A 20-nt long sequence, termed the D-sequence, in the adeno-associated virus (AAV) inverted terminal repeat was observed to share a partial sequence homology with the X-box in the regulatory region of the human leukocyte antigen DRA (HLA-DRA) promoter of the human major histocompatibility complex class II (MHC-II) genes. The D-sequence was also shown to specifically interact with the regulatory factor binding to the X-box (RFX), binding of which to the X-box is a critical step in the MHC-II gene expression, suggesting that D-sequence might compete for RFX transcription factor binding, thereby suppressing expression from the MHC-II promoter. In DNA-mediated transfection experiments, using a reporter gene under the control of the HLA-DRA promoter, D-sequence oligonucleotides were found to inhibit expression of the reporter gene expression in HeLa and 293 cells by ∼93% and 96%, respectively. No inhibition was observed when nonspecific synthetic oligonucleotides were used. D-sequence oligonucleotides had no effect on expression from the cytomegalovirus immediate-early gene promoter. Interferon-γ-mediated activation of MHC-II gene expression was also inhibited by D-sequence oligonucleotides as well as after infection with either the wild-type AAV or transduction with recombinant AAV vectors. These studies suggest that the D-sequence-mediated downregulation of the MHC-II gene expression may be exploited toward the development of novel AAV vectors capable of dampening the host humoral response, which has important implication in the optimal use of these vectors in human gene therapy.

中文翻译：

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腺相关病毒 D 序列介导的人类主要组织相容性 II 类基因表达的抑制：开发用于调节体液免疫反应的腺相关病毒载体的意义。

观察到腺相关病毒 (AAV) 反向末端重复序列中的 20 nt 长序列，称为 D 序列，与人类白细胞抗原 DRA (HLA) 调节区中的 X-box 共享部分序列同源性-DRA) 人类主要组织相容性复合体 II 类 (MHC-II) 基因的启动子。D-sequence 还被证明与结合 X-box (RFX) 的调节因子发生特异性相互作用，RFX 与 X-box 的结合是 MHC-II 基因表达的关键步骤，表明 D-sequence 可能竞争 RFX 转录因子结合，从而抑制 MHC-II 启动子的表达。在 DNA 介导的转染实验中，使用 HLA-DRA 启动子控制下的报告基因，发现 D 序列寡核苷酸分别抑制 HeLa 和 293 细胞中报告基因表达的约 93% 和 96%。当使用非特异性合成寡核苷酸时没有观察到抑制作用。D 序列寡核苷酸对巨细胞病毒立即早期基因启动子的表达没有影响。干扰素-γ 介导的 MHC-II 基因表达激活也受到 D 序列寡核苷酸的抑制，以及在感染野生型 AAV 或用重组 AAV 载体转导后。这些研究表明，D 序列介导的 MHC-II 基因表达下调可用于开发能够抑制宿主体液反应的新型 AAV 载体，这对这些载体在人类基因中的最佳应用具有重要意义。治疗。