My long career goal is to become an independent clinical research scientist and a leader in developing novel therapies for myotonic dystrophy. To continue my progress towards this goal, I propose to explore approaches to correct the mutant gene in Myotonic Dystrophy type 1 (DM1) induced pluripotent stem (iPS) cells. I have been trained in conducting scientific research in my MD, PhD student training and postdoctoral studies. I believe the K08 mentored career development award will greatly facilitate me to become an independent research scientist. I have developed a career development plan with my mentor. I have assembled a strong mentor committee. University of Florida, McKnight Brain Institute, Department of Neuroscience and Neurology, Center for Cellular Reprogramming and Center for NeuroGenetics provide an excellent environment for me to pursue the long term goal. My short-term goal is to use DM1 iPS cells as a platform to test our hypothesis that mutant gene in DM1 iPS cells can be efficiently corrected using transcription activator-like effector nuclease (TALEN) technology. I have developed a research project for this goal. DM1 is caused by CTG nucleotide repeat expansion within the dystrophia myotonica protein kinase gene 3'-untranslated region on chromosome 19. The expanded repeats encode toxic CUG RNA repeats, which sequester splicing factors and form intranuclear RNA foci, leading to aberrant gene splicing and subsequent clinical manifestations. Regenerative medicine holds hope for treatment of advanced myotonic dystrophy. Patient-specific iPS cells are making this realistic. However, patient-specific iPS cells still retain the pathogenic mutation and may undergo the same degenerative process after transplantation. To utilize these cells, the mutated gene needs to be corrected before transplantation. We have designed two approaches in correcting the mutant gene using TALEN technology and homologous recombination (HR): 1) targeted interruption of transcription of the expanded repeats. A sequence interrupting transcription will be inserted between the stop codon and expanded repeats through HR. 2) targeted deletion of the expanded repeats. LoxP sites will be inserted before and after the expanded repeat using HR. The expanded repeats flanked by LoxP sites will then be deleted following transient expression of Cre-recombinase. We will further evaluate in vitro and in vivo pluripotency of genome- corrected DM1 iPS cells by embryoid body-mediated differentiation and teratoma formation. We will evaluate RNA foci and downstream splicing events to confirm the successful correction of molecular phenotypes. We expect this study will provide proof of principle data that TALEN technology can be used to correct dominantly inherited mutant genes. This will overcome one of the hurdles in the development of autologous cell replacement therapy. The implementation of the proposed project and training under K08 mechanism will well- prepare me to compete successfully for R01 funding to become an independent clinical research scientist.
The project will develop approaches to correct mutant gene in stem cells developed from patient with myotonic dystrophy type 1. The successful completion of the study will overcome one of the hurdles for autologous cell transplantation therapy for muscular dystrophy type 1.