Abstract: Project 3: Somatic and germline gene therapy for male infertility Infertility impacts 10-15% of couples in the United States and a male factor is implicated alone or in combination with female factors in about 50% of cases. Infertility can be caused by hormonal, anatomical, immunological or chromosomal deficiencies, diseases or medical treatments, but is frequently of unknown origin (idiopathic). Idiopathic infertility is difficult to counsel and treatment options are limited. This program will focus on patients with the most severe form of male infertility, nonobstructive azoospermia (NOA). Improved knowledge about the genetic basis of NOA obtained in this program will aid in the counseling of infertile couples; justify the development of diagnostic screens; and may lead to new patient-specific treatment options. Project 3 will test the hypotheses that: 1) gene therapy/gene editing can be used to treat mouse models of human NOA that exhibit somatic cell or germ cell dysfunction; 2) gene editing can be performed in induced pluripotent stem cells (iPSCs) from mouse models of NOA, which can be differentiated into transplantable germ cells or sperm; and 3) gene editing can be performed in NOA patient-derived iPSCs that can be differentiated to primordial germ cell-like cells (PGCLCs). Project 3 will provide “gold standard” reproductive phenotyping in mouse models with NOA-associated variants that are discovered in Project 1 and validated with in vitro assays in Project 2. Mouse models that replicate the patient NOA phenotype will be used for development of gene therapies for male infertility. For Sertoli cell defects, Aim 1 will extend our previous results by demonstrating that adeno-associated viruses carrying corrective genes can be introduced to Sertoli cells in vivo and restore spermatogenesis with low toxicity and without integrating in the genome or modifying the germline. For germ cell defects, Aim 1 will prove the principle that NOA-associated variants in spermatogonial stem cells (SSCs) can be corrected ex vivo with CRISPR/Cas9 or Prime editing and transplanted to restore spermatogenesis and fertility in mouse models of NOA. Aim 2 will use CRISPR/Cas9 or Prime editing to correct NOA-associated variants mouse iPSCs followed by differentiation to transplantable PGCLCs or SSC-like cells (SSCLCs) to restore spermatogenesis and fertility. Aim 3 will use CRISPR/Cas9 or Prime editing to correct validated NOA-associated variants in NOA patient-derived iPSCs followed by differentiation to PGCLCs. This project will establish the safety and feasibility of gene therapies for male infertility in mouse models and human cells to support future translation to the human clinic.