OC 04.4 - A novel tailored correction approach for recurrent hemophilia-causing nonsense mutations through AntiCodon-Engineered suppressor tRNAs

Background: Suppression of premature termination codons (PTCs) in hemophilia A (HA) and B (HB) would rescue protein expression and ameliorate the severe phenotypes in patients with nonsense mutations. AntiCodon-Engineered tRNAs (ACE-tRNAs) are designed to introduce the original amino acid at PTCs, with potential therapeutic implications.

Aims: To rescue factor VIII (FVIII) and IX (FIX) biosynthesis by suppression of highly-frequent F8 and F9 nonsense mutations with ACE-tRNAs.

Methods: Recombinant expression levels of nonsense variants and luciferase or protein/activity assays after treatment with ACE-tRNAArg, introducing arginine at TGA F9/F8 PTCs.

Results: We selected highly-frequent F9 (n=6) and F8 (n=12) PTCs arising from CGA(arginine)>TGA changes. Cellular models expressing F9 PTC variants unravelled rescue efficiency of ACE-tRNAArg, which increased secreted protein (up to 24-60% of WT-FIX) and activity (13-30%) levels of full-length FIX, as detected in Western blotting.
This prompted us to challenge F8 PTCs. By taking advantage of an optimized recombinant platform based on FVIII-Gaussia luciferase (FVIII-GL) fusion to magnify full-length FVIII detection, we observed increased secreted luciferase values (corresponding to 60-95% of WT FVIII-GL) after ACE-tRNAArg-mediated PTC suppression.
Noticeably, the extent of rescue obtained for FVIII-PTCs in the luciferase-based system, mimicking FVIII secretion, was consistent with that obtained on secreted protein levels for FIX-PTCs in the native context, indicating significantly improved expression over the very low levels of F9/F8 PTC variants alone.
Intrigued by these data, the ACE-tRNAArg efficacy was further investigated on F8 PTCs in the native FVIII-coding context. Preliminary data support ACE-tRNAArg-mediated rescue of secreted FVIII levels (up to 30% of WT-FVIII), the activity of which is under investigation.

Conclusion(s): For the first time we provide experimental proof of tailored suppression of PTCs, thus opening the way for an innovative therapeutic approach in HA e HB caused by nonsense mutations, relatively frequent in severe disease forms.