Virtually all cells in our body possess an identical genetic code. However, the expression of this code is regulated by a multitude of factors that coordinate which portions of the genetic code can be read and interpreted. We further know that these so called epigenetic factors change drastically through development, and patterns of epigenetic marks control changes to cellular identity. The proposed research aims to site-specifically change key epigenetic marks, altering which DNA instructions are available to pluripotent cells and changing their behavior during cell fate commitment. We describe a technique to bind custom DNA binding proteins (TALES) to critical regulatory regions of known cell fate determining genes. TALE binding changes local DNA chromatin structures and implants instructions for becoming desirable cell types when given an appropriate environmental cue. This novel and potentially transformative approach, termed "epigenetic engineering" (EE), is proposed here to improve stem cell differentiation towards more pure and functionally mature cardiomyocytes. This will not only be the first example of guiding cell fate through gene-specific epigenetic manipulation, but will improve the generation of necessary cardiomyocyte types for cell replacement following heart attack. Lastly, EE also is likely to be adaptable to numerous differentiation strategies for routine production of many regenerative medicine cell types.
Statement of Benefit to California:
Heart disease and myocardial infarction represent the primary cause of death in industrialized nations. In California, greater than 1 out of 4 deaths are due to heart disease. The health and financial burdens are enormous. Currently, the most promising new strategies for heart regeneration involve replacement of dead or damaged tissue with cardiomyocytes (CMs) derived from pluripotent stem cells; however, the control of pluripotent cell fate is problematic. Pluripotent-derived-CMs are typically impure and functionally immature, so they may not contribute to heart contractility effectively or safely. We propose the development of a novel technology, termed “epigenetic engineering (EE),” to control cell fate and improve yields of mature CMs. EE involves the stable manipulation of stem cell chromatin structures, without DNA breaks or mutations, so as to “set the stage” for correct differentiation. When given an appropriate environmental cue, the engineered stem cells can then interpret these instructions to guide their cell fate commitment. This technology will improve the routine production of pure and functional CMs for cell replacement strategies that could significantly augment the current standard of care. Also, and highly important, EE is likely to be adaptable to alternative differentiation strategies (e.g. insulin producing beta-cells) and therefore potentially transformative for regenerative medicine.