Designing effective RNA therapies for oocyte maturation

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ABSTRACT Infertility is a major reproductive health issue that affects ~12% of reproductively aged women in the United States. Approximately 1-3% of infertile or subfertile women have oocytes that arrest in meiosis or shortly after fertilization due to genetic variants. Unfortunately, there are no therapies for women experiencing infertility due to oocyte arrest. Strategies to restore oocyte maturation in women with oocyte arrest are of dire need in order to give these women fertility options. An emerging class of therapies, called mRNA therapeutics, utilize in vitro synthesized mRNA as a treatment for diseases and for vaccines such as the SARS-CoV-2 mRNA vaccines, but the safety and efficacy has not been explored in infertility. Microinjection of RNA into oocytes is an established tool that has enabled discovery of critical aspects of oocyte biology, but it could also be used as a therapeutic, particularly in women with oocyte arrest. Two recent studies successfully generated blastocysts in oocytes from women with genetic variants causing oocyte arrest, following the injection of in vitro synthesized wild-type RNA during assisted reproductive procedures. RNA therapies represent a novel treatment strategy for women experiencing oocyte arrest, however, rigorous testing is needed before they become an assisted reproductive technology. Considering the unique RNA processing and transcriptional quiescence of fully grown oocytes it is critical to understand how oocytes process exogenous RNA therapeutic molecules. Furthermore, synthetic therapeutic mRNA contain RNA modifications that promote RNA stability, translation, and reduce immune stimulation. Recently, our work and others have implicated RNA modifications as playing an important regulatory role in RNA stability and translation in oocytes. However, the impact of multiple RNA modifications on RNA stability, translation, and oocyte maturation has not been examined. Our goal here is to test how RNA modifications impact the function of mRNA therapeutics designed to rescue oocyte maturation defects. To understand how RNA therapeutics are processed by the oocyte and how they impact oocyte maturation and fertility, we will use a genetic knockout mouse model of the Protein Associated with Topoisomerase II Homolog 2 (Patl2 gene), which results in oocyte maturation arrest. Mice lacking PATL2 protein phenocopy women with genetic defects in Patl2, and have oocytes that fail to mature, so we predict that microinjection of an RNA therapeutic for Patl2 will restore oocyte maturation, fertilization, and birth. We will determine the effects of RNA modifications on stability and translation of therapeutic Patl2 RNA. Our studies have the potential to reveal novel aspects of RNA modifications in oocyte RNA processing and translation, as well as establish groundwork for future studies testing the safety and efficacy of RNA therapeutics to treat female infertility due to oocyte arrest.