USING CAS9 ATFS TO ALTER TRANSCRIPTION NETWORKS AND CONVERT FIBROBLASTS TO GLIA

使用 CAS9 ATFS 改变转录网络并将成纤维细胞转化为胶质细胞

• 批准号: 8930207
• 负责人: JEFFREY D MILBRANDT
• 金额: $ 22.88万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8930207
• 关键词: Binding; Cardiac Myocytes; Cell Count; Cell Therapy; Cell Transplantation; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Computing Methodologies; Coupled; Demyelinating Diseases; Development; Fibroblasts; Future; Gene Activation; Genes; Genetic; Genetic Transcription; Genomics; Guide RNA; Health; Hematopoiesis; Methodology; Methods; Neuroglia; Neurons; Neuropathy; Oligodendroglia; Population; Production; Proteins; Protocols documentation; Regulation; Schwann Cells; Spinal cord injury; System; Technology; Transcriptional Activation; Transplantation; activating transcription factor; base; cell type; differential expression; functional genomics; gene repression; improved; induced pluripotent stem cell; mutant; novel; research study; small molecule; transcription factor; transdifferentiation

DESCRIPTION (provided by applicant): Schwann cell transplantation holds great promise for the treatment of spinal cord injuries and some neuropathies. In addition, Schwann cell functions are coming under wider scrutiny due to their potential importance in hematopoiesis. A major bottleneck hindering the progress of Schwann cell-based therapy and Schwann cell functional genomics is the lack of methods to produce large numbers of transplantable cells and the easy perturbation of their genetic network. Recently, it has become possible to reprogram fibroblasts into different cell types by expressing a small number of transcription factors. However, the efficiencies are typically low, and only a few cell types (e.g. neurons, cardiomyocytes, oligodendrocytes) have been produced to date. We propose to overcome these difficulties by creating artificial transcription factors (ATFs) based on the Cas9 protein. Cas9 can be directed to bind specific genomic sequences using "guide RNAs", so it will possible to specifically activate hundreds or even thousands of genes. We will use Cas9 ATFs to reprogram fibroblasts into neurons and Schwann cells by activating transcription factors that are specific to these cell types. We anticipate that this approach will substantially improve the efficiencies of existing transdifferentiation protocols (for conversion into neurons), as well as enable transdifferentiatio to previously unobtainable cell types (Schwann cells). Our preliminary experiments suggest our strategy is feasible. We have demonstrated that Cas9 ATFs can achieve potent gene activation (>100 fold), and we have developed computational methods to predict the sets of genes required for transdifferentiation. Our specific aims are as follows: 1) To determine the rules that govern gene activation by Cas9-based artificial transcription factors (ATFs). 2) To develop tunable Cas9 mutant proteins bearing transcriptional activation or repression domains wherein their activity can be controlled by addition of small molecules to enable regulable perturbation of large-scale genetic networks. 3) To transdifferentiate fibroblasts into Schwann cells or their precursors by simultaneously activating the expression of 75-100 transcription factors that are differentially expressed between these two cell types.

描述（由申请人提供）：许旺细胞移植对脊髓损伤和某些神经病的治疗有很大的希望。此外，由于许旺细胞在造血中的潜在重要性，许旺细胞功能正在受到更广泛的审查。阻碍基于雪旺细胞的治疗和雪旺细胞功能基因组学进展的主要瓶颈是缺乏产生大量可移植细胞的方法以及它们的遗传网络容易受到干扰。最近，通过表达少量转录因子将成纤维细胞重编程为不同的细胞类型已经成为可能。然而，效率通常较低，并且迄今为止仅产生了少数细胞类型（例如神经元、心肌细胞、少突胶质细胞）。我们建议通过创建基于Cas9蛋白的人工转录因子（ATF）来克服这些困难。Cas9可以使用“向导RNA”定向结合特定的基因组序列，因此它将有可能特异性激活数百甚至数千个基因。我们将使用Cas9 ATF通过激活对这些细胞类型特异性的转录因子将成纤维细胞重编程为神经元和许旺细胞。我们预计，这种方法将大大提高现有转分化方案（转化为神经元）的效率，并使转分化成为以前无法获得的细胞类型（许旺细胞）。初步实验表明，该策略是可行的。我们已经证明了Cas9 ATF可以实现有效的基因激活（>100倍），并且我们已经开发了计算方法来预测转分化所需的基因集。我们的具体目标如下：1）确定规则， 通过基于Cas9的人工转录因子（ATF）控制基因激活。2)为了开发携带转录激活或阻遏结构域的可调Cas9突变体蛋白，其中它们的活性可以通过添加小分子来控制，以使得能够对转录激活或阻遏结构域进行可调控的扰动。 大规模的基因网络。3)通过同时激活75-100种在这两种细胞类型之间差异表达的转录因子的表达，将成纤维细胞转分化为许旺细胞或其前体细胞。