| Nearly one third of all human genetic diseases are caused due to nonsense mutations that can result in truncated proteins. Nonsense suppressor tRNAs (NSTs) were proposed as valuable tools for gene therapy of genetic diseases caused by premature termination codons (PTCs). Although various strategies were adapted over the years to increase NST expression and efficacy, low suppression efficacies of NSTs and toxicity associated with stable expression of suppressor tRNAs have hampered development of NST mediated gene therapy. In this study, we have employed the U6 promoter to enhance Gln-Amber suppressor tRNA expression resulting in more effective suppression of PTCs. Since most tRNAs have cell specific differential expression, this technique will enable expression of different kinds of suppressor tRNAs in various cell types at high, functionally relevant levels. In an attempt to study toxic effects of NSTs, stable cell lines that constitutively express U6 promoter enhanced GlnUAG suppressor tRNAs were established. Proteomic analysis of these cells indicated that NST expression does not lead to significant read through of normal cellular proteins.;The other major objective of this study was to design a viral vector for in vivo functional expression of suppressor tRNAs. In that regard, first, we demonstrated that dsAAV2 vectors can carry a GlnUAG tRNA (dsAAV2-U6GlnUAG) and be propagated at high titers. Second, the efficacy of suppression in vitro was demonstrated by the rescue of the expression of a GFP gene inactivated by an amber nonsense mutation in 293 cells by a dsAAV2-U6GlnUAG vector. Finally, our initial in vivo study here restored full length GFP in one SEGFP mouse and suggests that dsAAV2-U6GlnUAG vectors injected into SEGFP mouse may result in modest in vivo functional correction of a mutant GFP gene. In conclusion, the techniques developed here may contribute to the further development of suppressor tRNA mediated gene therapy. |
