Enhanced transduction of mouse salivary glands with AAV5-based vectors
Enhanced transduction of mouse salivary glands with AAV5-based vectors
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DOI：
10.1038/sj.gt.3302691
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发表时间：
2006-04-01
期刊：
影响因子：
5.1
通讯作者：
Chiorini, JA
中科院分区：
文献类型：
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作者：
Katano, H;Kok, MR;Chiorini, JA
我们以前证明，重组腺相关病毒载体的基础上血清型2（rAAV 2）可以直接转基因表达在唾液腺细胞在体外和体内。然而，尚不清楚其他rAAV血清型在输注到唾液腺中时如何表现。血清型4和5的衣壳与rAAV 2不同，并且彼此不同，这表明它们可以指导结合并进入不同的细胞类型。在本研究中，我们研究了基于AAV血清型2、4和5的AAV载体的向性、转导效率和抗体应答。通过逆行导管滴注将rAAV 2 β-半乳糖苷酶（β gal）、rAAV 4 β gal或rAAV 5 β gal施用至小鼠下颂唾液腺导致唾液上皮细胞的有效转导，其中AAV 4和AAV 5产生比AAV 2多2.3和7.3倍的β gal活性。通过从腺体提取的DNA的QPCR和转基因表达的免疫组织化学染色证实了用AAV 5的改善的转导。与AAV 2一样，AAV 5主要转导纹状和闰管细胞。AAV 4转导在横纹、闰和排泄导管细胞以及曲颗粒小管中是明显的。与唾液腺的包囊性质一致，大多数持久性病毒基因组在腺体中而不是在其他组织中发现。在病毒输注动物血清中发现的中和抗体（NAB）具有血清型特异性，血清型之间无交叉反应性。在唾液中未检测到NAB，但唾液中存在的唾液酸缀合物可以在低稀释度下中和AAV 4。总之，我们的数据表明，由于受体结合和转导途径的差异，其他血清型可能已提高效用的基因转移载体在唾液腺和这些差异可以利用基因治疗的应用。
We previously demonstrated that recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) can direct transgene expression in salivary gland cells in vitro and in vivo. However, it is not known how other rAAV serotypes perform when infused into salivary glands. The capsids of serotypes 4 and 5 are distinct from rAAV2 and from each other, suggesting that they may direct binding and entry into different cell types. In the present study, we investigated the tropisms, transduction efficiencies, and antibody response to AAV vectors based on AAV serotypes 2, 4, and 5. Administration of rAAV2 beta-galactosidase (beta gal), rAAV4 beta gal, or rAAV5 beta gal to murine submandibular salivary glands by retrograde ductal instillation resulted in efficient transduction of salivary epithelial cells, with AAV4 and AAV5 producing 2.3 and 7.3 times more beta gal activity compared with AAV2. Improved transduction with AAV5 was confirmed by QPCR of DNA extracted from glands and immunohistochemical staining for transgene expression. Like AAV2, AAV5 primarily transduced striated and intercalated ductal cells. AAV4 transduction was evident in striated, intercalated, and excretory ductal cells, as well as in convoluted granular tubules. In keeping with the encapsulated nature of the salivary gland, the majority of persistent viral genomes were found in the gland and not in other tissues. Neutralizing antibodies (NABs) found in the serum of virus-infused animals were serotype specific and there was no cross-reactivity between serotypes. No NABs were detected in saliva but sialic acid conjugates present in saliva could neutralize AAV4 at low dilutions. Together our data suggest that because of differences in receptor binding and transduction pathways, other serotypes may have improved utility as gene transfer vectors in the salivary gland and these differences could be exploited in gene therapy applications.