Ex Vivo Gene Correction of Stem Leydig Cells as a Therapeutic Strategy for Hereditary Primary Hypogonadism

Leydig cells (LCs), located in the interstitial compartment of the testes among the seminiferous tubules, are primarily responsible for testosterone production, which is crucial for maintaining the masculine phenotype, endocrine homeostasis, and reproductive function. Hereditary primary hypogonadism (HPH) results from genetic defects impairing LC function, leading to low or absent testosterone levels, high gonadotropin levels, underdeveloped masculine features, and severely impaired spermatogenesis. A key player in LC function is the luteinizing hormone/choriogonadotropin receptor (LHCGR), which is critical for LC differentiation and testosterone synthesis. Mutations in the Lhcgr gene result in testosterone deficiency, impaired sexual development, and infertility, making it a prototype of HPH. While testosterone replacement therapy (TRT) is the standard treatment for HPH, it has several adverse effects and fails to mimic physiological testosterone secretion, prompting the need for alternative therapies.

Stem Leydig cells (SLCs) are capable of regenerating new LCs through proliferation and differentiation, thus playing a vital role in maintaining LC homeostasis in adult testes. SLCs from rodents have been successfully isolated, expanded, and differentiated ex vivo, and they can replace senescent or disrupted LCs to produce testosterone following transplantation in vivo. Studies have shown that autologous SLC transplantation can increase testosterone levels, improve spermatogenesis, and alleviate symptoms of primary hypogonadism in non-human primate models. Recently, human SLCs have been identified with clonogenic and self-renewal capabilities, exhibiting multi-lineage differentiation potential, including into functional LCs that produce testosterone in vitro and in vivo. However, SLC transplantation in HPH patients is challenged by the hereditary gene defects present in their SLCs.

Advances in genome-editing technologies have enabled precise manipulation of DNA sequences in various cell types, offering potential treatments for hereditary diseases. Ex vivo gene editing, which involves correcting genetic defects in patient-derived cells before transplantation, has shown promise in treating conditions like sickle cell disease, hereditary liver diseases, and permanent neonatal diabetes mellitus. These studies suggest that once the genetic mutations causing hypogonadism are corrected in SLCs, these cells could be suitable for autologous transplantation to rescue HPH by differentiating into functional LCs.

In this study, a mouse model harboring the LhcgrW495X mutation—a nonsense mutation observed in HPH patients—was generated using the CRISPR/Cas9 system. This mutation leads to a truncated, non-functional LHCGR protein. LhcgrW495X mice exhibited significantly lower testosterone levels, increased luteinizing hormone levels, impaired sexual development, and arrested spermatogenesis, mimicking the clinical features of human HPH, thus providing an ideal model for HPH research.

To investigate the potential of SLC-based therapy for HPH, SLCs from wild-type mice (WT-SLCs) were transplanted into the testes of LhcgrW495X mice. The WT-SLCs were transduced with a lentivirus vector expressing mCherry under the control of the CAG promoter, provided by PackGene, allowing for tracking of the transplanted cells in vivo. Following transplantation, WT-SLCs successfully differentiated into LCs that produced testosterone, leading to an increase in serum and intratesticular testosterone levels, normalization of sexual development, and recovery of spermatogenesis in LhcgrW495X mice. These findings demonstrate the feasibility of SLC transplantation for treating HPH.

Given the success of WT-SLC transplantation, we next explored ex vivo gene correction of SLCs from LhcgrW495X mice using prime editing (PE) technology. Prime editors were compared with adenine base editors (ABEs) in correcting the LhcgrW495X mutation. While ABEs showed limited effectiveness and unintended bystander edits, PEmax combined with engineered prime editing guide RNA (epegRNA) achieved the highest correction rate with minimal off-target effects. The PEmax system was split into two components to fit within the packaging capacity of lentivirus vectors, and these components were delivered into SLCs from LhcgrW495X mice (W495X-SLCs) using lentiviruses provided by PackGene. The PEmax system successfully corrected the LhcgrW495X mutation in W495X-SLCs, which subsequently differentiated into functional LCs in vitro, producing testosterone at levels comparable to WT-SLCs.

To assess the therapeutic potential of gene-corrected SLCs in vivo, PE-corrected W495X-SLCs were transplanted into the testes of LhcgrW495X mice. This treatment restored testosterone production, normalized sexual development, and rescued spermatogenesis. Furthermore, sperm derived from treated mice were capable of supporting fertilization and producing offspring. Notably, the offspring were fertile and capable of natural mating, highlighting the long-term efficacy and safety of the gene correction approach.

PackGene Biotech is a world-leading CRO and CDMO, excelling in AAV vectors, mRNA, plasmid DNA, and lentiviral vector solutions. Our comprehensive offerings span from vector design and construction to AAV, lentivirus, and mRNA services. With a sharp focus on early-stage drug discovery, preclinical development, and cell and gene therapy trials, we deliver cost-effective, dependable, and scalable production solutions. Leveraging our groundbreaking π-alpha 293 AAV high-yield platform, we amplify AAV production by up to 10-fold, yielding up to 1e+17vg per batch to meet diverse commercial and clinical project needs. Moreover, our tailored mRNA and LNP products and services cater to every stage of drug and vaccine development, from research to GMP production, providing a seamless, end-to-end solution.

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