Project 3

项目3

基本信息

批准号：
10270395
负责人：
Branden S Moriarity
金额：
$ 54.86万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-16 至 2026-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10270395
关键词：
Address Affect Animal Model Base Pairing Binding Sites Brain Neoplasms CAR T cell therapy CD19 gene CRISPR/Cas technology Cancer Patient Cancer Prognosis Cell Therapy Cell physiology Cells Collaborations Complementary DNA Complex Coupled DNA DNA Double Strand Break DNA Sequence Alteration Data Engineering Family Flow Cytometry Gene Delivery Gene Targeting Genes Genetically Engineered Mouse Genome engineering Human Human Engineering Immunocompetent Immunotherapy Knock-in Knock-out Laboratories Lead Malignant Neoplasms Mechanics Memory Methods Modality Modeling Mus NR4A1 gene NR4A2 gene Outcome Patients Phenotype Pre-Clinical Model Preclinical Testing Process Prognosis Publishing Recombinant adeno-associated virus (rAAV)Resolution Risk Safety Site Solid Neoplasm Survival Rate System T-Cell Receptor T-Lymphocyte Techniques Technology Testing Therapeutic Uses Three-Dimensional Imaging Work activating transcription factor antigen test base cancer cell cancer immunotherapy cell motility chimeric antigen receptor chimeric antigen receptor T cells clinical translation cost digital effective therapy engineered T cells ex vivo imaging genetically modified cells genome editing genotoxicity homologous recombination immunoengineering improved in vitro testing in vivo innovation interest intravital imaging knockout gene leukemia leukemia treatment live cell imaging mathematical model mesothelin mouse model new technology novel nuclease overexpression pancreatic cancer model pancreatic neoplasm pre-clinical pre-clinical assessment programmed cell death protein 1 programs promoter repaired success synergism targeted nucleases transcription factor tumor tumor microenvironment tumor-immune system interactions

项目摘要

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.

摘要：项目 3 在过去的几十年里，分离患者自身的细胞、对其进行改造和扩增已经成为可能在实验室中，并用它们来治疗现有的癌症，这项技术已在很大程度上发展到使用原发性癌症。人类 T 细胞经过基因改造以表达肿瘤特异性 T 细胞受体 (TCR) 或嵌合抗原受体 (CAR)。这种方法已证明可以持久治愈某些白血病，但在治疗白血病方面却取得了有限的成功。实体瘤治疗缺乏疗效被认为是由于 T 细胞在迁移过程中面临的挑战。进入复杂的实体瘤微环境和其中发现的多种免疫抑制方式。由于许多这些挑战已经被识别和机械研究，我们追求的是，我们可以工程 CAR T 细胞能够克服实体瘤微环境中发现的所有挑战，领先最近，许多研究小组发表了关于如何为预后最差的癌症患者提供持久治疗的研究成果。此外，使用 CRISPR/Cas9 改造 T 细胞的有效方法。相关病毒（rAAV）作为DNA供体分子与Cas9结合进行同源重组（HR）尽管这些方法的效率很高，但 T 细胞中的位点特异性基因传递率也令人难以置信。有效，但仍存在缺陷和问题有待解决，以提高基因编辑的能力和安全性例如，基于核酸酶的基因编辑仍然依赖于基因毒性双重的随机修复。幸运的是，新技术已经出现。以单碱基对分辨率对 DNA 进行基因编辑，产品纯度高、效率高，且无需靶向 DSB，称为 Cas9 碱基编辑器 (BE) 和引物编辑器 (PE)，我们已经证明了该技术。允许通过可编程酶促单碱基变化进行高效的多重基因组编辑，而无需创建有毒 DSB，即“数字编辑”，由于这项技术相对较新，因此也有发展的机会。新的和创新的基因组编辑策略，用于开发用于治疗用途的完全数字编辑的细胞因此，我们的具体目标是： 1）进一步发展多路复用。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) 部署多重数字化编辑以安装新颖的编辑，以增强 T 细胞迁移到机械复杂的肿瘤中和内部微环境和硬连线 T 细胞以维持 T 驻留记忆表型，3) 实施数字化通过数字敲除所有 3 个 NUR4A 进行编辑，开发具有增强实体瘤疗效的“现成”T 细胞总之，通过部署数字编辑，我们将实现对细胞的基因编辑。用于治疗用途更加复杂、安全，并导致针对实体瘤的有效疗法机械复杂和免疫抑制的肿瘤微环境。