Life-long phenotypic correction of CF airways

标题：CF气道的终生表型纠正

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.