Intracellular Trafficking of DNA for Gene Therapy

用于基因治疗的 DNA 细胞内运输

• 批准号: 10710840
• 负责人: David A Dean
• 金额: $ 39.81万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-22 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10710840
• 关键词: Affect; Affinity; Animals; Architecture; Binding; Binding Sites; CREB1 gene; Cell Nucleolus; Cell Nucleus; Cell division; Cell membrane; Cells; Complex; Cytoplasm; Cytoskeleton; DNA; DNA Binding; DNA Binding Domain; DNA Polymerase I; DNA Polymerase II; DNA Polymerase III; DNA Sequence; DNA cassette; DNA delivery; Disease; Gene Delivery; Gene Expression; Gene Order; Gene Transfer; Genes; Genetic Materials; Genetic Transcription; Goals; Host Defense; Immune response; Importins; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Innate Immune System; Interferons; Interphase Cell; Laboratories; Mediating; Messenger RNA; Methods; Microtubules; Motor; Movement; NF-kappa B; NFIA gene; Normal Statistical Distribution; Nuclear; Nuclear Envelope; Nuclear Import; Nuclear Localization Signal; Pathway interactions; Patients; Plasmids; Polymerase; Production; Proteins; RNA Polymerase II; RNA Splicing; Receptor Signaling; Ribosomal RNA; Signal Transduction; Site; Stimulator of Interferon Genes; Time; Tissues; Transfection; Travel; Viral; Work; cytokine; defense response; ds-DNA; effective therapy; gene therapy; improved; in vivo; mechanical force; plasmid DNA; promoter; release factor; residence; sensor; small hairpin RNA; therapeutic gene; trafficking; transcription factor; transgene expression; Affect; Affinity; Animals; Architecture; Binding; Binding Sites; CREB1 gene; Cell Nucleolus; Cell Nucleus; Cell division; Cell membrane; Cells; Complex; Cytoplasm; Cytoskeleton; DNA; DNA Binding; DNA Binding Domain; DNA Polymerase I; DNA Polymerase II; DNA Polymerase III; DNA Sequence; DNA cassette; DNA delivery; Disease; Gene Delivery; Gene Expression; Gene Order; Gene Transfer; Genes; Genetic Materials; Genetic Transcription; Goals; Host Defense; Immune response; Importins; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Innate Immune System; Interferons; Interphase Cell; Laboratories; Mediating; Messenger RNA; Methods; Microtubules; Motor; Movement; NF-kappa B; NFIA gene; Normal Statistical Distribution; Nuclear; Nuclear Envelope; Nuclear Import; Nuclear Localization Signal; Pathway interactions; Patients; Plasmids; Polymerase; Production; Proteins; RNA Polymerase II; RNA Splicing; Receptor Signaling; Ribosomal RNA; Signal Transduction; Site; Stimulator of Interferon Genes; Time; Tissues; Transfection; Travel; Viral; Work; cytokine; defense response; ds-DNA; effective therapy; gene therapy; improved; in vivo; mechanical force; plasmid DNA; promoter; release factor; residence; sensor; small hairpin RNA; therapeutic gene; trafficking; transcription factor; transgene expression

