the direct local injection exerted a greater performance, the systemic administration also delivered a measurable amount of siRNA to the brain, which led to a significant knockdown of the target gene. In summary, results demonstrate that this peptide-based siRNA carrier system can be a promising strategy for neurotargeted siRNA delivery both in vitro and in vivo.