A streamlined base editor reengineering platform to enhance gene editing specificity and selectivity

About This Grant

PROJECT SUMMARY Base editing (BE) can permanently correct over half of known human pathogenic genetic variants without requiring a repair template, thus serving as a promising therapeutic tool to treat a broad spectrum of genetic diseases. However, the broad activity windows and substantial off-target activities of current base editors pose a major challenge to their therapeutic application. Current methods to improve base editing specificity rely on large-scale saturation mutagenesis of the key residues involved in substrate interactions, which can be labor- intensive and time-consuming. The main goal of this project is to establish a streamlined base editor engineering platform to accelerate the development of therapeutic base editing. Recently, we developed a base editor re- engineering strategy by integrating a naturally occurring oligonucleotide binding module into the deaminase active center to enhance its editing specificity. Preliminary editing studies demonstrate that this engineering platform yielded a suite of base editors (iBEs) with refined activity windows and decreased off-target activities, enabling versatile yet precise single-nucleotide conversions. Encouragingly, iABE outperforms all the existing ABEs in correcting one of the most common CFTR nonsense mutations. Building upon this work, we will further advance this base editor re-engineering platform to develop base editors with minimized off-target activities and programmable editing selectivity, and assess its broad application in directing the evolution of alternative deaminases with enhanced specificity (Aim 1). Further, we will validate the editing efficiency, specificity, and outcomes of iBEs in correcting diverse CFTR nonsense mutations in cystic fibrosis cell and animal models (Aim 2). Successful completion of this project will provide a roadmap for designing and engineering therapeutic base editors for correcting disease-causing point mutations and establish a lung-targeted gene correction toolkit tailored for CF, laying the groundwork for future CF gene therapy development.