AAV-mediated gene therapy restores natural fertility and improves physical function in the Lhcgr-deficient mouse model of Leydig cell failure.

Leydig cell failure (LCF) caused by gene mutations leads to testosterone deficiency, infertility and reduced physical function. Adeno-associated virus serotype 8 (AAV8)-mediated gene therapy shows potential in treating LCF in the Lhcgr-deficient (Lhcgr-/-) mouse model. However, the gene-treated mice still cannot naturally sire offspring, indicating the modestly restored testosterone and spermatogenesis in AAV8-treated mice remain insufficient to support natural fertility. Recognizing this, we propose that enhancing gene delivery could yield superior results. Here, we screened a panel of AAV serotypes through in vivo transduction of mouse testes and identified AAVDJ as an impressively potent vector for testicular cells. Intratesticular injection of AAVDJ achieved markedly efficient transduction of Leydig cell progenitors, marking a considerable advance over conventional AAV8 vectors. AAVDJ-Lhcgr gene therapy was well tolerated and resulted in significant recovery of testosterone production, substantial improvement in sexual development, and remarkable restoration of spermatogenesis in Lhcgr-/- mice. Notably, this therapy restored fertility in Lhcgr-/- mice through natural mating, enabling the birth of second-generation. Additionally, this treatment led to remarkable improvements in adipose, muscle, and bone function in Lhcgr-/- mice. Collectively, our findings underscore AAVDJ-mediated gene therapy as a promising strategy for LCF and suggest its broader potential in addressing various reproductive disorders.

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 (LhcgrW495X ) 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 LhcgrW495X 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 LhcgrW495X mice ex vivo. Gene-corrected SLCs from LhcgrW495X 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 LhcgrW495X 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.

AAV-mediated gene therapy produces fertile offspring in the Lhcgr-deficient mouse model of Leydig cell failure.

Leydig cell failure (LCF) caused by gene mutation results in testosterone deficiency and infertility. Serum testosterone levels can be recovered via testosterone replacement; however, established therapies have shown limited success in restoring fertility. Here, we use a luteinizing hormone/choriogonadotrophin receptor (Lhcgr)-deficient mouse model of LCF to investigate the feasibility of gene therapy for restoring testosterone production and fertility. We screen several adeno-associated virus (AAV) serotypes and identify AAV8 as an efficient vector to drive exogenous Lhcgr expression in progenitor Leydig cells through interstitial injection. We observe considerable testosterone recovery and Leydig cell maturation after AAV8-Lhcgr treatment in pubertal Lhcgr-/- mice. Of note, this gene therapy partially recovers sexual development, substantially restores spermatogenesis, and effectively produces fertile offspring. Furthermore, these favorable effects can be reproduced in adult Lhcgr-/- mice. Our proof-of-concept experiments in the mouse model demonstrate that AAV-mediated gene therapy may represent a promising therapeutic approach for patients with LCF.