Novel approaches for disrupting gene expression in mammalian oocytes

描述(申请人提供)：这个R21项目寻求开发哺乳动物卵母细胞基因操作的新方法，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干扰和小鼠基因敲除。虽然RNAi是在卵母细胞中进行RNA消融和随后的蛋白质敲除的一种非常成功的方法，但RNAi方法也不是没有局限性，因为敲除可能效率低下。此外，必须通过显微注射将双链RNA和siRNAs导入卵母细胞，这是劳动和时间密集型的，因此使用大规模RNAi方法(例如，基于siRNA文库的筛选)是不切实际的。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，但基因敲除方法也不是没有陷阱，包括获得基因敲除所涉及的时间和费用。该项目寻求开发这些方法的替代品，以新的组合利用不同的已建立的基于核酸的方法，并针对这里提出的应用进行专门的修改。在目标1中，我们将增加siRNAs用于转录后基因沉默的另一种试剂，一种被称为三链形成寡核苷酸(TFO)的短单链核酸，用于转录前沉默。TFOS在双链DNA中与同型尿路结合，已被用来调节培养细胞和体内的基因表达。目标1的假设是，TFOS将抑制目标基因的转录，而siRNAs将介导转录的任何剩余mRNAs的降解。这将在体外用分离的卵母细胞和封闭卵泡的卵母细胞进行体外测试，以进行长期培养。在目标2中，我们将开发将药物输送到卵母细胞的方法。我们将通过筛选适体文库(具有1.2×1018寡聚-2‘-脱氧核糖核苷酸序列异构体)来分离将与卵母细胞的透明带(ZP)相互作用的适体，从而确定一种用于卵母细胞特异性递送的新试剂。适配子是以核酸为基础的分子，与靶分子具有高亲和力。适配子可用于将siRNA等试剂输送到细胞内；这种输送在体内有效，目前正在开发适配子作为靶向药物和其他药物的治疗药物，以特定类型的细胞治疗各种疾病(13个适配子正在进行临床试验)。此外，作为替代工具，我们还将测试一种细胞穿透肽，用于卵母细胞内的传递。我们将把siRNAs或TFOS偶联到ZP结合适配子和/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ZP靶向适体系统地递送siRNAs和TFOS，作为体内卵母细胞特异性敲除的一种手段，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.