Development of AAV vectors for CF therapy

摘要 囊性纤维化是由囊性纤维化跨膜电导调节基因突变引起的。 呼吸道疾病是发病率和死亡率的主要原因。基因治疗为CF提供了一种潜在的治疗方法。 在基因传递载体中，腺相关病毒载体已在临床上取得了成功。 审判。AAV载体也已经通过呼吸道输送在CF患者中进行了基因添加的测试，但没有 可衡量的成功可能是由于物理上的呼吸道障碍和低效的转导。研究表明， AAV对呼吸道上皮细胞的高转导作用发生在体外的基底膜和我们的 初步研究表明，全身注射AAV载体可诱导更有效的肺功能。 在小鼠体内的转导比呼吸道给药更有效。这些发现有力地表明AAV可以转导呼吸道。 上皮细胞通过全身给药。甲型肝炎病毒在呼吸道上皮细胞中的转导效率 AAV载体的全身递送通常受到血管屏障和高度流行的 AAV中和抗体(NAB)。在这个提案中，我们将在我们之前的研究的基础上，探索不同的 利用定向进化和AAV病毒粒子特异性结合肽通过 噬菌体展示技术增加AAV载体血管通透性以增强呼吸道转导 全身性给药后，对上皮细胞有保护作用，并能避开NABS。最后，开发的新方法 这项建议将用于将针对CFTRdel508的优化CRISPR/Cas9传递给CF小鼠并进行研究 长期的表型修正。这些研究产生的结果将使我们能够开发出一种 AAV载体介导的基因转移治疗慢性萎缩性胃炎的有效途径 。

Abstract Cystic fibrosis (CF) is caused by mutations of the CF transmembrane conductance regulator (CFTR) gene. Airway disease is the leading cause of morbidity and mortality. Gene therapy offers a potential cure for CF. Among gene delivery vehicles, adeno-associated virus (AAV) vectors have demonstrated success in clinical trials. AAV vectors have also been tested in CF patients via airway delivery for gene addition but without measurable success perhaps due to physical airway barriers and inefficient transduction. Studies have shown that higher AAV transduction of airway epithelial cells occur via the basolateral membrane in vitro and our preliminary study demonstrated that systemic administration of AAV vectors induced much more efficient lung transduction than airway delivery in mice. These findings strongly suggest that AAV can transduce airway epithelial cells via systemic administration. The efficiency of AAV transduction in airway epithelial cells after systemic delivery of AAV vectors is usually restricted by the blood vascular barrier and a high prevalence of AAV neutralizing antibodies (Nabs). In this proposal, based on our previous studies, we will explore different strategies to develop novel AAV vectors using directed evolution and AAV virion specific binding peptides via phage display technology to increase AAV vector vascular permeability for enhanced transduction in airway epithelial cells and to evade Nabs after systemic administration. Finally, the novel approaches developed in this proposal will be used to deliver optimized CRISPR/Cas9 specific for CFTR del508 to CF mice and study the long-term phenotypic correction. The results generated from these studies will allow us to develop an effective approach to treat CF patients using AAV vector mediated gene delivery. .