Life-long phenotypic correction of CF airways

标题：CF Airways的终身表型矫正 项目概要/摘要： 囊性纤维化（CF）是由囊性纤维化传导调节因子（CFTR）基因突变引起的。 自1989年发现CFTR以来，对CFTR功能和细胞类型表达的认识已经有了很大的进展， 近五年来的重大发现。虽然小分子已经取得了重大进展， 调节剂治疗恢复大多数CFTR突变类型的功能，约10%的CF患者没有 受益于这些战略。我们有一个使用许多类别的病毒和 用于将基因递送至气道的非病毒基试剂。我们的目标是实现终身矫正， 单剂量雾化病毒载体。因此，治疗性基因或基因编辑的有效递送 呼吸道祖细胞的机制是至关重要的。在这个建议中，我们提出了两种基因治疗技术， 两种策略都利用了令人印象深刻的转导效率， 大包装容量的腺病毒（Ad）载体。广告载体的两个潜在缺点是 瞬时表达和免疫应答，这两者都将得到解决。在目标1中，我们设计基于广告的 具有改进的祖细胞靶向和校正的病毒载体。我们比较嵌合载体的基础上， Ad 5与来自B种腺病毒的纤维。此外，我们还发现Ad具有穿透气道的能力， 粘液屏障，但研究可进一步改善载体递送的粘液溶解剂。在目标2中，我们对比 使用基因递送和体外基因编辑的CFTR功能校正的效率。实现基因 为了校正，将使用与Ad（Ad-Cas9-ABE）一起递送的腺嘌呤碱基编辑器（ABE）来校正细胞中的CFTR。 作为原理的证明，我们专注于CFTR无义突变R553 X。这种突变会导致早产 终止密码子，并且不响应任何小分子调节剂。在载体投放之后，我们将 使用下一代测序和Cl-电流测量的组合来确认基因编辑。的 将所达到的表型校正水平与PB/Ad-CFTR基因添加策略进行比较。我们 假设无论基因治疗策略如何，都可以实现氯电流的最大阈值水平。 该电流在杂合（CFTR+/-）或野生型（CFTR+/+）细胞中是相似的。在目标3中，我们对比基因 在猪气道中的递送和基因编辑效率。我们将产生CFTRG 551 D/R553 X复合杂合 猪模型筛选设计用于校正CF突变的前导载体， 治疗是可用的。我们的目标是提供一种终生的基因修复策略， 大量CF导致突变。这项研究具有很强的创新性。所述试剂、方法、 这些实验产生的数据可以应用于其他基因的基因添加或碱基编辑。 单基因疾病，从而显著推进基因治疗领域。

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.