MICRORNA AND NEURAL FACTOR-MEDIATED DIRECT REPROGRAMMING OF CELL FATES

微小RNA和神经因子介导的细胞命运直接重编程

• 批准号: 8355743
• 负责人: Andrew Yoo
• 金额: $ 228万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8355743
• 关键词: Adult; Affect; Animal Model; Cells; Characteristics; Dermal; Disease; Disease model; Fibrinogen; Fibroblasts; Gene Mutation; Genes; Human; Individual; Inherited; Mediating; Methods; MicroRNAs; Mitotic; Modeling; Nerve Degeneration; Neuroglia; Neuronal Injury; Neurons; Pathogenesis; Pathology; Patients; Process; Protocols documentation; Skin; Therapeutic; abstracting; base; cell type; embryonic stem cell; human ASCL1 protein; in vivo; insight; nerve stem cell; nervous system disorder; novel; novel therapeutic intervention; public health relevance; relating to nervous system; restoration; tissue culture; tool; transcription factor; tumorigenic

DESCRIPTION (Provided by the applicant) Abstract: The ability to obtain neurons directly from the patients with neurological disorders will offer opportunities to study pathogenesis from the affected cells and develop novel therapeutic approaches. In hereditary neurological diseases, patient-specific neurons reprogrammed from non-neuronal cell types will harbor the same genetic mutation, thus offering valuable tools to study cell-autonomous pathology. For the disorders of somatic neurodegeneration, the isogenic, induced neurons may be used for cell replacement-based therapies. Most of current approaches towards deriving human individual-specific neurons have focused on transforming a differentiated cell type (for instance, dermal fibroblasts) to a pluripotent state and further differentiating them into neurons. This method requires forced expression of tumorigenic transcription factors that are highly expressed in embryonic stem cells. Moreover, differentiation of multipotent neural progenitors into specific subtypes of neurons is a difficult process to control. I have devised an alternative strategy of non-invasively obtaining neurons from adult human skin cells by converting their cell fate without going through pluripotent state (thus not requiring the expression of tumorigenic genes) and directly into post-mitotic neurons (direct reprogramming). I recently discovered that neuron-specific microRNAs (miR-9/9* and miR-124) could promote the switching of a non-neuronal cell fate into neurons when ectopically expressed with as few as one neural factor. The reprogramming efficiency was higher with more neural factors, and I devised a protocol in which miR-9/9* and -124 with neural factors, NeuroD2, ASCL1 and Myt1l generated neurons with cortical neuron-like characteristics. In this proposal, I will develop strategies to directly reprogram non-neuronal cells into various subtype-specific neurons. In developing tissue culture models of neurological diseases, it is important to obtain the type of neurons affected by the disease. Because the miR-9/9* and miR-124 are expressed pan-neuronally, I hypothesize that neuronal reprogramming can be customized for different subtypes of neurons by using transcription factors specific for the desired cell type in the background of miR-9/9*-124 expression. I will extend these studies to apply the direct reprogramming method in vivo from non-neuronal cells and hope to gain insights into possible restoration of the neuronal function lost in animal models of neuronal injury. Public Health Relevance: The ability to non-invasively obtain neurons from human individuals with neurological disorders will offer novel directions towards disease modeling and cell replacement-based therapeutic approaches. Here, we prose to devise methods to directly reprogram non-neuronal cell fates into subtype-specific neurons and develop in vivo application of this method in animal models of neuronal injury.

描述（由申请人提供） 翻译后摘要：直接从神经系统疾病患者获得神经元的能力将 为研究受影响细胞的发病机制和开发新的治疗方法提供了机会。在遗传性神经系统疾病中，从非神经元细胞类型重编程的患者特异性神经元将具有相同的基因突变，从而为研究细胞自主病理学提供了有价值的工具。对于躯体神经变性的病症，同基因的诱导神经元可用于基于细胞增殖的疗法。目前大多数用于获得人类个体特异性神经元的方法都集中在将分化的细胞类型（例如，真皮成纤维细胞）转化为多能状态并进一步将其分化为神经元。该方法需要强制表达在胚胎干细胞中高度表达的致瘤性转录因子。此外，多能神经祖细胞分化成特定亚型的神经元是一个难以控制的过程。我已经设计了一种替代策略，即通过转换其细胞命运而不经过多能状态（因此不需要表达致瘤基因）并直接转化为有丝分裂后的神经元（直接重编程），非侵入性地从成人皮肤细胞中获得神经元。我最近发现，神经元特异性microRNA（miR-9/9* 和miR-124）可以促进非神经元细胞的命运转换为神经元时，异位表达少至一个神经因子。随着神经因子的增加，重编程效率更高，我设计了一种方案，其中miR-9/9* 和-124与神经因子NeuroD 2，ASCL 1和Myt 1 l一起产生具有皮质神经元样特征的神经元。在这个建议中，我将开发策略，直接将非神经元细胞重编程为各种亚型特异性神经元。在开发神经系统疾病的组织培养模型时，重要的是获得受疾病影响的神经元的类型。由于miR-9/9* 和miR-124在全神经元表达，因此我假设可以通过在miR-9/9*-124表达的背景下使用对所需细胞类型特异性的转录因子来针对不同神经元亚型定制神经元重编程。我将扩展这些研究，应用直接重编程方法在体内从非神经元细胞，并希望获得洞察神经元损伤的动物模型中失去的神经元功能的可能恢复。 公共卫生相关性：从患有神经系统疾病的人类个体中非侵入性地获得神经元的能力将为疾病建模和基于细胞增殖的治疗方法提供新的方向。在这里，我们散文设计的方法，直接重编程非神经元细胞的命运亚型特异性神经元和开发这种方法在动物模型中的体内应用神经元损伤。