Novel nanoscale approaches to whole tissue reprogramming

摘要： 这一应用侧重于开发新的血管生成细胞疗法来治疗脑血流 阿尔茨海默病(AD)的脑血流(CBF)缺陷。我们的目标是通过预先编程成纤维细胞来改善AD患者的CBF 转化为诱导内皮细胞(IECs)，以在脑和干细胞中驱动新血管组织的形成 阿尔茨海默病是痴呆症最常见的形式，目前影响着数百万人 全世界。目前，AD尚无有效的治疗方法，治疗主要集中在管理上 症状。大量研究表明，脑血流量不足与患者和小鼠AD模型之间存在明显的联系 广告。CBF缺陷先于淀粉样β斑块和磷酸化tau缠结的形成，以及 与阿尔茨海默病相关的认知衰退。此外，最近对阿尔茨海默病小鼠模型的研究表明， 旨在增加CBF的药物策略会导致行为缺陷的快速改善。已被占用 综上所述，这些观察表明CBF受损在AD中起着关键作用，而CBF增加可能 是治疗或预防阿尔茨海默病的可行策略。在我的新创新者奖(DP2)下，我们一直在努力 基于纳米转染法(即非病毒)、重编程驱动的细胞治疗缺血性疾病的研究进展 卒中。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，用于治疗骨肉瘤 缺血性中风。在这项研究中，我们发现已经被预先编程为转化为IECs的成纤维细胞， 在体内，可以改善缺血性中风小鼠的皮质血流，促进中风的恢复， 与对照成纤维细胞处理的小鼠相比，减少了运动损伤。IECS可以派生自 通过纳米转染转录因子基因ETV2、FOXC2和FLI1(Eff)诱导成纤维细胞。纳米转染体 细胞还可以释放细胞外小泡(EVS)，这些囊泡具有强大的血管生成/血管生成功能 效果，并有助于放大IEC指令的转换。前景看好的试点研究正在进行，以回应令人兴奋的 中风数据支持这样的科学前提，即预先编程生成IECs的成纤维细胞也可以增加 并减少阿尔茨海默病小鼠模型的认知缺陷。在这里，我们提议在这些打击的基础上 首次研究IEC引导的成纤维细胞重编程和/或EFF负载的观察 EVS可以成为一种可行的策略来对抗AD下的CBF缺陷和病理进展。目前，有 关于血管生成细胞或EV疗法的研究很少，旨在解决CBF缺陷以预防、延迟、 或者逆转AD。因此，本文提出的工作具有很高的创新性和潜在的变革性。vt.在.的基础上 完成后，我们希望已经严格地证明了EFF-纳米转基因成纤维细胞或EFF- EVS在AD防治中的应用这项研究的结果将被用来追求未来 使用患者来源的细胞进行纳米转基因细胞/EV治疗AD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.