Next generation CRISPR/Cas9-RNAi mouse models for accelerated drug discovery research

摘要 意义：新疗法的快速识别和早期临床前验证的新方法 目标对于做出重要的“通过/不通过”决定和控制开发新癌症的成本至关重要。 治疗。基因工程小鼠模型(GEMM)是研究疾病发生的强大平台 肿瘤微环境和癌症对已知或新事物的反应性 治疗；然而，开发、交叉和维护模型所需的长准备时间和高成本 不同的致癌基因组合限制了它们在药物发现中的实用价值 进程。最近，我们发现小鼠体内的RNA干扰(RNAi)可以作为基因的快速变种 缺失，并被实验利用来沉默几乎任何基因靶点，通过表达合成的Short 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拟了 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 病变诱发疾病的发病机制。与RNAi技术协同，基于复杂多等位基因的ESC 可以在没有大量交叉的情况下产生GEMM。使用CRISPR/Cas9和RNAi的这种组合 技术，我们不仅能够模拟疾病的发病机制，而且能够在小鼠身上模拟药物治疗，给予 美国前所未有的在体内进行临床前研究的能力。假设：我们假设 利用新的基因组编辑技术可以快速开发癌症的CRISPR/Cas9-RNAi-GEMM (CRISPR)引入额外的致敏病变和重组酶介导盒交换(RMCE) 用于精确整合四环素诱导的shRNA，以沉默特定的基因靶点。初步数据：WE 以前曾使用CRISRP/Cas9和RMCE在没有任何育种的情况下产生RNAi-GEMM。特定的 目的：作为概念验证，我们将利用CRISPR/CAS9建立肺腺癌模型 将条件性KrasG12D等位基因引入内源基因座并原位传递sgRNAs的系统 靶向TrP53，它将被有条件表达的Cas9等位基因激活。我们将进一步调整 突变的Kras或MEK1/2活性通过引入四环素诱导的shRNA来模拟治疗抑制。 最后，我们将通过生产有效的、现成的病毒载体来扩展我们的灵活平台 针对非小细胞肺癌常见改变基因的组合sgRNAs。总而言之，这些研究将定义一个新的 通过创建一个灵活的RNAi生成平台来规范和加速药物发现研究- GEMM将作为创新的研究工具，指导创新和有效的开发 治疗学。

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.