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: 10430257
• 负责人: Isaac Hilton
• 金额: $ 19.03万
• 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/10430257
• 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; design-build-test; 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.

项目摘要/摘要 当前设计基于人类细胞的治疗剂的策略依赖于交付和随后的策略 转基因有效载荷的基因组整合。尽管这些方法催化了变革性 医学进步，转基因DNA的整合永久破坏自然基因组序列 并可能导致意外的后果。另外，综合转基因DNA 经常表达不可预测，并且很容易随着时间的流逝而表观遗传沉默 人免疫细胞。此外，现有的方法以验证大型转基因基因组 - 集成的DNA货物效率低下且昂贵。这些关键障碍限制了人类的程度 细胞可以作为基于细胞的治疗剂重新利用和设计，这些挑战正在阻止 生物技术和临床创新。非整合，双链DNA病毒已经发展 这些关键障碍的复杂解决方案，它们可以稳定在人类细胞中，因为 跨细胞分裂和感染宿主的生命周期的循环自包含的偶发。这些 病毒通过调整自己的基因表达模式来实现这一非凡的持久性， 同步其基因组复制，并通过重塑内源性转录网络 宿主细胞。在此提案中，我们将利用这些自然能力，并使用临床级来完善它们 基因治疗载体试验台。我们的方法将建立一种全新的使用循环的方式 人类细胞中的正交性偶发性DNA。 在该提案的目标1中，我们设计，构建，测试和优化了遗传编码的偶发模块 启用i）特定于位点特异性和可调的基因组定位，ii）可编程偶发复制和iii） 多层安全开关，在经过临床验证的集成酶缺陷型慢病毒（IDLV）和高位中 容量腺病毒（HCADV）基因治疗载体测试床。在该提案的目标2中，我们将建立遗传 IDLV和HCADV基因治疗载体中的电路，感知低氧环境和/或小的电路 分子并通过产生荧光诊断和/或实时响应这些信号 基于合成CRISPR/CAS9的转录因子，以驱动治疗上至关重要的表达 细胞因子或生物医学相关的表型变化。在每个独立目标中，我们将使用 功能基因组学，基因组工程和合成的界面的实验技术 生物学。保留和最大化结果的治疗效用，并确保适用于 该提案的范围，两个目标都将使用原代人T细胞和间质进行 基质细胞。总的来说，该项目将结合生物医学的工程原理和课程 刺激进步的科学对生物医学研究人员将非常有用，对临床医生可行， 对需要复杂的基于细胞的治疗剂的未来患者有意义。