White April F, Mazur Marina, Sorscher Eric J, Zinn Kurt R, Ponnazhagan Selvarangan
Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Hum Gene Ther. 2008 Dec;19(12):1407-14. doi: 10.1089/hum.2008.117.
Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.
囊性纤维化（CF）是一种常见的遗传疾病，其特征在于囊性纤维化跨膜传导调节因子（CFTR）基因表达存在缺陷。基因治疗为CF的治疗带来了更大的希望。腺相关病毒（AAV）载体能够稳定表达且免疫原性低。尽管它们在CF基因治疗中有潜力，但由于这些患者存在病理生理障碍，AAV的基因转移效率受到限制。虽然与基于AAV2的载体相比，少数AAV血清型显示出更好的转导效果，但在人气道上皮中的基因转移效率仍未达到治疗水平。为了构建更好的AAV载体以增强在人气道上皮中的基因递送，我们通过基因衣壳修饰开发并表征了突变型AAV载体，以特征明确的AAV2血清型为模型。我们在AAV2衣壳蛋白的GH环区域上基因整合了与人气道上皮具有假定高亲和力的肽配体。构建了六个独立的突变型AAV，它们含有先前报道的与人气道上皮细胞上已知和未知受体具有高亲和力结合的肽配体。对这些载体在非气道细胞以及非极化和极化的人气道上皮细胞上进行测试，以评估其增强的感染性。其中一个具有肽序列THALWHT的突变载体不仅在未分化的人气道上皮细胞中显示出最高的转导效率，而且在极化细胞中也显示出显著的转导。有趣的是，这种修饰后的载体还能够独立于硫酸乙酰肝素蛋白聚糖受体感染细胞。将这种配体整合到在人气道上皮中已显示出提高的基因转移效率的其他AAV血清型上，可能会增强AAV载体在CF基因治疗中的应用。
