Mechanism-based approach in designing patient-specific combination therapies for nonsense mutation diseases

Premature termination codon (PTC) diseases account for ∼12% of all human disease mutations. Although there are no FDA approved treatments for increasing PTC readthrough, one readthrough inducing drug, ataluren, has conditional approval for treatment of Duchenne muscular dystrophy elsewhere. Ataluren displays low toxicity in clinical trials for treatment of PTC diseases, but its therapeutic effects are inconsistent. The messenger RNA (mRNA) sequence context of a PTC is a major determinant of PTC readthrough efficiency. We have shown that ataluren stimulates readthrough exclusively by competitively inhibiting release factor complex (RFC) catalysis of translation termination. Here, using an in vitro reconstituted system, we demonstrate that PTC identity and the immediately adjacent mRNA sequence contexts modulate RFC activity in terminating peptide elongation. Such modulation largely determines the effectiveness of ataluren in stimulating readthrough, whether added alone or in combination with either the aminoglycoside G418 or an anticodon edited aa-tRNA, which stimulate readthrough by mechanisms orthogonal to that of ataluren. Our results suggest a potential rationale for the variability of ataluren effectiveness in stimulating readthrough. We hypothesize that patients harboring a PTC mutation within a sequence context promoting strong interaction with RFC will be resistant to ataluren, but that ataluren treatment will be more effective for patient sequences conferring weaker interaction with RFC.