Suppressing oncogenic RNA regulons using engineered zinc finger ribonucleases

使用工程锌指核糖核酸酶抑制致癌 RNA 调节子

• 批准号: 10369661
• 负责人: Gerald M. Wilson
• 金额: $ 17.7万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-09 至 2024-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10369661
• 关键词: 3' Untranslated Regions; Address; Anti-Inflammatory Agents; Apoptotic; Attenuated; Binding; Biological; Biological Models; Breast Cancer Cell; Breast Cancer cell line; Bypass; Cell Cycle; Cell Proliferation; Cell Survival; Cell model; Cells; Cellular biology; Chimera organism; Chimeric Proteins; DNA Damage; Development; Disease; Disease model; Elements; Endoribonucleases; Engineering; Exhibits; Family; Future; Gene Expression; Genes; Genetic Transcription; Goals; Guide RNA; Human; In Vitro; Individual; Inflammation; Inflammatory; Investigation; Link; Malignant Neoplasms; Mammary Neoplasms; Mediating; Messenger RNA; Methods; MicroRNAs; Mitogen-Activated Protein Kinases; Modification; Molecular; Neoplasm Metastasis; Oncogenic; Pathway interactions; Phenotype; Phosphorylation; Pilot Projects; Process; Property; Protein Engineering; Proteins; RNA; RNA Binding; RNA Degradation; RNA Interference; RNA Recognition Motif; RNA Sequences; RNA-Binding Proteins; Reagent; Regulation; Regulator Genes; Regulatory Pathway; Regulon; Resistance; Ribonucleases; Role; Signal Transduction; Site-Directed Mutagenesis; Specificity; Substrate Specificity; System; TIS11 protein; Techniques; Technology; Testing; Tissues; Transfection; Tumor Cell Biology; Zinc Fingers; aggressive breast cancer; angiogenesis; anti-cancer; anti-cancer therapeutic; base; cancer cell; cancer gene expression; cell transformation; cell type; combinatorial; design; endonuclease; experimental study; gene network; high reward; high risk; improved; in vivo; in vivo Model; innovation; interest; mRNA Decay; mRNA Expression; mRNA Transcript Degradation; migration; neoplastic cell; novel; patient prognosis; prevent; prototype; response; restoration; stable cell line; stemness; tool; transcriptome; tumor; tumorigenic; uptake

Gene expression is extensively reprogrammed in cancer. These dysregulated genes include subpopulations called RNA regulons that are coordinately regulated by posttranscriptional mechanisms at the RNA level and control key features of tumor aggressiveness. To understand the molecular consequences of dysregulated RNA regulons in cancer, the goal of this exploratory, high-risk/high-reward R21 proposal is to develop a zinc finger-directed RNA-cleaving agent to suppress RNA regulons that are upregulated in many tumors. Our prototype links the tandem zinc finger (TZF) domain from tristetraprolin (TTP) to the endoribonuclease RNase4 (R4). In cells, the chimeric TZF-R4 protein is expected to bind and rapidly degrade mRNA substrates of TTP, but our design will allow substrate specificity to be systematically modified. The TTP TZF domain was chosen for our prototype because it has been evolutionarily selected to target an RNA regulon containing AU-rich elements (AREs), which includes many mRNAs that encode regulators of the cell cycle, angiogenesis, and metastasis. Furthermore, TTP expression or activity is frequently suppressed in human cancers; in particular, low TTP levels in breast tumors are associated with poor patient prognosis. This proposal is aimed at providing the “proof of concept” that a TZF-R4 chimera can function as a guided RNA degradation system in cancer cells to suppress a pro-tumorigenic RNA regulon and attenuate associated tumor cell phenotypes. Purified TZF-R4 shows selective RNA recognition and cleavage in vitro, and suppressed two known TTP substrate mRNAs when transiently transfected into cells. Furthermore, TZF-R4 dramatically slows cell proliferation when expressed in a clonal, stably-transfected breast cancer cell line. Building upon these key preliminary results, two specific aims will be pursued. First, we will use transcriptome- wide approaches to define the RNA regulon that is targeted and destabilized by TZF-R4 when stably expressed in aggressive breast cancer cell models and demonstrate that TZF-R4 suppresses multiple mRNA targets more efficiently than current technologies. Second, we will assess the impact of TZF-R4 on breast cancer cell proliferation, stemness, invasion, migration, and in vivo tumor development. Several future applications of this technology are envisioned, including: (i) discovery tools for characterizing RNA-mediated biological pathways, (ii) developing methods for promoting uptake of purified TZF-R4 into cells, bypassing transfection and opening possibilities for direct in vivo administration of this reagent, (iii) restoration or augmentation of TTP function to suppress tumor aggressiveness or inflammatory signaling, and (iv) expanding the specificity of the TZF-R4 platform by altering its RNA-targeting specificity. Strategies to broaden the scope include the iterative or combinatorial modification of the TZF moiety and substitution of other RNA-binding domains to `guide' the chimeric protein, creating a tunable family of targeted ribonucleases with long-term impact.

