Novel approaches for disrupting gene expression in mammalian oocytes

描述（由申请人提供）：本R21项目旨在开发哺乳动物卵母细胞遗传操作的新方法，我们预计这将加速推进我们对卵母细胞功能和生殖健康的了解。哺乳动物卵母细胞的遗传操作主要使用两种方法：RNA干扰和敲除小鼠。虽然RNAi是一种非常成功的卵母细胞RNA消融和随后的蛋白敲除方法，但RNAi方法并非没有局限性，因为敲除可能效率低下。此外，双链RNA和siRNA必须通过显微注射引入卵母细胞，这是人工和时间密集型的，并且使得使用大规模RNAi方法（例如，基于siRNA文库的筛选）不切实际。基因敲除小鼠当然也为哺乳动物卵母细胞生物学提供了重要的见解，但基因敲除方法也并非没有缺陷，包括获得基因敲除所涉及的时间和费用。该项目旨在开发替代这些方法，利用不同的已建立的基于核酸的新组合方法，并针对本文提出的应用进行专门修改。在Aim 1中，我们将使用另一种试剂增加sirna在转录后基因沉默中的使用，这种试剂是一种称为三聚体形成寡核苷酸（TFO）的短单链核酸，用于转录前沉默。tfo与双链DNA中的同嘌呤束结合，并被用于调节培养细胞和体内的基因表达。Aim 1的假设是，tfo会抑制靶基因的转录，而sirna会介导任何被转录的残余mrna的降解。这将在体外用分离的卵母细胞以及卵泡封闭的卵母细胞进行长期培养。在目标2中，我们将开发药物进入卵母细胞的方法。我们将鉴定一种用于卵母细胞特异性递送的新型药物，使用适配体文库（具有1.2 X 1018 oligo-2'-脱氧核糖核苷酸序列异构体）筛选分离出一种与卵母细胞透明带（ZP）相互作用的适配体。适配体是基于核酸的分子，与靶分子具有高亲和力。适配体可用于将sirna等试剂输送到细胞中；这种递送在体内起作用，适配体目前正被开发为针对特定细胞类型的药物和其他药物的治疗方法，以治疗各种疾病（13种适配体正在临床试验中）。此外，作为一种替代工具，我们还将测试用于卵母细胞内递送的细胞穿透肽。我们将把sirna或TFOs偶联到zp结合适配体和/或细胞穿透肽上，并测试它们在卵母细胞中的作用。这项工作的未来方向将是测试针对zp的适体，用于sirna和TFOs的全身递送，作为一种体内卵母细胞特异性敲除的手段，作为敲除/转基因方法的替代方法，以及潜在的新型女性避孕药的基础。

DESCRIPTION (provided by applicant): This R21 project seeks to develop new approaches for the genetic manipulation of mammalian oocytes, which we envision will accelerate advancing our knowledge of oocyte function and reproductive health. Genetic manipulation of mammalian oocytes has primarily used two methods: RNA interference and knockout mice. While RNAi has been a highly successful method of RNA ablation and subsequent protein knockdown in oocytes, RNAi approaches are not without limitations, as knockdown can be inefficient. Furthermore, double-stranded RNA and siRNAs have to be introduced into oocytes by microinjection, which is labor- and time-intensive and makes it impractical do use a large-scale RNAi approach (e.g., siRNA library-based screens). Knockout mice certainly have provided significant insights into mammalian oocyte biology as well, but knockout approaches also are not without pitfalls, including the time and expense involved in obtaining a knockout. This project seeks to develop alternatives to these methods, utilizing different established nucleic acid-based methods in novel combinations and with specialized modifications for the applications proposed here. In Aim 1, we will augment the use of siRNAs for post-transcriptional gene silencing with another reagent, a short single-stranded nucleic acid called a triplex-forming oligonucleotide (TFO) for pre-transcriptional silencing. TFOs bind to homopurine tracts in double-stranded DNA, and have been used to regulate gene expression in cultured cells and in vivo. The hypothesis for Aim 1 is that TFOs will inhibit transcription of a targeted gene, while siRNAs will mediate degradation of any residual mRNAs that were transcribed. This will be tested in vitro with isolated oocytes as well as with follicle-enclosed oocytes for longer-term culture. In Aim 2, we will develop methods for delivery of agents into oocytes. We will identify a novel agent for oocyte- specific delivery, using a screen of an aptamer library (with 1.2 X 1018 oligo-2'-deoxyribonucleotide sequence isomers) to isolate an aptamer that will interact with the oocyte's zona pellucida (ZP). Aptamers are nucleic acid-based molecules that bind with high affinity to target molecules. Aptamers can be used for delivery of agents such as siRNAs into cells; this delivery works in vivo, and aptamers currently are being developed as therapeutics to target drugs and other agents to specific cell types for treatment of a variety of diseases (13 aptamers are in clinical trials). Additionally, as an alternative tool, we will also test a cell-penetrating peptide for intra-oocyte delivery. We will couple siRNAs or TFOs to ZP-binding aptamers and/or a cell-penetrating peptide, and test these for their actions in oocytes. The future direction of this work will be to test the ZP-targeting aptamer for systemic delivery of siRNAs and TFOs, as a means of in vivo oocyte-specific knockdown as an alternative to knockout/transgenic methodologies, as well as potentially the foundation of a novel female contraceptive. PUBLIC HEALTH RELEVANCE: Investigations of the biology of the mammalian oocyte are of significant value, both for increasing our basic biological knowledge and for applications to human reproductive health. This project will accelerate the pace of research in this important area of reproductive health by developing new research methods for use in studying oocyte biology and female fertility and infertility, and for development of new female contraceptive methods.