Neural Tract-tracing Nucleic Acid Carriers

描述（由申请人提供）：“神经束示踪核酸载体”现有的转染试剂在体外成熟神经元中的表现非常差，并且不适合于体内使用。该第一阶段项目将测试创新表面技术公司正在开发的新型核酸载体的性能。（ISurTec）用于体外和体内神经元的通用转染。该技术是基于平台的，由用于高效非病毒基因和siRNA转染的聚合物纳米颗粒组成，通过表面修饰作为神经束示踪剂进行化学掩蔽。选择神经束示踪表面化学用于载体，因为束示踪剂已经反复证明了有效的试剂摄取和逆行颗粒转运，这被认为是神经元转染的主要障碍。由于神经元过程的高度伸长性质，逆行运输对于用基因构建体转染成熟神经元特别重要。所提出的载体还包括用于靶向配体的可定制连接的表面官能团。本研究项目的目标是优化试剂配方，以实现在成熟哺乳动物神经元中超过50%的转染效率，通过配体附着证明神经元亚型靶向能力，并可预测地修改动物模型中CNS回路的功能特性。该技术的成功开发将使得能够使用分子构建体在完整的神经系统中有针对性地调节/观察神经可塑性和回路功能。一个前所未有的水平的转染效率和成熟神经元的靶向能力，预计从这种核酸载体通过纳入纳米颗粒神经束示踪剂的基本特征。高效转染和精确的体内靶向能力的组合将作为分子神经科学领域与系统、行为和临床前神经科学之间的桥梁，通过使当今使用的日益强大的分子生物学工具能够广泛应用于神经科学研究，极大地增加了我们理解和操纵神经可塑性和脑功能的手段。最终，这一策略有望为各种神经系统疾病和障碍提供更有效的治疗方法，包括帕金森病，脑损伤和慢性药物成瘾。 公共卫生相关性：神经系统疾病和障碍，从帕金森病到慢性药物成瘾，由于大脑、其神经元和其回路的复杂性和独特挑战而极难治疗。此外，神经元对核酸转染具有高度抵抗性，这阻碍了当今日益强大的分子生物学工具在神经科学研究中的应用。在这个第一阶段的提案中，我们将测试一种新的纳米颗粒技术，旨在通过大脑回路的遗传调节来显着增加我们对神经系统功能的理解，并使人们能够更有效地治疗各种困扰人类神经系统的疾病。

DESCRIPTION (provided by applicant): "Neural Tract-tracing Nucleic Acid Carriers" Existing transfection reagents perform very poorly in mature neurons in vitro, and are not suitable for in vivo use. This Phase I project will test the performance of a novel nucleic acid carrier under development at Innovative Surface Technologies, Inc. (ISurTec) for universal transfection of neurons in vitro and in vivo. This technology is platform-based and consists of a polymeric nanoparticle for high-efficiency non-viral gene and siRNA transfection, chemically masked as a neural tract-tracer through surface modification. Neural tract-tracing surface chemistry was chosen for the carrier because the tract-tracers have repeatedly demonstrated efficient reagent uptake and retrograde particle transport, which are believed to be the major barriers to neuronal transfection. Retrograde transport is particularly important for transfection of mature neurons with gene constructs, due to the highly elongated nature of neuronal processes. The proposed carrier also includes surface functional groups for customizable attachment of targeting ligands. The objectives of this research project are to optimize the reagent formulation to achieve a transfection efficiency of greater than 50% in mature mammalian neurons, demonstrate neuronal sub-type targeting capability through ligand attachment, and predictably modify the functional properties of a CNS circuit in an animal model. Successful development of this technology will enable the targeted regulation/observation of neuroplasticity and circuit function in the intact nervous system using molecular constructs. An unprecedented level of transfection efficiency and targeting capability for mature neurons is expected from this nucleic acid carrier by incorporating the essential features of nanoparticulate neural tract-tracers. A combination of high efficiency transfection and precise in vivo targeting capability would serve as a bridge between the field of molecular neurosciences and the systems, behavioral, and preclinical neurosciences, greatly increasing our means to understand and manipulate neuroplasticity and brain function, by enabling the increasingly powerful molecular- biological tools in use today to be broadly applied to neuroscience research. Ultimately, this strategy is expected to lead to more effective therapeutic treatments for a variety of neurological diseases and disorders, including Parkinson's disease, brain injury, and chronic drug addiction. PUBLIC HEALTH RELEVANCE: Neurological diseases and disorders, ranging from Parkinson's disease to chronic drug addiction, are extremely difficult to treat due to the complexity and unique challenges of the brain, its neurons, and its circuits. Moreover, neurons are highly resistant to nucleic acid transfection, which hinders the application of today's increasingly powerful molecular-biological tools in neuroscience research. In this Phase I proposal we will test a novel nanoparticle technology designed to significantly increase our understanding of nervous system function through the genetic regulation of brain circuits, and enable more effective treatments for variety of ailments afflicting the human nervous system.