Molecularly self-assembled multi-functional DNA Origami nanostructure for safe and effective siRNA delivery

说明（申请人提供）：RNA干扰（RNAi）介导的基因沉默已成为操纵细胞表型、绘制遗传通路和发现治疗靶点的重要技术，具有治疗潜力。实现RNAi技术广泛潜力的关键挑战是需要安全有效的方法将小干扰RNA（siRNA）递送到靶细胞中。尽管已经探索了多种用于siRNA递送的材料和方法，但是目前的材料和方法的性能仍然不令人满意，因为它们通常不能很好地与原代细胞或非粘附细胞类型一起工作，并且经常导致一定程度的细胞毒性和细胞生物学的改变，从而严重限制了适合于发现研究并且不适合于人类治疗的细胞类型。在这个项目中，我们建议研究一种新的，分子设计的多功能DNA折纸纳米结构（DONs），用于在靶细胞内有效递送siRNA。我们的假设是，生物相容性的，分子定义的，单分散的DON与siRNA和细胞穿透肽（CPP）的结合，将使有效的siRNA摄取细胞和有效的siRNA释放细胞内，导致高基因敲低，即使当使用低siRNA浓度。我们的最终目标是开发一种安全有效的DONS-siRNA治疗疾病的药物;本I期项目的短期目标是开发具有最佳结构的基于DONS-siRNA的多功能siRNA递送和转染试剂，以辅助siRNA沉默研究。第一阶段的概念验证示范项目将侧重于1）开发用于构建DONS-siRNA的化学和方案，以及2）体外评估DONS-siRNA用于细胞转染和基因敲减的有效性。本研究拟采用一锅法构建萤火虫荧光素酶（Luc）siRNA基因和绿色荧光蛋白（GFP）siRNA基因的多功能DONs，并评价其对表达Luc的HeLa细胞和表达GFP的人前列腺癌（PC-3）细胞的沉默效率。在第二阶段，我们将彻底优化与细胞靶向配体结合的多功能DON-siRNA的制备，研究DON-siRNA的体内递送和基因敲除的有效性，DON-siRNA在荷瘤裸鼠中的药代动力学特征和器官生物分布。

DESCRIPTION (provided by applicant): The RNA interference(RNAi)-mediated gene silencing has become an important technology for manipulating cellular phenotypes, mapping genetic pathways and discovering therapeutic targets, and has therapeutic potential. A key challenge to realizing the broad potential of RNAi technology is the need for safe and effective methods for delivery Small interfering RNA (siRNA) into targeted cell. Although a variety of materials and approaches have been explored for siRNA delivery, the performance of current materials and approaches remain unsatisfactory because they generally do not work well with primary cells or non-adherent cell types and often result in some degree of cytotoxicity and alterations in cell biology, thereby severely limiting the cell types amenable to discovery research and not suitable for human therapeutics. In this project, we propose to investigate a novel, molecularly designed multi-functional DNA Origami nanostructures (DONs) for effective delivery of siRNA inside targeted cells. Our hypothesis is that the biocompatible, molecularly defined, monodispersed DONs incorporated with siRNAs, and cell-penetrating peptides (CPP) would enables effective siRNA uptake by cells and efficient release of siRNA inside cells, resulting in high gene knockdown even when using low siRNA concentrations. Our ultimate goal is to develop a safe and effective DONs-siRNA therapeutic for diseases; the short-term goal of this Phase I project is to develop DONs-based multi-functional siRNA delivery and transfection reagent with optimal structure to aid siRNA silencing research. The Phase I proof-of-concept demonstration project will focus on 1) developing chemistry and protocol for constructing DONs-siRNA and 2) in vitro assessing the effectiveness of DONs-siRNA for cell transfection and gene knockdown. We plan to develop one-pot approach to create multi-functional DONs incorporated with firefly luciferase(Luc) siRNA gene and green fluorescent protein (GFP) siRNA gene, and evaluate their silencing efficiency to Luc-expressed HeLa cells and GFP-expressed Human prostate carcinoma (PC-3) cells. In Phase II we will thoroughly optimize the preparation of multi-functional DON-siRNA incorporated with cell-target ligands, investigate the in vivo delivery of DON-siRNA and the effectiveness of gene knockdown, the pharmacokinetic profile and organ biodistribution of DON-siRNA in nude mice bearing tumors.