Reversal of Ovarian Aging in Mice Through AAV-mediated Oocyte Reprogramming in vivo

项目摘要 衰老是一个复杂的多因素的生物过程，所有生物体共享。在卵巢中，它表现为 正常生殖功能的逐渐下降，在绝经期达到高潮，此时卵母细胞的数量 福尔斯低于阈值。更年期之前，从30年代中期开始， 由于衰老导致的卵母细胞功能障碍的发生率，其导致减数分裂分离错误，非整倍体和不良的 胚胎发育这种细胞功能障碍在临床上表现为不孕症、辅助生殖失败 技术（抗逆转录病毒疗法）、（复发性）流产和出生缺陷。目前没有有效的治疗方法， 改善高龄生育妇女卵母细胞功能和新的方法学策略， 急需的。许多参与卵母细胞减数分裂机制的因素与年龄有关， 相关的卵母细胞功能障碍然而，卵巢衰老的根本原因以及为什么女性生殖细胞 相对于体细胞经历加速老化仍然是未知的。时间依赖性累积 细胞损伤被广泛认为是衰老的基础并且有证据表明衰老的速度是受控的， 至少在某种程度上是通过进化中保守的遗传途径。最近的研究表明， 表观遗传景观的侵蚀（表观基因组漂移）导致细胞功能障碍可能主要是 负责衰老。有趣的是，这些研究表明，通过异位表达的“部分重编程”， 一组有限的多能性因子可以恢复年轻的表观遗传模式，并逆转衰老的迹象， 体细胞然而，部分重编程对减数分裂细胞的细胞身份的影响， 独特的基因调控网络和保持多能性的特征，是未知的。 这项提案研究了卵巢衰老的全球机制，挑战其不可逆性，并制定了一项 为未来开发减缓或逆转卵巢衰老的治疗方法提供了新途径。我们将雇用一名 转基因小鼠模型，以检验卵丘细胞-卵母细胞的部分重编程 复合物可以逆转卵巢衰老，恢复生殖功能。我们将使用综合多组学， 明确甲基化时钟和表观遗传漂变在卵巢衰老中的作用， 卵巢由于衰老而导致卵母细胞功能障碍。我们将研究部分重编程是否可以 逆转卵母细胞-卵丘复合体中与年龄相关的转录和表观遗传变化， 在不改变卵母细胞的细胞特性的情况下，然后我们将检查 这种方法的翻译潜力，使用一种新的AAV递送系统，能够穿过血液， 卵泡屏障和体内靶卵母细胞和颗粒细胞。这项工作将增加我们对 卵巢衰老的生物学，并为开发表观遗传药物提供新的分子靶点， 扭转人类卵巢生殖功能的下降，减少流产、反复妊娠 与高龄生育有关的损失和出生缺陷。

Project Summary Aging is a complex multifactorial biological process shared by all living organisms. In the ovary, it manifests as a gradual decline of normal reproductive function which culminates in menopause when the number of oocytes falls below a threshold. Menopause is preceded, from the mid-30s onward, by a dramatically increased incidence of oocyte dysfunction due to aging which results in meiotic segregation errors, aneuploidy and poor embryo development. This cellular dysfunction clinically presents as infertility, failure of assisted reproduction technologies (ART), (recurrent) pregnancy loss and birth defects. There are currently no effective treatments to improve oocyte function in women with advanced reproductive age and novel methodological strategies are critically needed. Many factors involved in the meiotic machinery in oocytes have been implicated in age- related oocyte dysfunction. However, the underlying cause of ovarian aging and why the female germline undergoes accelerated aging relative to somatic cells remains unknown. Time-dependent accumulation of cellular damage is widely considered to underlie aging and there is evidence that the rate of aging is controlled, at least to some extent, by genetic pathways conserved in evolution. Recent studies indicate that in somatic cells an erosion of the epigenetic landscape (epigenomic drift) resulting cellular dysfunction may be primarily responsible for aging. Interestingly, these studies show that ‘partial reprogramming’ through ectopic expression of a limited set of pluripotency factors can restore youthful epigenetic patterns and reverse signs of aging in somatic cells. However, the effect of partial reprogramming on the cell identity of meiotic cells, which exhibit unique gene regulatory networks and maintain features of pluripotency, is unknown. This proposal examines the global mechanism of ovarian aging, challenges its irreversibility and develops a novel pathway for the future development of treatments to slow or reverse ovarian aging. We will employ a transgenic mouse model to examine the hypothesis that partial reprogramming of the cumulus cell-oocyte complex can reverse ovarian aging and restore reproductive function. We will use integrative multi-omics to define the role of the methylation clock and epigenetic drift in ovarian aging and whether non-oocyte cells of the ovary contribute to oocyte dysfunction due to aging. We will investigate whether partial reprogramming can reverse age-related transcriptional and epigenetic changes in the oocyte-cumulus-complex to more youthful patterns and restore fertility in vivo without altering the cellular identity of oocytes. We will then examine the translational potential of this approach using a novel AAV delivery system with the ability to cross the blood- follicle barrier and target oocytes and granulosa cells in vivo. This work will increase our understanding of the biology of ovarian aging and provide novel molecular targets for the development of epigenetic drugs that reverse the decline in human reproductive ovarian function and reduce the miscarriages, recurrent pregnancy loss and birth defects associated with advanced reproductive age.