Decoding and reprogramming T cells through synthetic biology for cancer immunotherapy

通过合成生物学解码和重编程 T 细胞用于癌症免疫治疗

基本信息

批准号：
10568704
负责人：
Alexander Marson
金额：
$ 77.15万
依托单位：
J. DAVID GLADSTONE INSTITUTES
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-01-01 至 2027-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10568704
关键词：
Acceleration Address Adoptive Cell Transfers Adverse event Antigens Biological CD28 gene CRISPR interference CRISPR-mediated transcriptional activation CRISPR/Cas technology CTLA4 gene Cancer Model Cell Therapy Cell physiology Cells Cellular immunotherapy Chromatin Chronic Clustered Regularly Interspaced Short Palindromic Repeats Cues DNA Sequence Development Engineering Environment Evaluation Face Functional disorder Genes Genetic Genetic Complementation Test Genetic Engineering Genetic Screening Genetic Transcription Genetic study Genome Goals Human In Vitro Interferon Type II Interleukin-2 Knock-in Knock-out Learning Libraries Locales Malignant Neoplasms Methods Pre-Clinical Model Preclinical Testing Production Regulation Regulator Genes Repression Resistance Safety Science Site Synthetic Genes T cell therapy T-Cell Activation T-Lymphocyte Technology Testing Therapeutic Transgenic Organisms Translating Treatment Efficacy Tumor Antigens VAV1 gene Xenograft Model Xenograft procedure antigen-specific T cells cancer immunotherapy cancer therapy candidate validation chimeric antigen receptor T cells cytokine design engineered T cells fitness functional genomics gain of function gene discovery gene network genetic element genome wide screen genome-wide high throughput technology improved in vivo insight knock-down loss of function member mouse model next generation novel overexpression pre-clinical programs promoter rational design receptor response single-cell RNA sequencing small hairpin RNA synthetic biology synthetic construct therapeutic gene tool transcription factor tumor tumor microenvironment

项目摘要

ABSTRACT Engineered T cell-based cancer therapies are a major advancement in cancer treatment; however the majority of cancers still do not respond to adoptive cellular therapy. We need to “design” new T cell therapies with increased potency, and we need to overcome cell dysfunction that occurs as T cells face chronic tumor antigen stimulation. We and others have screened for genes that can be “knocked out” in antigen-specific T cells to enhance their functions, but enormous opportunities still remain to “knock-in” new synthetic DNA sequences at targeted genome sites. This proposal is focused on detailed evaluation of genes and inducible gene programs that will enable next-generation cellular therapies for cancer. We have developed several complementary technologies to discover synthetic gene programs that can be “inserted” into T cell genomes to enhance therapeutic functions. We developed a CRISPR technology for high throughput pooled knock-ins to accelerate discovery of synthetic knock-in programs (Roth et al., Cell, 2020), and have now have conducted two screens with ~100-member libraries that include transcription factors and synthetic chimeric receptors (“switch receptors”) to discover programs that make chronically stimulated T cells resistant to dysfunction. In addition, we have optimized a complementary robust platform for genome-wide CRISPR activation (CRISPRa) gain-of-function forward genetic screens in human T cells, and have already completed systematic discovery of factors that regulate stimulation-dependent cytokine production (Schmidt and Steinhart et al., Science, 2022). We propose to translate insights from these high-throughput discovery efforts into preclinical testing of novel knock-in designs with screen hits in vivo using xenotransplanted mouse models. In this proposal, we will test validated candidates from gain-of-function CRISPR PoKI (Aim 1) and CRISPRa (Aim 2) screens to discover new components of knock-in constructs that improve cell-based T cell therapies. We also recognize that these genetic components may be more beneficial if they are not expressed constitutively. In Aim 3, we draw on the power of synthetic biology to engineer synthetic circuits that can induce or repress genetic programs in response to antigen stimulation. This precise and dynamic regulation of genetic elements has great potential to further enhance efficacy and safety of next-generation immune cell therapies. Taken together, we present a proposal that leverages recent discoveries from CRISPR discovery platforms and deep expertise in synthetic biology to engineer powerful “knock-in” circuits that we will validate and study in preclinical cancer models. We leverage functional genomics, CRISPR engineering and synthetic cell program design expertise to address insufficient T cell potency and T cell dysfunction, which remain significant barriers to adoptive cell therapy for cancer.

抽象的基于工程化 T 细胞的癌症疗法是癌症治疗的一项重大进步；的癌症仍然对过继细胞疗法没有反应，我们需要“设计”新的 T 细胞疗法。效力增加，我们需要克服 T 细胞面对慢性肿瘤抗原时发生的细胞功能障碍我们和其他人筛选了可以在抗原特异性 T 细胞中“敲除”的基因。增强它们的功能，但仍然存在巨大的机会来“敲入”新的合成 DNA 序列该提案的重点是基因和诱导基因程序的详细评估。这将使下一代癌症细胞疗法成为可能。我们已经开发了几种互补的方法。发现可“插入”T 细胞基因组以增强功能的合成基因程序的技术我们开发了一种用于高通量基因敲入的 CRISPR 技术，以实现治疗功能。加速合成敲入程序的发现（Roth 等人，Cell，2020），并且现已进行两次筛选，包含约 100 个成员的文库，其中包括转录因子和合成嵌合体受体（“开关受体”）来发现使长期刺激的 T 细胞产生抵抗力的程序此外，我们还优化了用于全基因组 CRISPR 的互补强大平台。人类T细胞的功能获得性前向遗传筛选，并已完成系统地发现调节刺激依赖性细胞因子产生的因素（Schmidt 和 Steinhart 等人，Science，2022）。我们建议转化这些高通量发现的见解。使用异种移植小鼠进行体内屏幕点击的新型敲入设计的临床前测试工作在本提案中，我们将测试来自功能获得性 CRISPR PoKI 的经过验证的候选模型（目标 1）和 CRISPRa（目标 2）筛选发现可改善基于细胞的 T 细胞的敲入结构的新成分我们还认识到，如果这些遗传成分不表达，可能会更有益。在目标 3 中，我们利用合成生物学的力量来设计能够实现的合成电路。诱导或抑制基因程序以响应抗原刺激。遗传元件的调控具有进一步提高下一代疗效和安全性的巨大潜力综上所述，我们提出了一项利用最近发现的提案。 CRISPR 发现平台和合成生物学方面的深厚专业知识可设计强大的“敲入”电路我们将利用功能基因组学、CRISPR 在临床前癌症模型中进行验证和研究。工程和合成细胞程序设计专业知识可解决 T 细胞效力和 T 细胞不足的问题功能障碍，这仍然是癌症过继细胞疗法的重大障碍。