Adeno-associated virus (AAV) has been investigated to transfer the cystic fibrosis transmembrane conductance regulator (CFTR) to airways. Inhaled AAV2-CFTR in people with cystic fibrosis (CF) is safe, but inefficient. In vitro, AAV2 transduction of human airway epithelia on the apical (luminal) side is inefficient, but efficient basolaterally. We previously selected AAV2.5T, a novel capsid that apically transduces CF human airway epithelia and efficiently restores CFTR function. We hypothesize the AAV receptor (AAVR) is basolaterally localized, and that AAV2.5T utilizes an alternative apical receptor. We found AAVR in human airway epithelia by western blot and RNA-Seq analyses. Using immunocytochemistry we did not find endogenous AAVR at membranes but overexpression localized AAVR to the basolateral membrane, where it preferentially increased transduction. Anti-AAVR antibodies blocked transduction by AAV2 from the basolateral side but not AAV2.5T from the apical side, suggesting a unique apical receptor. Finally, we found infection by AAV2 but not AAV2.5T was blocked by CRISPR knockout of AAVR in cell lines. Our data suggest the absence of apical AAVR is rate limiting for AAV2, and efficient transduction by AAV2.5T is accomplished using an AAVR independent pathway. Our findings inform the development of gene therapy for CF, and AAV vectors in general.

中文翻译：

---

极化的AVR表达式决定了AAV基因治疗载体的感染性。

已经研究了腺伴随病毒（AAV）将囊性纤维化跨膜电导调节剂（CFTR）转移至气道。在患有囊性纤维化（CF）的人中吸入AAV2-CFTR是安全的，但效率低下。在体外，顶侧（管腔）的人气道上皮的AAV2转导效率低下，但在基底外侧有效。我们先前选择了AAV2.5T，这是一种新颖的衣壳，可在顶端传导CF人气道上皮并有效恢复CFTR功能。我们假设AAV受体（AAVR）位于基底外侧，并且AAV2.5T利用了一种替代性的顶端受体。通过蛋白质印迹和RNA-Seq分析，我们在人的气道上皮细胞中发现了AAVR。使用免疫细胞化学，我们在膜上未发现内源性AAVR，但过表达将AVR定位于基底外侧膜，优先增加转导。抗AAVR抗体从基底外侧阻止了AAV2的转导，但从顶端阻止了AAV2.5T的转导，提示了独特的顶端受体。最后，我们发现AAV2感染，但CRISPR敲除AAVR在细胞系中并未阻断AAV2.5T。我们的数据表明，不存在根尖的AAVR限制了AAV2的速率，并且使用AAVR独立的途径可以实现AAV2.5T的有效转导。我们的发现总体上指导了CF和AAV载体基因治疗的发展。我们的数据表明，不存在根尖的AAVR限制了AAV2的速率，并且使用AAVR独立的途径可以实现AAV2.5T的有效转导。我们的发现总体上指导了CF和AAV载体基因治疗的发展。我们的数据表明，不存在根尖的AAVR限制了AAV2的速率，并且使用AAVR独立的途径可以实现AAV2.5T的有效转导。我们的发现总体上指导了CF和AAV载体基因治疗的发展。