Efficient base editing in cystic fibrosis airway epithelial cells

A fundamental challenge for cystic fibrosis (CF) gene therapy is ensuring sufficient transduction of airway epithelial cells to achieve therapeutic correction. Gene editing is a method to permanently correct pathogenic variants of the CFTR gene in airway epithelial cells. Our overall goal is to repair CFTR mutations that are unresponsive to modulator therapies, such as R553X and W1282X. Although CRISPR/Cas9 is a powerful gene editing tool, it requires a double stranded break of the genome. Adenine base editors (ABE) offer a safer approach by generating a single stranded break through the nickase activity of a deactivated Cas9 (dCas9)to convert an A>G nucleotide (present in all nonsense and many missense variants). Here we employ new editing tools delivered by a single AAV vector with enhanced tropism for airway epithelia in a G551D CF pig model. Although G551D is responsive to small molecule therapies, establishing gene editing proof of concept in a large animal model would widely advance applications of base editing tools, paving the way for other target mutations.   Recently, we made an exciting discovery that formulating viral vectors with hypertonic saline remarkably enhances gene transfer. Given the safety track record with inhaled hypertonic saline (3-7% NaCl), this is a formulation that could be immediately implemented in a clinical trial design. This simple, but innovative formulation increases ionic strength, enhances stem cell transduction, and improves overall delivery efficacy. Although G551D is a druggable mutation type, using this large animal model to establish proof of concept will advance our knowledge on the efficacy of base editing in vivo. This innovative work combines multiple factors including a base editor delivered by a single AAV vector, a boost in delivery using hypertonic saline, and a large animal model that recapitulates CF lung disease.

Learning Objectives:

1. Explain how adenine base editors (ABE) differ from CRISPR/Cas9 in their mechanism and potential safety advantages for correcting CFTR mutations.

2. Describe how a single AAV vector and hypertonic saline formulation enhance gene transfer and editing efficiency in airway epithelial cells.

3. Discuss the role of the G551D CF pig model in establishing proof of concept for base editing approaches in cystic fibrosis.