Multiplexed and Logical Control of the Mammalian Transcriptome Using Cas13

使用 Cas13 对哺乳动物转录组进行多重逻辑控制

• 批准号: 10279442
• 负责人: Wilson Wong
• 金额: $ 39.69万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-20 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10279442
• 关键词: Antitumor Response; Biological; CAR T cell therapy; Cell Therapy; Cell physiology; Cells; Chemicals; Chromatin Structure; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Cues; Dangerousness; Dimerization; Disease; Engineering; FDA approved; Gene Expression; Genes; Genetic; Genetic Engineering; Genome engineering; Guide RNA; Human; Immune; In Vitro; Intelligence; Investigation; Knock-out; Lead; Light; Malignant Neoplasms; Mammalian Cell; Modeling; Mus; Names; Orthologous Gene; Pharmaceutical Preparations; Production; Public Health; Publishing; RNA Degradation; RNA Interference; Ribonucleases; Safety; Sensitivity and Specificity; Signal Pathway; Signal Transduction; Site; Specificity; Surface Antigens; Work; Xenograft procedure; chimeric antigen receptor T cells; combat; cytokine; cytokine release syndrome; design; extracellular; flexibility; genome editing; improved; interest; novel; novel diagnostics; novel therapeutics; prevent; programs; response; sensor; side effect; small molecule; success; therapy development; tool; transcription factor; transcriptome; tumor

The mammalian transcriptome integrates diverse extracellular and intracellular signals and controls numerous critical cell functions. Furthermore, the ability to flexibly control endogenous gene expression in response to external and internal signals will lead to breakthrough cell therapies with enhanced safety and efficacy. However, we lack tools that can reversibly and effectively modulate the transcriptome. Genome editing tools, RNA inter- ference, and programmable transcription factors are powerful genetic engineering tools. However, they all have some deficiencies, either they permanently disrupt the gene of interest, which prevents investigation of the dy- namic impact of the gene, or they lack specificity and activity. Therefore, novel tools are urgently needed to advance our ability to reprogram the mammalian transcriptome. Recently, a new class of RNA-guided RNA nuclease, Cas13, has been discovered. Some Cas13 orthologs, such as Cas13d and Cas13b, have been shown to have higher and more specific RNA degradation activity than RNAi in mammalian cells. Cas13 is also not affected by the chromatin structure, a challenge commonly encoun- tered by programmable transcription factors for transcriptome engineering. It also doesn't have the collateral cleavage activity in mammalian cells that other Cas13 has. Therefore, Cas13 has the potential to be the best tool for mammalian transcriptome engineering. Similar to other genome engineering tools, the full potential of Cas13 can only be realized if regulatory mecha- nisms and genetic circuits have been incorporated that afford facile and intelligent control. Currently, there are no tools specifically designed to regulate Cas13 activity. Therefore, the objective of this proposed work is to develop a set of groundbreaking tools with Cas13 that can inducibly and reversibly modulate the mammalian transcriptome in response to exogenous small molecules (and FDA approved drugs) or biological cues. Also, to showcase their clinical potential, we will leverage our engineered Cas13 to improve the safety of chimeric antigen receptor (CAR) T cell therapy. CAR T cell therapies have shown tremendous promise and efficacy against vari- ous cancers, but they can also lead to cytokine release syndrome (CRS), a dangerous adverse side effect. The factors produced by CAR T cells lead to CRS are also necessary for the function of the therapy. Therefore, inducible and tunable control of these critical factors, as opposed to knockout, are required to develop safe and effective CAR T cell therapy. We will achieve our objectives through the following aims: Aim 1: Develop a collection of split inducible Cas13 Aim 2: Establish cell-autonomous control of Cas13 in response to biological signals Aim 3: Modulate cytokine production in CAR T cells to combat CRS Success from this work will have a transformative impact on cell therapy development.

哺乳动物转录组整合了多种细胞外和细胞内信号，并控制着许多 关键的细胞功能。此外，灵活控制内源基因表达的能力，以响应 外部和内部信号将导致具有增强的安全性和功效的突破性细胞疗法。然而，在这方面， 我们缺乏可以可逆地和有效地调节转录组的工具。基因组编辑工具，RNA间 参考和可编程转录因子是强大的基因工程工具。然而，他们都有 一些缺陷，要么他们永久性地破坏感兴趣的基因，这阻止了对疾病的研究， 或者它们缺乏特异性和活性。因此，迫切需要新的工具， 提高我们重新编程哺乳动物转录组的能力。 最近，发现了一类新的RNA引导的RNA核酸酶，Cas13。一些Cas13直系同源物， 例如Cas13d和Cas13b，已经显示出比Cas13d和Cas13b具有更高和更特异的RNA降解活性。 哺乳动物细胞中的RNAi。Cas13也不受染色质结构的影响，这是一个常见的挑战。 由用于转录组工程化的可编程转录因子所控制。它也没有抵押品 其他Cas13在哺乳动物细胞中具有的切割活性。因此，Cas13有潜力成为最好的 哺乳动物转录组工程的工具。 与其他基因组工程工具类似，Cas13的全部潜力只有在监管机制发挥作用的情况下才能实现。 已经结合了寄生虫和遗传电路，提供了方便和智能的控制。目前有 没有专门设计用于调节Cas13活性的工具。因此，本拟议工作的目标是 开发一套具有Cas13的开创性工具，可以诱导和可逆地调节哺乳动物 转录组响应外源小分子（和FDA批准的药物）或生物学线索。我们还会利用这些信息来 展示其临床潜力，我们将利用我们的工程化Cas13来提高嵌合抗原的安全性 受体（CAR）T细胞疗法。CAR T细胞疗法已经显示出巨大的前景和对各种瓦里的有效性。 它们可以治疗癌症，但也可能导致细胞因子释放综合征（CRS），这是一种危险的不良副作用。的 由CAR T细胞产生的导致CRS的因子也是治疗功能所必需的。因此，我们认为， 与敲除相反，需要对这些关键因子进行可诱导和可调的控制，以开发安全且 有效的CAR T细胞疗法。我们将通过以下目标实现我们的目标： 目的1：开发裂解诱导型Cas13的集合 目的2：建立响应生物信号的Cas13的细胞自主控制 目的3：调节CAR T细胞中的细胞因子产生以对抗CRS 这项工作的成功将对细胞疗法的发展产生变革性的影响。