Person:

Levy, Jonathan

Base editors are widely used engineered proteins that introduce targeted point mutations into DNA without creating double-stranded DNA breaks. The application of base editors to study and treat genetic diseases is dependent on their in vivo delivery into relevant cell types. Delivery by adeno-associated virus (AAV), a clinically validated delivery method, poses special challenges because the size of base editors exceeds the AAV packaging limit. Here we describe the development and application of in vivo delivery methods for both cytosine base editors (CBEs) and adenine base editors (ABEs). Dual AAVs each provide one half of the editor and trans-splicing inteins reconstitute full base editor activity, circumventing the AAV packaging limit. We optimized each AAV component to greatly improve editing efficiency. The resulting AAVs enable in vivo base editing for the first time in mouse brain, retina, and heart, as well as the most efficient base editing to date in muscle and liver, with therapeutically relevant efficiencies at viral dosages known to be tolerated in humans. A single intravenous injection of split CBE in PHP.eB AAV resulted in editing of up to 59% of unsorted mouse cortical tissue. Intravenous injection of split CBE or ABE in AAV9 mediated up to 38%, 20%, and 9% base editing in unsorted mouse liver, heart, and skeletal muscle, respectively. Subretinal and intracerebroventricular injections of split CBE and ABE in Anc80, PHP.B/PHP.eB, and AAV9 viruses mediated mouse retina and brain editing in up to 38% and 50% of unsorted cells, respectively. We applied this system to directly correct in mouse brain tissue a mutation that causes the neurodegenerative ataxia Niemann-Pick disease type C (NPC), slowing neurodegeneration and increasing lifespan consistent with expectations based on mosaic NPC mice. These findings establish a broadly useful AAV platform for the efficient introduction of targeted point mutations into multiple tissues of therapeutic interest for which in vivo base editing has not been previously reported.

Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BE–PACE) to improve their editing efficiency and target sequence compatibility. We used BE–PACE to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.