Histidylated poly(lysine) highly branched polymers for siRNA delivery

基因沉默在人类治疗中的应用已经引起了人们的极大兴趣。这源于小干扰RNA(siRNA，21-23个核苷酸的RNA片段)诱导RNA干扰(RNAi)的能力，RNAi是真核细胞中导致互补mRNA破坏的一种途径。RNAi途径可以用来沉默可能导致癌症等疾病的内源性基因。对于引起传染病的生物体的增殖所必需的基因，也可以使基因沉默或击倒，因此，这种实现基因沉默的方法可以用于疾病预防或治疗。从理论上讲，基因沉默可以在任何组织中诱导，而且据报道，在肝硬变、乙肝(乙肝)、卵巢癌和骨癌等疾病中也能实现基因沉默。这种效应在快速分裂的细胞中可以持续几天，在不分裂的细胞中可以持续几周，因此需要重复给药。到目前为止，开发安全有效的体内siRNA递送系统以实现细胞内的生物利用度是实现RNAi广泛用于疾病预防和治疗的主要挑战。SiRNA的传递可以在当地或系统内实现。SiRNA制剂策略通常基于颗粒相关方法(例如，脂类或聚合物胶体)。如果系统地给药，大多数siRNA制剂往往会在肝、肺或肾(网状内皮系统的器官)中蓄积。为了针对其他器官，可以使用局部给药(如眼睛)，也可以通过淋巴系统给药。除了促进siRNA进入细胞外，siRNA载体的一个关键特性是避免诱导免疫反应。因此，需要一些方法来获得最大限度地减少免疫系统激活的载体。在这项工作中，我们建议通过首先检测阳离子脂质和聚合物辅料来开发电荷中性粒子，以建立复杂的siRNA模型。通过电荷-电荷相互作用使siRNA预络合可导致设计用于细胞内解离的颗粒。复杂的siRNA可以通过皮下注射进一步配制成淋巴吸收，并在眼睛内(眼内)局部使用。最初的努力将利用基于赖氨酸-组氨酸氨基酸的超支化聚合物来制备siRNA，这将降低细胞毒性。我们还将利用最先进的表征技术，包括动态光散射(DLS)、纳米颗粒跟踪分析(NTA)和电子显微镜来评估颗粒大小和尺寸分布，因为这是细胞摄取颗粒、流式细胞仪和用于功能测试的体外细胞分析的关键。特征化将允许确定相关性，以通过迭代过程优化配方策略。这项工作将涉及以下阶段，这些阶段与CDT的核心主题：先进产品设计、制药工艺工程和复杂产品表征相一致：-超支化聚合物-siRNA颗粒的配制：这将需要确定聚合物与siRNA的最佳比例。在这一阶段，将使用等温滴定量热法来研究颗粒的组装、形成和结合特性的机理。-颗粒的表征：这包括颗粒大小分析(DLS、NTA)、表面特性表征。-体外研究：细胞培养和转基因研究，通过适当的分析来确定纳米颗粒的摄取和细胞活力。-体内研究。该项目将以一项简短的体内研究结束，希望在合适的小鼠模型中探索铅配方的效力。

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.