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.
Schwann cell transplantation could, in the future, become a treatment for spinal cord injuries and some neuropathies. However, it is not currently possible to produce large numbers of cells suitable for transplantation. This proposal seeks to develop methods to efficiently reprogram fibroblasts by creating Cas9-based artificial transcription factors, which would enable the simultaneous and targeted activation of hundreds of genes. We anticipate that this approach will substantially improve the efficiencies of existing reprogramming protocol, as well as enable reprogramming to previously unobtainable cell types, such as Schwann cells.
