525. Directed Molecular Evolution of Transcription Factors in Mammalian Cells for Enhanced Directed Cell Differentiation
Authors: Ami M. Kabadi, Tyler S. Klann, Malathi Chellappan, Jennifer Kwon, Timothy E. Reddy, Charles A. Gersbach
Journal: Molecular Therapy (2016)

The advent of cellular reprogramming has introduced new possibilities for regenerative medicine, drug discovery, and controlling cellular behavior. Recent studies have identified many natural transcription factors that are capable of directing lineage-specific differentiation in adult somatic cells. However, genetic reprogramming is often inefficient and highly variable, making it difficult to obtain sufficient quantities of completely reprogrammed cells, and thus unrealistic as a reliable source for cellular therapies and regenerative medicine applications. Directed evolution presents a well-established and currently unexplored approach for improving the intrinsic properties of reprogramming transcription factors. MyoD is the master transcription factor that defines the myogenic lineage. Overexpression of MyoD in certain cell types upregulates the myogenic gene network and induces differentiation to a myogenic phenotype. This process is well characterized and therefore provides an ideal model for developing a directed evolution scheme for mammalian transcription factors. Here we present a mammalian-based directed evolution system that enhances transcription factor-mediated genetic reprogramming in mammalian cells. A quantitative and high-throughput selection system was engineered using a cell reporter assay to isolate enhanced MyoD variants from a library of mutated gene sequences. During skeletal myoblast differentiation, MyoD directly activates expression of the myogenin transcription factor. Myogenin is one of the first markers of muscle maturation and marks commitment to myocyte differentiation. It is also important for cell fusion into functional multinucleated myotubes. Therefore, we hypothesized that engineering MyoD variants with an enhanced ability to induce expression of myogenin would lead to more effective and efficient genetic reprogramming. For this purpose, we engineered an isogenic myogenin-eGFP reporter cell line. The library of MyoD mutants were packaged into a lentiviral vector and delivered to the reporter cell line for selection. Cells with high GFP expression are indicative of hyperactive mutants with an enhanced ability to activate the target promoter and were isolated using fluorescence activated cell sorting. The variants were recovered from the genomic DNA of these sorted cells by PCR. This new library of mutants was recloned into the lentiviral vector for subsequent rounds of selection. Following ten rounds of selection, the resulting libraries were evaluated for enrichment of amino acid substitutions through next generation sequencing. Variants of interests were isolated and tested individually for their ability to induce myogenic differentiation in human cells. In this study, we demonstrate that directed evolution can be used to identify single amino acid substitutions within the master transcription factor MyoD that improve the conversion of human fibroblasts to a skeletal myocyte phenotype. This study presents a generalizable approach for enhancing the activity of mammalian transcription factors.