Sperm and offspring production in a nonobstructive azoospermia mouse model via testicular mRNA delivery using lipid nanoparticles

Abstract

Microsurgical testicular sperm extraction (microTESE) with intracytoplasmic sperm injection (ICSI) represents the current standard treatment for nonobstructive azoospermia (NOA). However, cures remain unavailable for NOA patients lacking retrievable haploid cells. mRNA supplementation could be a potential treatment for genetic defects leading to impaired spermatogenesis. Lipid nanoparticles (LNPs) have emerged as mRNA delivery vehicles with minimal risk of genome integration; however, their ability to selectively deliver mRNA to specific cell types remains limited. To overcome this, microRNA (miRNA) target sequences were incorporated into mRNA constructs to restrict expression specifically to germ cells. Using pyruvate dehydrogenase E1 subunit alpha 2 (PDHA2) knockout mice as an NOA model with meiotic arrest, we demonstrate that LNP-mediated delivery of Pdha2 mRNA enables the resumption and completion of meiosis, restores sperm production, and facilitates the generation of healthy fertile offspring via ICSI. Whole-genome sequencing of the offspring confirmed the absence of large-scale genomic abnormalities. Our results provide proof of concept for a safe and effective chemically synthesized LNP-based mRNA therapy with miRNA-regulated germ cell specificity, offering a promising therapeutic approach to treating male infertility caused by spermatogenesis arrest.

Keywords: LNP therapy; azoospermia; germline therapy; lipid nanoparticle; sterility.

Fig. 1.

mRNA delivery into testicular cells via LNP. (A) Schematic illustration of LNPs. (B) Schematic illustration of rete testis injection. (C) Testis after EGFP mRNA injection via LNP. (D) Fluorescence image of the whole testis after LNP-EGFP injection. (E) Fluorescence image of testis sections after LNP-EGFP injection. (F) Fluorescence image of testis sections after LNP-mScarlet injection in GBGS mice, in which the EGFP signal first appears in the cytoplasm of spermatocytes and later localizes into the acrosome (18). Sc: Spermatocyte, Rs: Round spermatid, Es: Elongated spermatid.

Fig. 2.

Germ cell-specific expression strategy. (A) Fluorescence images of testis sections after LNP-mediated delivery of histone H3.3-mCherry mRNA (Green arrowheads) and Acr3-EGFP mRNA (Yellow arrowheads). Sc: Spermatocyte, Rs: Round Spermatid. (B) Observation of the acrosome after LNP-mediated delivery of Acr3-EGFP mRNA. The yellow box shows the enlarged image. (C) Schematic illustration of the strategy for germ cell-specific expression using miRNA. (D) Comparison of fluorescence expression with or without the miR-471 recognition sequence.

Fig. 3.

Rescue of Pdha2 KO Testes. (A) Observation of chromosome spreads. Green indicates SCP3, and red indicates γH2AX. The spread nuclei of prophase spermatocytes collected from adult Pdha2^−/− and injected Pdha2^−/− male mice were stained with anti-SYCP3 (green) and γH2AX (red) antibodies. XY indicates the sex chromosomes encircled by γH2AX signal during the pachytene and diplotene stages. (Scale bar, 10 µm.) Experiments were repeated at least twice with consistent results. (B) Graph showing the proportion of Prophase I stages. (C) Image of testis sections stained with H-PAS.

Fig. 4.

Acquisition of offspring by TESE-ICSI. (A) Representative image of testicular cells at 2 wk postinjection (2 wpi). (B) Sperm image at 3 wpi. (C) Results of TESE-ICSI. (D) Image of the obtained offspring. (E) Genome-wide visualization of lcWGS data using Integrative Genomics Viewer (IGV).