RNA-programmable cell-type targeting, editing, and therapy

RNA 可编程细胞类型靶向、编辑和治疗

• 批准号: 10483215
• 负责人: Z JOSH HUANG
• 金额: $ 112.7万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-07 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10483215
• 关键词: Ablation; Anatomy; Animals; Base Pairing; Biological Process; Biology; Biomedical Research; Biotechnology; Birds; Brain; Cell Culture System; Cell physiology; Cells; Cerebral cortex; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; Devices; Disease; Engineering; Enhancers; Enzymes; Ethical Issues; Genes; Genetic Transcription; Genome; Health; Human; Industrialization; Libraries; Link; Macaca; Medicine; Methods; Molecular Genetics; Monitor; Mus; Neurons; Organ; Organism; Physiological; Population; Primates; RNA; RNA Editing; Ribonucleoproteins; Role; Somatic Cell; Specimen; System; Technology; Tissue Engineering; Tissues; Validation; Visualization; adenosine deaminase; base; cell type; combinatorial; cost; design; genetic manipulation; genetic technology; new technology; next generation; novel strategies; programs; sensor; transcriptome

Josh Huang Sept 6, 2020 RNA-programmable cell-type targeting, editing, and therapy Abstract Systematic identification and manipulation of cell types is necessary for dissecting mechanisms of biological functions in health and disease. Although large-scale, single-cell transcriptome profiling now enables identification of all major transcription-defined cell types in many organisms, easy and systematic experimental access to all major cell populations is needed to physiologically and anatomically validate these statistical “transcriptional clusters” as cell types and, more importantly, to interrogate their roles in tissue organization and function. The difficulty of selectively manipulating cell types remains a critical barrier to such studies. Current approaches to this problem mostly rely on germline DNA engineering, which is slow and expensive and poses ethical issues, especially in humans and other primates. Cell-type transcriptional enhancers afford a non- germline approach, but their identification and validation remain effort-intensive and costly. To overcome these barriers, all of biomedical research urgently needs a novel approach to manipulate cell types in a way that is specific, easy yet comprehensive, affordable, and generalizes across organs and species, akin to CRISPR- based manipulation of genes. Here I propose to develop a paradigm-shifting technology that will enable RNA- programmable cell-type targeting and manipulation based on the fundamental biology of RNA editing. To achieve this breakthrough, I will harness a set of next-generation, multi-functional ribonucleoprotein devices, which can detect the presence of specific RNAs in somatic cells and trigger the expression of effector genes for cell visualization, monitoring, and manipulation. This method builds upon the universal RNA sensing and editing system within all metazoan cells, centered around the editing enzyme adenosine deaminase acting on RNA (ADAR). I term this method CellREADR (Cell access through RNA sensing by Endogenous ADAR). As CellREADR leverages endogenous cellular machinery and is built with a single modular RNA molecule that functions through Watson-Crick base pairing, it is highly specific, inherently programmable, fast, affordable, easy to use, and widely applicable. I propose to build and optimize CellREADR devices in cell-culture systems and validate the method in a highly complex organ - the brain - by targeting and manipulating a large set of neuronal cell types in the mouse cerebral cortex. We will extend CellREADR across species by targeting cell types in ex vivo human brain specimens, and in the macaque and avian brain. Further, we will design intersectional strategies for targeting increasingly specific cell types, and combinatorial strategies for simultaneous and differential manipulation of multiple cell types in a tissue. By linking cell-type RNA sensors to a variety of effector genes that alter cell functions, ranging from ablation to subtle physiological modulation, we aim to edit cell composition and function for next-generation tissue engineering. This technology will result in large arrays of CellREADR libraries for targeting all major cell types across diverse species, akin to CRISPR-based gene editing of diverse genomes. Thus, CellREADR will have a broad impact in basic biology, medicine, and biotechnology.

乔什·黄2020年9月6日 RNA可编程的细胞类型靶向，编辑和治疗 抽象的 对于生物学的不同意，需要系统的识别和操纵细胞类型是必要的 尽管大规模，单细胞的转录组分析可以实现健康和疾病 识别许多生物体中所有主要转录定义的细胞类型，易于实验 需要访问所有主要的单元组人口 “转录簇”作为细胞类型，更重要的是，询问其在组织组织中的作用 功能。 解决问题的方法主要依赖于种系DNA工程，这是缓慢而昂贵的，姿势 道德问题，尤其是人类和其他灵长类动物。 种系方法，但是识别和验证仍然是付​​出的努力和昂贵的 障碍，所有的生物医学研究都需要一种新颖的方法来操纵细胞类型的方式 特定，容易但全面，负担得起，并且遍布器官和物种，类似于crispr- 基因的操纵。我建议开发一种范式转移技术 基于RNA编辑的基本生物学的可编程细胞类型靶向。 这个突破，我将利用一组下一代多功能核糖核蛋白设备，可以 检测体细胞中特定RNA的存在并触发细胞有效基因的表达 可视化，监视和操纵。 在RNA上的所有后生细胞中的系统中，以编辑酶腺苷脱氨酶为中心 （ADAR） CellReadr利用内源性细胞机制，并用单个模块化RNA分子构建 通过Watson-Crick Base配对的功能，它具有高度特定的，固有的可编程，快速，可容纳，容易 使用，并广泛适用。 通过靶向和操纵大型神经元来验证高度复杂的器官中的方法 - 大脑 - 大脑 小鼠大脑皮层中的细胞类型，我们将通过靶向EX的细胞类型来扩展细胞读 体内人脑标本，在猕猴和鸟类的大脑中。 针对越来越具体的细胞类型的策略，以及同时和 通过将细胞类型的RNA传感器与多种效应器联系起来的多种细胞类型的差异操纵 从ABR到亚曲生理学调制的基因，细胞功能明显的函数，我们的目标是编辑细胞 下一代组织工程的组成和功能。 CellReadR库，用于针对各种物种的所有主要细胞类型，类似于CRISPR-BASS的基因编辑 因此，各种基因组。