Next generation CRISPR/Cas9-RNAi mouse models for accelerated drug discovery research
用于加速药物发现研究的下一代 CRISPR/Cas9-RNAi 小鼠模型
基本信息
- 批准号:9282298
- 负责人:
- 金额:$ 65.66万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2014
- 资助国家:美国
- 起止时间:2014-08-04 至 2019-03-31
- 项目状态:已结题
- 来源:
- 关键词:AdultAllelesAnimalsBiological ModelsBreathingBreedingCRISPR/Cas technologyCancer ModelCancer-Predisposing GeneComplexDataDevelopmentDiseaseDisease modelDoxycyclineDrug TargetingES Cell LineEngineeringEvaluationGene CombinationsGene DeletionGene SilencingGene TargetingGenerationsGenesGeneticGenetic EngineeringGenetically Engineered MouseGoalsGuide RNAIn SituInjection of therapeutic agentKnock-outLeadLesionLung AdenocarcinomaLung AdenomaMaintenanceMalignant NeoplasmsMeasuresMediatingModelingMusMutagenesisMutationNon-Small-Cell Lung CarcinomaOncogenesOncogenicPathogenesisPathologyPharmacotherapyPhaseProcessProductionPublishingRNA InterferenceResearchSafetySideSmall Business Innovation Research GrantSystemTP53 geneTechniquesTechnologyTetracyclinesTherapeuticTimeToxic effectTreatment EfficacyValidationViral Vectorbaseblastocystcancer therapycohortcostcost effectivedrug discoveryembryonic stem cellfight againstflexibilitygenome editingin vivoinnovationmouse modelmutantnew therapeutic targetnext generationnovelnovel strategiesnovel therapeuticspre-clinicalpreclinical studyrecombinase-mediated cassette exchangerepairedsmall hairpin RNAsmall moleculesuccesssynergismtooltumor microenvironmentvector
项目摘要
Abstract
Significance: New approaches for rapid identification and early preclinical validation of novel therapeutic
targets are crucial to make important “go/no-go” decisions and curb the cost of developing new cancer
treatments. Genetically engineered mouse models (GEMMs) are a powerful platform to study disease initiation
and maintenance, the tumor microenvironment and the responsiveness of cancers to known or novel
therapeutics; however, the long lead times and high costs required to develop, intercross and maintain models
with various cancer predisposing gene combinations have limited their practical utility in the drug discovery
process. Recently, we have shown RNA interference (RNAi) in mice can serve as a fast alterative to gene
deletion and be exploited experimentally to silence nearly any gene target, by the expression of synthetic short
hairpin RNAs (shRNAs). Importantly, because it is reversible, gene silencing by RNAi better mimics the
dynamics of small molecule inhibition than permanent genetic knockouts. Furthermore, with the advent of new
genome editing techniques, such as CRISPR/Cas9 technology, we are able to introduce additional sensitizing
lesions to induce disease pathogenesis. In synergy with RNAi technology, complex multi-allelic ESC based
GEMMs can be generated without extensive intercrossing. Using this combination of CRISPR/Cas9 and RNAi
technologies, we are able to not only model disease pathogenesis, but also mimic drug therapy in mice, giving
us unprecedented capabilities to perform preclinical studies in vivo. Hypothesis: We hypothesize that
CRISPR/Cas9-RNAi-GEMMs of cancer can be developed rapidly using new genome editing technologies
(CRISPRs) to introduce additional sensitizing lesions and recombinase-mediated cassette exchange (RMCE)
for precise integration of tetracycline inducible shRNAs to silence specific gene targets. Preliminary data: We
have previously used CRISRP/Cas9 and RMCE to generate RNAi-GEMMs without any breeding. Specific
Aims: As a proof-of-concept, we will develop a model of lung adenocarcinoma by using the CRISPR/Cas9
system to introduce a conditional KrasG12D allele into the endogenous locus and in situ delivery of sgRNAs
targeting Trp53 which will be activated by a conditionally expressed Cas9 allele. We will further modulate
mutant Kras or Mek1/2 activity by introducing tetracycline inducible shRNAs to model therapeutic inhibition.
