Genetic engineering RGD-containing siRNA protein carrier

siRNA已经显示出在治疗诸如癌症的不可治疗的疾病中的潜力。尽管 siRNA的潜在的治疗应用由于缺乏安全和有效的药物而受到极大的阻碍。 有效的传递载体。病毒载体存在安全性问题，已排除用于临床 应用程序.目前大多数非病毒载体包括脂质复合物、聚合物、无机 纳米颗粒、胶束和细胞穿透肽是带正电荷的， 血清稳定性、脱靶效应、细胞毒性和批间变异。此外，无机 纳米颗粒和聚合物由于不可降解和差的生物相容性而具有严重的安全性问题。 间隙因此，目前的载体不适合siRNA的全身递送。前几 研究中，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）肽在人源dsRNA结合结构域（dsRBD）蛋白上的表达。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， 不带电荷，在酸性条件下带电荷，如核内体。18他能给予足够的 缓冲能力驱动货物内体逃逸。然而，目前的siRNA载体，包括 dsRBD-18 his缺乏细胞类型特异性，靶向分子如抗体、适体或 必须将配体添加到每个货物中。这是一个耗时和昂贵的过程。RGD（精氨酸- Gly-Asp）肽已被充分表征为整联蛋白αvβ3的结合配体， 在肿瘤新生血管和一些肿瘤细胞上高度表达，但在静息状态下不存在。 内皮细胞和正常器官系统。RGD肽是一种经过充分验证的肿瘤靶向药物， 分子，并已用于指导肿瘤诊断的成像剂和药物， 临床环境中的治疗。在这个项目中，我们将基因工程三个肿瘤和肿瘤血液 通过将RGD肽融合到dsRBD的N末端、C末端或N末端和C末端两者中， 18 His蛋白质新的载体将具有三种功能，包括siRNA结合，内体逃逸， 和肿瘤靶向。新的载体将是不带电的、低毒性的、可生物降解的、细胞类型特异性的， 并且易于大规模生产。该提案的具体目标是：1）克隆，表达， RGD载体的表征，和2）肿瘤和肿瘤新血管系统靶向的评估 癌症异种移植模型的能力。具有装载到运行能力的新载体将简化siRNA 在体内递送，并且任何siRNA都可以被装载并靶向递送至肿瘤或肿瘤血液 船舶.

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.