Restoration of a functional spermatogenesis in infertile mice by in vivo therapy. – SPERMREPAIR
mRNA and episomal plasmids were directly injected into the seminiferous tubules via the rete testis, followed by electroporation. The progress of the injection was monitored using a dye, and only a portion of the testis was filled to preserve tissue integrity. Protein expression was monitored over a full spermatogenesis cycle (35 days), and testes and epididymides were collected to analyze sperm morphology and production.
Sperm were evaluated for motility and morphology, and fertilization was tested via IVF or ICSI. The absence of residual plasmids or mRNA was confirmed through molecular analyses.
The approach was structured into three main tasks:
Comparing the efficiency of mRNA and EEV in producing fluorescent sperm.
Restoring spermatogenesis and fertility (switching from the Spink2⁻/⁻ model to the Armc2⁻/⁻ model).
Assessing biosafety and the quality of restored sperm.
Each step was designed to ensure safety, reproducibility, and relevance for future applications.
We demonstrated that mRNA can be efficiently expressed in the testis, covering a full spermatogenesis cycle, with transfection rates and expression levels higher than EEV plasmids, despite a shorter duration (~21 days vs. ~120 days for EEV).
In the Armc2⁻/⁻ model, injection of mRNA encoding ARMC2 led, five weeks later, to the appearance of motile, morphologically normal sperm capable of fertilizing oocytes via IVF or ICSI. Embryos reached the two-cell stage, and some progressed to the blastocyst stage. These results provide the first evidence that naked mRNA can transiently but effectively restore spermatogenesis and fertility in a severe mouse infertility model.
The SPERMREPAIR results open the door to new targeted therapies for certain severe forms of genetic male infertility. In the short term, we will improve mRNA vectors by encapsulating them in lipid nanoparticles (LNPs), which should increase efficacy and duration while ensuring safety and eliminating the need for electroporation. In the medium term, these approaches will be tested in animal models closer to humans, such as non-human primates, to verify clinical applicability. In the long term, this work could lead to innovative treatments restoring fertility in men currently without solutions. Finally, this strategy could be adapted to other genes involved in spermatogenic disorders, paving the way for personalized treatments and precision medicine in reproductive health.
Infertility affects 1 in 7 couples and has become a public health concern. In 50% of cases, it is of male origin and is mainly due to abnormalities in spermatogenesis leading to defects in sperm production. One of the most severe sperm abnormalities is non-obstructive azoospermia (NOA), defined by impaired spermatogenesis resulting in the absence of sperm in the ejaculate. It is generally of genetic origin and affects around 10% of infertile men. As part of the management of NOA it is possible to perform a testicular biopsy to look for living sperm which can be used to perform an intra-cytoplasmic injection (ICSI). However, sperm are found in less than half of the cases and there are questions about the long-term health of the children born from immature sperm generated by a highly altered gametogenesis. We therefore believe that it is important to identify and test new therapeutic strategies in order to better manage these infertile men, especially when a genetic diagnosis has been obtained.
This project aims to carry out a preclinical trial on murine models presenting with NOA in order to test new therapeutic strategies aiming to correct genetic defects leading to the loss of a protein necessary for spermatogenesis. I will determine the feasibility (effectiveness) and the associated risks (toxicity) of the tested protocols. My goal is to express the missing protein without altering the gamete’s genomic DNA, because France is a signatory to the Oviedo convention which prohibits the practice of genetic modifications transmissible to offspring. Two approaches will be tested: injecting the mRNA coding for the missing protein and injecting a non-integrative plasmid (episome) containing the coding sequence for this protein. The mRNA / plasmids will be injected into the seminiferous tubules by the rete testis and transfected into the germ cells by an overall electroporation of the testes. The sperm created will then be used to generate embryos and pups. My project is divided into 3 parts: in the 1st part I will compare the effectiveness of the 2 approaches using reporter genes in order to identify the best protocol; in the second part, using ANO mouse models already available in the laboratory, I will verify that the chosen protocol allows to revive the spermatogenesis of these infertile mice; finally, I will assess the effectiveness and safety of this technique. To do this, I will assess the physiology and quality of the gametes created and ensure that the embryos generated do not contain any exogenous sequence, in particular from the plasmid used.
I am convinced that this project has a good chance of success because: 1 / A transient restoration of spermatogenesis is sufficient as the gametes can be produced only once (and be cryopreserved for later use), 2 / the maximum duration required for restoration is short since a complete cycle of spermatogenesis lasts only 74 days in humans, 3 / the success of the treatment can be easily measured by a simple spermogram and 4 / my preliminary results show that the injection of mRNA or episomes in the retis testis of mice allows transient protein translation in all of the seminiferous tubules, compatible with the duration of spermatogenesis.
The financial assistance requested from the ANR will cover the cost of a doctoral student and the scientific means necessary for the project. The work carried out during this project will allow me to create my own research group on sperm therapy, research complementary to the theme of the genetics of infertility developed by my host laboratory.
Project coordination
Jessica Escoffier (Institut pour l'Avancée des Biosciences)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Partnership
IAB Institut pour l'Avancée des Biosciences
Help of the ANR 269,358 euros
Beginning and duration of the scientific project:
December 2020
- 48 Months
