Designing effective RNA therapies for oocyte maturation

摘要 不孕不育是一个主要的生殖健康问题，影响到美国约12%的育龄妇女 各州。大约1-3%的不孕不育或不育妇女的卵母细胞在减数分裂或减数分裂后不久停止。 由于遗传变异而导致的受精。不幸的是，对于经历不孕不育的妇女来说，由于 导致卵母细胞停滞。恢复卵母细胞停滞妇女卵母细胞成熟的策略是迫切需要的。 为这些女性提供生育选择。一种名为信使核糖核酸疗法的新兴疗法在体外使用 合成的信使核糖核酸用于治疗疾病和疫苗，如SARS-CoV-2信使核糖核酸疫苗， 但它在不孕症中的安全性和有效性还没有被研究过。将RNA显微注射到卵母细胞中是一种 已经建立的工具，使人们能够发现卵母细胞生物学的关键方面，但它也可以用作 治疗，特别是对卵母细胞受阻的妇女。最近的两项研究成功地在 来自基因变异导致卵母细胞停滞的妇女的卵母细胞，注射体外合成的 辅助生殖过程中的野生型RNA。RNA疗法代表了一种新的治疗策略 然而，对于经历卵母细胞停滞的妇女来说，在成为辅助卵母细胞之前需要进行严格的测试。 生殖技术。考虑到完全生长的植物独特的RNA加工和转录静止 卵母细胞理解卵母细胞如何处理外源RNA治疗分子是至关重要的。此外， 合成的治疗性信使核糖核酸含有促进核糖核酸稳定性、翻译和减少的核糖核酸修饰 免疫刺激。最近，我们和其他人的工作表明，RNA修饰在一个重要的 卵母细胞中RNA稳定性和翻译的调节作用。然而，多重RNA修饰的影响 对RNA稳定性、翻译和卵母细胞成熟度的影响还没有得到检验。我们的目标是测试RNA如何 修饰会影响旨在挽救卵母细胞成熟缺陷的信使核糖核酸疗法的功能。至 了解RNA疗法是如何被卵母细胞处理的，以及它们如何影响卵母细胞的成熟和 生育能力，我们将使用与拓扑异构酶II同源蛋白相关的基因敲除小鼠模型 2(Patl2基因)，导致卵母细胞成熟停滞。缺乏PATL2蛋白表型的小鼠 Patl2中的遗传缺陷，并有无法成熟的卵母细胞，所以我们预测显微注射RNA 治疗Patl2将恢复卵母细胞成熟、受精和出生。我们将确定RNA的影响 治疗性Patl2 RNA的稳定性和翻译修饰。我们的研究有可能揭示 在卵母细胞RNA加工和翻译中RNA修饰的新方面，以及建立基础 在未来的研究中测试RNA疗法治疗卵细胞所致女性不孕症的安全性和有效性 逮捕。

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.