Developing a platform for human somatic cell rejuvenation, expansion and genetic engineering using synthetic RNA molecules

项目摘要 包括成体干细胞在内的人类体细胞在离体环境中的短寿命是一个重要的挑战。 许多临床和研究应用的障碍。因此，开发新的方法， 促进成体细胞的扩增，同时恢复这些细胞的类神经元特征 没有永久永生是再生医学的高度优先事项。端粒的磨损是 导致体细胞增殖和功能下降的特征性机制， 培养并可以被端粒酶的活性所抵消。线粒体功能障碍， 表观遗传变化也是导致细胞老化、衰老和衰老的机制之一。 功能.在我们的初步实验中，我们开发了一种正在申请专利的基于非整合RNA的 这些因子的混合物在转染到人体细胞中后将端粒的长度增加到 在多能干细胞中观察到，提高细胞的增殖率，恢复线粒体DNA含量， 并允许单独接种的成年人成纤维细胞（FB）的克隆扩增。我们现在建议使用 这种恢复活力的RNA鸡尾酒开发了两种新的细胞技术平台。一个将改善 扩增人原代体细胞并以临床相关方式促进其再生 没有永久永生化，同时保持增殖能力，功能和正常 这些原始细胞的特征。第二个平台将允许有效的临床相关遗传学 直接在原代体细胞中进行工程化。在后者中，使用我们的再生RNA鸡尾酒将允许 基因修饰的原代体细胞的克隆扩增，同时保留这些细胞的功能， 用于后续移植。为了开发细胞扩增平台，在具体目标1和2中，我们将 进一步表征我们的鸡尾酒对研究和临床中常用的三种人类细胞类型的影响， 背景：FB、角质形成细胞（KC）和间充质基质/干细胞（MSC）。我们将进行详细的 低代和高代人FB、KC和MSC的分子和功能表征 我们的鸡尾酒，重点是恢复这些细胞的类神经元特征。在具体目标3中，我们 使用我们的恢复活力的鸡尾酒来开发一个平台， 体细胞系使用Cas9介导的基因靶向，我们将产生一组原代人FB、KC 以及具有荧光报告基因和荧光素酶的位点特异性敲入的MSC系。导出的线将是 进行了体外和体内测试，以确认其功能性和安全性，为开发提供可行性数据 新型体细胞基因疗法如果成功，这些研究将证明 为其他体细胞类型开发复壮策略和基因工程平台的原则 用于移植，如造血和肌肉干细胞。

Project Summary The short lifespan of human somatic cells, including adult stem cells, in ex vivo settings is a significant hurdle for many clinical and research applications. Therefore, the development of novel approaches that can facilitate the expansion of adult cells and at the same time restore the young-like characteristics of these cells without permanent immortalization is of high priority for regenerative medicine. Telomere attrition is one of the well-characterized mechanisms responsible for the decline in proliferation and function of somatic cells in culture and can be counteracted by the activity of the enzyme telomerase. Mitochondrial dysfunctions and epigenetic changes are also among mechanisms responsible for cellular aging, senescence and decline in functionality. In our preliminary experiments, we developed a patent-pending non-integrating RNA-based cocktail of factors that upon transfection into human somatic cells increases the length of telomeres to that observed in pluripotent stem cells, improves the proliferation rate of cells, restores mitochondrial DNA content and allows for a clonal expansion of individually seeded adult human fibroblasts (FBs). We now propose to use this rejuvenating RNA cocktail to develop two novel cellular technology platforms. One will improve the expansion of human primary somatic cells and promote their rejuvenation in a clinically relevant manner without permanent immortalization, while maintaining the proliferative capacity, functionality and normal characteristics of these primary cells. The second platform will allow for the efficient clinically relevant genetic engineering directly in primary somatic cells. In the latter, the use of our rejuvenating RNA cocktail will allow for the clonal expansion of genetically modified primary somatic cells, while preserving the functionality of these cells for subsequent transplantation. To develop the cell expansion platform, in Specific Aims 1 and 2, we will further characterize the effect of our cocktail on three human cell types commonly used in research and clinical settings: FBs, keratinocytes (KCs) and mesenchymal stromal/stem cells (MSCs). We will perform detailed molecular and functional characterizations of low and high passage human FBs, KCs and MSCs treated with our cocktail with a focus on the restoration of young-like characteristics of these cells. In Specific Aim 3, we will employ our rejuvenating cocktail to develop a platform for the generation of genetically modified human somatic cell lines. Using Cas9-mediated gene targeting, we will generate a panel of primary human FB, KC and MSC lines with the site-specific knock-in of fluorescence reporters and luciferase. The derived lines will be tested ex vivo and in vivo to confirm their functionality and safety, providing feasibility data for the development of novel somatic cell gene therapies for many diseases. If successful, the studies will serve as a proof of principle for developing rejuvenating strategies and genetic engineering platforms for other somatic cell types used for transplantation, such as hematopoietic and muscle stem cells.