(595) ACE-tRNA and small molecule combinatorial treatment of CF nonsense mutations

Background: Cystic fibrosis (CF) is an inherited disorder, of which ~10% result from single nucleotide changes that encode premature termination codons (PTCs), classified as type 1 mutations. The presence of the PTC results in the activation of the nonsense-mediated mRNA decay (NMD) pathway that significantly reduced steady-state transcript expression and production of truncated, nonfunctional protein. Current FDA-approved therapies for CF target the CF transmembrane conductance regulator (CFTR) protein and modulate its trafficking and activity. Because there is very little CFTR protein to act on, the current small molecule therapies do not work for type 1 mutations. There are currently several therapeutic methods being developed to edit the genetic misstep within the genome, inhibit NMD, replace the CFTR gene and readthrough or suppress PTCs. Our lab is focused on a gene therapy approach that utilizes anticodon engineered transfer RNA (ACE-tRNA) to suppress PTCs, inhibit NMD by promoting the completion of the pioneer round of translation and results in the expression of full-length, wild-type CFTR protein. Our preliminary data indicates that ACE-tRNAs are very efficient at PTC suppression, however our current efforts are focused on determining if ACE-tRNA-dependent rescue of CFTR protein can be augmented by already FDA-approved drugs that act on the expressed CFTR protein.

Methods: Immortalized human bronchial epithelial cells (16HBE14o-) and gene-edited 16HBE14o- cells with CFTR nonsense mutations (16HBEge) were transduced with adenovirus carrying ACE-tRNAs. The ability of ACE-tRNAs to rescue CFTR transcript and protein expression was accessed with quantitative reverse transcription PCR and Western blot, respectively. Functional CFTR channel level after delivery of ACE-tRNAs was determined using Ussing chamber recordings. In addition, cells transduced with ACE-tRNAs were treated with NMD inhibitor and CFTR modulators to understand the possibility of enhancement in ACE-tRNA-mediated CFTR rescue.

Results: Delivery of ACE-tRNAs to 16HBEge cells with common CF nonsense mutations significantly increased not only CFTR transcript and protein levels, but also functional CFTR channel level at the cell surface. Further, combination treatment of ACE-tRNAs with NMD inhibitor and CFTR modulator drugs had synergistic effects on the CFTR level.

Conclusions: These findings suggest that ACE-tRNAs, especially in combination with other pharmacological agents that enhance the overall transcript pool, protein stability and channel activity, may be a potential therapeutic option for patients with CF-causing nonsense mutations.

Acknowledgements: This work was supported by a Cystic Fibrosis Foundation Research Grant (LUECK20GO) and NHLBI 1 R01 HL153988-01A1 to J.D.L.

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