The genetically-engineered stem cell therapy of huntington disease: spt4 knockout hd patient ipsc-npcs transplantation rescue abnormal neuronal dysfunction in the YAC128 model

Abstract

Background & Aim The induced pluripotent stem cell (iPSC) technology has raised the possibility that patient-specific iPSCs may become a renewable source of autologous cells for cell therapy without the concern of immune rejection. However, the iPSC of gene mutation patients have same gene mutation. Huntington's disease (HD), one of the gene mutation disease has mutation that extended CAG repeat sequences at the N-terminus of huntingtin protein. This study was examined whether the transplantation of genetic edited HD iPSC-NPCs influenced cell therapy for HD patient. Our genetic editing target, Spt4 is transcription elongation factor that needed for expanded CAG repeats. Methods, Results & Conclusion We first were defected the Spt4 in Q57 HD iPSC-NPCs using CRISPR/Cas9. Spt4 knockout using CRISPR/Cas9 had 80∼90% indel effect. The prepared Spt4 knockout Q57 HD iPSC-NPCs, non-genetic edited Q57 HD iPSC-NPC, and normal iPSC-NPCs were transplanted in the striatum of 6 month old HD YAC128 mice. Using hNu staining, the survival of transplanted cells was identified at 1 week after transplantation. All groups that were cell transplant detected hNu positive cells. Interestingly, the hNu positive cells of non-genetic edited Q57 HD iPSC-NPCs transplantation group expressed mutant huntingtin protein (EM48); however, the hNu positive cells of Spt4 knockout Q57 HD iPSC-NPCs transplantation group no expressed EM48. The genetic editing of Spt4 removed huntingtin protein aggregation by CAG repeat mutation in Q57 HD iPSC-NPCs. We examined whether Spt4 knockout Q57 HD iPSC-NPCs transplantation influenced behavior recovery. The rotarod test and grip strength test as motor function were difference between Q57 HD iPSC-NPCs transplantation and Spt4 knockout Q57 HD iPSC-NPCs group from 2 months after transplantation. In addition, we observed the elevated plus maze test for emotion function. At 3 months after transplantation, Spt4 knockout Q57 HD iPSC-NPCs transplantation was increased the frequency of open arm/total arm compare with Q57 HD iPSC-NPCs transplantation. Taken together, these results strongly suggest that the inherent mutation of HD disappeared in Q57 HD iPSC-NPCs using the genetic editing of Spt4, providing a new possibility that genetic editing of Spt4 may play a major role in the autologous cell therapy of HD. In addition, the other suggest that the genetic editing of SPT4 without deficiency of normal huntingtin protein may be the most ideal genetic editing in HD.