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Enes AKYUZ, Pawan Shahi, Bikash R Pattnaik; Anticodon-Engineered transfer RNA as a novel therapy for nonsense mutation. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4944.
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© ARVO (1962-2015); The Authors (2016-present)
About 10-15% of inherited diseases are caused due to nonsense mutations, leading to a truncated protein with severe pathological outcomes. Gene augmentation, genome editing, and read through drugs are some of the leading therapeutic approaches in spite of several limitations. Nonsense mutation (c.158G>A, W53X) in the KCNJ13 gene (Kir7.1 protein) results in an autosomal recessive Leber congenital amaurosis (LCA16). In the present study, we test a novel anticodon-engineered tRNA (ACE-tRNA-Trp that targets the UAG codon) approach to overcome the LCA16 nonsense mutation phenotype.
Human Embryonic Kidney (HEK293-T) cells were co-transfected at a 1:1 ratio with a plasmid carrying ACE-tRNA- 3X-Trp (PMID: 30778053) and pLV-eGFP-W53X-KCNJ13 plasmid using TransIT-LT1 (Mirus Bio). All clones were sequence verified. After 48 hours of transfection, the cells were subjected to live-cell confocal imaging to assess GFP fused Kir7.1 protein localization. The Kir7.1 channel function after the ACE-tRNA treatment was determined using whole-cell patch-clamp electrophysiology. Cells transfected with pLV-eGFP-W53X-KCNJ13 alone served as the experimental control.
We found GFP fluorescence localized to HEK293-T cell membrane domains in several cells transfected with both ACE-tRNA and GFP-W53X. A subpopulation of cells also had a cytoplasmic distribution of GFP fluorescence similar to cells transfected with GFP-W53X alone. The plot of current-voltage showed inward rectifying current in the cells with membrane localization of Kir7.1 but not in the cells with cytoplasmic GFP expression. The inward current measured at -150 mV was -221 ± 63 pA, which increased to 1499 ± 121pA (~ 7 folds) when Rb+ was used as a permeable ion. In comparison, non-ACE-tRNA with cytoplasmic localization of Kir7.1 measured -68 ± 12 pA.
After treatment with ACE-tRNA, both membrane localization and current measurement confirm the presence of a full-length Kir7.1 protein.As ACE-tRNA are codon specific, nonsense mutations harboring other codons will require specific designed ACE-tRNA.Increased efficiency in functional recovery, as well as both in vivo delivery and cell-specific expression, remains to be tested.
This is a 2020 ARVO Annual Meeting abstract.
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