Novel nanoscale approaches to whole tissue reprogramming

摘要： 这项申请的重点是开发新的血管生成细胞疗法来治疗脑血流 (CBF)阿尔茨海默病（AD）的缺陷。目的是通过预编程成纤维细胞以改善AD中的CBF， 转化为诱导内皮细胞（iECs），以驱动大脑和干细胞中新血管组织的形成。 AD是最常见的痴呆症，目前影响着数百万人， 国际吧目前，AD没有可用的治疗方法，治疗主要集中在管理 症状许多研究表明，在患者和小鼠模型中，CBF缺陷和AD之间存在明确的联系。 AD. CBF缺陷先于淀粉样β蛋白斑块和磷酸化tau蛋白缠结的形成， 与AD相关的认知能力下降。此外，最近在AD小鼠模型中的研究表明， 旨在增加CBF的药理学策略导致行为缺陷的快速改善。采取 总之，这些观察结果表明，受损的CBF在AD中起关键作用，增加CBF可以 是治疗或预防AD的可行策略。在我的新创新者奖（DP 2）下，我们一直致力于 基于纳米转染的开发（即，非病毒）、重编程驱动的细胞疗法 中风最近，我们报道了一种新的基于重编程的血管生成细胞疗法，用于治疗 缺血性中风在这项研究中，我们发现，已经预先编程转化为iEC的成纤维细胞， 在体内，可以改善患有缺血性中风小鼠的皮质灌注，增强中风恢复， 并且与用对照成纤维细胞处理的小鼠相比减少运动损伤。IEC可以从以下公式推导出： 通过纳米转染转录因子基因ETV 2、FOXC 2和FLI 1（EFF），在成纤维细胞中表达。纳米转染 细胞还可以释放细胞外囊泡（EV）负载的EFF，其具有有效的血管生成/血管生成功能。 效果，并有助于放大IEC指导的转换。有希望的试点研究是为了应对令人兴奋的 中风数据支持这样的科学前提，即预编程产生iEC的成纤维细胞也可以增加 CBF和减少AD小鼠模型中的认知缺陷。在这里，我们建议建立在这些惊人的 首次观察研究了iEC指导的成纤维细胞重编程和/或细胞负载的细胞重编程， EV可以是对抗AD下CBF缺陷和病理进展的可行策略。目前 缺乏关于血管生成细胞或EV疗法的研究，旨在解决CBF缺陷，以预防，延迟， 或反向AD。因此，本文提出的工作具有高度创新性和潜在的变革性。后 完成后，我们希望严格证明了EFF纳米转染成纤维细胞或EFF的益处， 在预防/治疗AD中的负载EV。本研究的结果将用于未来 使用患者来源的细胞对AD进行纳米转染驱动的细胞/EV疗法的安全性和有效性的研究。

ABSTRACT: This application focuses on the development of novel vasculogenic cell therapies to treat cerebral blood flow (CBF) deficits in Alzheimer’s disease (AD). The goal is to improve CBF in AD by pre-programming fibroblasts to convert into induced endothelial cells (iECs) to drive the formation of new vascular tissue, in the brain, and stem the progression of AD, which is the most common form of dementia, currently affecting millions of people worldwide. Currently, there is no available therapy for AD, and treatments are mainly focused on managing symptoms. Numerous studies show a clear link between CBF deficits and AD in patients and murine models of AD. CBF deficits precede the formation of amyloid beta plaques and phosphorylated tau tangles, as well as the cognitive decline associated with AD. Moreover, recent studies in murine models of AD show that pharmacological strategies aimed at increasing CBF lead to a rapid improvement in behavioral deficits. Taken together, these observations suggest that impaired CBF plays a key role in AD, and that increasing CBF could be a viable strategy to treat or prevent AD. Under my New Innovator Award (DP2), we have been working on the development nanotransfection-based (i.e., non-viral), reprogramming-driven cell therapies for ischemic stroke. Recently we reported on a novel reprogramming-based vasculogenic cell therapy for the treatment of ischemic stroke. In this study we showed that fibroblasts that have been pre-programmed to convert into iECs, in vivo, can improve cortical perfusion in mice that have suffered an ischemic stroke, enhance stroke recovery, and reduce motor impairment compared to mice treated with control fibroblasts. iECs can be derived from fibroblasts via nanotransfection of transcription factor genes, ETV2, FOXC2, and FLI1 (EFF). Nanotransfected cells can also release extracellular vesicles (EVs) loaded EFF, which have potent vasculogenic/angiogenic effects, and can help to amplify iEC-directed conversions. Promising pilot studies run in response to the exciting stroke data support the scientific premise that fibroblasts pre-programmed to generate iECs can also increase CBF and reduce cognitive deficits in a mouse model of AD. Here we are proposing to build upon these striking observations to study, for the first time, whether iEC-directed reprogramming of fibroblasts, and/or EFF-loaded EVs, can be a viable strategy to counteract CBF deficits and pathology progression under AD. Currently, there is a paucity of research on vasculogenic cell or EV therapies aimed at addressing CBF deficits to prevent, delay, or reverse AD. As such, the work proposed herein is highly innovative and potentially transformative. Upon completion, we expect to have rigorously demonstrated the benefits of EFF-nanotransfected fibroblasts or EFF- loaded EVs in the prevention/treatment of AD. Outcomes from this study will be leveraged to pursue future studies on the safety and efficacy of nanotransfection-driven cell/EV therapies for AD using patient-derived cells.