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

项目概要 本研究的目标是开发一种靶向、可注射的质粒递送系统，该系统能够有效地 体内心脏成纤维细胞直接重编程为心肌细胞 (CMC)，有助于心脏组织 心肌梗塞（MI）或“心脏病发作”后的再生。心血管疾病（CVD）是 是全球主要死亡原因，仅 2016 年就导致 1,790 万人死亡。心脏纤维化是一个重要的疾病 CVD 的诱因是由 MI 等外伤引起的。一种有前景的治疗途径是直接治疗 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 Gata4、Mef2c 和 Tbx5 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 的分化现在还没有进展。 一种可能性是，这种方法作为 CVD 治疗的实施并不可行。基因传递 Ma 实验室开发的载体（复合物）已被证明可以在体内驱动骨组织再生， 开创了本研究的重要先例。使用改进的聚合体的初步数据 迄今为止，在体外将心脏成纤维细胞重编程为 CMC，这是一个有希望的概念验证结果。在 在拟议的研究中，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) 通过核靶向进行表达。初步结果表明 与单独的聚合复合物相比，这提高了基因表达。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 聚合体。假设是通过添加 NLS 并使用 可注射水凝胶，核定位和递送的质粒数量将增加，从而导致 增加体内成纤维细胞重编程为 CMC 的能力。这将使用 PCR 进行评估，并 免疫组织化学比较基因表达，随后进行 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.