Histidylated poly(lysine) highly branched polymers for siRNA delivery

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There has been a great interest in the research into the therapeutic applications of gene silencing in humans. This stems from the ability of small interfering RNA (siRNA, RNA fragments of 21-23 nucleotides) to induce RNA interference (RNAi), a pathway in eukaryotic cells that leads to destruction of complementary mRNA. The RNAi pathway can be used to silence endogenous genes that can cause diseases such as cancer. Silencing or knocking down of genes is also possible for the genes that are necessary for the proliferation of organisms that cause infectious diseases, and thus this approach of achieving gene silencing can be used for disease prevention or treatment. Gene silencing can be induced in any tissue, in theory, and knocking down genes has been reported to be achieved in diseases such as liver cirrhosis, hepatitis B (HBV), and ovarian and bone cancers. This effect can last from few days in rapidly dividing cells to several weeks in non-dividing cells and thus requires repeated administration. To date, developing safe and effective delivery systems for siRNA in vivo that can achieve intracellular bioavailability is the main challenge for achieving a widespread use of RNAi for disease prevention and treatment. The delivery of siRNA can be achieved either locally or systemically. siRNA formulation strategies are often based on particulates associated approaches (e.g. lipid or polymer-based colloids). If delivered systemically, most siRNA formulations tend to accumulate in organs such as liver, lungs, or kidneys (organs of the reticuloendothelial system). To target other organs either local delivery can be used (such as to the eye) or via the lymphatic system. A key property of siRNA delivery vehicles, in addition to promoting the delivery of siRNA into the cells, is avoiding inducing immune responses. Thus, approaches that result in obtaining vehicles that minimise the activation of the immune system is required.In this work we propose to develop charge-neutral particles by first examining cationic lipid and polymeric excipients to complex model siRNA. Pre-complexation of siRNA by charge-charge interactions can result in particulates designed to dissociate intracellularly. The complexed siRNA can be further formulated for lymphatic uptake via subcutaneous administration and for local use within the eye (intraocular). Initial efforts will utilise lysine-histidine amino acid-based hyperbranched polymers for siRNA, which would reduce cytotoxicity. We will also utilise state of the art characterisation techniques including dynamic light scattering (DLS), Nanoparticle Tracking Analysis (NTA) and electron microscopy to assess the particle size and size distribution as this is key in particle uptake by the cells, flow cytometry and in vitro cell assays for functionality testing. Characterisation will allow correlations to be determined to optimise formulation strategies by an iterative process. The work will involve the following stages, which align with the CDT core themes of: Advanced Product Design, Pharmaceutical Process Engineering and Complex Product Characterisation:- Formulation of hyperbranched polymer-siRNA particles: This will require determining the optimum ratio of polymer to siRNA ratio. Isothermal titration calorimetry will be utilised in this stage to study the mechanism of particle assembly, formation, and binding properties. - Characterisation of particles: This includes particle size analysis (DLS, NTA), surface properties characterisation.- In-vitro studies: cell culture and transfection study with suitable assays to determine nanoparticle uptake and cell viability.- In-vivo studies. The project will end with a short in-vivo study, looking to explore the potency of the lead formulation in a suitable mouse model.