Under almost all conditions using any method, the levels of gene transfer to any cell or tissue are low because many barriers exist for the efficient delivery of genes to cells. The primary goal of our laboratory is to identify and overcome the intracellular barriers to promote effective gene delivery and therapy. Exogenous viral or non- viral DNA must cross the plasma membrane, travel through the cytoplasm and the cytoskeletal networks, cross the nuclear envelope, localize to specific regions within the nucleus, and be transcribed in order for gene therapy to be successful. We have shown that once in the cytoplasm, plasmids carrying DNA nuclear targeting sequences (DTS) that are required for nuclear import in non-dividing cells rapidly associate with transcription factors that mediate movement along microtubules and across the nuclear envelope. NF-kB is one such factor that binds to several ubiquitously active DTSs and is required for DNA nuclear import, but in the cytoplasm it is maintained in a sequestered state, unable to bind DNA. The question then is how is NF-kB activated to bind to plasmids and mediate their cytoskeletal movement and nuclear import? In the case of NF-kB, a major pathway for its activation is through a set of cytoplasmic dsDNA sensors, such as cGAS-STING, that are part of the innate immune system and drive inflammatory responses. When dsDNA binds to cGAS, signaling cascades are initiated that result in activation of key pro-inflammatory transcription factors (including NF-kB) and ultimately production of pro-inflammatory cytokines. Thus, a major focus in the gene therapy space has been to block activation of these sensors to reduce inflammation. However, we have observed that when cGAS is silenced, cytoplasmically injected plasmids fail to traffic to the nucleus. We hypothesize that limited activation of one or more of these sensors is actually needed for low level activation of key transcription factors in order to facilitate DNA nuclear import in non-dividing cells. If we can find ways to limit sensor activation, but not abolish it, this will allow for enhanced gene delivery with limited accompanying inflammation. We have also spent considerable effort detailing the distribution of plasmids inside the nucleus and have found that the subnuclear mislocalization of plasmids can affect their transcriptional activity. We have found that plasmids localize to discrete transcriptional domains within the nucleus based on the type of promoter (Pol I, Pol II, or Pol III) they carry and that when two different promoter types are placed on one plasmid, not only is the intranuclear distribution of the DNA different that either promoter type alone, but transgene expression is significantly reduced. We will dissect the pathways used for DNA movement within the nucleus and exploit them to improve transgene expression based on the subnuclear localization of the transfected DNA. Our specific aims are to (1) determine whether cytosolic dsDNA sensors are required for DNA nuclear import; (2) evaluate whether residence time of DNA in the cytoplasm affects sensor activation and transfection efficiency; and (3) characterize how subnuclear organization affects exogenous DNA expression.

在几乎所有条件下使用任何方法，基因转移到任何细胞或组织的水平都很低，因为 有许多障碍可以有效地传递基因向细胞。我们实验室的主要目标是确定 并克服细胞内屏障以促进有效的基因递送和治疗。外源性病毒或非 病毒DNA必须穿过质膜，穿过细胞质和细胞骨架网络，交叉 核包膜，位于核内的特定区域，并进行转录以进行基因 疗法成功。我们已经表明，一旦在细胞质中，携带DNA核靶向的质粒 在非分裂细胞中核进口所需的序列（DTS）迅速与转录相关联 介导沿着微管运动和跨核包膜运动的因素。 NF-KB就是这样的因素之一 与几个无处不在活性的DTS结合，是DNA核进口所必需的，但在细胞质中是 保持在隔离状态，无法结合DNA。那么问题是如何激活NF-KB以结合到 质粒并介导其细胞骨架运动和核进口？对于NF-KB，是主要途径 因为它的激活是通过一组细胞质dsDNA传感器（例如CGAS） 先天免疫系统并推动炎症反应。当DSDNA与CGA结合时，信号级联 启动会导致关键促炎转录因子（包括NF-KB）和 最终产生促炎性细胞因子。因此，基因治疗空间的主要重点是 阻止这些传感器的激活以减少炎症。但是，我们观察到，当CGA为 沉默的细胞质注射质粒未能传播到核。我们假设有限的激活 实际上，一个或多个传感器的一个或多个是要按顺序进行关键转录因子的低水平激活 促进在非分裂细胞中进口DNA核的。如果我们能找到限制传感器激活的方法，但不能 废除它，这将允许增强基因递送，而伴随炎症有限。我们也有 花费了巨大的努力，详细介绍了细胞核内质粒的分布，并发现 质粒的核下核错误定位会影响其转录活性。我们发现质粒 基于启动子类型（Pol I，Pol II或 他们携带的是，当将两种不同的启动子类型放在一个质粒上时， 单独启动子类型的DNA的核内分布不同，但是转基因表达是 大幅减少。我们将剖析用于核内DNA运动的途径并利用 它们基于转染的DNA的亚核定位来改善转基因表达。我们的 具体目的是（1）确定DNA核进口是否需要胞质DSDNA传感器； （2） 评估DNA在细胞质中的停留时间是否会影响传感器的激活和转染效率； （3）表征亚核组织如何影响外源DNA表达。