In Vivo Delivery and Therapeutic Applications of Base Editors

Abstract

Base editors (BEs) mediate targeted point mutations in living cells and have the potential to correct many mutations that cause genetic diseases. Robust and therapeutically relevant in vivo delivery methods are needed for BEs to realize their potential as therapeutics. Adeno-associated viruses (AAVs) can deliver proteins with high efficiency and in a cell-type specific manner. However, AAVs have a packaging capacity size limit that is too small to accommodate full-length BEs. To address this challenge, we developed a dual AAV strategy in which a BE is divided into two halves, each fused to a trans-splicing intein, and packaged into two separate AAVs. Following co-infection, the BE is reconstituted into its full-length protein by trans-splicing of the protein halves. This dual-AAV delivery method supports efficient base editing (up to 59%) in an AAV-serotype specific manner, across various tissue types (brain, liver, retina, heart and skeletal muscle) in the mouse. Next, we explored whether we could apply base editing in non-dividing cells in vivo since most genetic diseases occur in cells that do not proliferate after birth. We sought to correct a recessive, loss-of-function point mutation in transmembrane channel-like 1 (Tmc1) that causes deafness in the Baringo mouse model. We screened base editors with different deaminases and found that activation-induced cytidine deaminase (AID) was the most efficient in targeting the Baringo mutation. We then packaged and injected AID-BE using the dual-AAV strategy into the inner ears of Baringo mice postnatally. Injected mice showed up to 51% reversion of the Tmc1 point mutation compared to wild type sequence in Tmc1 transcripts. Repair of Tmc1 in vivo restored inner hair-cell sensory transduction, hair-cell morphology, and partial low-frequency auditory function at four weeks post-injection. These studies provide a foundation for a potential one-time treatment of certain genetic diseases and further advances the application of base editing in vivo to correct pathogenic point mutations.