Incorporation of adenovirus in calcium phosphate precipitates enhances gene transfer to airway epithelia in vitro and in vivo
Incorporation of adenovirus in calcium phosphate precipitates enhances gene transfer to airway epithelia in vitro and in vivo
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DOI：
10.1172/jci2732
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发表时间：
1998-07-01
影响因子：
15.9
通讯作者：
Welsh, MJ
中科院分区：
文献类型：
--
作者：
Fasbender, A;Lee, JH;Welsh, MJ
腺病毒(Ad)介导的基因转移到呼吸道上皮细胞是无效的，因为顶膜缺乏结合腺病毒纤维蛋白的受体活性。磷酸钙(CAPI)沉淀物已被用来向培养的细胞系运送质粒DNA。然而，这种沉淀物在许多原代培养物或体内都不起作用。在这里，我们表明，将重组腺病毒整合到CAPI共沉淀物中，显著增强了对腺病毒感染具有抵抗力的细胞中的转基因表达，增强需要将病毒包含在沉淀物中，并要求病毒蛋白增加表达，Ad：CAPI共沉淀物通过增加病毒与细胞的纤维非依赖性结合来增加基因转移。对于体外培养的分化囊性纤维化(CF)呼吸道上皮细胞，应用编码囊性纤维化(CF)跨膜电导调节剂的Bd：CAPI共沉淀物20min产生的CF跨膜电导调节剂Cl-电流与单独应用腺病毒24 h的作用相当。我们发现，在体内，Ad：CAPI共沉淀物也增加了转基因在小鼠肺中的表达；重要的是，在呼吸道上皮细胞中的表达尤其显著。这些结果提示了一种新的基因转移机制，该机制可能适用于许多不同的基因转移应用，并可能在体内向CF气道上皮细胞转移基因。
Adenovirus (Ad)-mediated gene transfer to airway epithelia is inefficient because the apical membrane lacks the receptor activity to bind adenovirus fiber protein. Calcium phosphate (CaPi) precipitates have been used to deliver plasmid DNA to cultured cell lines. However, such precipitates are not effective in many primary cultures or in vivo. Here we show that incorporating recombinant adenovirus into a CaPi coprecipitate markedly enhances transgene expression in cells that are resistant to adenovirus infection, Enhancement requires that the virus be contained in the precipitate and viral proteins are required to increase expression, Ad: CaPi coprecipitates increase gene transfer by increasing fiber-independent binding of virus to cells. With differentiated cystic fibrosis (CF) airway epithelia in vitro, a 20-min application of Bd:CaPi coprecipitates that encode CF transmembrane conductance regulator produced as much CF transmembrane conductance regulator Cl- current as a 24-h application of adenovirus alone. We found that Ad:CaPi coprecipitates also increased transgene expression in mouse lung in vivo; importantly, expression was particularly prominent in airway epithelia. These results suggest a new mechanism for gene transfer that may be applicable to a number of different gene transfer applications and could be of value in gene transfer to CF airway epithelia in vivo.