Intracellular Trafficking of DNA for Gene Therapy

在几乎所有使用任何方法的条件下，基因转移到任何细胞或组织的水平都很低，因为 要有效地将基因输送到细胞，存在许多障碍。我们实验室的主要目标是确定 并克服细胞内的障碍，促进有效的基因传递和治疗。外源病毒或非病毒 病毒DNA必须穿过质膜，穿过细胞质和细胞骨架网络， 核膜，定位于核内的特定区域，并被转录以用于基因 治疗要成功。我们已经证明，一旦进入细胞质，携带DNA核靶向的质粒 未分裂细胞中核输入所需的序列(DTS)与转录迅速相关 调节沿微管和跨核被膜运动的因素。核因子-kB就是这样一个因素 它与几个普遍存在的活性DTS结合，是DNA核输入所必需的，但在细胞质中是 保持隔离状态，不能结合DNA。那么问题是，核因子-kB是如何被激活以结合到 并调节它们的细胞骨架运动和核进口？在核因子-kB的情况下，一个主要途径 因为它的激活是通过一系列细胞质dsDNA传感器，如cGAS-STING，这是 与生俱来的免疫系统，并驱动炎症反应。当dsDNA与cGAS结合时，信号级联 启动导致关键的促炎性转录因子(包括核因子-kB)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 阻断这些感应器的激活以减少炎症。然而，我们观察到，当cGAS是 沉默的、以细胞质注射的质粒不能进入细胞核。我们假设有限的激活 关键转录因子的低水平激活实际上需要这些传感器中的一个或多个 以促进未分裂细胞中的DNA核进口。如果我们能找到限制传感器激活的方法，但不能 废除它，这将允许在有限的伴随炎症的情况下增强基因传递。我们还有 花了相当大的努力详细描述了细胞核内的质粒分布，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)携带，并且当两种不同类型的启动子放置在同一质粒上时，不仅 DNA在核内的分布不同于任何一种启动子类型，但转基因表达 显著减少。我们将剖析DNA在细胞核内移动的路径，并利用 根据转基因DNA的亚核定位，提高转基因表达水平。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感受器的激活和转染率； 以及(3)亚核组织如何影响外源DNA表达。

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.