基于功能化外泌体的靶向递送策略在基因治疗异位骨化中的应用

Heterotopic ossification (HO) is a debilitating condition common in cases of inflammation associated with severe injury, surgery, or disease. HO most commonly occurs in muscle, tendons, and ligaments, although it is unclear as to why these specific tissue environments foster the formation of ectopic bone. Activin-like kinase 2 (ALK2) is a transforming growth factor-beta (TGF-β)/bone morphogenetic protein (BMP) type I receptor known to be activated by cytokines such as BMP2, BMP4, BMP7, and TGF-β2 in various cell types.Vascular endothelial cells have emerged as the leading candidate for the cellular origin of heterotopic cartilage and bone in fibrodysplasia ossificans progressive (FOP) . Approximately 50% of the cartilage and bone cells found in heterotopic lesions appear to be of endothelial origin based on Tie2-Cre lineage tracing and expression of various endothelial in these cells. Endothelial-mesenchymal transition (EndMT) is an important pathological mechanism of heterotopic ossification.This project has proved that EndMT as well as HO could be inhibited by over-expression of Smad-7 gene which exists in the TGF-β signal transduction pathway in the previous study. So the hypothesis is EndMT and HO in the local tissue could be inhibited by the increasing of Smad-7 plasmid DNA. Two steps are involved in EndMT which include vascular endothelial cell-mesenchymal transformation and mesenchymal differentiate into osteoblast. So we propose a strategy to deliver the gene to the different target cells, vascular endothelial cell and mesenchymal cell for precision drug dosing. CD34 is chosen for target vascular endothelial cell. Target molecule for mesenchymal cell will be screened by SELEX technique. CD34 will be connected with lipid molecule and the aptamer will be connected with cholesterol molecule. These two kinds “connected molecules” will be introduced into the exosomes derived from red blood cell and mesenchymal stem cell for the “functional exosomes” for cell targeting. This project intends to validate the hypothesis preliminary by in vivo and in vitro experiments, also further discuss the mutual interaction and molecular mechanism between TGF-β and EndMT as well as HO in cells, tissues and animals, including the downstream signal of TGF-β signal transduction pathway. The proposed research will clarify the molecular mechanism of HO and lead to new strategy for prevention and gene therapy of HO by exosomes in precision drug dosing level.

异位骨化(HO)的发病原因及机制尚未清楚，有研究提示内皮细胞的间质转化(EndMT)可能是HO的主要发病机制之一。根据前期预实验发现慢病毒介导的Smad7 plasmid DNA(p DNA)可以明显抑制EndMT过程。据此推断局部增强Smad7基因表达，能够通过抑制EndMT治疗HO，但已形成的间质细胞可以继续转化成骨细胞。所以本项目拟通过功能化外泌体来实现对Smad7 pDNA的精准递送，以防治HO。选取红细胞和间充质干细胞来源的两种不同的外泌体作为载体，通过CD34抗体修饰实现对血管内皮细胞的靶向。通过SELEX技术筛选间质细胞的适配体来修饰外泌体，实现对间质细胞的靶向。从细胞水平和动物水平考察这种具有靶向不同细胞功能的外泌体在HO基因治疗中的效果。评价这种载药体系的基因装载能力的优势及基因靶向治疗效果。这种基于功能化外泌体的基因递送体系将为HO的防治及靶向载药提供新的技术及思路。

根据课题研究方向和任务计划，本研究提供了一种制备装载SMAD7基因的工程化外泌体的创新方法，显著地提高了外泌体转染血管内皮细胞的效率和SMAD7基因在细胞中的表达，从而更为有效地抑制了内皮-间充质细胞转化过程（EndMT），减少了异位骨化的发生。本研究成果为开发更为高效的异位骨化的预防和基因治疗方案提供了创新思路和实验依据。.为了提高SMAD7基因转染血管内皮细胞的效率，课题组制备了包含SMAD7表达质粒的外泌体Exosome-DSPE-PEG-AbCD34-SMAD7。通过将其与小鼠主动脉内皮细胞（MAECs）共培养，课题组利用qPCR和Western blot方法检测发现细胞中SMAD7的表达量比以前报道的外泌体方法有了显著的提高。 这些结果说明 Exosome-DSPE-PEG-AbCD34外泌体能够更加靶向、有效地将目标基因序列转送至靶向细胞中。毒性试验结果表明，Exosome-DSPE-PEG-AbCD34外泌体对细胞的毒性很小，是一种安全性高的基因转载工具。同时，Exosome-DSPE-PEG-AbCD34-SMAD7可以在TGF-β1刺激的小鼠主动脉内皮细胞模型中有效诱导过表达SMAD7并降低TGF-β1的表达水平，并通过升高CD31和VE-Cadherin表达和降低N-Cadherin及Vimintin的表达而抑制细胞模型中的EndMT转化。对异位骨化小鼠模型在造模后给予Exosome-DSPE-PEG-AbCD34-SMAD7治疗，可有效减少异位骨化的产生。综上，Exosome-DSPE-PEG-AbCD34-SMAD7诱导的SMAD7过表达可有效逆转内皮细胞中TGF-β1介导的EndMT转化并减少体内异位骨化的产生。本课题制备的Exosome-DSPE-PEG-AbCD34-SMAD7工程化外泌体为预防和治疗异位骨化提供了一定的理论创新和实验依据。 .课题研究者按照年度计划要点的计划进行，完成预期课题目标。 .目前该课题发表SCI论文3篇， 发表中文论文1篇。本课题研究培养研究生3名，申请并申请国家专利1项。