Effects of Self-Complementarity, Codon Optimization, Transgene, and Dose on Liver Transduction with AAV8
Effects of Self-Complementarity, Codon Optimization, Transgene, and Dose on Liver Transduction with AAV8
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DOI：
10.1089/hgtb.2016.039
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发表时间：
2016-12-01
影响因子：
--
通讯作者：
Wilson, James M.
中科院分区：
文献类型：
--
作者：
Bell, Peter;Wang, Lili;Wilson, James M.
已经采用了许多载体设计和递送方法来尝试在基于AAV的基因治疗后增加转基因表达。在此，在小鼠中进行基因转移研究，以比较载体自身互补性（双链或单链DNA）、转基因的密码子优化和载体剂量对肝脏中转基因表达水平的影响。使用了两种不同的报告基因：通过免疫荧光检测的人鸟氨酸转氨甲酰酶（hOTC）和通过直接荧光检测的增强型绿色荧光蛋白（EGFP）。选择AAV 8衣壳用于所有实验，因为其强烈的肝嗜性。虽然EGFP已经是原始基因的密码子优化版本，但在本研究中比较了hOTC转基因的野生型（WT）和密码子优化（co）版本。此外，该研究评估了两种hOTC修饰--密码子优化或自身互补--中的哪一种在给定剂量下会赋予表达水平最高的增加。有趣的是，基于形态测量图像分析，观察到自身互补（sc）和单链（ss）hOTCco载体之间可检测表达水平的差异是剂量依赖性的，在每只小鼠3 × 10（9）个基因组拷贝（GC）的剂量下使用sc载体时，OTC阳性面积增加了7倍，但在1 × 10（10）个GC/小鼠的剂量下没有显著差异。相比之下，用EGFP作为转基因，当使用sc载体时，在3x 10（9）GC/小鼠和1x 10（10）GC/小鼠剂量下均观察到表达水平的增加。此外，hOTC转基因的密码子优化比使用自身互补性产生更显著的表达改善。总的来说，结果表明，通过使用sc载体代替ss载体获得的表达水平的增加可以在不同的转基因之间变化，并且转基因的密码子优化可以对所得的表达水平具有甚至更强大的影响。
Numerous methods of vector design and delivery have been employed in an attempt to increase transgene expression following AAV-based gene therapy. Here, a gene transfer study was conducted in mice to compare the effects of vector self-complementarity (double- or single-stranded DNA), codon optimization of the transgene, and vector dose on transgene expression levels in the liver. Two different reporter genes were used: human ornithine transcarbamylase (hOTC) detected by immunofluorescence, and enhanced green fluorescent protein (EGFP) detected by direct fluorescence. The AAV8 capsid was chosen for all experiments due to its strong liver tropism. While EGFP is already a codon-optimized version of the original gene, both wild-type (WT) and codon-optimized (co) versions of the hOTC transgene were compared in this study. In addition, the study evaluated which of the two hOTC modificationscodon optimization or self-complementaritywould confer the highest increase in expression levels at a given dose. Interestingly, based on morphometric image analysis, it was observed that the difference in detectable expression levels between self-complementary (sc) and single-stranded (ss) hOTCco vectors was dose dependent, with a sevenfold increase in OTC-positive area using sc vectors at a dose of 3x10(9) genome copies (GC) per mouse, but no significant difference at a dose of 1x10(10) GC/mouse. In contrast, with EGFP as a transgene, the increases in expression levels when using the sc vector were observed at both the 3x10(9) GC/mouse and 1x10(10) GC/mouse doses. Furthermore, codon optimization of the hOTC transgene generated a more significant improvement in expression than the use of self-complementarity did. Overall, the results demonstrate that increases in expression levels gained by using sc vectors instead of ss vectors can vary between different transgenes, and that codon optimization of the transgene can have an even more powerful effect on the resulting expression levels.