Decoding and reprogramming T cells through synthetic biology for cancer immunotherapy

摘要 基于工程化T细胞的癌症疗法是癌症治疗的重大进步;然而，大多数癌症治疗方法都是基于T细胞的。 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， 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， 刺激.我们和其他人已经筛选出可以在抗原特异性T细胞中“敲除”的基因， 增强它们的功能，但仍有巨大的机会“敲入”新的合成DNA序列， 靶向基因组位点。这项建议的重点是详细评估基因和诱导基因程序 这将使下一代癌症细胞疗法成为可能。我们开发了几种互补的 技术，以发现可以“插入”到T细胞基因组中的合成基因程序， 治疗功能。我们开发了用于高通量合并敲入的CRISPR技术， 加速合成敲入程序的发现（Roth等，细胞，2020年），现在已经进行了 两次筛选，使用约100个成员的文库，包括转录因子和合成嵌合体， 受体（“开关受体”），以发现程序，使长期刺激的T细胞抵抗 功能障碍此外，我们还优化了一个互补的强大平台，用于全基因组CRISPR 在人类T细胞中进行CRISPRa激活（CRISPRa）功能获得性正向遗传筛选，并已完成 调节刺激依赖性细胞因子产生的因子的系统发现（施密特和 Steinhart等人，Science，2022）。我们建议将这些高通量发现的见解 使用异种移植小鼠在体内筛选命中的新型敲入设计的临床前测试的努力 模型在本提案中，我们将测试来自功能获得性CRISPR PoKI（Aim 1）的经验证候选物， CRISPRa（Aim 2）筛选发现敲入构建体的新组分，可改善基于细胞的T细胞 治疗我们也认识到，这些遗传成分如果不表达，可能更有益 本质上。在目标3中，我们利用合成生物学的力量来设计合成电路， 诱导或抑制响应抗原刺激遗传程序。这种精确而动态的 基因元件的调控具有进一步增强下一代药物的有效性和安全性的巨大潜力。 免疫细胞疗法总之，我们提出了一个建议，利用最近的发现， CRISPR发现平台和在合成生物学方面的深厚专业知识，可设计强大的“敲入”电路 我们将在临床前癌症模型中验证和研究。我们利用功能基因组学、CRISPR 工程和合成细胞程序设计专业知识，以解决T细胞效力不足和T细胞 功能障碍，这仍然是癌症过继细胞疗法的重大障碍。

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.