Intracellular Trafficking of DNA for Gene Therapy

用于基因治疗的 DNA 细胞内运输

基本信息

  • 批准号:
    10710840
  • 负责人:
  • 金额:
    $ 39.81万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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 对于基因向细胞的有效递送存在许多障碍。我们实验室的主要目标是 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, 核被膜定位于细胞核内的特定区域,并被转录以形成基因 治疗是成功的。我们已经证明,一旦进入细胞质,携带DNA核靶向的质粒 在非分裂细胞中,核输入所需的序列(cDNAs)迅速与转录相关, 介导沿沿着微管和穿过核膜运动的因子。NF-kB就是这样一个因子 它与几种普遍活跃的DTS结合,是DNA核输入所需的,但在细胞质中它是 保持在隔离状态,无法结合DNA。接下来的问题是NF-kB是如何被激活以结合到 质粒并介导其细胞骨架运动和核输入?在NF-kB的情况下, 因为它的激活是通过一组细胞质dsDNA传感器,如cGAS-STING,这是细胞质dsDNA传感器的一部分。 先天免疫系统和驱动炎症反应。当dsDNA与cGAS结合时, 启动,导致关键促炎转录因子(包括NF-κ B)的激活, 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 来阻断这些传感器的激活以减少炎症。然而,我们已经观察到,当CGAS是 沉默的、胞质注射的质粒不能运输到细胞核。我们假设有限的激活 这些传感器中的一个或多个实际上需要低水平激活关键转录因子, 以促进非分裂细胞中的DNA核输入。如果我们能找到限制传感器激活的方法, 废除它,这将允许增强的基因传递与有限的伴随炎症。我们还 花了相当大的努力详细说明质粒在细胞核内的分布,并发现, 质粒的亚核错误定位可影响它们的转录活性。我们发现质粒 基于启动子的类型(Pol I、Pol II或Pol III)定位于细胞核内的离散转录结构域。 Pol III),并且当两种不同的启动子类型被置于一个质粒上时, DNA的核内分布与单独的启动子类型不同,但转基因表达是 大幅减少。我们将剖析DNA在细胞核内运动的途径, 它们基于转染DNA的亚核定位来改善转基因表达。我们 具体目的是(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活化和转染效率; 以及(3)表征亚核组织如何影响外源DNA表达。

项目成果

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David A Dean其他文献

David A Dean的其他文献

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{{ truncateString('David A Dean', 18)}}的其他基金

A multimodal delivery and treatment approach for Acute Lung Injury
急性肺损伤的多模式递送和治疗方法
  • 批准号:
    10378509
  • 财政年份:
    2020
  • 资助金额:
    $ 39.81万
  • 项目类别:
Mitigating Acute Lung Injury by Cell-specific Targeting of MTOR
通过细胞特异性靶向 MTOR 减轻急性肺损伤
  • 批准号:
    10187645
  • 财政年份:
    2020
  • 资助金额:
    $ 39.81万
  • 项目类别:
Mitigating Acute Lung Injury by Cell-specific Targeting of MTOR
通过细胞特异性靶向 MTOR 减轻急性肺损伤
  • 批准号:
    10631224
  • 财政年份:
    2020
  • 资助金额:
    $ 39.81万
  • 项目类别:
Mitigating Acute Lung Injury by Cell-specific Targeting of MTOR
通过细胞特异性靶向 MTOR 减轻急性肺损伤
  • 批准号:
    10414888
  • 财政年份:
    2020
  • 资助金额:
    $ 39.81万
  • 项目类别:
Gene therapy for GERD-associated esophageal epithelial barrier dysfunction
GERD相关食管上皮屏障功能障碍的基因治疗
  • 批准号:
    10372106
  • 财政年份:
    2020
  • 资助金额:
    $ 39.81万
  • 项目类别:
A multimodal delivery and treatment approach for Acute Lung Injury
急性肺损伤的多模式递送和治疗方法
  • 批准号:
    10593959
  • 财政年份:
    2020
  • 资助金额:
    $ 39.81万
  • 项目类别:
Mitigating Acute Lung Injury by Cell-specific Targeting of MTOR
通过细胞特异性靶向 MTOR 减轻急性肺损伤
  • 批准号:
    10056811
  • 财政年份:
    2020
  • 资助金额:
    $ 39.81万
  • 项目类别:
Novel Peptide/siRNA Nanoparticles for Treatment of Acute Lung Injury
用于治疗急性肺损伤的新型肽/siRNA纳米颗粒
  • 批准号:
    9376455
  • 财政年份:
    2017
  • 资助金额:
    $ 39.81万
  • 项目类别:
Development of a gene therapy approach to treat acute lung injury using a preclinical, large animal model
使用临床前大型动物模型开发治疗急性肺损伤的基因治疗方法
  • 批准号:
    9044084
  • 财政年份:
    2016
  • 资助金额:
    $ 39.81万
  • 项目类别:
Cell-specific gene delivery methods for expression and silencing in the lung
用于肺部表达和沉默的细胞特异性基因递送方法
  • 批准号:
    8978332
  • 财政年份:
    2014
  • 资助金额:
    $ 39.81万
  • 项目类别:

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