Selection-fr ee pr ecise gene r epair using high-capacity adeno v ect or deliv ery of adv anced prime editing systems rescues dystrophin synthesis in DMD muscle cells

Abstract

Prime editors have high potential for disease modelling and regenerative medicine efforts including those directed at the muscle-wasting disorder Duchenne muscular dy stroph y (DMD). Ho w e v er, the large siz e and multicomponent nature of prime editing systems pose substantial production and deliv ery issues. Here, w e report that packaging optimized full-length prime editing constructs in adeno v ector particles (A dVPs) permits installing precise DMD edits in human m y ogenic cells, namely, m y oblasts and mesench ymal stem cells (up to 80% and 64%, respectiv ely). A dVP transductions identified optimiz ed prime-editing reagents capable of correcting DMD reading frames of ∼14% of patient genotypes and restoring dystrophin synthesis and dystrophin- β-dystroglycan linkages in unselected DMD muscle cell populations. AdVPs were equally suitable f or correcting DMD iPSC-deriv ed cardiom y ocytes and deliv ering dual prime editors tailored for DMD repair through targeted e x on 51 deletion. Moreo v er, b y e xploiting the cell cy cle-independent A dVP transduction process, w e report that 2- and 3-component prime-editing modalities are both most active in cycling than in post-mitotic cells. Finally, we establish that combining AdVP transduction with seamless prime editing allo ws f or stac king c hromosomal edits through successiv e deliv ery rounds. In conclusion, A dVPs permit v ersatile in v estigation of adv anced prime editing systems independently of their size and component numbers, which should facilitate their screening and application.