Optimization of Recombinant Adeno-Associated Virus-Mediated Expression for Large Transgenes, Using a Synthetic Promoter and Tandem Array Enhancers
Optimization of Recombinant Adeno-Associated Virus-Mediated Expression for Large Transgenes, Using a Synthetic Promoter and Tandem Array Enhancers
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DOI：
10.1089/hum.2015.001
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发表时间：
2015-06-01
影响因子：
4.2
通讯作者：
Engelhardt, John F.
中科院分区：
文献类型：
--
作者：
Yan, Ziying;Sun, Xingshen;Engelhardt, John F.
重组腺相关病毒（rAAV）载体的包装能力限制了可用于表达4.43-kb囊性纤维化跨膜传导调节因子（CFTR）cDNA的启动子的大小。为了规避这一限制，我们筛选了一组100-mer合成增强子元件，其由10个10-bp重复序列组成，在设计用于囊性纤维化（CF）雪貂模型的rAAV载体的背景下，其能够从短的83-bp合成启动子增强CFTR转基因表达。我们的初步研究评估在单层（非极化）培养的人气道细胞系和原代雪貂气道细胞的转录活性显示，这些合成的增强子（F1，F5和F10）显着促进荧光素酶转基因的转录在质粒转染的背景下。在人和雪貂气道上皮细胞在气液界面（ALI）的极化培养物中以及在雪貂气道体内的进一步分析表明，在AAV载体的背景下，F5增强子产生最高水平的转基因表达。此外，我们证明了将病毒基因组的大小从4.94 kb增加到5.04 kb并不显著影响载体的颗粒产量，但由于延伸到5.04 kb超大基因组的CFTR表达盒中的小的末端缺失而显著降低了rAAV-CFTR载体的功能。由于大小大于5 kb的rAAV-CFTR载体在载体功效方面显著受损，我们使用在R结构域中具有159-bp缺失的缩短的雪貂CFTR小基因来构建rAAV载体（AV 2/2.F5tg83-fCFTRR）。该载体使CFTR的表达增加了约17倍，并显著改善了CF ALI培养物中的Cl-电流。我们的研究已经确定了一个小的增强子/启动子组合，可能有广泛的用途rAAV介导的CF基因治疗的气道。
The packaging capacity of recombinant adeno-associated viral (rAAV) vectors limits the size of the promoter that can be used to express the 4.43-kb cystic fibrosis transmembrane conductance regulator (CFTR) cDNA. To circumvent this limitation, we screened a set of 100-mer synthetic enhancer elements, composed of ten 10-bp repeats, for their ability to augment CFTR transgene expression from a short 83-bp synthetic promoter in the context of an rAAV vector designed for use in the cystic fibrosis (CF) ferret model. Our initial studies assessing transcriptional activity in monolayer (nonpolarized) cultures of human airway cell lines and primary ferret airway cells revealed that three of these synthetic enhancers (F1, F5, and F10) significantly promoted transcription of a luciferase transgene in the context of plasmid transfection. Further analysis in polarized cultures of human and ferret airway epithelia at an air-liquid interface (ALI), as well as in the ferret airway in vivo, demonstrated that the F5 enhancer produced the highest level of transgene expression in the context of an AAV vector. Furthermore, we demonstrated that increasing the size of the viral genome from 4.94 to 5.04 kb did not significantly affect particle yield of the vectors, but dramatically reduced the functionality of rAAV-CFTR vectors because of small terminal deletions that extended into the CFTR expression cassette of the 5.04-kb oversized genome. Because rAAV-CFTR vectors greater than 5 kb in size are dramatically impaired with respect to vector efficacy, we used a shortened ferret CFTR minigene with a 159-bp deletion in the R domain to construct an rAAV vector (AV2/2.F5tg83-fCFTRR). This vector yielded an approximate to 17-fold increase in expression of CFTR and significantly improved Cl- currents in CF ALI cultures. Our study has identified a small enhancer/promoter combination that may have broad usefulness for rAAV-mediated CF gene therapy to the airway.