目的:由于腺相关病毒的体积较小,AAV,囊性纤维化电导调节因子,CFTR,cDNA太大以至于不能容纳在AAV中并且必须被截短。我们在这里报告CFTR的两个截断版本,which,当插入AAV1并用于感染气道细胞时,通过反式互补拯救F508-delCFTR。这项研究的目的是阐明细胞中发生反式互补的位置以及它如何导致内源性F508-del和截短的CFTR之间的紧密联系。
方法:我们用含有CFTR截短形式的AAV2/1(AAV2反向末端重复序列/AAV1衣壳)处理CF气道细胞(CFBE41o-),Δ264和Δ27-264CFTR,谁可以通过反式互补恢复F508-del的功能。我们使用共聚焦显微镜和短路电流测量的组合来解决研究的目的。对于后者,CF支气管上皮细胞(CFBE)在可渗透的支持物上生长。
结果:我们显示F508del和截短突变体共同定位在ER中,并且获救的F508-del和反式互补突变体一起到达质膜。F508-del和内质网(ER)内的反式互补突变体之间存在显著的荧光共振能量转移(FRET),表明反式互补是通过双分子相互作用发生的。我们发现,在稳定表达额外wt-CFTR或F508-del的CFBE41o细胞中和在表达内源水平的F508-del的亲本CFBE41o细胞中,反式互补可以增加Isc。
结论:我们得出结论,通过反式互补作用对F508-del的功能性拯救是通过双分子相互作用发生的,该相互作用很可能始于ER并在质膜上继续。这些结果是在开发CF基因疗法的适当时机出现的,并为广泛的CF患者提供了新的治疗选择。
OBJECTIVE: Because of the small size of adeno-associated virus, AAV, the cystic fibrosis conductance regulator, CFTR, cDNA is too large to fit within AAV and must be truncated. We report here on two truncated versions of CFTR, which, when inserted into AAV1 and used to infect airway cells, rescue F508-del CFTR via
transcomplementation. The purpose of this study is to shed light on where in the cell
transcomplementation occurs and how it results in close association between the endogenous F508-del and truncated CFTR.
METHODS: We treated CF airway cells (CFBE41o-) with AAV2/1 (AAV2 inverted terminal repeats/AAV1 capsid) containing truncated forms of CFTR, ∆264 and ∆27-264 CFTR, who can restore the function of F508-del by transcomplementation. We addressed the aims of the study using a combination of confocal microscopy and short circuit currents measurements. For the latter, CF bronchial epithelial cells (CFBE) were grown on permeable supports.
RESULTS: We show that both F508del and the truncation mutants colocalize in the ER and that both the rescued F508-del and the transcomplementing mutants reach the plasma membrane together. There was significant fluorescence resonance energy transfer (FRET) between F508-del and the transcomplementing mutants within the endoplasmic reticulum (ER), suggesting that
transcomplementation occurs through a bimolecular interaction. We found that
transcomplementation could increase the Isc in CFBE41o- cells stably expressing additional wt-CFTR or F508-del and in parental CFBE41o- cells expressing endogenous levels of F508-del.
CONCLUSIONS: We conclude that the functional rescue of F508-del by
transcomplementation occurs via a bimolecular interaction that most likely begins in the ER and continues at the plasma membrane. These results come at an opportune time for developing a gene therapy for CF and offer new treatment options for a wide range of CF patients.