Project 3

摘要：项目3 在过去的几十年里，已经有可能分离患者自己的细胞，设计并扩大它们 在实验室里，并用它们来治疗现有的癌症。这项技术已经在很大程度上进步到使用初级 基因修饰的人T细胞表达肿瘤特异性T细胞受体(TCR)或嵌合抗原受体 (汽车)。这种方法在一些白血病中显示了持久的治愈作用，但在治疗白血病方面的成功有限 实体肿瘤的治疗。这种疗效的缺乏被认为是由于T细胞在迁移过程中面临的挑战 进入和进入复杂的固体肿瘤微环境和在那里发现的多种免疫抑制方式。 随着这些挑战中的许多已经被识别和机械地研究，我们假设我们可以 工程CAR T细胞能够克服在固体肿瘤微环境中发现的所有挑战，领先 为预后最差的癌症患者提供持久的治疗。最近，一些团体发表了高水平的文章 使用CRISPR/CAS9设计T细胞的有效方法。此外，重组腺病毒的使用 相关病毒(RAAV)作为DNA供体分子与Cas9结合进行同源重组 还显示了T细胞中令人难以置信的位点特异性基因传递速度。尽管这些方法是高度 虽然有效，但在提高基因编辑的能力和安全性方面仍存在一些缺陷和问题需要解决 用于治疗的细胞。例如，基于核酸酶的基因编辑仍然依赖于遗传毒性双链的随机修复 链断裂(DSB)，在编辑结果中几乎没有一致性。幸运的是，新技术已经出现。 在单碱基对分辨率下进行DNA基因编辑，产物纯度高、效率高、无靶向 DSB，称为Cas9基本编辑者(BES)和初级编辑者(PE)。我们已经证明了这项技术 允许通过可编程的酶促单碱基改变进行高效的多重基因组编辑，而无需 制造有毒的DSB，即“数字编辑”。由于这项技术相对较新，也有发展的机会 新的和创新的基因组编辑策略，以开发用于治疗的完全数字化编辑的细胞 以成本效益、快速和准确的方式。因此，我们的具体目标是：1)进一步发展多元化 在小鼠和人类T细胞中进行数字编辑并探索数字编辑的新用途，2)部署多路数字编辑 编辑以安装新的编辑，以增强T细胞在机械复杂的肿瘤中的迁移 微环境和硬连接T细胞，以保持T驻留的记忆表型，3)实现数字化 编辑通过数字敲除所有3个NUR4A来开发具有增强实体瘤疗效的“现成”T细胞 转录因子家族。总而言之，通过部署数字编辑，我们将使细胞的基因编辑成为预期的 用于更复杂、更安全的治疗用途，并导致针对实体肿瘤的有效治疗 机械复杂和免疫抑制的肿瘤微环境。

ABSTRACT: PROJECT 3 Over the last several decades, it has become possible to isolate a patient’s own cells, engineer and expand them in the laboratory, and use them to treat an existing cancer. This technology has largely advanced to using primary human T cells genetically modified to express a tumor specific T cell receptor (TCR) or chimeric antigen receptor (CAR). This approach has demonstrated durable cures in some leukemias, but has had limited success in the treatment of solid tumors. This lack of efficacy is believed to be due to challenges faced by T cells in migrating into and within complex solid tumor microenvironments and multiple immunosuppressive modalities found there. As many of these challenges have been identified and mechanistically studied, we hypothesize that we can engineer CAR T cells capable of overcoming all challenges found in the solid tumor microenvironment, leading to durable cures for cancer patients with the worst prognosis. Recently, a number of groups have published high efficiency methods for engineering T cells using CRISPR/Cas9. Moreover, the use of recombinant adeno associated virus (rAAV) as a DNA donor molecule for homologous recombination (HR) combined with Cas9 has also demonstrated incredible rates of site-specific gene delivery in T cells. Although these approaches are highly effective, there are still drawbacks and issues to be resolved to improve capabilities and safety of gene editing cells for therapy. For instance, nuclease-based gene editing still relies on stochastic repair of genotoxic double strand breaks (DSBs) with little uniformity in the editing outcome. Fortunately, new technologies have emerged to gene edit DNA at single base pair resolution with high product purity and efficiency and without a targeted DSB, termed Cas9 base editors (BEs) and primer editors (PEs). We have demonstrated that this technology allows for highly efficient multiplex genome editing via programmable enzymatic single base changes without creating toxic DSBs, i.e. “digital editing”. As this technology is relatively new, there is also opportunity to develop new and innovative genome editing strategies to develop fully digitally edited cells intended for therapeutic use in a cost effect, rapid, and accurate manner. Thus, our specific aims are as follows: 1) Further develop multiplex digital editing in murine and human T cells and explore novel uses for digital editing, 2) Deploy multiplex digital editing to install novel edits in order to enhance T cells migration into and within mechanically complex tumor microenvironment and also hardwire T cells to maintain a T resident memory phenotype, 3) Implement digital editing to develop “off-the-shelf” T cells with enhanced solid tumor efficacy via digital knockout of all 3 NUR4A transcription factor family. In summary, by deploying digital editing, we will make gene editing of cells intended for therapeutic use more sophisticated, safe and lead to effective therapies against solid tumor tumors with mechanically complex and immunosuppressive tumor microenvironments.