Ribozyme-activated mRNA trans-ligation enables large gene delivery to treat muscular dystrophies

Science. 2024 Nov 15;386(6723):762-767. doi: 10.1126/science.adp8179. Epub 2024 Nov 14.

Abstract

Ribozymes are small catalytic RNA sequences capable of nucleotide-specific self-cleavage found widespread in nature. Ribozyme cleavage generates distinct 2',3'-phosphate and 5'-hydroxyl termini that resemble substrates for recently characterized RNA repair pathways in cells. We report that ribozyme cleavage of two separate mRNAs activated their scarless trans-ligation and translation into full-length protein in eukaryotic cells, a process that we named StitchR (for Stitch RNA). Optimization of StitchR activity in mammalian cells resulted in a ~900-fold increase in protein expression that approached levels observed for genes expressed from single vectors. We demonstrate that StitchR can be harnessed for effective dual adeno-associated virus gene therapies to correct muscular dystrophies by restoring large functional muscle proteins to endogenous levels in vivo.

MeSH terms

  • Animals
  • Dependovirus
  • Dysferlin* / genetics
  • Female
  • Gene Transfer Techniques
  • Genetic Therapy* / methods
  • Genetic Vectors
  • HEK293 Cells
  • Humans
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Muscular Dystrophies* / genetics
  • Muscular Dystrophies* / therapy
  • Protein Biosynthesis
  • RNA Cleavage
  • RNA, Catalytic* / genetics
  • RNA, Catalytic* / metabolism
  • RNA, Messenger* / genetics
  • RNA, Messenger* / metabolism

Substances

  • DYSF protein, human
  • Dysferlin
  • RNA, Catalytic
  • RNA, Messenger