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
基本信息
- 批准号:10369661
- 负责人:
- 金额:$ 17.7万
- 依托单位:
- 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:2021-03-09 至 2024-02-28
- 项目状态:已结题
- 来源:
- 关键词:3&apos Untranslated RegionsAddressAnti-Inflammatory AgentsApoptoticAttenuatedBindingBiologicalBiological ModelsBreast Cancer CellBreast Cancer cell lineBypassCell CycleCell ProliferationCell SurvivalCell modelCellsCellular biologyChimera organismChimeric ProteinsDNA DamageDevelopmentDiseaseDisease modelElementsEndoribonucleasesEngineeringExhibitsFamilyFutureGene ExpressionGenesGenetic TranscriptionGoalsGuide RNAHumanIn VitroIndividualInflammationInflammatoryInvestigationLinkMalignant NeoplasmsMammary NeoplasmsMediatingMessenger RNAMethodsMicroRNAsMitogen-Activated Protein KinasesModificationMolecularNeoplasm MetastasisOncogenicPathway interactionsPhenotypePhosphorylationPilot ProjectsProcessPropertyProtein EngineeringProteinsRNARNA BindingRNA DegradationRNA InterferenceRNA Recognition MotifRNA SequencesRNA-Binding ProteinsReagentRegulationRegulator GenesRegulatory PathwayRegulonResistanceRibonucleasesRoleSignal TransductionSite-Directed MutagenesisSpecificitySubstrate SpecificitySystemTIS11 proteinTechniquesTechnologyTestingTissuesTransfectionTumor Cell BiologyZinc Fingersaggressive breast cancerangiogenesisanti-canceranti-cancer therapeuticbasecancer cellcancer gene expressioncell transformationcell typecombinatorialdesignendonucleaseexperimental studygene networkhigh rewardhigh riskimprovedin vivoin vivo ModelinnovationinterestmRNA DecaymRNA ExpressionmRNA Transcript Degradationmigrationneoplastic cellnovelpatient prognosispreventprototyperesponserestorationstable cell linestemnesstooltranscriptometumortumorigenicuptake
项目摘要
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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Gerald M. Wilson其他文献
Nitroxyl and “Forbidden Disulfides”: Phospholamban Cysteines are Targeted to Enhance SERCA2a Activity
- DOI:
10.1016/j.bpj.2009.12.4189 - 发表时间:
2010-01-01 - 期刊:
- 影响因子:
- 作者:
Nazareno Paolocci;Carlo G. Tocchetti;James E. Mahaney;Gizem Keceli;Dong I. Lee;Jeff D. Ballin;Iain K. Farrance;Evangelia Kranias;Wei Dong Gao;Gerald M. Wilson;David A. Kass;John P. Toscano - 通讯作者:
John P. Toscano
The search for trans-acting factors controlling messenger RNA decay.
寻找控制信使 RNA 衰变的反式作用因子。
- DOI:
- 发表时间:
1999 - 期刊:
- 影响因子:0
- 作者:
Gerald M. Wilson;Gary Brewer - 通讯作者:
Gary Brewer
Reciprocal regulation of tristetrapoline and RNase-L modulates the induction of proinflammatory cytokines
- DOI:
10.1016/j.cyto.2009.07.316 - 发表时间:
2009-10-01 - 期刊:
- 影响因子:
- 作者:
Xiao-Ling Li;Sarah E. Brennan;Gerald M. Wilson;Bret A. Hassel - 通讯作者:
Bret A. Hassel
An episomal expression vector system for monitoring sequence-specific effects on mRNA stability in human cell lines.
一种附加型表达载体系统,用于监测人类细胞系中 mRNA 稳定性的序列特异性影响。
- DOI:
10.1006/plas.1995.1021 - 发表时间:
1995 - 期刊:
- 影响因子:2.6
- 作者:
Gerald M. Wilson;R. Deeley - 通讯作者:
R. Deeley
Gerald M. Wilson的其他文献
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{{ truncateString('Gerald M. Wilson', 18)}}的其他基金
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:
10571941 - 财政年份:2021
- 资助金额:
$ 17.7万 - 项目类别:
ULTRASENSITIVE RNA SENSING USING SURFACE PLASMON COUPLED EMISSION
使用表面等离子体耦合发射的超灵敏 RNA 传感
- 批准号:
7182003 - 财政年份:2005
- 资助金额:
$ 17.7万 - 项目类别:
BIOPHYSICAL ANALYSES OF INTERACTIONS BETWEEN A ZINC-FINGER PEPTIDE AND MRNA-DEST
锌指肽与 mRNA-DEST 之间相互作用的生物物理学分析
- 批准号:
7181987 - 财政年份:2005
- 资助金额:
$ 17.7万 - 项目类别:
REGULATION OF PROTEIN BINDING BY ION-DEPENDENT CHANGES IN RNA CONFORMATION
通过 RNA 构象的离子依赖性变化来调节蛋白质结合
- 批准号:
7181998 - 财政年份:2005
- 资助金额:
$ 17.7万 - 项目类别:
BIOPHYSICAL ANALYSES OF INTERACTIONS BETWEEN A ZINC-FINGER PEPTIDE AND MRNA-DEST
锌指肽与 mRNA-DEST 之间相互作用的生物物理学分析
- 批准号:
6978338 - 财政年份:2004
- 资助金额:
$ 17.7万 - 项目类别:
Mechanisms directing oncoprotein and cytokine mRNA decay
指导癌蛋白和细胞因子 mRNA 衰减的机制
- 批准号:
6895416 - 财政年份:2003
- 资助金额:
$ 17.7万 - 项目类别:
Mechanisms directing oncoprotein and cytokine mRNA decay
指导癌蛋白和细胞因子 mRNA 衰减的机制
- 批准号:
7622813 - 财政年份:2003
- 资助金额:
$ 17.7万 - 项目类别:
Mechanisms directing oncoprotein and cytokine mRNA decay
指导癌蛋白和细胞因子 mRNA 衰减的机制
- 批准号:
8248659 - 财政年份:2003
- 资助金额:
$ 17.7万 - 项目类别:
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