A shortened adeno-associated virus expression cassette for CFTR gene transfer to cystic fibrosis airway epithelia
A shortened adeno-associated virus expression cassette for CFTR gene transfer to cystic fibrosis airway epithelia
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DOI：
10.1073/pnas.0409845102
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发表时间：
2005-02-22
影响因子：
11.1
通讯作者：
Welsh, MJ
中科院分区：
文献类型：
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作者：
Ostedgaard, LS;Rokhlina, T;Welsh, MJ
腺相关病毒（AAVs），如AAV5，从气道顶端表面转导气道上皮，是囊性纤维化（CF）基因转移的有吸引力的载体。然而，它们在CF中的应用受到了限制，因为当插入的长度超过大约4,900-5,000 bp时，其封装效率就会降低。为了部分规避这种尺寸限制，我们之前开发了一种CIF跨膜电导调节剂（CFTR）转基因，该转基因删除了一部分R结构域（CFTRDeltaR）。在本研究中，我们着重于缩短AAV表达盒中的其他元件。我们发现CMV即时/早期（CMVie）增强子/启动子的部分可以被删除而不取消活性。我们还测试了各种中间序列、多聚(A)信号和内含子，以开发满足AAV大小限制的表达盒。然后我们将这些缩短的元件与CFTRDeltaR转基因一起包装到AAV5中，并将其应用于分化的CF气道上皮的顶表面。2 ~ 4周后，AAV5载体部分纠正了CF Cl-转运缺陷。这些结果表明，单个AAV载体可以补充分化的气道上皮中的CF缺陷，从而进一步发展有效的CF基因转移。
Adeno-associated viruses (AAVs) such as AAV5 that transduce airway epithelia from the apical surface are attractive vectors for gene transfer in cystic fibrosis (CF). However, their utility in CF has been limited because packaging of the insert becomes inefficient when its length exceeds approximate to4,900-5,000 bp. To partially circumvent this size constraint, we previously developed a CIF transmembrane conductance regulator (CFTR) transgene that deleted a portion of the R domain (CFTRDeltaR). In this study, we focused on shortening the other elements in the AAV expression cassette. We found that portions of the CMV immediate/early (CMVie) enhancer/promoter could be deleted without abolishing activity. We also tested various intervening sequences, poly(A) signals, and an intron to develop an expression cassette that meets the size restrictions imposed by AAV. We then packaged these shortened elements with the CFTRDeltaR transgene into AAV5 and applied them to the apical surface of differentiated CF airway epithelia. Two to 4 weeks later, the AAV5 vectors partially corrected the CF Cl- transport defect. These results demonstrate that a single AAV vector can complement the CF defect in differentiated airway epithelia and thereby further the development of effective CF gene transfer.