Induced pluripotent stem cells (iPSC) have tremendous potential for regenerative medicine in future. However, there are obstacles to overcome before the iPSC can be used therapeutically. One major hurdle is the tool used to generate iPSC from adult somatic cells. We believe that the delivery of reprogramming proteins directly rather than the genes that encode these proteins is the safest means for generating iPSC. We propose to engineer Clostridium difficile toxin TcdB as a cytosolic delivery vehicle for embryonic transactivators that can reprogram adult somatic cells to induced pluripotent stem cells (iPSC) and/or tissue specific cells. C. difficile toxin TcdB, exhibits multiple modular domains that provide all the mechanisms that allow the toxin efficiently deliver its N-terminal enzymatic domain (GT domain) to cytosol. We will engineer TcdB as a universal protein delivery vehicle by replaceing the enzymatic domain with a protein """"""""cargo"""""""" to provide therapeutic benefits. We will initially append a chimeric transacivator to the N-terminus of the toxin or replace the GT domain with the chimeric transactivator. Using a high-throughput reporter cell line system, we will first examine how effectively the engineered recombinant protein can deliver a biologically active """"""""cargo."""""""" We will then test if the engineered TcdB can deliver biologically active reprogramming factors (Oct4 and Sox2) to another reporter cell line and assess their ability to differentiate mouse embryonic stem cells with the help of our collaborators. Finally, we will use the TcdB delivery vehicle to generate of iPSC from mouse embryonic fibroblasts after we demonstrate that we can deliver those reprogramming factors efficiently to the well-characterized reporter cells. Other methods of delivering proteins, such as the TAT protein transduction domain, will be tested in parallel to compare the efficiency of our novel system to established techniques. We believe this TcdB-based protein delivery vehicle will be a valuable tool for generating iPSC for research purposes, drug development, and toxicology studies. We also anticipate the development of TcdB-based biologics for regenerative medicine.
This project will investigate the use of a new protein delivery vehicle to introduce reprogramming factors to adult skin cells in order to convert them to induced pluripotent stem cells (iPSC). This approach relies on proteins rather than genes to create iPSC, which avoids the potential dangers of genetic modification. These iPSC have enormous potential to replace embryonic stem cells for regenerative medicine.
