Project 3

项目3

• 批准号: 10700941
• 负责人: Branden S Moriarity
• 金额: $ 53.91万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-16 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10700941
• 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; 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; Tumor Promotion; Work; activating transcription factor; antigen test; base; base editing; base editor; 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; lentivirally transduced; leukemia; leukemia treatment; live cell imaging; mathematical model; mesothelin; migration; mouse model; new technology; novel; nuclease; overexpression; pancreatic cancer model; pancreatic neoplasm; pre-clinical; pre-clinical assessment; prime editing; programmed cell death protein 1; promoter; repaired; success; synergism; targeted nucleases; transcription factor; tumor; tumor microenvironment; tumor-immune system interactions

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，设计和扩增它们已经成为可能 在实验室里，用它们来治疗现有的癌症。这项技术在很大程度上已经发展到使用主 经遗传修饰以表达肿瘤特异性T细胞受体（TCR）或嵌合抗原受体的人T细胞 （CAR）.这种方法已经证明在一些白血病中具有持久的治愈效果，但是在一些白血病患者中成功有限。 治疗实体瘤。这种缺乏功效被认为是由于T细胞在迁移中面临的挑战。 复杂的实体瘤微环境和在那里发现的多种免疫抑制方式。 由于这些挑战中的许多已经被确定并进行了机制研究，我们假设我们可以 工程CAR T细胞能够克服实体瘤微环境中发现的所有挑战， 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 使用CRISPR/Cas9工程化T细胞的有效方法。此外，使用重组腺病毒， 与Cas9组合的作为用于同源重组（HR）的DNA供体分子的rAAV相关病毒（rAAV）已经 也证明了T细胞中位点特异性基因传递的惊人速度。虽然这些方法都是高度 有效，但仍存在缺陷和问题需要解决，以提高基因编辑的能力和安全性 用于治疗的细胞例如，基于核酸酶的基因编辑仍然依赖于基因毒性双链的随机修复。 链断裂（DSB），编辑结果几乎没有一致性。幸运的是，新技术已经出现 以单碱基对分辨率进行基因编辑DNA，具有高产品纯度和效率，并且没有靶向的 DSB，称为Cas9碱基编辑器（BE）和引物编辑器（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变化进行高效的多重基因组编辑， 创建有毒的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，以开发用于治疗用途的完全数字编辑的细胞 以成本有效、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细胞迁移到机械复杂的肿瘤中和肿瘤内 微环境以及硬连线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种CD 4A，编辑开发具有增强实体瘤疗效的“现成”T细胞 转录因子家族总之，通过部署数字编辑，我们将使细胞的基因编辑 对于治疗用途，更复杂，安全，并导致针对实体瘤肿瘤的有效疗法， 机械复杂和免疫抑制肿瘤微环境。