Nanoparticles for siRNA delivery to mammalian neurons

描述（由申请人提供）：将外源性核酸引入中枢神经系统（CMS）细胞的能力是一项强大的技术，可用于神经生物学研究和神经系统疾病的治疗。RNA干扰（RNAi）是目前最有效和特异性的阻断基因表达的方法；然而，到目前为止，还没有一个成功的RNAi在哺乳动物神经系统疾病模型中的体内应用。实现RNAi对CMS的全部治疗价值的主要挑战在于将核酸递送到靶细胞。合成纳米颗粒提供体内保护，低免疫原性，相对容易制造和扩大规模，并已用于传递质粒和寡核苷酸到各种类型的培养细胞。然而，由于传递效率低，该技术在体外和体内神经细胞传递中的成功应用受到限制。这项研究计划的主要目标是开发纳米颗粒，以介导介导RNA干扰的短干扰RNA （siRNA）分子的有效神经元传递。我们提出了一种新的策略，通过设计纳米颗粒来克服细胞内运输障碍，这些纳米颗粒可以“搭便车”在向细胞体运输囊泡的运动蛋白上。这一目标可以通过实现以下目标来实现：(i)合成纳米颗粒配方，整合神经元靶向、囊泡释放和运动蛋白辅助、逆行运输的成分，并通过评估有丝分裂后神经元样pc12细胞的传递效率来优化配方；（ii）证明siRNA的传递和原代神经元中转基因表达的特异性下调；（iii）通过脊髓注射的逆行运输实现纳米颗粒递送到脑内的神经元细胞体。有效的中枢神经系统输送系统对研究和临床应用都至关重要；因此，这个项目的成功完成将是朝着实现这项技术的全部潜力迈出的重要一步。

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.