Engineered mRNA-packaging virus-like particles enable efficient and precise mitochondrial base editing in vitro and in vivo

Jang, H.; Rodrigues Menezes, C.; Palczewski, K.; Raguram, A.

doi:10.64898/2026.10.04.756548

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bioRxiv · 10.64898/2026.10.04.756548

Engineered mRNA-packaging virus-like particles enable efficient and precise mitochondrial base editing in vitro and in vivo

Jang, H.·Rodrigues Menezes, C.·Palczewski, K.·Raguram, A.

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Abstract

Mitochondrial DNA (mtDNA) base editors (mtBEs) are promising tools for studying and correcting pathogenic mtDNA mutations. However, the application of mtBEs is often hindered by substantial mtDNA-wide off-target editing that accumulates with prolonged mtBE expression resulting from plasmid DNA or viral delivery. To overcome this limitation, we developed mtBE-VLPs, a virus-like-particle (VLP) platform for transiently delivering mtBEs in vitro and in vivo with minimal off-target effects. While canonical protein-packaging VLPs yielded undetectable mtDNA base editing, engineered mRNA-packaging VLPs enabled efficient but transient mtBE cargo delivery. By iteratively engineering mRNA-packaging VLP compositions, we improved mtBE-VLP-mediated base editing efficiencies from less than 1% to greater than 50% across multiple target mtDNA loci in bulk populations of treated cultured cells. To further improve the packaging and delivery of certain mtBEs, we developed a strategy to selectively reduce mtBE cargo expression in VLP producer cells but not transduced cells, which increased VLP-mediated base editing efficiencies for certain mtBEs by 7.1-fold. Compared to plasmid DNA delivery in vitro, mtBE-VLPs exhibited equivalent on-target editing but 23- to 41-fold lower mtDNA-wide off-target editing, highlighting the improved editing specificity offered by transient mtBE delivery. mtBE-VLPs efficiently installed a pathogenic mtDNA mutation in mouse embryonic fibroblasts (88% efficiency) and corrected a pathogenic mtDNA mutation in a human patient-derived cell line (41% efficiency). Additionally, a single subretinal injection of mtBE-VLPs yielded 6-20% (average 12%) mtDNA base editing in the mouse retinal pigment epithelium with minimal mtDNA-wide off-target editing. These findings establish mRNA-packaging mtBE-VLPs as promising vehicles for efficient and precise mitochondrial base editing via transient mtBE delivery.

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Jang, H., Rodrigues Menezes, C., Palczewski, K., Raguram, A.. 2026-10-05. Engineered mRNA-packaging virus-like particles enable efficient and precise mitochondrial base editing in vitro and in vivo. https://doi.org/10.64898/2026.10.04.756548

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