Development and Application of Adenine Base Editors for Precision Gene Therapy

Abstract

CRISPR-Cas technology has revolutionized functional genomics, and gene therapy by enabling precise and programmable manipulation of the genome. Early CRISPR-Cas nucleases relied on the generation of double-strand breaks (DSBs) to disrupt genes or introduce exogenous DNA. More recently, the development of base editors and prime editors has expanded the genome editing toolbox, enabling precise single-nucleotide substitutions as well as targeted insertions and deletions without requiring DSBs. By avoiding DSB-associated toxicity and unpredictable repair outcomes, these next-generation editors have substantially improved the precision and safety of therapeutic genome editors. This dissertation focuses on the therapeutic application and engineering of adenine base editors (ABEs). We first demonstrated that a compact ABE based on an evolved Neisseria meningitidis Cas9 could be packaged into a single adeno-associated virus (AAV) and achieve therapeutic genome editing in two mouse models of alpha-1 antitrypsin disease (AATD). Treatment resulted in therapeutically relevant levels of editing and significant improvements in both liver and lung pathology. We next sought to further optimize ABEs through rational engineering of the terminal regions of the TadA deaminase. This work generated a panel of base editors with altered editing windows and efficiencies, thereby expanding the repertoire of available genome editing tools. Collectively, this work advances both the therapeutic application and engineering of ABEs by establishing a compact platform for in vivo genome editing and identifying protein terminal engineering as a modulator of base editor activity.