Stable In Vivo Transgene Expression in Endothelial Cells with Helper-Dependent Adenovirus: Roles of Promoter and Interleukin-10
Stable In Vivo Transgene Expression in Endothelial Cells with Helper-Dependent Adenovirus: Roles of Promoter and Interleukin-10
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DOI：
10.1089/hum.2016.134
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发表时间：
2017-03-01
影响因子：
4.2
通讯作者：
Dichek, David A.
中科院分区：
文献类型：
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作者：
Dronadula, Nagadhara;Wacker, Bradley K.;Dichek, David A.
我们的长期目标是通过从稳定转导的内皮细胞（EC）中递送基因治疗来预防或逆转动脉粥样硬化。我们先前报道了用表达载脂蛋白A-I（apo A-I）的辅助依赖性腺病毒（HDAd）进行EC定向基因治疗，在1个月的时间间隔内延缓了兔颈动脉粥样硬化的发展。然而，在此期间apo A-I表达下降了70%，这引起了人们对这种方法长期有效性的担忧。在这里，我们报告了几种方法的使用，旨在防止这种下降或在所有时间点增加HDAd的apo A-I表达：密码子优化、删除3'非翻译序列、用合成的哺乳动物启动子（4XETE）取代巨细胞病毒（CMV）启动子，以及与表达白细胞介素-10的HDAd共转导。我们使用质粒转染培养的EC和兔颈动脉EC的体内转导来测试这些方法。密码子优化没有增加apo A-I的表达。3'端非翻译序列的缺失使apo A-I表达消失。4XETE替换CMV启动子和白细胞介素-10的表达都稳定了apoA-I在体内的表达，尽管以较低的早期（3天）表达水平为代价。令人惊讶的是，这两种干预措施都稳定了载脂蛋白A-I的表达，而没有改变HDAd基因组丢失的速率。这些数据证实，在EC中来自HDAd的转基因表达在体内是固有稳定的，并且表明来自HDAd转导的EC的CMV启动子驱动的表达的早期下降既不是由于转录的主动下调也不是由于HDAd基因组的丢失。相反，CMV启动子表达的明显丧失似乎是CMV启动子活性通过炎症途径早期（3天）上调的结果。我们的研究结果产生了新的范例来解释体内基因转移后基因组和转基因表达的早期丢失。这些新的范式将重新调整策略，以实现转基因在EC中的高水平、稳定表达。
Our long-term goal is to prevent or reverse atherosclerosis by delivering gene therapy from stably transduced endothelial cells (EC). We previously reported that EC-directed gene therapy with a helper-dependent adenovirus (HDAd) expressing apolipoprotein A-I (apo A-I) retarded development of atherosclerosis in rabbit carotid arteries over a 1-month interval. However, a 70% decline in apo A-I expression during this time raised concerns about long-termefficacy of this approach. Here we report use of several approaches aimed either at preventing this decline or at increasing apo A-I expression from HDAd at all time points: codon optimization, deletion of 3' untranslated sequences, substitution of a synthetic mammalian-based promoter (4XETE) for the cytomegalovirus (CMV) promoter, and co-transduction with an HDAd expressing interleukin-10. We tested these approaches using plasmid transfection of cultured EC and in vivo transduction of rabbit carotid artery EC. Codon optimization did not increase apo A-I expression. Deletion of 3' untranslated sequences extinguished apo A-I expression. Both substitutionof 4XETE for the CMV promoter and expression of interleukin-10 stabilized apoA-I expression in vivo, although at the cost of lower early (3-day) expression levels. Surprisingly, both interventions stabilized apo A-I expression without altering the rate at which HDAd genomes were lost. These data establish that transgene expression from HDAd in EC is inherently stable in vivo and suggest that the early decline of CMV promoter-driven expression from HDAd-transduced EC is due neither to active downregulation of transcription nor to loss of HDAd genomes. Instead, apparent loss of expression from the CMV promoter appears to be a consequence of early (3-day) upregulation of CMV promoter activity via inflammatory pathways. Our results yield new paradigms to explain the early loss of genomes and transgene expression after in vivo gene transfer. These new paradigms will redirect strategies for achieving high-level, stable expression of transgenes in EC.