Nanoparticles for siRNA delivery to mammalian neurons

描述(由申请人提供)：将外源核酸引入中枢神经系统(CMS)细胞的能力是一项强大的技术，在神经生物学研究和神经疾病治疗中具有应用价值。RNA干扰(RNAi)是目前最有效和最特异的阻断基因表达的方法；然而，到目前为止，还没有成功的RNAi在哺乳动物神经疾病模型中的应用。要实现RNAi对CMS的全部治疗价值，主要挑战在于将核酸输送到靶细胞。人工合成的纳米粒子具有体内保护、低免疫原性、相对容易制造和放大的特点，已被用于将质粒和寡核苷酸输送到各种类型的培养细胞。然而，由于递送效率低，这项技术在体外和体内的神经细胞递送方面的成功应用一直受到限制。这项研究计划的主要目标是开发纳米颗粒，以介导高效的神经元递送短的干扰RNA(SiRNA)，即介导RNA干扰的分子。我们建议实施一种新的策略来克服细胞内的运输障碍，方法是设计一种纳米颗粒，这种纳米颗粒可以“搭便车”在马达蛋白上将囊泡输送到细胞体。这一目标可通过实现以下目标来实现：(I)合成集成了神经元靶向、囊泡释放和运动蛋白辅助的逆行转运成分的纳米制剂，并通过评估有丝分裂后神经元样PC 12细胞的递送效率来优化配方，(Ii)展示siRNA递送和特异性下调原代神经元中转基因表达，以及(Iii)通过脊髓注射逆行递送纳米颗粒至脑内神经元胞体。高效的中枢神经系统给药系统对于研究和临床应用都是至关重要的；因此，该项目的成功完成将导致朝着实现这项技术的全部潜力迈出重要的一步。

DESCRIPTION (provided by applicant): The ability to introduce exogenous nucleic acids to cells in the central nervous system (CMS) is a powerful technique with applications in neurobiology research and in treatment of neurological disease. RNA interference (RNAi) is currently the most potent and specific method for blocking gene expression; however, there is not to date a successful in vivo application of RNAi to a mammalian model of neurological disease. The major challenge to realizing the full therapeutic value of RNAi for the CMS lies in the delivery of the nucleic acids to target cells. Synthetic nanoparticles offer in vivo protection, low immunogenicity, and relative ease of manufacturing and scale up, and have been used to deliver plasmid and oligonucleotides to various types of cultured cells. However, successful application of this technology for neuronal cell delivery both in vitro and in vivo has been limited due to low delivery efficiencies. The major goal of this research proposal is to develop nanoparticles that mediate efficient neuronal delivery of short, interfering RNA (siRNA), molecules that mediate RNA interference. We propose to implement a novel strategy to overcome intracellular transport barriers by designing nanoparticles that "hitchhike" on motor proteins that transport vesicles toward the cell body. This goal can be achieved by realizing the following aims: (i) synthesizing nanoparticle formulations that integrate components for neuron targeting, vesicle release, and motor protein-assisted, retrograde transport, and optimizing formulations by evaluating the delivery efficiency in postmitotic neuron-like PC 12 cells, (ii) demonstrating siRNA delivery and specific downregulation of transgene expression in primary neurons, and (iii) achieving nanoparticle delivery to neuronal cell bodies in the brain by retrograde transport from spinal cord injection. Efficient delivery systems for the CNS are crucial for both research and clinical applications; thus, successful completion of this project would result in a major step toward realizing the full potential of this technology.