Development of a Polymeric Delivery System for Efficient in vivo Cellular Reprogramming for Cardiac Regeneration

项目摘要 本研究的目的是开发一种靶向的、可注射的质粒递送系统， 在体内将心脏成纤维细胞直接重编程为心肌细胞（CMC）， 心肌梗死（MI）或“心脏病发作”后的再生。心血管疾病（CVD）是 全球主要死因，仅2016年就导致1790万人死亡。心脏纤维化， 心血管疾病的主要原因是创伤性损伤，如心肌梗死。一个有希望的治疗途径是直接 将心脏成纤维细胞重编程为CMC以再生心肌组织并恢复功能 梗塞后的心脏组织最近的几项研究表明，转录因子的组合， 同时递送的Gata 4、Mef 2c和Tbx 5可以直接将心脏成纤维细胞重编程为CMC， 在体外和体内诱导CMC表型。不幸的是，虽然心脏成纤维细胞向CMC分化， 作为一种可能性，这种方法作为CVD治疗的实施是不可行的。基因递送 Ma实验室开发的载体（聚合复合物）已显示在体内驱动骨组织再生， 这是本研究所依据的一个重要先例。使用改进的复合物的初步数据 迄今为止，驱动了心脏成纤维细胞体外重编程为CMC，这是一个有希望的概念验证结果。在 在这项拟议的研究中，研究人员将通过改进基因来进一步改进最近开发的系统。 使用核定位信号（NLS）通过核靶向表达。初步结果表明 与单独的复合物相比，这改善了基因表达。此外，研究人员将直接推动 使用新开发的水凝胶支架进行体内重编程，以实现持续递送 复合物假设通过添加NLS和使用质粒载体持续递送质粒DNA， 当使用可注射水凝胶时，递送的质粒的核定位和量将增加，从而导致细胞内的细胞分裂。 成纤维细胞体内重编程为CMC的增加。这将使用PCR进行评价， 免疫组化比较基因表达，随后MRI评估心脏功能变化。 这项研究旨在为患有心脏病的患者建立第一个再生治疗方案。 攻击这项工作可能为有效的非病毒基因传递系统奠定框架，用于 治疗其他丢失或患病的组织，特别是其他组织/器官系统的纤维化。

PROJECT SUMMARY The goal of this study is to develop a targeted, injectable plasmid delivery system that is capable of efficient direct reprogramming of cardiac fibroblasts to cardiomyocytes (CMCs) in vivo, aiding in cardiac tissue regeneration following a myocardial infarction (MI) or ‘heart attack’. Cardiovascular disease (CVD) is the leading cause of death worldwide and resulted in 17.9 million deaths in 2016 alone. Cardiac fibrosis, a major contributor to CVD is which results from traumatic injury such as MI. A promising treatment avenue is direct reprogramming of cardiac fibroblasts into CMCs to regenerate the myocardial tissue and regain functional post-infarct heart tissue. Several recent studies have demonstrated that a combination of transcription factors, Gata4, Mef2c, and Tbx5 delivered simultaneously can directly reprogram cardiac fibroblasts to CMCs and induce CMC phenotype in vitro and in vivo. Unfortunately, while cardiac fibroblast to CMC differentiation is now a possibility, implementation of this approach as a CVD treatment has not been viable. The gene delivery vehicles (polyplexes) developed in the Ma Lab have been shown to drive bone tissue regeneration in vivo, setting an important precedent on which this study is based. Preliminary data using an improved polyplex has thus far driven in vitro reprogramming of cardiac fibroblasts into CMCs, a promising proof-of-concept result. In the proposed study, researchers will be further improving the recently developed system by improving the gene expression through nuclear targeting using a Nuclear Localization Signal (NLS). Preliminary results indicate this improves gene expression compared to polyplexes alone. Further, researchers will be driving direct reprogramming in vivo using a newly developed hydrogel scaffold to implement sustained delivery of the polyplexes. The hypothesis is that through addition of a NLS and sustained delivery of plasmid DNA using an injectable hydrogel, the nuclear localization and amount of plasmid delivered will be increased, resulting in an increase in in vivo reprogramming of fibroblasts into CMCs. This will be evaluated using PCR and immunohistochemistry to compare gene expression and later MRI to evaluate functional changes in the heart. This research aims to establish the first regenerative treatment option for patients who have suffered a heart attack. This work may likely lay the framework for an effective non-viral gene delivery system for use in treatment of other lost or diseased tissues, especially the fibrosis of other tissue/organ systems.