Cox, G. A. | bioRxiv Science

bioRxiv2025

Efficient Cas9 nuclease-based editing in skeletal muscle via lipid nanoparticle delivery

Gene editing holds great promise for muscular dystrophy treatment, but the rapid evaluation of different editing modalities in skeletal muscle in vivo remains challenging due to lack of simple, effective delivery tools. Here we demonstrate that selective organ targeting (SORT) lipid nanoparticles (LNP) encapsulating optimized Cas9 cargo can facilitate efficient, local delivery to skeletal muscles achieving editing rates [≥]35% and restore protein expression for a proof-of-concept muscular dystrophy target. Interestingly, efficient editing in skeletal muscle was observed despite a strong adaptive immune response to repeat dosing of the Cas9 LNPs. High efficiency editing mediated by LNP-based delivery of Cas9 to skeletal muscle permitted detailed analysis of insertion and deletion (InDel) outcomes in vivo for a set of potential therapeutic target sites, which differed substantially from InDel outcomes observed in proliferating cells in one specific instance. Overall, our findings on enhanced LNP delivery of Cas9, platform-specific immune responses, and differential editing patterns observed between in vitro and in vivo models provide valuable insights that should inform the development of gene editing therapeutics for neuromuscular diseases. One Sentence SummarySORT LNPs permitted efficient Cas9-mediated repair of a pathogenic allele in skeletal muscle in a mouse model of LGMDR7.

molecular biology↗

bioRxiv2024

Poly-alanine-tailing is a modifier of neurodegeneration caused by Listerin mutation

The surveillance of translation is critical for the fitness of organisms from bacteria to humans. Ribosome-associated Quality Control (RQC) is a surveillance mechanism that promotes the elimination of truncated polypeptides, byproducts of ribosome stalling during translation. In canonical mammalian RQC, NEMF binds to the large ribosomal subunit and recruits the E3 ubiquitin ligase Listerin, which marks the nascent-chains for proteasomal degradation. NEMF additionally extends the nascent-chains C-terminus with poly-alanine ( Ala-tail), exposing lysines in the ribosomal exit tunnel for ubiquitination. In an alternative, Listerin-independent RQC pathway, released nascent-chains are targeted by Ala-tail-binding E3 ligases. While mutations in Listerin or in NEMF selectively elicit neurodegeneration in mice and humans, the physiological significance of Ala-tailing and its role in disease have remained unknown. Here, we report the analysis of mice in which NEMFs Ala-tailing activity was selectively impaired. Whereas the Nemf homozygous mutation did not affect lifespan and only led to mild motor defects, genetic interaction analyses uncovered its synthetic lethal phenotype when combined with the lister neurodegeneration-causing mutation. Conversely, the lister phenotype was markedly improved when Ala-tailing capacity was partially reduced by a heterozygous Nemf mutation. Providing a plausible mechanism for this striking switch from early neuroprotection to subsequent neurotoxicity, we found that RQC substrates that evade degradation form amyloid-like aggregates in an Ala-tail dependent fashion. These findings uncover a critical role for Ala-tailing in mammalian proteostasis, and deepen our molecular understanding of pathophysiological roles of RQC in neurodegeneration.

biochemistry↗