Genetic engineering RGD-containing siRNA protein carrier

SiRNA已显示出在治疗癌症等无法治愈的疾病方面的潜力。尽管它的 由于缺乏安全性和安全性，siRNA的潜在治疗应用受到极大阻碍 有效的传递载体。病毒载体有安全问题，已被排除在临床之外 申请。目前大多数非病毒载体包括脂复合物、聚合物、无机载体 纳米粒子、胶束和细胞穿透肽都带正电荷， 血清稳定性、脱靶效应、细胞毒性和批次间差异。此外，无机盐 纳米粒子和聚合物由于不可降解和较差而存在严重的安全问题 通行证。因此，目前的载体不适合系统地传递siRNA。在以前的 通过研究，我们开发了一种基于非阳离子蛋白的适体-siRNA嵌合体载体 18组氨酸(His)多肽位于人源性dsRNA结合域(DsRBD)蛋白上。DsRBD-18HIS 不依赖于高正电荷与rna分子相互作用；相反，它结合dsrna。 通过特定的三维构象。组氨酸分子的pKa值约为6，在中性pH下，它们 不带电，在酸性条件下带电，如内吞体体。他可以给予足够的 缓冲能力，以驱动货物内体逃逸。然而，目前的siRNA载体包括 DsRBD-18缺乏细胞类型特异性和靶向分子，如抗体、适体或 每件货物中都必须添加配基。这是一个既耗时又昂贵的过程。RGD(参数- Gly-Asp)肽被认为是整合素αvβ3的结合配体，它是 在肿瘤新生血管和一些肿瘤细胞上高表达，但在静止状态下不表达 内皮细胞和正常器官系统。RGD多肽是一种有效的肿瘤靶向药物 分子，并已被用于指导显像剂和药物的肿瘤诊断和 临床环境下的治疗。在这个项目中，我们将对三种肿瘤和肿瘤血液进行基因工程 RGD多肽融合到dsRBD-N-、C-或N-和C-末端的血管靶向载体 18His蛋白。新载体将具有三种功能，包括siRNA结合、内体逃逸、 和肿瘤靶向。新的载体将不带电荷，低毒，可生物降解，细胞类型特异， 以及大规模生产的便利性。这项提议的具体目标是：1)克隆、表达和 RGD载体的特性，以及2)肿瘤和肿瘤新生血管靶向性的评价 在癌症异种移植模型中的能力。具有即装即用功能的新载体将简化siRNA 体内递送，任何siRNA都可以被加载并靶向递送到肿瘤或肿瘤血液中 船只。

siRNA has shown the potential in treating undruggable diseases such as cancer. Despite its potential, therapeutic application of siRNA has been greatly impeded by the lack of safe and effective delivery vectors. Viral vectors have safety concern and have been precluded for clinical application. Current most non-viral vectors including lipoplexes, polymers, inorganic nanoparticles, micelles, and cell penetrating peptide are positively charged and suffer from low serum stability, off-target effect, cytotoxicity and batch-to-batch variation. Additionally, inorganic nanoparticles and polymers have serious safety concerns due to non-degradable and poor clearance. Therefore, current carriers are unsuitable for systemic delivery of siRNA. In previous study, we have developed a non-cationic protein based aptamer-siRNA chimera carrier by adding 18 Histidine (His) peptide on human origin dsRNA binding domain (dsRBD) protein. DsRBD-18his does not rely on high positive charges to interact with RNA molecules; instead, it binds dsRNA via specific 3-D conformation. Histidine molecules have pKa value about 6, at neutral pH, they are uncharged, and charged in acidic condition such as endosome. 18His can confer sufficient buffering capacity to drive cargo endosomal escape. However, current siRNA vectors including dsRBD-18his lacking cell type specificity, and targeting molecules such as antibody, aptamer, or ligand have to be added into each cargo. That is a time consuming and costly process. RGD (Arg- Gly-Asp) peptide has been well characterized as a binding ligand of an integrin αvβ3, which is highly expressed on tumor neo-vasculature as well as some tumor cells, but not present in resting endothelial cells and normal organ systems. RGD peptide is a well validated tumor targeting molecule, and has been used for guiding imaging agents and drugs for tumor diagnosis and therapy in clinical setting. In this project, we will genetic engineer three tumor and tumor blood vessel targeted vectors by fusing RGD peptide into N-, C-, or both N-and C termini of dsRBD- 18His protein. New vectors will have three functions including siRNA binding, endosomal escape, and tumor targeting. New vectors will be uncharged, low toxicity, biodegradable, cell type specific, and ease of mass production. The specific aims of this proposal are 1) cloning, expression, and characterization of RGD vectors, and 2) evaluation of tumor and tumor neo-vasculature targeting capabilities in cancer xenograft models. New vectors with load-to-go capability will simplify siRNA delivery in vivo, and any siRNAs can be loaded and targeting delivered to tumors or tumor blood vessels.