Cardiac Revascularization with Direct Reprogramming Approaches

项目摘要 缺血性心脏病是发病率和死亡率的主要原因。这些潜在的问题 疾病是血管的损失或功能障碍以及新血管形成不足。虽然细胞疗法 作为促进血管生长的有希望的选择出现，但临床上还没有可用的治疗方法， 还有很大的改进空间。骨髓（BM）衍生细胞的潜力被证明是最小的9， 胚胎或诱导多能干细胞（ESC/iPSC）衍生的EC难以维持，生产成本高， 并且可能引起副作用。为了避免这些问题，一种新的方法，称为直接重编程或直接 已经开发了转化，其中体细胞通过过表达转化为其它谱系细胞 谱系或细胞类型特异性转录因子（TF）。这种方法允许更简单和更安全的靶细胞 生成，并具有更方便的临床翻译的潜力。我们已经尝试直接 使用七种内皮相关TF的组合对EC进行重编程，并首次证明 ETV 2单独足以将人成纤维细胞转化为EC的时间。然而，由于我们使用了慢病毒 与其他载体一样，这些rEC在临床适用性方面具有限制。 用于治疗的直接EC重编程方法有两种选择：细胞治疗或直接体内重编程。 对于临床应用，两者都需要更安全的递送载体以最小化基因组整合的可能性。 因此，我们开发了腺病毒-ETV 2（Ad-ETV 2）载体。细胞疗法的另一个重要障碍是短- 移植细胞的长期存活。为了克服这个问题，我们研究了 用于延长细胞存活和治疗效果的生物材料。我们最近发现， 两亲性（PA）纳米基质凝胶非常有效，将人iPSC衍生的EC的存活延长超过10 个月，并在体内诱导连续的血管形成。 在这项研究中，首先，我们将首先开发基于细胞的治疗。我们将产生临床上相容的rEC， 并产生将这些rEC与各种类型PA组合的最佳PA构建体 纳米基质凝胶，包括新开发的一氧化氮（NO）释放PA。然后我们将确定 所选rEC-PA纳米基质构建体对啮齿动物缺血心脏的血管化和治疗作用 疾病模型第二，我们将研究Ad-ETV 2直接递送到心肌缺血模型中是否 可以直接将成纤维细胞重编程为功能性内皮细胞并在体内诱导血管形成。我们将使用 转基因小鼠来跟踪体内成纤维细胞向EC的命运。该项目的目标是开发 临床上适用的心脏血运重建策略，采用新型直接重编程方法， 组织工程技术。如果成功，这项研究将为广泛的 缺血性心脏病的研究和治疗领域。

Project Summary Ischemic heart diseases are the leading cause of morbidity and mortality. The underlying problems of these diseases are loss or dysfunction of blood vessels and insufficient new vessel formation. While cell therapy has emerged as a promising option to promote blood vessel growth, no therapy is yet clinically available and there is much room for improvement. The potential of bone marrow (BM)-derived cells turned out to be minimal9, and embryonic or induced pluripotent stem cell (ESC/iPSC)-derived ECs are difficult to maintain, costly to produce, and may cause side effects. To avoid these problems, a new approach, called direct reprogramming or direct conversion has been developed, in which somatic cells are converted into other lineage cells by overexpression of lineage- or cell-type specific transcription factors (TFs). This approach allows simpler and safer target cell generation and has the potential for more convenient clinical translation. We have attempted this direct reprogramming toward ECs using combinations of seven endothelial-related TFs and demonstrated for the first time that ETV2 alone is sufficient to convert human fibroblasts into ECs. However, since we used a lentiviral vector like others, these rECs have restrictions in clinical applicability. The direct EC reprogramming approach for therapy has two options: cell therapy or direct in vivo reprogramming. For clinical application, both require a safer delivery vector to minimize the possibility of genomic integration. Thus, we developed an adenoviral-ETV2 (Ad-ETV2) vector. Another important obstacle for cell therapy is short- term survival of the transplanted cells. To overcome this problem, we have investigated the potential of biomaterial for prolongation of the cell survival and therapeutic effects. We recently showed that peptide amphiphile (PA) nanomatrix gel is very effective, extending survival of human iPSC-derived ECs longer than 10 months and inducing continuous vessel formation in vivo. In this study, first, we will first develop cell-based therapy. We will generate clinically compatible rECs from human fibroblasts and generate an optimal PA construct combining these rECs with various types of PA nanomatrix gel including a newly developed nitric oxide (NO)-releasing PA. We will then determine the vascularization and therapeutic effects of the selected rEC-PA nanomatrix constructs on rodent ischemic heart disease models. Second, we will investigate whether direct delivery of Ad-ETV2 into cardiac ischemia models can directly reprogram fibroblasts into functional endothelial cells and induce vascularization in vivo. We will use genetically modified mice to track the fate of fibroblasts toward ECs in vivo. The goal of this project is to develop clinically applicable cardiac revascularization strategy using a novel direct reprogramming approach combined with tissue engineering technologies. If successful, this study will provide next-generation platforms for broad areas of research and therapy for ischemic heart diseases.