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

• 批准号: 9282298
• 负责人: Prem Khovabutr Premsrirut
• 金额: $ 65.66万
• 依托单位: MIRIMUS, INC.
• 依托单位国家: 美国
• 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: https://reporter.nih.gov/project-details/9282298
• 关键词: Adult; Alleles; Animals; Biological Models; Breathing; Breeding; CRISPR/Cas technology; Cancer Model; Cancer-Predisposing Gene; Complex; Data; Development; Disease; Disease model; Doxycycline; Drug Targeting; ES Cell Line; Engineering; Evaluation; Gene Combinations; Gene Deletion; Gene Silencing; Gene Targeting; Generations; Genes; Genetic; Genetic Engineering; Genetically Engineered Mouse; Goals; Guide RNA; In Situ; Injection of therapeutic agent; Knock-out; Lead; Lesion; Lung Adenocarcinoma; Lung Adenoma; Maintenance; Malignant Neoplasms; Measures; Mediating; Modeling; Mus; Mutagenesis; Mutation; Non-Small-Cell Lung Carcinoma; Oncogenes; Oncogenic; Pathogenesis; Pathology; Pharmacotherapy; Phase; Process; Production; Publishing; RNA Interference; Research; Safety; Side; Small Business Innovation Research Grant; System; TP53 gene; Techniques; Technology; Tetracyclines; Therapeutic; Time; Toxic effect; Treatment Efficacy; Validation; Viral Vector; base; blastocyst; cancer therapy; cohort; cost; cost effective; drug discovery; embryonic stem cell; fight against; flexibility; genome editing; in vivo; innovation; mouse model; mutant; new therapeutic target; next generation; novel; novel strategies; novel therapeutics; pre-clinical; preclinical study; recombinase-mediated cassette exchange; repaired; small hairpin RNA; small molecule; success; synergism; tool; tumor microenvironment; vector

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.

抽象的 意义：快速识别和早期临床前验证的新方法的新方法 目标对于做出重要的“走/不执行”决定至关重要，并遏制发展新癌症的成本 治疗。基因工程的小鼠模型（GEMM）是研究疾病启动的强大平台 和维护，肿瘤微环境以及癌症对已知或新颖的反应性 治疗;但是，发展，互变和维护模型所需的漫长的交货时间和高成本 具有各种癌症的基因组合限制了它们在药物发现中的实际实用性 过程。最近，我们在小鼠中显示了RNA干扰（RNAi）可以作为基因的快速改变 删除并通过实验探索几乎所有基因靶标的沉默，通过合成简短的表达 发夹RNA（shrnas）。重要的是，因为它是可逆的，所以RNAi的基因沉默更好地模仿 小分子抑制的动力学比永久性遗传基因敲除。此外，随着新冒险 基因组编辑技术，例如CRISPR/CAS9技术，我们能够引入其他敏感性 诱导疾病发病机理的病变。与RNAi技术协同作用，基于复杂的多行ESC 可以在不大规模间交叉的情况下生成宝石。使用CRISPR/CAS9和RNAi的组合 技术，我们不仅能够建模疾病发病机理，而且还可以模仿小鼠的药物治疗，从而给予药物治疗。 美国在体内进行临床前研究的空前能力。假设：我们假设 CRISPR/CAS9-RNAI癌症可以使用新的基因组编辑技术迅速开发 （CRISPR）引入其他敏化病变和重组酶介导的盒式磁带交换（RMCE） 为了精确整合四环素诱导的shRNA，以使特定基因靶标保持沉默。初步数据：我们 以前已经使用CRISRP/CAS9和RMCE生成RNAi-GEMM，而无需任何繁殖。具体的 目的：作为概念证明，我们将使用CRISPR/CAS9开发肺腺癌模型 系统将有条件的KRASG12D等位基因引入内源性基因座和SGRNA的原位传递 靶向TRP53，该TRP53将被有条件表达的Cas9等位基因激活。我们将进一步调制 突变的KRAS或MEK1/2活性通过引入四环素诱导的shRNA来建模治疗抑制作用。 最后，我们将通过生产经过验证的“现成”病毒向量来扩展我们的灵活平台 NSCLC中通常改变基因的SGRNA组合。这些研究将共同​​定义一个新的 通过创建一个灵活的平台来生成RNAi-，范式和加速药物发现研究 将作为创新研究工具的宝石，指导新颖有效的发展 疗法。