Life-long phenotypic correction of CF airways

标题：CF Airways的终生表型矫正 项目摘要/摘要： 囊性纤维化是由囊性纤维化传导调节基因突变引起的。 自1989年发现CFTR以来，对它的功能和细胞类型表达的知识有了很大的进步 在过去的5年中有显著的发现。虽然在小分子方面已经取得了重大进展 调节疗法可恢复大多数CFTR突变类别的功能，约10%的CF患者尚未恢复功能 受益于这些战略。我们有一个证明的记录，使用许多类别的病毒和 用于将基因输送到呼吸道的非病毒试剂。我们的目标是实现终生修正 单剂雾化病毒载体。因此，有效地传递治疗性基因或基因编辑 气道祖细胞的机械是至关重要的。在这项提案中，我们提出了两项基因治疗技术和 比较他们的利弊。这两种策略都利用了令人印象深刻的转导效率和 大包装容量的腺病毒(Ad)为基础的载体。广告载体的两个潜在缺点是 瞬时表达和免疫反应，这两个问题都将得到解决。在目标1中，我们设计了基于广告的 具有改进的祖细胞靶向和校正的病毒载体。我们比较嵌合向量基于 Ad5和来自B类腺病毒的纤维。此外，我们还证明了广告具有穿透呼吸道的能力 粘液屏障，但研究可能进一步改善媒介传递的粘液溶解物质。在目标2中，我们对比 使用基因传递和基因编辑的CFTR功能矫正的体外效率。为了获得基因 更正，将使用随Ad(Ad-Cas9-ABE)提供的腺嘌呤碱基编辑程序(ABE)来更正细胞中的CFTR。 作为原理的证明，我们将重点放在CFTR无义突变R553X上。这种突变导致早产 终止密码子，对任何小分子调节剂都不反应。在媒介传播之后，我们将 使用下一代测序和氯电流测量相结合的方法确认基因编辑。这个 已实现的表型校正水平将与PB/Ad-CFTR基因添加策略进行比较。我们 假设无论采用何种基因治疗策略，氯离子电流的最大阈值水平都是可以实现的。 这一电流在杂合型(CFtr+/-)或野生型(CFtr+/+)细胞中类似。在目标3中，我们对比了基因 猪呼吸道的递送和基因编辑效率。我们将产生一个CFTRG551D/R553X复合杂合子 猪模型用于筛选旨在纠正当前没有小分子的CF突变的前导载体 治疗方法是可用的。我们的目标是提供一种终生基因修复策略，这种策略可以适用于 大量的CF导致突变。这项研究具有很强的创新性。试剂，方法， 这些实验产生的数据可以用于其他基因的添加或碱基编辑 单基因紊乱，从而大大推进了基因治疗领域。

Title: Life-Long Phenotypic Correction of CF Airways Project Summary/Abstract: Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis conductance regulator (CFTR) gene. Knowledge of CFTR function and cell type expression has advanced greatly since its discovery in 1989, with notable discoveries in the last 5 years. While significant advances have been made with small molecule modulator therapies to restore function for most CFTR mutation classes, ~10% of people with CF have not benefited from these strategies. We have a demonstrated track record of using many categories of viral and non-viral based reagents for gene delivery to the airways. Our goal is to achieve life-long correction from a single dose of aerosolized viral vector. As such, efficient delivery of a therapeutic gene or gene editing machinery to airway progenitor cells is critical. In this proposal, we advance two gene therapy technologies and compare their pros and cons. Both strategies take advantage of the impressive transduction efficiency and large packaging capacity of Adenoviral (Ad)-based vectors. Two potential drawbacks of Ad vectors are transient expression and immune response, both of which will be addressed. In Aim 1 we engineer Ad-based viral vectors with improved progenitor cell targeting and correction. We compare chimeric vectors based on Ad5 with fibers from species B adenoviruses. In addition, we show that Ad has the capacity to penetrate airway mucus barriers but investigate mucolytics that may further improve vector delivery. In Aim 2 we contrast efficiency of CFTR functional correction using gene delivery and gene editing in vitro. To achieve gene correction, will use an adenine base editor (ABE) delivered with Ad (Ad-Cas9-ABE) to correct CFTR in cells. As a proof of principle, we focus on the CFTR nonsense mutation R553X. This mutation results in premature termination codon and does not respond to any small molecule modulator. Following vector delivery, we will confirm gene editing using a combination of next generation sequencing and Cl- current measurements. The achieved levels of phenotypic correction will be compared to the PB/Ad-CFTR gene addition strategy. We hypothesize that regardless of gene therapy strategy, a maximum threshold level of Cl- current is achievable. This current is similar in heterozygous (CFTR+/-) or wild-type (CFTR+/+) cells. In Aim 3 we contrast gene delivery and gene editing efficiency in pig airways. We will generate a CFTRG551D/R553X compound heterozygous pig model to screen leading vectors designed to correct a CF mutation for which no current small molecule treatments are available. Our goal is to provide a life-long gene repair strategy that could be adapted for a great number of CF causing mutations. This proposed research is highly innovative. The reagents, methods, and data generated by these experiments could be applied to gene addition or base editing for other monogenic disorders, thereby significantly advancing the gene therapy field.