Advanced Delivery Platforms for Base Editing In Vivo

Project Summary The Cas9 platform has enabled genome editing, base editing (BE), and prime editing to induce gene knockouts as well as tailor-made sequence alterations. CRISPR-Cas9 systems have the potential to similarly revolutionize clinical practice through precise editing of disease loci. We have developed Nme2Cas9 as an editing platform with (1) compact size, facilitating single adeno-associated virus (AAV) delivery; (2) a dinucleotide (N4CC) protospacer-adjacent motif (PAM) that affords high target site density; and (3) exceptional accuracy. More recently, we developed Nme2Cas9 adenine base editor (ABE) systems as among the first to be validated in vivo for single-AAV delivery. AAV is a potent editing delivery modality in vivo, especially in extrahepatic tissues such as the central nervous system (CNS). Nonetheless, the therapeutic promise of base editing systems will hinge upon improving editing efficiency, limiting bystander edits (or their consequences), maximizing PAM-dependent targeting scope, and minimizing immunogenicity, toxicity, and prolonged deaminase expression (which can compromise editing efficiency and lead to safety risks such as hepatotoxicity and the accumulation of unwanted edits). Here we propose to capitalize on our establishment of single-vector Nme2-ABE systems to develop novel base editing capabilities with increased effectiveness, targeting scope, utility, and safety, and to validate these systems in the treatment of CNS disease models in mice. The goals of this proposal are (1) to develop next-generation, single-AAV, deaminase-inlaid Nme2-ABEs and guides with increased efficiency, a single-nucleotide PAM, and greater control over bystander editing; (2) to use next- generation, single-AAV Nme2-ABE systems for therapeutic editing of disease genes in the CNS of mouse models of amyotrophic lateral sclerosis and Batten disease; and (3) to develop systems that use repression by endogenous microRNAs and drug-dependent splicing systems to enhance the safety of AAV-delivered Nme2- ABE in vivo. These safety enhancements will reduce anti-Nme2-ABE immune responses, ameliorate potential toxic effects on the liver and on specific CNS cell types, and limit the off-target mutagenesis that can arise from sustained expression of the editing machinery. Successful completion of these aims will provide invaluable enhancements to the delivery, efficacy, and specificity of in vivo genome editing.