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

描述(申请人提供)：雪旺细胞移植在治疗脊髓损伤和一些神经疾病方面前景看好。此外，雪旺细胞的功能正受到更广泛的审查，因为它们在造血方面具有潜在的重要性。阻碍雪旺细胞治疗和雪旺细胞功能基因组学进展的一个主要瓶颈是缺乏产生大量可移植细胞的方法及其遗传网络的容易扰动。最近，通过表达少量的转录因子，可以将成纤维细胞重新编程为不同的细胞类型。然而，效率通常很低，到目前为止只培养出了几种细胞类型(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可以被指示使用“引导RNA”来结合特定的基因组序列，因此将有可能特异性地激活数百甚至数千个基因。我们将使用Cas9 ATF通过激活这些细胞类型特有的转录因子，将成纤维细胞重新编程为神经元和雪旺细胞。我们预计，这种方法将大大提高现有转分化方案(用于转化为神经元)的效率，并使转分化成为以前无法获得的细胞类型(雪旺细胞)。我们的初步实验表明我们的策略是可行的。我们已经证明了Cas9ATF可以实现强大的基因激活(&gt；100倍)，我们已经开发出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)确定符合以下条件的规则 通过基于Cas9的人工转录因子(ATF)控制基因激活。2)开发具有转录激活或抑制结构域的可调节的Cas9突变蛋白，其中它们的活性可以通过添加小分子来控制，以实现对 大规模的遗传网络。3)通过同时激活在这两种细胞类型之间差异表达的75-100转录因子的表达，将成纤维细胞转化为雪旺细胞或其前体细胞。

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.