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 该提案描述了一个五年的研究职业发展项目，旨在评估 通过细胞外蔬菜（EV）传递的基因编辑是一种新颖的避孕方法。几乎一半 美国怀孕是意想不到的，可用的避孕药通常具有不良的副作用，并且 只有在正确使用时才有效。因此，新的易于使用的避孕产品较少 需要效果。对产后妇女的调查表明，约有50％的人喜欢永久性灭菌 与其他避孕药具相比。可用的唯一永久避孕药是手术灭菌。尽管 经常执行，有麻醉风险，高手术成本，需要设施和人员 在大多数低收入地点不存在。目前不可用的永久性违规选择。 通过分泌允许精子电容，受精和早期胚胎发育的蛋白质， 输卵管（输卵管）对于生育力至关重要。卵形上皮细胞孕酮受体（PGR）和产卵 小鼠的特异性糖蛋白（OVGP1）基因敲除分别诱导不育症并降低垃圾大小。 使用CRISPR-CAS9的基因编辑最近在镰状细胞贫血的临床试验中广受欢迎，并且具有 用于编辑肿瘤基因，导致体内啮齿动物模型中肿瘤收缩。但是，特定于组织 靶向CRISPR-CAS9核糖核蛋白（RNP）和体内转染是挑战的。电动汽车包含 脂质双层中的蛋白质和核酸自然分泌用于细胞间通信。 由于电动汽车是在体内产生的用于细胞运输的，因此它们会绕过免疫通量，避免过敏 反应，并进入可能仅用于外国化合物可能无法访问的细胞。总体目标 该建议是评估电动汽车将RNP靶向卵子细胞的效用。具体来说，我们将评估 在小鼠模型中沉积宫内的电动汽车的体内生物分布以确定电动汽车的传播（AIM 1）。然后，我们将通过设计指南RNA敲出带有RNP的电动汽车的EV的体外基因编辑 在评估体内基因编辑之前 在小鼠模型之后，进行了繁殖试验，以评估效用作为永久避孕药（AIM 2.2）。 除了产生影响产卵功能的机制的知识外，该项目将导致 有效递送CRISPR-CAS9 RNP到产卵细胞的条件优化。调查结果可以放置 为妇女提供非手术，永久性违约的基础。 候选人是科罗拉多州立大学的博士后研究员，并组建了一支多元化的团队 在她的咨询委员会任职的专家。该提案建立在候选人之前的研究基础上 背景并将通过基因编辑专门培训EV来增强她在生殖生物学方面的专业知识 表征和工程以及鼠标菌落管理。此外，培训和发展 计划是全面的，并根据她的需求量身定制，这将促进她向独立研究人员的过渡。