Developing Nanopieces, a Platform RNAi Delivery Technology for Treatment of Multiple Diseases

STTR_开发纳米片 7.项目总结/摘要 开发新型RNAi（RNA Interference）药物的瓶颈是缺乏高效、无毒的 RNA递送至体内非肝组织。递送带负电荷的核酸是特别具有挑战性的。 进入包括关节软骨在内的难以到达的组织中的无血管、致密和带负电荷的基质。NanoDe 治疗公司（NanoDe）是一家处于开发阶段的公司，致力于开发RNAi的递送， 治疗，从而创造更有效的药物。NanoDe是建立在一个新的平台RNA交付 一种名为NanopiecesTM的技术，一种源自小分子JBAK的新型仿生纳米材料， 含胺或赖氨酸的Janus碱（K）。通过其生物相容性Janus-Base单元的自我相互作用， DNA碱基对，JBAK形成非共价纳米管（NT）与带正电荷的胺或赖氨酸上的 面JBAK NT进一步与siRNA组装形成JBAK NP，从而包封带负电荷的 siRNA插入带正电荷的NP中。我们的初步数据表明，1）纳米粒子可以渗透到基质丰富的 组织，并且在细胞内高效释放siRNA治疗剂， 修饰否则无法治疗的疾病; 2）NP递送的RNAi疗法已经取得了成功的结果 用于治疗多种疾病，包括类风湿性关节炎，一种称为软骨肉瘤的罕见实体瘤， 和创伤后骨关节炎（PTOA）的动物模型; 3）NP具有良好的 生物相容性和生物降解性，这对于维持体内最小毒性至关重要。这个目标 I期申请是通过使用PTOA作为“用例”疾病来进一步开发NP递送技术。的 中心假设是NP的最佳正电荷是使其能够实现的关键参数之一。 渗透到带负电荷的软骨基质中，内吞到软骨细胞中，并释放siRNA， 抑制疾病基因表达，从而对PTOA有显著的治疗效果。这一假设将 测试有两个目的：1）确定NP的最佳电荷，以使其渗透和保留 在软骨组织内，转染软骨细胞，抑制基质蛋白酶ADAMTS-5基因， 分别在软骨细胞中表达;和2）确定NP的最佳电荷，以使其在软骨细胞内表达。 关节输送和关节内的长半衰期，并在PTOA动物中实现显著的治疗效果 模型本研究具有创新性，因为：1）与传统分娩方式有本质区别 纳米片是一种非共价载体，具有独特的优势，如尺寸的多功能性， 和表面电荷、对细胞外基质的亲和性、优异的生物降解性和生物相容性。2)为 我们首次确定了成功递送siRNA所需的NP的重要技术参数，包括 基质渗透、细胞转染和基因敲除的最佳载体表面电荷。3)的 技术突破为多种疾病的治疗提供了一种治疗方法 包括PTOA。

STTR_Developing Nanopieces 7. Project Summary/Abstract The bottleneck of developing new RNAi (RNA Interference) drugs is the lack of highly efficient and non-toxic RNA delivery to non-liver tissues in vivo. It is especially challenging to deliver negatively-charged nucleic acid into avascular, dense, and negatively-charged matrix in hard-to-reach tissues including joint cartilage. NanoDe Therapeutics, Inc. (NanoDe) is a development stage company dedicated to developing delivery of RNAi therapeutics, thereby creating more effective drugs. NanoDe is founded on a novel platform RNA delivery technology termed NanopiecesTM, a novel biomimetic nanomaterial derived from a small molecule JBAK, Janus-Base with Amine or lysine (K). Through self-interaction of its biocompatible Janus-Base units mimicking DNA base pairs, JBAK forms non-covalent nanotubes (NT) with positively charged amine or lysine on the surface. JBAK NT further assembles with siRNA to form JBAK NP, thereby encapsulating negatively charged siRNA into positively charged NP. Our preliminary data has shown that 1) NPs can penetrate matrix-rich tissues that conventional vehicles cannot, and release siRNA therapeutics intracelluarly in high efficiency to modify otherwise untreatable diseases; 2) NP delivered RNAi therapeutics has achieved successful outcomes in the treatment of multiple diseases including rheumatoid arthritis, a rare solid tumor called chondrosarcoma, and post-traumatic osteoarthritis (PTOA) in animal models respectively; and 3) NP has excellent biocompatibility and biodegradability, which are critical for maintaining minimal toxicity in vivo. The goal of this Phase I application is to further develop NP delivery technology by using PTOA as a “use case” disease. The central hypothesis is that the optimal positive charge of NP is one of the critical parameters to enable its penetration into negatively charged cartilage matrix, endocytosis into chondrocytes, and release siRNA to inhibit disease gene expression, thereby achieving significant therapeutic effects on PTOA. This hypothesis will be tested with the two aims: 1) Determining the optimal charge of NP to enable its penetration and retention within cartilage tissue, transfection into chondrocytes, and inhibiting matrix proteinase ADAMTS-5 gene expression in chondrocytes, respectively; and 2) Determining the optimal charge of NP to enable its intra- articular delivery and long half-life within joint, and achieve significant therapeutic outcomes in a PTOA animal model. The proposed research is innovative because: 1) Essentially different from conventional delivery vehicles, Nanopiece is a non-covalent vehicle presenting unique advantages, such as versatility in dimensions and surface charge, affinity to extracellular matrix, excellent biodegradability and biocompatibility. 2) For the first time, we identify important technology parameters of NP required for successful siRNA delivery including the optimal vehicle surface charge for matrix penetration, cell transfection, and gene knockdown. 3) The technology breakthrough enlightens a therapeutic approach to deliver RNAi for treatment of multiple diseases including PTOA.