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Abstract：
Nuclear proteins, like histone H2A, are promising non-viral carriers for gene delivery since they are biocompatible, biodegradable, bear intrinsic nuclear localization signal, and are easy to modify. The addition of surface-protein-binding ligand to histone H2A may increase its DNA delivery efficiency. Tumor microenvironment (TME) is a promising target for gene therapy since its surface protein repertoire is more stable than that of cancer cells. Cancer-associated fibroblasts (CAFs) are important components of TME, and one of their surface markers is beta-type platelet-derived growth factor receptor (PDGFRβ). In this study, we fused histone H2A with PDGFRβ-binding peptide, YG2, to create a novel non-viral fibroblast-targeting DNA carrier, H2A-YG2. The transfection efficiency of histone complexes with pDNA encoding a bicistronic reporter (enhanced green fluorescent protein, EGFP, and firefly luciferase) in PDGFRβ-positive and PDGFRβ-negative cells was estimated by luciferase assay and flow cytometry. The luciferase activity, percentage of transfected cells, and overall EGFP fluorescence were increased due to histone modification with YG2 only in PDGFRβ-positive cells. We also estimated the internalization efficiency of DNA-carrier complexes using tetramethyl-rhodamine-labeled pDNA. The ligand fusion increased DNA internalization only in the PDGFRβ-positive cells. In conclusion, we demonstrated that the H2A-YG2 carrier targeted gene delivery to PDGFRβ-positive tumor stromal cells.
摘要：
核蛋白,像组蛋白H2A，是有前途的基因传递的非病毒载体，因为它们是生物相容的，可生物降解，承受固有的核定位信号，并且易于修改。向组蛋白H2A添加表面蛋白结合配体可以提高其DNA递送效率。肿瘤微环境（TME）是基因治疗的有希望的靶标，因为其表面蛋白库比癌细胞更稳定。癌相关成纤维细胞（CAFs）是TME的重要组成部分，它们的表面标志物之一是β型血小板衍生生长因子受体(PDGFRβ)。在这项研究中,我们将组蛋白H2A与PDGFRβ结合肽融合，YG2，以创建一种新型的非病毒成纤维细胞靶向DNA载体，H2A-YG2。具有编码双顺反子报告子的pDNA的组蛋白复合物的转染效率（增强的绿色荧光蛋白，EGFP,和萤火虫荧光素酶）通过荧光素酶测定和流式细胞术估计PDGFRβ阳性和PDGFRβ阴性细胞。荧光素酶活性，转染细胞的百分比，由于仅在PDGFRβ阳性细胞中用YG2进行组蛋白修饰，因此整体EGFP荧光增加。我们还使用四甲基-罗丹明标记的pDNA估算了DNA-载体复合物的内化效率。配体融合仅在PDGFRβ阳性细胞中增加DNA内化。总之,我们证明H2A-YG2载体靶向基因递送至PDGFRβ阳性肿瘤基质细胞。
