Modified cyclodextrins as novel non-viral vectors for neuronal siRNA delivery: focus on Huntington’s disease

Huntington’s Disease (HD) is a rare autosomal dominant neurodegenerative disease caused by the expression of a mutant Huntingtin (muHTT) protein. Therefore, preventing the expression of muHTT by harnessing the specificity of the RNA interference (RNAi) pathway is a key research avenue for developing novel therapies for HD. However, the biggest caveat in the RNAi approach is the delivery of short interfering RNA (siRNAs) to neurons, which are notoriously difficult to transfect. Indeed, despite the great advances in the field of nanotechnology, there remains a great need to develop more effective and less toxic carriers for siRNA delivery to the Central Nervous System (CNS). Thus, the aim of this thesis was to investigate the utility of modified amphiphilic β-cyclodextrins (CDs), oligosaccharide-based molecules, as non-viral vectors for siRNA delivery for HD. Modified CDs were able to bind and complex siRNAs forming nanoparticles capable of delivering siRNAs to ST14A-HTT120Q cells and to human HD fibroblasts, and reducing the expression of the HTT gene in these in vitro models of HD. Moreover, direct administration of CD.siRNA nanoparticles into the R6/2 mouse brain resulted in significant HTT gene expression knockdown and selective alleviation of rotarod motor deficits in this mouse model of HD. In contrast to widely used transfection reagents, CD.siRNA nanoparticles only induced limited cytotoxic and neuroinflammatory responses in multiple brain-derived cell-lines, and also in vivo after single direct injections into the mouse brain. Alternatively, we have also described a PEGylation-based formulation approach to further stabilise CD.siRNA nanoparticles and progress towards a systemic delivery nanosystem. Resulting PEGylated CD.siRNA nanoparticles showed increased stability in physiological saltconditions and, to some extent, reduced protein-induced aggregation. Taken together, the work outlined in this thesis identifies modified CDs as effective, safe and versatile siRNA delivery systems that hold great potential for the treatment of CNS disorders, such as HD.

Gene delivery for the treatment of prostate cancer

Prostate cancer is one of the most common cancers diagnosed in men. Whilst treatments for early-stage disease are largely effective, current therapies for metastatic prostate cancer, particularly for bone metastasis, offer only a few months increased lifespan at best. Hence new treatments are urgently required. Small interfering RNA (siRNA) has been investigated for the treatment of prostate cancer where it can ‘silence’ specific cancer-related genes. However the clinical application of siRNA-based gene therapy is limited due to the absence of an optimised gene delivery vector. The optimisation of such gene delivery vectors is routinely undertaken in vitro using 2D cell culture on plastic dishes which does not accurately simulate the in vivo bone cancer metastasis microenvironment. The goal of this thesis was to assess the potential of two different targeted delivery vectors (gold or modified β-cyclodextrin derivatives) to facilitate siRNA receptor-mediated uptake into prostate cancer cells. Furthermore, this project aimed to develop a more physiologically relevant 3D in vitro cell culture model, to mimic prostate cancer bone metastasis, which is suitable for evaluating the delivery of nanoparticulate gene therapeutics. In the first instance, cationic derivatives of gold and β-cyclodextrin were synthesized to complex anionic siRNA. The delivery vectors were targeted to prostate cancer cells using the anisamide ligand which has high affinity for the sigma receptor that is overexpressed by prostate cancer cells. The gold nanoparticle demonstrated high levels of uptake into prostate cancer PC3 cells and efficient gene silencing when transfection was performed in serum-free media. However, due to the absence of a poly(ethylene glycol) (PEG) stabilising group, the formulation was unsuitable for use in serum-containing conditions. Conversely, the modified β-cyclodextrin formulation demonstrated enhanced stability in the presence of serum due to the inclusion of a PEG chain onto which the anisamide ligand was conjugated. However, the maximum level of gene silencing efficacy from three different prostate cancer cell lines (DU145, VCaP and PC3 cells) was 30 %, suggesting that further optimisation of the formulation would be required prior to application in vivo. In order to develop a more physiologically-relevant in vitro model of prostate cancer bone metastasis, prostate cancer cells (PC3 and LNCaP cells) were cultured in 3D on collagenbased scaffolds engineered to mimic the bone microenvironment. While the model was suitable for assessing nanoparticle-mediated gene knockdown, prostate cancer cells demonstrated a phenotype with lower invasive potential when grown on the scaffolds relative to standard 2D cell culture. Hence, prostate cancer cells (PC3 and LNCaP cells) were subsequently co-cultured with bone osteoblast cells (hFOB 1.19 cells) to enhance the physiological relevance of the model. Co-cultures secreted elevated levels of the MMP9 enzyme, a marker of prostate cancer metastasis, relative to prostate cancer cell monocultures (2D and 3D) indicating enhanced physiological relevance of the model. Furthermore, the coculture model proved suitable for investigating nanoparticle-mediated gene silencing. In conclusion, the work outlined in this thesis identified two different sigma receptor-targeted gene delivery vectors with potential for the treatment of prostate cancer. In addition, a more physiologically relevant model of prostate cancer bone metastasis was developed with the capacity to help optimise gene delivery vectors for the treatment of prostate cancer.