Reviewer #3 (Public Review): Sperm motility in mice with Oligo-astheno-teratozoospermia restored by in vivo injection and electroporation of naked mRNA

Oligo-astheno-teratozoospermia (OAT), a recurent cause of male infertility, is the most frequent disorder of spermatogenesis with a probable genetic cause. Patients and mice bearing mutations in the ARMC2 gene have a decreased sperm concentration, and individual sperm show multiple morphological defects and a lack of motility – a canonical OAT phenotype. Intra Cellular Sperm Injection (ICSI) is required to treat such a condition but it has limited efficacy and was associated with a small increase in birth defects. Consequently, new targeted treatments are needed to restore spermatogenesis. Here, a combination of in vivo injection and electroporation of capped and poly-A-tailed naked mRNA is tested as a strategy to treat ARMC2-related infertility in mouse. mRNAs coding for several reporter genes are tested and the efficiency and the kinetic of expression are assessed using in vivo and in vitro 2D and 3D imaging experiments. We show that mRNA-coded reporter proteins are detected for up to 3 weeks mostly in germ cells, making the use of mRNA possible to treat infertility. We compare these results with those obtained with a more conventional DNA plasmid vector. In contrast, the use of the non-integrative plasmid Enhanced Episomal Vector (EEV) shows low and transient expression in spermatogenic cells. Consequently, injection and electroporation of naked mRNA-Armc2 into the testes of Armc2-deficient males were performed and we show the presence of normal and motile sperm in the epididymis. This study shows for the first time that mRNA-Armc2 efficiently restores spermatogenesis and opens new paths for male infertility treatment.All procedures involving animals were performed in line with the French guidelines for the use of live animals in scientific investigations. The study protocol was approved by the local ethics committee (ComEth Grenoble #318) and received governmental authorization (ministerial agreement #38109-2022072716142778).