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

使用合成 RNA 分子开发人类体细胞再生、扩增和基因工程平台

基本信息

批准号：
10623161
负责人：
Igor Kogut
金额：
$ 47.65万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-05-01 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10623161
关键词：
Acceleration Adult Aging Autologous Transplantation Biological Assay Birth Burn injury Cell Aging Cell Line Cell Proliferation Cell Transplantation Cell division Cell physiology Cells Characteristics Clinical Clonal Expansion Complex Connective Tissue Diseases DNA Damage Data Development Disease Elongation Factor Epigenetic Process Fibroblasts Fluorescence Foundations Future Gene Targeting Generations Genes Genetic Genetic Engineering Growth Guide RNA Hematologic Neoplasms Hematological Disease Hematopoietic stem cells Human Human Engineering Individual Knock-in Legal patent Length Life Longevity Luciferases Mediating Mesenchymal Messenger RNA Mitochondrial DNA Molecular Mus Muscle satellite cell Phenotype Ploidies Pluripotent Stem Cells Premature aging syndrome Proliferating Proteins RNA Regenerative Medicine Rejuvenation Reporter Research Safety Site Skin graft Somatic Cell Source Stromal Cells System T-Lymphocyte Technology Telomerase Telomere Maintenance Telomere Shortening Testing Time Tissue Engineering Tissue Model Tissues Transcription Coactivator Transfection Transplantation Treatment Efficacy Xenograft Model adult stem cell aged cell type cellular engineering chimeric antigen receptor T cells chronic wound clinically relevant drug testing enzyme activity epigenome epithelial stem cell experimental study gene therapy genetically modified cells healing human adult stem cell human disease improved in vivo induced pluripotent stem cell induced pluripotent stem cell technology keratinocyte mitochondrial dysfunction mitochondrial metabolism novel novel strategies nuclease preservation prevent response restoration senescence stem stem cells technology platform telomere transplant model

项目摘要

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.

项目概要人类体细胞（包括成体干细胞）在离体环境中的寿命较短是一个重要原因许多临床和研究应用的障碍。因此，开发新方法可以促进成体细胞的扩张，同时恢复这些细胞的年轻特征没有永久的永生是再生医学的首要任务。端粒磨损是其中之一体细胞增殖和功能下降的明确机制培养物，并且可以被端粒酶的活性抵消。线粒体功能障碍和表观遗传变化也是导致细胞衰老、衰老和衰退的机制之一功能。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 RNA 的非整合转染人体细胞后，端粒长度会增加至在多能干细胞中观察到，提高细胞增殖率，恢复线粒体 DNA 含量并允许单独接种的成人成纤维细胞 (FB) 进行克隆扩增。我们现在建议使用这种恢复活力的 RNA 混合物可开发两个新颖的细胞技术平台。一将改善扩增人类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、功能和正常这些原代细胞的特征。第二个平台将允许有效的临床相关遗传直接在原代体细胞中进行工程改造。在后者中，使用我们的复兴 RNA 鸡尾酒将允许转基因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的细胞。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设置：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传代和高传代人 FB、KC 和 MSC 处理后的分子和功能特征我们的鸡尾酒专注于恢复这些细胞的年轻特征。在具体目标 3 中，我们将使用我们的恢复活力鸡尾酒开发一个用于生成转基因人类的平台体细胞系。使用 Cas9 介导的基因打靶，我们将生成一组原代人类 FB、KC 以及具有荧光报告基因和荧光素酶位点特异性敲入的 MSC 系。导出的线将是进行离体和体内测试，确认其功能性和安全性，为开发提供可行性数据针对许多疾病的新型体细胞基因疗法。如果成功的话，这些研究将作为证据为其他体细胞类型开发复兴策略和基因工程平台的原则用于移植，如造血干细胞和肌肉干细胞。