基因表达在癌症中被广泛重新编程。这些失调的基因包括 称为 RNA 调节子的亚群，由转录后机制协调调节 RNA水平和控制肿瘤侵袭性的关键特征。了解分子后果 癌症中的 RNA 调节子失调，这项探索性、高风险/高回报的 R21 提案的目标是 开发一种锌指导向的 RNA 切割剂来抑制在许多细胞中上调的 RNA 调节子 肿瘤。我们的原型将三四脯氨酸 (TTP) 的串联锌指 (TZF) 结构域连接到 核糖核酸内切酶 RNase4 (R4)。在细胞中，嵌合 TZF-R4 蛋白预计会结合并快速降解 TTP 的 mRNA 底物，但我们的设计将允许系统地修改底物特异性。 TTP 选择 TZF 结构域作为我们的原型是因为它经过进化选择来靶向 RNA 含有富含 AU 元件 (ARE) 的调节子，其中包括许多编码细胞调节因子的 mRNA 周期、血管生成和转移。此外，TTP 表达或活性经常受到抑制 人类癌症；特别是，乳腺肿瘤中 TTP 水平低与患者预后不良相关。 该提案旨在提供 TZF-R4 嵌合体可以充当引导器的“概念证明” 癌细胞中的 RNA 降解系统抑制促肿瘤 RNA 调节子并减弱相关 肿瘤细胞表型。纯化的 TZF-R4 在体外显示出选择性 RNA 识别和切割，并且 当瞬时转染到细胞中时，抑制了两种已知的 TTP 底物 mRNA。此外，TZF-R4 当在克隆、稳定转染的乳腺癌细胞系中表达时，会显着减缓细胞增殖。 在这些关键的初步结果的基础上，将追求两个具体目标。首先，我们将使用转录组- 广泛的方法来定义 TZF-R4 稳定时靶向和不稳定的 RNA 调节子 在侵袭性乳腺癌细胞模型中表达并证明 TZF-R4 抑制多种 mRNA 比现有技术更有效地瞄准目标。其次，我们将评估TZF-R4对乳房的影响 癌细胞增殖、干性、侵袭、迁移和体内肿瘤发展。 设想了该技术的几种未来应用，包括：(i) 表征 RNA 介导的生物途径，(ii) 开发促进纯化的吸收的方法 TZF-R4 进入细胞，绕过转染并为直接体内施用提供了可能性 试剂，(iii)恢复或增强TTP功能以抑制肿瘤侵袭性或炎症 (iv) 通过改变 RNA 靶向特异性来扩展 TZF-R4 平台的特异性。 扩大范围的策略包括 TZF 部分的迭代或组合修饰以及 替换其他RNA结合域来“引导”嵌合蛋白，创建一个可调节的靶向家族 具有长期影响的核糖核酸酶。