Boston University Boston, Massachusetts, United States
Introduction: Base editors enable the therapeutic correction of pathogenic point mutations in the genomic DNA of living organisms. While various strategies have been used to deliver base editors in vivo, a method that delivers base editor ribonucleoproteins (RNPs) into tissues in animals would offer important safety advantages over existing approaches that deliver DNA or mRNA. We report the extensive engineering and application of a DNA-free platform that mediates efficient delivery of base editor RNPs in vivo. Our platform enables therapeutic levels of on-target base editing across multiple tissues that are comparable to levels achieved with current state-of-the-art viral and non-viral DNA or mRNA delivery methods, but with virtually undetectable off-target editing and no possibility of unwanted DNA integration. The efficiency and high target site specificity of this in vivo delivery platform supports therapeutic rescue of disease phenotypes in multiple mouse models. We anticipate that this platform will be broadly useful for transient delivery of base editor RNPs in vivo with therapeutically relevant efficiencies and minimal off-target activity.
Materials and
Methods: We hypothesized that retroviruses could serve as effective scaffolds for base editor virus-like particles (BE-VLPs). Initially, we designed the v1BE-VLPs by fusing ABE8e, a highly active adenine base editor, to C-terminus of the Friend murine leukemia virus (FMLV) gag polyprotein via a linker peptide cleavable by the FMLV protease upon particle maturation. The v1 BE-VLPs achieved 73% editing efficiency at the BCL11A enhancer locus at high doses, but editing efficiency dropped steeply with decreasing doses, indicating room for improvement. To enhance BE-VLP efficiency, we systematically engineered each component of the VLPs. First, we hypothesized that cleavage of the gag–ABE8e linker by the FMLV protease post-maturation limit BE-VLP efficiency. We constructed a series of v2BE-eVLPs with various protease-cleavable linker between the FMLV gag and ABE8e and we identified several that improved editing efficiencies by 1.2–1.5-fold compared to v1BE-VLPs. Next, we speculated that nuclear localization sequences (NLS) within the gag–BE fusion could hinder gag–BE localization to the outer membrane, impeding BE incorporation into VLPs. To promote cytosolic gag–cargo localization in producer cells, we designed v3eVLPs incorporating nuclear export signals (NES) alongside NLS. This modification enhanced cytoplasmic localization during eVLP production, improving editing efficiency 4-fold. Finally, we optimized the stoichiometry between the cargo and structural proteins, resulting in a v4BE-eVLP. These v4BE-eVLPs exhibited spherical morphology with a diameter of approximately 100-150 nm, confirmed by TEM. We also validated the delivery efficiency of these BE-eVLPs across various primary cell types and in vivo in mouse models targeting the brain, eye, and liver.
Results, Conclusions, and Discussions: Methods to deliver gene editing agents in vivo as ribonucleoproteins could offer safety advantages over nucleic acid delivery approaches. We report the engineering and application of eVLPs, engineered DNA-free virus-like particles that efficiently package and deliver base editor or Cas9 ribonucleoproteins. By engineering VLPs to overcome cargo packaging, release, and localization bottlenecks, we developed v4eVLPs that mediate efficient base editing in several primary mouse and human cell types. Our work showed over 50%-90% editing in therapeutically relevant primary cells. We also showed ability to program targeting with eVLPs by using different glycoproteins. Importantly, a single injections of eVLPs into mice support therapeutic levels of base editing, reducing serum Pcsk9 levels 78% following 63% liver editing. We also showed that eVLPs can partially restore visual function in a mouse model of genetic blindness. Notably, eVLPs is one of the first non-viral delivery systems that shows high levels of editing in the neo-cortex after local delivery. Both in vitro and in vivo off-target editing from eVLPs was virtually undetected, an improvement over AAV or plasmid delivery. These results establish eVLPs as promising vehicles for therapeutic macromolecule delivery that combines advantages of both viral and non-viral delivery for precision genome editing technologies.
