Anticodon Edited Transfer RNAs (ACE-tRNAs) Encoded as Therapeutic Nonviral Minimal DNA Vectors

Nonsense mutations, resulting from a premature termination codon (PTC), make up ~11% of all genetic lesions causing disease, affecting millions of people worldwide. Nonsense suppressor anticodon-edited tRNAs (ACE-tRNAs) have emerged as a therapeutic modality for the rescue of PTCs. Delivery of ACE-tRNAs in vivo has been achieved by adeno-associated viral vector and RNA-lipid nanoparticle, however due to drawbacks associated with these approaches, DNA delivery remains an attractive approach. DNA-based approaches afford ease of manufacturing at a relatively low cost and exhibit improved therapeutic durability and safety as compared to viral vector- or RNA-based approaches. Due to the small size of human tRNA genes employed as ACE-tRNAs, in principle, DNA vectors <200 base pairs (bp) in size (minivectors) could be utilized for delivery of actively transcribed ACE-tRNAs. Here we demonstrate that linear DNA ACE-tRNA vectors as small as 200 bp effectively suppress several nonsense mutations in CFTR and REP1, and that ACE-tRNA minivectors display significantly improved bioavailability, reduced innate immune burden, and superior biostability as compared to conventional plasmid DNA vectors.