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

研究通过细胞外囊泡传递的输卵管基因编辑作为一种新型避孕药

• 批准号: 10806665
• 负责人: Riley Thompson
• 金额: $ 12.53万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10806665
• 关键词: Address; Advisory Committees; Anesthetics; Autopsy; Biodistribution; Breeding; CRISPR/Cas technology; Cardiovascular system; Cattle; Cell Culture Techniques; Cell secretion; Cells; Clinical; Clinical Trials; Colorado; Contraceptive Agents; Contraceptive Availability; Contraceptive Usage; Contraceptive methods; Data; Deposition; Development; Development Plans; Doctor of Philosophy; Electroporation; Embryonic Development; Endometrial; Endometrium; Engineering; Epithelial Cells; Exhibits; FVB/N Mouse; Fellowship; Fertility; Fertilization; Fluorescent Dyes; Foundations; Gene Expression; Genes; Glycoproteins; Goals; Guide RNA; Hemorrhage; Hormones; Human Resources; Hypersensitivity; In Vitro; Infertility; K-Series Research Career Programs; Knock-out; Knowledge; Label; Lipid Bilayers; Lipid Binding; Litter Size; Location; Low income; Mammalian Oviducts; Mammals; Mediating; Membrane Proteins; Mentors; Methods; Microscopy; Mouse Strains; Mus; Nucleic Acids; Operative Surgical Procedures; Organoids; Positioning Attribute; Postdoctoral Fellow; Postpartum Women; Pregnancy; Procedures; Progesterone Receptors; Protein Secretion; Proteins; Reaction; Reproduction; Reproductive Biology; Research; Research Personnel; Ribonucleoproteins; Risk; Rodent Model; Scientist; Serum; Sickle Cell Anemia; Sperm Capacitation; Sterilization; Surface; Surveys; Texas; Therapeutic Trials; Tissues; Training; Transfection; Tropism; United States; Universities; Uterus; Viral Vector; Woman; body system; career development; cost; design; endometrial organoid; extracellular vesicles; fluorescence imaging; imaging system; immune clearance; immunoreaction; in vivo; in vivo Model; innovation; intercellular communication; interest; knockout gene; monolayer; mouse model; neoplastic; neoplastic cell; novel; novel strategies; preference; protein expression; reproductive; reproductive tract; side effect; skills; sperm cell; tissue fixing; treatment group; tumor; unintended pregnancy; uptake

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.

项目摘要/摘要 该提案描述了一个为期五年的指导研究职业发展项目，重点是评估 通过细胞外囊泡（EV）递送的基因编辑作为避孕的新方法。几乎一半 在美国，怀孕是意外的，可用的避孕药往往有不良的副作用， 只有正确使用才有效。因此，新的避孕产品使用方便，方少 效果是必须的。一项对产后妇女的调查表明，约50%的妇女倾向于永久绝育。 与其他避孕药相比。唯一可用的永久避孕方法是外科绝育; 常规进行，存在麻醉风险，手术成本高，并且需要 在大多数低收入地区，目前没有永久避孕的非手术选择。 通过分泌使精子获能、受精和早期胚胎发育的蛋白质， 输卵管（输卵管）是生育所必需的。输卵管上皮细胞孕酮受体与输卵管 特异性糖蛋白（OVGP 1）基因敲除分别诱导小鼠不育和减少窝仔数。 使用CRISPR-cas9的基因编辑最近在镰状细胞贫血的临床试验中获得了普及， 用于编辑肿瘤基因，导致体内啮齿动物模型中的肿瘤缩小。然而，组织特异性 CRISPR-cas9核糖核蛋白（RNP）的靶向和体内转染具有挑战性。电动汽车包含 脂质双层内的蛋白质和核酸，并且天然分泌用于细胞间通讯。 由于EV是在体内产生的，用于细胞运输，它们绕过免疫清除，避免超敏反应， 反应，并获得进入细胞，可能无法访问的外来化合物单独。的总目标 该建议是评估EV用于将RNP靶向输卵管细胞的效用。具体来说，我们将评估 沉积在子宫内EV在小鼠模型中的体内生物分布，以确定EV的传播（目的 1）。然后，我们将通过设计指导RNA来敲除负载RNP的EV的体外基因编辑， 在评估体内基因编辑之前，输卵管类器官中的必需生育力基因（PGR，OVGP 1）（Aim 2.1） 在小鼠模型中，随后进行繁殖试验，以评估其作为永久避孕药的效用（目标2.2）。在 除了产生关于影响输卵管功能的机制的知识外，该项目还将导致 CRISPR-cas9 RNP有效递送至输卵管细胞的条件的优化。调查结果可能会奠定 妇女非手术永久避孕发展基金会。 这位候选人是科罗拉多州立大学的博士后，组建了一个多元化的团队 她的顾问委员会的专家。这个建议建立在候选人以前的研究基础上 背景，并将通过基因编辑，EV 表征和工程化以及小鼠群体管理。此外，培训和发展 该计划是全面的，适合她的需要，这将有助于她过渡到一个独立的研究人员。