Generating transplantable neurons by in vivo combinatorial screening of transcrip

通过体内转录组合筛选产生可移植神经元

• 批准号: 8142682
• 负责人: Mehmet Fatih Yanik
• 金额: $ 80.25万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8142682
• 关键词: Address; Adult; Affect; Animals; Biological; Biological Assay; Brain; Cell Lineage; Cell Transplants; Cells; Chemicals; Clinical; Clinical Trials; Cues; Degenerative Disorder; Disease; Epilepsy; Evaluation; Eye diseases; Genetic; Human; Huntington Disease; In Vitro; Individual; Laboratories; Mediating; Methodology; Modification; Molecular; Neuronal Injury; Neurons; Parkinson Disease; Physical therapy; Preparation; Process; RNA; Recovery of Function; Rodent; Screening procedure; Signal Transduction; Site; Solutions; Staging; Stroke; Symptoms; Technology; Testing; Tissue Transplantation; Tissues; Transplantation; Xenograft procedure; aging population; cell type; combinatorial; fetal; functional improvement; high throughput screening; in vivo; induced pluripotent stem cell; innovation; neuronal growth; neuronal survival; nuclear reprogramming; release factor; research study; restoration; retina transplantation; synaptogenesis; transcription factor

Problem: Neuronal injuries, degenerative diseases, and disorders such as Parkinson¿s and Huntington¿s diseases, epilepsy, and stroke affect tens of millions of individuals in the USA alone, and are becoming a more severe problem with the aging population. Although significant effort is being invested for identification of the molecular processes involved, existing chemical and physical therapies do not promise restoration of lost neuronal circuits beyond the short term remedy of symptoms. A potential solution could be the use of tissue transplantation to restore neuronal function. There have been human trials where transplantations, although variably, have resulted in functional recovery in Parkinson¿s (PD) and Huntington¿s (HD) diseases. Cell replacement for epilepsy and stroke has shown promise in several rodent studies, and the transplantation of retinal cells to treat degenerative eye diseases is under evaluation in several laboratories. Although there are still many unknowns, in various cases, it has been observed that functional improvements occurred owing to the integration of grafted neurons into existing neuronal networks, and was not simply due to trophic factors released by the transplanted cells. Several studies have also shown that the adult brain is remarkably capable of providing signaling cues that guide the growth of neuronal processes and induce formation of synapses with desired targets when correct cell types are present within the grafts. Xenograft studies with animals larger than rodents have shown that these cues can function over long distances. However, while tissue transplantation may have significant potential, there are many scientific unknowns and several fundamental challenges exist as outlined in this proposal. Handling complexity of these challenges is currently beyond the capabilities of the largest laboratories in the world, and will likely require deployment of systematic high-throughput approaches that will not only address fundamental biological questions and rapidly test various hypotheses without bias, but also provide results that are, if promising, translatable to clinical trials. Challenges: (1) Human fetal or iPS-derived cells are either too scarce or tumorogenic for clinical use, (2) Transplanted cells are too heterogeneous, (3) Preparation of transplanted cells in the correct and synchronized stages is currently impossible, (4) Physical site of transplantation significantly varies from experiment to experiment, (5) Existing in vivo transplantation assays are too slow for screening of multitudes of different hypotheses. Innovation & Methodology: Here, we propose a systematic, unbiased, in vivo, large-scale, and high throughput approach for overcoming these challenges to in vitro differentiation and in vivo testing of transplanted neuronal tissues. The proposed methodologies here are applicable to most transplantation paradigms. The key technologies and strategies we will develop include: (A) RNA-mediated nuclear reprogramming without genetic modification, (B) Reprogramming human cell lineages by systematic ultra-high-throughput screening of RNA transcription factor cocktails using a massively parallel technology, (C) High-throughput transplantation of human cells into rodents (with minimal rodent sacrifice) and in vivo analysis of neuronal survival and integration.

问题：神经元损伤，退化性疾病以及诸如帕金森氏症和亨廷顿疾病，癫痫病和中风等疾病，仅在美国就会影响数千万个人，并且正在成为老龄化人口的更严重的问题。尽管正在投入大量努力来识别所涉及的分子过程，但现有的化学和物理疗法并不能保证恢复损失的神经元电路以外的短期疗法。潜在的解决方案可能是使用组织移植来恢复神经元功能。有人类试验，尽管移植可变，但导致帕金森（PD）和亨廷顿疾病（HD）疾病导致功能恢复。在几项啮齿动物研究中，癫痫和中风的细胞置换已显示出希望，并且在几个实验室中评估了残留细胞以治疗退行性眼部疾病的残留细胞。尽管仍然有许多未知数，但在各种情况下，已经观察到，功能性改进发生在将移植神经元整合到现有神经元网络中，这不仅是由于移植细胞释放的营养因子所致。几项研究还表明，成年大脑非常能够提供信号提示，以指导神经元过程的生长并在移植物中存在正确的细胞类型时诱导具有所需靶标的突触的形成。对大于啮齿动物的动物的异种移植研究表明，这些提示可以长距离运行。但是，尽管组织移植可能具有巨大的潜力，但仍有许多科学的未知数，如本提案中概述的那样存在一些基本挑战。目前，处理这些挑战的复杂性超出了世界上最大的实验室的能力，并且很可能需要部署系统的高通量方法，这些方法不仅可以解决基本的生物学问题，并迅速测试没有偏见的各种假设，而且还提供了结果，如果可以，如果可以，如果可以（如果可以）转换为临床试验。挑战：（1）人类胎儿或IPS衍生的细胞过于稀缺或无法进行临床使用，（2）移植细胞过于异质，（3）当前不可能在正确和同步阶段进行移植细胞的准备，目前是不可能的，（4）实验的较大范围是较大的群体变化（5）实验的变化（5）（5）VIV（5）VIV（5）VIV（5）VIV（5）VIV（5）VIV（5）VIV（5）假设。创新与方法论：在这里，我们提出了一种系统的，无偏见的体内，大规模和高吞吐量方法，以克服这些挑战以在体外分化和体内测试移植的神经元时间。这里提出的方法适用于大多数移植范例。我们将制定的关键技术和策略包括：（a）RNA介导的核重编程而无需遗传修饰，（b）通过系统的超高通行筛查对RNA转录因子鸡尾酒的重新编程，使用大量平行技术对RNA转录因子鸡尾酒进行鸡尾酒，（c）将人类细胞的高直接转移到Minim andar andim andar andal andal vivo和vivo vivo和vivo中。