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

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 将基因有效传递至细胞存在许多障碍。我们实验室的主要目标是确定 并克服细胞内障碍，促进有效的基因传递和治疗。外源病毒或非 病毒DNA必须穿过质膜，穿过细胞质和细胞骨架网络，穿过 核膜，定位于细胞核内的特定区域，并被转录以便基因 治疗才能成功。我们已经证明，一旦进入细胞质，携带 DNA 的质粒就会进行核靶向 非分裂细胞核输入所需的序列（DTS）快速与转录相关 介导沿微管和穿过核膜运动的因素。 NF-kB 就是这样的因素之一 与几种普遍存在的活性 DTS 结合，并且是 DNA 核输入所必需的，但在细胞质中它是 保持在隔离状态，无法结合DNA。那么问题是 NF-kB 如何被激活并结合 质粒并介导其细胞骨架运动和核输入？就 NF-kB 而言，一个主要途径 因为它的激活是通过一组细胞质 dsDNA 传感器（例如 cGAS-STING）来实现的，这些传感器是 先天免疫系统并驱动炎症反应。当 dsDNA 与 cGAS 结合时，信号级联 被启动，导致关键促炎转录因子（包括 NF-kB）的激活和 最终产生促炎细胞因子。因此，基因治疗领域的一个主要焦点是 阻止这些传感器的激活以减少炎症。然而，我们观察到，当 cGAS 为 沉默的、注入细胞质的质粒无法运输到细胞核。我们假设有限的激活 实际上需要这些传感器中的一个或多个来低水平激活关键转录因子，以便 促进非分裂细胞中 DNA 入核。如果我们能找到限制传感器激活的方法，但不能 废除它，这将允许增强基因传递并限制伴随的炎症。我们还有 花费了大量的精力来详细描述质粒在细胞核内的分布，并发现 质粒的亚核错误定位会影响其转录活性。我们发现质粒 根据启动子的类型（Pol I、Pol II 或 Pol III）它们携带，当两种不同的启动子类型放置在一个质粒上时，不仅 DNA 的核内分布与单独的启动子类型不同，但转基因表达是 显着减少。我们将剖析DNA在细胞核内运动的途径并利用 它们基于转染 DNA 的亚核定位来改善转基因表达。我们的 具体目标是 (1) 确定 DNA 核输入是否需要胞质 dsDNA 传感器； (2) 评估DNA在细胞质中的停留时间是否影响传感器激活和转染效率； (3) 描述亚核组织如何影响外源 DNA 表达。