Engineering Therapeutic Human Immune Cells with Modular Self-contained Genetic Circuits

具有模块化独立遗传电路的工程治疗性人类免疫细胞

• 批准号: 10303600
• 负责人: Isaac Hilton
• 金额: $ 22.93万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10303600
• 关键词: Adenovirus Vector; Adoption; Area; Behavior; Biotechnology; CRISPR/Cas technology; Cell Therapy; Cells; Clinical; Clinical Trials; Complex; DNA; Development; Diagnostic; Double Stranded DNA Virus; Engineered Gene; Engineering; Ensure; Environment; Episome; Exploratory/Developmental Grant; Funding; Future; Gene Expression; Gene Expression Profile; Gene Transduction Agent; Genetic; Genetic Transcription; Genome; Genome engineering; Genomics; Goals; Health; Human; Human Cell Line; Human Engineering; Human Genome; Hypoxia; Immune; Immunologic Receptors; Insertional Mutagenesis; Integrase; Kinetics; Lead; Life; Medical; Medicine; Mission; Modernization; National Institute of Biomedical Imaging and Bioengineering; Nuclear; Pathologic; Patients; Phenotype; Proteins; Research Personnel; Safety; Savings; Science; Signal Transduction; Site; Stimulus; T-Lymphocyte; Techniques; Technology; Testing; Therapeutic; Time; Transgenic Organisms; United States National Institutes of Health; Vertebral column; Viral Genes; Viral Genome; Virus; Work; base; biological systems; biomedical scientist; cellular engineering; chimeric antigen receptor; clinical application; clinically relevant; cost; cytokine; design; drug production; epigenetic silencing; functional genomics; gene therapy; genetic payload; high risk; immunoregulation; in-vivo diagnostics; innovation; mesenchymal stromal cell; next generation; patient safety; preservation; prevent; programs; prophylactic; receptor density; response; small molecule; synthetic biology; tool; transcription factor; viral DNA

PROJECT SUMMARY/ABSTRACT Current strategies to engineer human cell-based therapeutics rely upon the delivery and subsequent genomic integration of transgenic payloads. Although these approaches have catalyzed transformative medical advances, the integration of transgenic DNA permanently disrupts natural genomic sequences and can lead to unexpected and even hazardous consequences. In addition, integrated transgenic DNA is often unpredictably expressed and is prone to epigenetic silencing over time, especially within primary human immune cells. Furthermore, existing approaches to validate large transgenic genomically- integrated DNA cargoes are inefficient and costly. These critical barriers limit the extent to which human cells can be repurposed and engineered as cell-based therapeutics and these challenges are preventing biotechnological and clinical innovations. Non-integrating, double-stranded DNA viruses have evolved sophisticated solutions to these critical barriers, and they can stably persist within human cells as circularized self-contained episomes across cellular divisions and for the lifetime of infected hosts. These viruses accomplish this remarkable persistence by tailoring their own gene expression patterns, synchronizing their genomic replication, and by reshaping the endogenous transcriptional networks of host cells. In this proposal, we will harness these natural abilities and refine them using clinical-grade gene therapy vector testbeds. Our approach will establish an entirely new way to use of circular, orthogonal episomal DNA within human cells. In Aim 1 of this proposal, we design, build, test, and optimize genetically-encoded episomal modules to enable i) site-specific and tunable genomic localization, ii) programmable episomal replication, and iii) multi-layered safety switches, within clinically validated integrase-deficient lentiviral (IDLV) and high- capacity adenoviral (HcAdV) gene therapy vector testbeds. In Aim 2 of this proposal, we will build genetic circuits within IDLV and HcAdV gene therapy vectors that sense hypoxic environments and/or small molecules and respond to these signals in real time by producing fluorometric diagnostics and/or synthetic CRISPR/Cas9-based transcription factors to drive the expression of therapeutically crucial cytokines or biomedically relevant phenotypic changes. In each independent Aim, we will use experimental techniques at the interface of functional genomics, genome engineering, and synthetic biology. To preserve and maximize the therapeutic utility of our results and to ensure applicability beyond the scope of this proposal, both Aims will be carried out using primary human T cells and mesenchymal stromal cells. Collectively, this project will combine engineering principles and lessons from biomedical sciences to spur advances that will be broadly useful to biomedical researchers, actionable for clinicians, and meaningful to future patients in need of sophisticated cell-based therapeutics.

项目概要/摘要 当前设计基于人类细胞的疗法的策略依赖于递送和后续 转基因有效负载的基因组整合。尽管这些方法已经促进了变革 医学进步，转基因 DNA 的整合永久破坏了自然基因组序列 并可能导致意想不到的甚至危险的后果。此外，整合的转基因DNA 通常表达不可预测，并且随着时间的推移容易发生表观遗传沉默，尤其是在原代体内 人类免疫细胞。此外，现有的验证大型转基因基因组的方法- 整合 DNA 货物效率低下且成本高昂。这些关键障碍限制了人类 细胞可以被重新利用和设计为基于细胞的疗法，这些挑战正在阻止 生物技术和临床创新。非整合双链DNA病毒已经进化 针对这些关键障碍的复杂解决方案，它们可以稳定地存在于人体细胞内 跨细胞分裂和受感染宿主的一生循环化独立附加体。这些 病毒通过定制自己的基因表达模式来实现这种非凡的持久性， 同步它们的基因组复制，并通过重塑内源转录网络 宿主细胞。在本提案中，我们将利用这些自然能力并使用临床级的技术对其进行改进 基因治疗载体试验台。我们的方法将建立一种全新的循环利用方式， 人类细胞内的正交游离DNA。 在本提案的目标 1 中，我们设计、构建、测试和优化基因编码的附加型模块，以 实现 i) 位点特异性和可调节的基因组定位，ii) 可编程游离复制，以及 iii) 多层安全开关，在临床验证的整合酶缺陷型慢病毒（IDLV）和高 容量腺病毒（HcAdV）基因治疗载体试验台。在本提案的目标 2 中，我们将建立遗传 IDLV 和 HcAdV 基因治疗载体内的电路可感知缺氧环境和/或小 分子并通过产生荧光诊断和/或实时响应这些信号 基于 CRISPR/Cas9 的合成转录因子可驱动治疗关键因子的表达 细胞因子或生物医学相关的表型变化。在每个独立的目标中，我们将使用 功能基因组学、基因组工程和合成领域的实验技术 生物学。保留并最大限度地提高我们结果的治疗效用，并确保其适用性超出 在该提案的范围内，这两个目标都将使用原代人类 T 细胞和间充质细胞来实现 基质细胞。总的来说，该项目将结合工程原理和生物医学的经验教训 科学推动进步，对生物医学研究人员广泛有用，对临床医生可采取行动， 对未来需要复杂的细胞疗法的患者有意义。