Development of AAV vectors for CF therapy

摘要 囊性纤维化（CF）是由CF跨膜传导调节因子（CFTR）基因突变引起的。 气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。 在基因递送载体中，腺相关病毒（AAV）载体已经在临床上显示出成功。 审判AAV载体也已经在CF患者中通过气道递送进行了基因添加的测试，但没有 可测量的成功可能是由于物理气道屏障和低效的转导。研究表明 在体外，气道上皮细胞通过基底外侧膜进行更高的AAV转导， 初步研究表明，AAV载体的全身给药诱导了更有效的肺 在小鼠中，转导比气道递送更有效。这些发现强烈提示AAV可以阻断气道 上皮细胞通过全身给药。AAV转导在气道上皮细胞中的效率 AAV载体的全身递送通常受到血管屏障的限制，并且AAV载体的高患病率是一个很大的障碍。 AAV中和抗体（Nabs）。在这个建议中，基于我们以前的研究，我们将探索不同的 利用定向进化和AAV病毒体特异性结合肽开发新型AAV载体的策略， 噬菌体展示技术增加AAV载体血管通透性以增强气道中的转导 上皮细胞，并在全身给药后逃避Nabs。最后，新的方法， 该提案将用于向CF小鼠递送对CFTR del 508特异性的优化CRISPR/Cas9，并研究 长期的表型校正。从这些研究中产生的结果将使我们能够制定一个 使用AAV载体介导的基因递送治疗CF患者的有效方法。 .

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. .