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

• 批准号: 10399980
• 负责人: Igor Kogut
• 金额: $ 47.18万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 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/10399980
• 关键词: Adult; Aging; Autologous Transplantation; Biological Assay; Birth; Burn injury; Cell Aging; Cell Line; Cell Proliferation; Cell Transplantation; Cell division; Cell physiology; Cells; Characteristics; Clinical; Clinical Research; Clonal Expansion; Complex; Connective Tissue Diseases; DNA Damage; Data; Development; Disease; Epigenetic Process; Fibroblasts; Fluorescence; Foundations; Future; Gene Targeting; Generations; Genes; Genetic; Genetic Engineering; Growth; Guide RNA; Hematologic Neoplasms; Hematological Disease; Hematopoietic; 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; Proteins; RNA; Regenerative Medicine; Rejuvenation; Reporter; Research; Safety; Site; Skin graft; Somatic Cell; Source; Stromal Cells; System; 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; base; cell type; cellular engineering; chimeric antigen receptor T cells; chronic wound; clinically relevant; drug testing; enzyme activity; epigenome; epithelial stem cell; experimental study; functional decline; gene therapy; genetically modified cells; healing; human adult stem cell; human disease; improved; improved functioning; 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; telomere; transplant model

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.

项目总结