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