Investigating oviductal gene editing delivered via extracellular vesicles as a novel contraceptive

项目摘要/摘要 这份提案描述了一个为期五年的有指导的研究职业发展项目，重点是评估 通过细胞外小泡(EVS)传递基因编辑作为一种新的避孕方法。几乎一半的人 美国的怀孕是意外的，而且现有的避孕药具往往有不良的副作用和 只有在正确利用的情况下才有效。因此，新的易于使用的避孕产品具有更少的副作用 效果是必需的。一项对产后妇女的调查显示，约50%的人倾向于永久绝育 与其他避孕药相比。唯一可用的永久避孕措施是手术绝育；而 常规手术有麻醉风险，手术费用高，需要的设施和人员 在大多数低收入地区都没有。目前还没有永久避孕的非手术选择。 通过分泌允许精子获能、受精和早期胚胎发育的蛋白质， 输卵管(输卵管)对生育至关重要。输卵管上皮细胞孕激素受体与输卵管 小鼠的特异性糖蛋白(OVGP1)基因敲除分别导致不孕和减少产仔数。 使用CRISPR-Cas9的基因编辑最近在镰状细胞性贫血的临床试验中变得流行起来，并已 在活体动物模型中被用来编辑导致肿瘤缩小的肿瘤基因。然而，组织特异性 靶向CRISPR-Cas9核糖核蛋白(RNPs)和体内转基因是具有挑战性的。电动汽车包含 脂双层中的蛋白质和核酸，为细胞间通讯而自然分泌。 由于EV是在体内产生的，用于细胞运输，它们绕过了免疫清除，避免了过敏 反应，并获得进入的细胞，可能无法进入的外来化合物单独。的总目标是 该建议旨在评估EVS将RNPs靶向输卵管细胞的效用。具体地说，我们将评估 EVS在宫内沉积的小鼠体内的生物分布以确定EVS的扩散(目的 1)。然后，我们将通过设计引导RNA敲除来确定携带RNPs的EV的体外基因编辑 在评估体内基因编辑之前，输卵管器官中的必需生育基因(PGR、OVGP1)(AIM 2.1) 在小鼠模型中，随后进行育种试验，以评估作为永久避孕药的效用(目标2.2)。在……里面 除了产生关于影响输卵管功能的机制的知识外，这个项目还将导致 CRISPR-Cas9 RNPs高效输送至输卵管细胞的条件优化这一发现可能会为 为妇女开发一种非手术、永久避孕方法的基金会。 这位候选人是科罗拉多州立大学的博士后研究员，他组建了一个多元化的团队 在她的顾问委员会任职的专家。这项建议是在候选人先前研究的基础上提出的 背景，并将通过基因编辑的专门培训来增强她在生殖生物学方面的专业知识，EV 特征化和工程化，以及鼠群管理。此外，培训和发展 计划是全面的，并根据她的需要量身定做，这将促进她向独立研究人员的过渡。

PROJECT SUMMARY/ABSTRACT This proposal describes a five-year mentored research career development project focused on evaluating gene editing delivered via extracellular vesicles (EVs) as a novel approach for contraception. Almost half of United States pregnancies are unintended, and available contraceptives often have undesirable side effects and are effective only when utilized properly. Therefore, new easy-to-use contraceptive products with less side effects are required. A survey of postpartum women indicated about 50% prefer permanent sterilization compared with other contraceptives. The only permanent contraceptive available is surgical sterilization; while routinely performed, there are anesthetic risks, high surgical cost, and requires facilities and personnel that are absent in most low income locations. Non-surgical options for permanent contraception are currently unavailable. By secreting proteins that allow sperm capacitation, fertilization, and early embryo development, the oviduct (fallopian tube) is essential for fertility. Oviductal epithelial cell progesterone receptor (PGR) and oviduct specific glycoprotein (OVGP1) gene knockouts in mice induce infertility and reduce litter sizes, respectively. Gene editing using CRISPR-cas9 has recently gained popularity in a clinical trial for sickle cell anemia and has been used to edit neoplastic genes resulting in tumor shrinkage in rodent models in vivo. However, tissue-specific targeting of CRISPR-cas9 ribonucleoproteins (RNPs) and in vivo transfection are challenging. EVs contain proteins and nucleic acids within a lipid bilayer and are naturally secreted for intercellular communication. Because EVs are produced in vivo for cell transport, they circumvent immune clearance, avoid hypersensitivity reactions, and gain entry to cells that may not be accessible for foreign compounds alone. The overall goal of this proposal is to evaluate the utility of EVs for targeting RNPs to oviductal cells. Specifically, we will evaluate in vivo biodistribution of EVs deposited intrauterine in a mouse model to determine dissemination of EVs (Aim 1). Then, we will determine in vitro gene editing of EVs loaded with RNPs by designing guide RNAs to knock out essential fertility genes (PGR, OVGP1) in oviductal organoids (Aim 2.1) prior to assessing in vivo gene editing in a mouse model followed by a breeding trial to evaluate utility as a permanent contraceptive (Aim 2.2). In addition to generating knowledge on mechanisms influencing oviductal function, this project will lead to optimization of conditions for efficient delivery of CRISPR-cas9 RNPs to oviductal cells. Findings could lay the foundation for development of a non-surgical, permanent contraception for women. The candidate is a postdoctoral fellow at Colorado State University and has assembled a diverse team of experts to serve on her advisory committee. This proposal builds upon the candidate’s previous research background and will augment her expertise in reproductive biology with specialized training in gene editing, EV characterization and engineering, and mouse colony management. Furthermore, the training and development plan is comprehensive and tailored to her needs, which will facilitate her transition to an independent researcher.