Precise Correction of Lhcgr Mutation in Stem Leydig Cells by Prime Editing Rescues Hereditary Primary Hypogonadism in Mice.

Hereditary primary hypogonadism (HPH), caused by gene mutation related to testosterone synthesis in Leydig cells, usually impairs male sexual development and spermatogenesis. Genetically corrected stem Leydig cells (SLCs) transplantation may provide a new approach for treating HPH. Here, a novel nonsense-point-mutation mouse model (Lhcgr W495X ) is first generated based on a gene mutation relative to HPH patients. To verify the efficacy and feasibility of SLCs transplantation in treating HPH, wild-type SLCs are transplanted into Lhcgr W495X mice, in which SLCs obviously rescue HPH phenotypes. Through comparing several editing strategies, optimized PE2 protein (PEmax) system is identified as an efficient and precise approach to correct the pathogenic point mutation in Lhcgr. Furthermore, delivering intein-split PEmax system via lentivirus successfully corrects the mutation in SLCs from Lhcgr W495X mice ex vivo. Gene-corrected SLCs from Lhcgr W495X mice exert ability to differentiate into functional Leydig cells in vitro. Notably, the transplantation of gene-corrected SLCs effectively regenerates Leydig cells, recovers testosterone production, restarts sexual development, rescues spermatogenesis, and produces fertile offspring in Lhcgr W495X mice. Altogether, these results suggest that PE-based gene editing in SLCs ex vivo is a promising strategy for HPH therapy and is potentially leveraged to address more hereditary diseases in reproductive system.