Non-viral gene delivery to neural progenitor cells in the SVZ

描述（由申请人提供）：在脑室管膜下区（SVZ）中发现的神经干细胞和祖细胞（分别为NSC和NPC）由于其通过细胞分裂更新的能力，具有替代受损神经元群体的潜力。因此，这些细胞是治疗神经退行性疾病的治疗操作的有趣靶点。虽然从SVZ产生新的神经元是自然发生的，但很明显，为了达到治疗相关的神经发生水平，需要诸如生长因子的局部递送以增加来自干细胞区的新神经元的分化、迁移和存活的机制。这项工作的目标是开发一种合成的，生物相容性载体，有效的基因传递到SVZ的NPC。聚合载体将并入多种生物活性肽以辅助NPC靶向和细胞内运输。具体地，肽官能化聚合物将包括：（i）用于DNA缩合、细胞靶向和内体逃逸的生物活性肽，（ii）用于生物相容性的HPMA（N-（2-羟丙基）甲基丙烯酰胺）骨架，（iii）用于颗粒稳定的聚乙二醇（PEG），和（iv）用于细胞内化后降解的二硫键。将通过体外转染筛选优化聚合物组合物以用于高效基因递送至NPC，并通过流式细胞术、共聚焦显微镜和FRET分析研究递送机制。此外，质粒载体将被工程化用于在NPC中持续的转基因表达，其在NPC分化成成熟神经元后关闭。最有前途的载体将通过脑室内施用至小鼠来评估，以进一步优化载体用于有效的体内递送。将通过脑切片的共聚焦显微镜对给药后双标记（质粒和聚合物）载体的分布进行成像。此外，将通过脑切片的荧光抗体染色来确定转染后不同时间点表达递送的转基因的细胞类型。最后，通过局部生长因子递送来扩增分裂细胞的数量，以及通过递送BDNF（脑源性神经营养因子）质粒来促进神经元迁移和存活，将进一步提高体内基因转移的效率。如果成功，这个项目的潜在影响是广泛的。所开发的载体可用作运载体以递送用于治疗神经变性CNS病症的治疗基因。 公共卫生相关性：脑的神经变性疾病，例如阿尔茨海默病、亨廷顿病和帕金森病，影响全世界超过2000万人，并且目前对这些疾病的大多数治疗是姑息性的而不是恢复性的。神经变性疾病的一种有希望的治疗方法是操纵成人神经系统中的神经干细胞和祖细胞以恢复丢失的神经元群体。这项工作的目标是开发合成基因递送载体，可用于特异性地将生长因子递送到大脑中的神经祖细胞，以刺激神经发生。所提出的方法包括将生物活性肽掺入聚合物载体中以促进靶向神经祖细胞和有效的细胞内递送。所开发的技术将具有广泛的意义，作为车辆提供治疗基因的中枢神经系统疾病。

DESCRIPTION (provided by applicant): The neural stem and progenitor cells (NSCs and NPCs, respectively) found in the subventricular zone (SVZ) of the brain have the potential to replace damaged neuronal populations due to their ability to renew through cell division. These cells are therefore interesting targets for therapeutic manipulation in treatment of neurodegenerative diseases. Although the generation of new neurons from the SVZ occurs naturally, it is clear that in order to achieve therapeutically-relevant levels of neurogenesis, mechanisms such as localized delivery of growth factors to increase differentiation, migration and survival of new neurons from stem cell zones is required. The goal of this work is to develop a synthetic, biocompatible vector for efficient gene delivery to NPCs in the SVZ. The polymeric vector will incorporate multiple bioactive peptides to assist in NPC targeting and intracellular trafficking. Specifically, the peptide-functionalized polymer will include: (i) bioactive peptides for DNA condensation, cell targeting and endosomal escape, (ii) HPMA (N-(2- hydroxypropyl)methacrylamide) backbone for biocompatibility (iii) polyethylene glycol (PEG) for particle stabilization, and (iv) disulfide bonds for degradation after cellular internalization. The polymer composition will be optimized for high efficiency gene delivery to NPCs through in vitro transfection screening, and the mechanism of delivery investigated through flow cytometry, confocal microscopy and FRET analyses. In addition, the plasmid vector will be engineered for sustained transgene expression in NPCs that shuts down after the NPCs differentiate into mature neurons. The most promising vectors will be evaluated by intraventricular administration to mice in order to further optimize vectors for efficient in vivo delivery. The distribution of dual-labeled (plasmid and polymer) vectors after administration will be imaged by confocal microscopy of brain sections. In addition, the type of cells expressing the delivered transgene at various time points after transfection will be determined by fluorescent antibody staining of brain sections. Finally, the efficiency of in vivo gene transfer will be further enhanced by amplifying the number of dividing cells through localized growth factor delivery and by promoting neuron migration and survival by delivery of the BDNF (brain- derived neurotrophic factor) plasmid. If successful, the potential impact of this project is broad. The developed vectors could be used as vehicles to deliver therapeutic genes for treatment of neurogenerative CNS disorders. PUBLIC HEALTH RELEVANCE: Neurogenerative diseases of the brain, such as Alzheimer's Disease, Huntington's Disease, and Parkinson's Disease, affect over 20 million people worldwide, and most current treatments for these diseases are palliative rather than restorative. One promising treatment approach to neurodegenerative disorders is the manipulation of neural stem and progenitor cells in the adult human nervous system to restore lost neuronal populations. The goal of this work is to develop synthetic gene delivery vectors that can be used to specifically deliver growth factors to neural progenitor cells in the brain in order to stimulate neurogenesis. The proposed approach involves incorporating bioactive peptides in the polymeric vector to promote targeting to the neural progenitor cells and efficient intracellular delivery. The developed technology would have broad significance as vehicles to deliver therapeutic genes for CNS disorders.