DNA Nuclear Import Sequences for Cell-Specific Gene and Drug Delivery

描述（由申请人提供）：限制基因传递和表达到特定细胞类型的能力对基因治疗至关重要，因为转基因的异位表达可能导致有害的宿主炎症反应或正常细胞功能失调。药物和化疗药物以及显像剂的输送也是如此。目前，只有三种方法来限制药物、探针和/或基因的递送/表达到特定的细胞类型和组织：物理递送到所需的靶器官，使用配体或抗体作为细胞表面受体，以及细胞特异性启动子来驱动所需细胞类型的转录。基于我们对质粒DNA核输入机制的阐明，我们开发了第四种非病毒DNA载体的细胞特异性递送方法。我们已经证明，在没有细胞分裂的情况下，质粒的核定位是序列特异性的，需要转录因子与细胞质中的这些序列结合并促进DNA核输入。我们已经确定了一些DNA序列，显示出细胞核进口的细胞特异性，因为它们结合细胞特异性转录因子。迄今为止，我们已经确定了在平滑肌细胞、成骨细胞、内皮细胞和肺泡2型上皮细胞中起作用的细胞特异性DNA核靶向序列。这些序列仅在体外和活体动物的各自细胞中支持DNA核输入和随后的质粒基因表达。我们现在的目标是使用一组细胞类型筛选大量潜在的细胞特异性DNA核靶向活性DNA序列，并开发一个细胞特异性基因和药物传递文库。在这个应用中，我们建议构建和筛选1000个细胞特异性哺乳动物启动子库，用于从全身组织中提取100种细胞类型的核输入活性。量子点标记的质粒将被电穿孔到细胞中，并在ImageStream流式细胞仪上进行定量图像分析。潜在的DNA核靶向序列将使用传统的显微注射方法进行验证，其细胞特异性和核输入的机制将被表征。最后，从先前的目标验证命中将通过电穿孔传递到体内单个器官，以测试细胞和组织特异性基因传递。

DESCRIPTION (provided by applicant): The ability to restrict gene delivery and expression to particular cell types is of paramount importance for gene therapy, since ectopic expression of a transgene could lead to deleterious host inflammatory responses or dysregulation of normal cellular functions. The same is true for delivery of drugs and chemotherapeutic agents, as well as imaging agents. At present there are only three methods to limit delivery/expression of drugs, probes, and/or genes to specific cell types and tissues: physical delivery to a desired target organ, use of ligands or antibodies for cell surface receptors, and cell-specific promoters to drive transcription in desired cell types. We have developed a fourth approach for cell-specific delivery of nonviral DNA-based vectors based on our elucidation of the mechanisms of plasmid DNA nuclear import. We have shown that the nuclear localization of plasmids in the absence of cell division is sequence-specific and requires transcription factors that bind to these sequences in the cytoplasm and facilitate DNA nuclear import. We have identified a number of DNA sequences that show cell-specificity of nuclear import because they bind to cell-specific transcription factors. To date, we have identified cell-specific DNA nuclear targeting sequences that act in smooth muscle cells, osteoblasts, endothelial cells, and alveolar type 2 epithelial cells. These sequences support DNA nuclear import and subsequent gene expression from the plasmids only in their respective cells in vitro and in living animals. Our goal now is to screen a large number of potential DNA sequences for cell-specific DNA nuclear targeting activity using a panel of cell types and develop a library for cell-specific gene and drug delivery. In this application, we propose to construct and screen a library of 1000 cell-specific mammalian promoters for nuclear import activity in an array of 100 cell types from tissues throughout the body. Quantum dot-labeled plasmids will be electroporated into cells and assayed by quantitative image analysis on an ImageStream flow cytometer. Potential DNA nuclear targeting sequences will be validated using a traditional microinjection approach and the mechanisms of their cell-specificity and nuclear import will be characterized. Finally, validated hits from the previous aims will be delivered by electroporation to individual organs in vivo to test for cell- and tissue-specific gene delivery. PUBLIC HEALTH RELEVANCE: Gene therapy is an exciting and potentially very useful approach to treat a number of diseases at the molecular level. Unfortunately, when targeting genes or drugs to any tissue, one would like the delivery to be targeted and specific for the desired cell type; delivery of agents to the wrong cells could be disasterous. We have developed a new approach to limit gene delivery to desired cell types by focusing on how genes move within cells. In this proposal, we will screen a large number of DNA sequences to identify a panel of elements that can direct cell-specific delivery of genes and drugs to a set of 100 different cell types that represent tissues throughout the body. This study will create a new group of agents that can promote gene and drug delivery to specific cells while avoiding others.