Finally, we will expand our flexible platform by producing validated, ‘off-the-shelf’ viral vectors carrying
combination sgRNAs targeting commonly altered genes in NSCLC. Together, these studies will define a new
paradigm and accelerate drug discovery research by creating a flexible platform for the generation of RNAi-
GEMMs that will serve as innovative research tools, guiding the development of novel and effective
therapeutics.
摘要
意义:快速鉴定和早期临床前验证新型治疗药物的新方法
目标对于做出重要的“去/不去”决定和控制发展新癌症的成本至关重要
治疗。基因工程小鼠模型(GEMM)是研究疾病发生的有力平台
肿瘤微环境和癌症对已知或新的抗肿瘤药物的反应性,
然而,开发、交叉和维持模型所需的时间长,成本高,
限制了它们在药物发现中的实际效用
过程最近,我们在小鼠中发现RNA干扰(RNAi)可以作为一种快速的基因改变剂,
通过表达合成的短片段,
发夹RNA(shRNAs)。重要的是,因为它是可逆的,RNAi的基因沉默更好地模拟了基因沉默。
与永久性基因敲除相比,小分子抑制的动力学。此外,随着新的
基因组编辑技术,如CRISPR/Cas9技术,我们能够引入额外的敏化
病变诱发疾病发病机制。与RNAi技术协同作用,基于复杂多等位基因ESC的
GEMM可以在没有广泛交叉的情况下产生。使用这种CRISPR/Cas9和RNAi的组合
技术,我们不仅能够模拟疾病的发病机制,而且还能在小鼠中模拟药物治疗,
我们前所未有的能力,进行临床前研究在体内。假设:我们假设
癌症的CRISPR/Cas9-RNAi-GEMM可以使用新的基因组编辑技术快速开发
(CRISPR)引入额外的致敏病变和重组酶介导的盒交换(RMCE)
用于精确整合四环素诱导的shRNA以沉默特异性基因靶。初步数据:我们
先前使用CRISRP/Cas9和RMCE来产生RNAi-GEMM而没有任何育种。具体
目的:作为概念验证,我们将通过使用CRISPR/Cas9构建肺腺癌模型。
将条件KrasG 12 D等位基因引入内源基因座并原位递送sgRNA的系统
靶向Trp 53,其将被条件性表达的Cas9等位基因激活。我们将进一步调整
通过引入四环素诱导的shRNA来模拟治疗抑制,从而获得突变的Kras或Mek 1/2活性。
最后,我们将通过生产经过验证的“现成”病毒载体来扩展我们的灵活平台,
靶向NSCLC中常见改变基因的组合sgRNA。总之,这些研究将定义一个新的
通过创建一个灵活的RNAi生成平台,
GEMM将作为创新的研究工具,指导开发新颖有效的
治疗学
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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Prem Khovabutr Premsrirut其他文献
Prem Khovabutr Premsrirut的其他文献
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{{ truncateString('Prem Khovabutr Premsrirut', 18)}}的其他基金
Preclinical Evaluation of a Novel ADAM10 Modulator to Treat ColorectalCancer
新型 ADAM10 调节剂治疗结直肠癌的临床前评估
- 批准号:
10697653 - 财政年份:2023
- 资助金额:
$ 65.66万 - 项目类别:
CRISPR/Cas-mediated development of an RNAi rat model system
CRISPR/Cas介导的RNAi大鼠模型系统的开发
- 批准号:
9908231 - 财政年份:2018
- 资助金额:
$ 65.66万 - 项目类别:
High efficiency platform for rapid RNAi rat model development
用于快速RNAi大鼠模型开发的高效平台
- 批准号:
9557373 - 财政年份:2018
- 资助金额:
$ 65.66万 - 项目类别:
CRISPR/Cas-mediated development of an RNAi rat model system
CRISPR/Cas介导的RNAi大鼠模型系统的开发
- 批准号:
10160974 - 财政年份:2018
- 资助金额:
$ 65.66万 - 项目类别:
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