Identification of 2,6-Diaminopurine As a Candidate for Hemophilia a Ribosomal Readthrough Therapy

Nonsense mutations introduce premature termination codons (PTCs) and are responsible for 11% hereditary genetic disorders including hemophilias. Drug-induced ribosomal readthrough over PTCs is an emerging strategy to treat the genetic disorders caused by nonsense mutations. Ribosomal readthrough therapy aims to allow translation beyond the PTC, creating a full-length protein and possible phenotypic rescue. Ataluren (PTC124), which induces readthrough by binding to the ribosomal 60S subunit, was recently granted conditional approval in the European Union for the treatment of Duchenne muscular dystrophy caused by nonsense mutations. Most nonsense mutations in FVIII and FIX lead to severe diseases. In theory, hemophilias are ideally suited for drug-induced readthrough therapy, because even a small increase in FVIII or FIX activity to over 1% could significantly improve bleeding symptoms in patients with nonsense mutations. Thus, ribosomal readthrough therapy has the potential to provide a small-molecule, personalized treatments based on patient's mutation profile. We previously investigated different readthrough drugs on different FVIII nonsense mutations and found that geneticin, but not gentamicin and Ataluren, is an effective readthrough agent for a number of such mutations. Recently, 2,6-diaminopurine (DAP), a natural compound found in extracts of the edible mushroom Lepista inversa, has shown high-efficiency correction of UGA nonsense mutations and low toxicity. DAP is thought to act as an inhibitor of a tRNA-specific 2'-O-methyltransferase and promotes the interaction of the near-cognate tRNATrp with the UGA stop codon. We introduced 22 UGA mutations, including the most common recurring mutations from hemophilia A (HA) patients into the B domain deleted FVIII-Gassia luciferase fusion (BDD-Gluc) protein. Cells stably expressing these mutant fusion proteins were treated with DAP and geneticin. DAP induced higher secreted luciferase activity, FVIII antigen and cofactor activity levels in cell culture media than geneticin treatments. A majority of DAP-treated mutants produced FVIII co-factor activities and antigens that are over 2% of wild-type level, with several mutants reaching 10-15% levels. Using an immunoprecipitation-immunoblotting assay, we detected full-length BDD-Gluc fusion proteins in culture media and cell lysates of both DAP- and geneticin-treated cells expressing nonsense mutants. Two major factors affect the outcome of readthrough therapies are the readthrough efficiency of drugs and the nature of replacement of amino acid (AA) residues. Studies had shown that a cytosine at +4 position is a favorable nucleotide sequence context. Readthrough efficiency of F8 mRNA is influenced, but not determined by nucleotide sequence contexts of the PTC. It was reported that only tryptophan was replaced with DAP-induced readthrough while three potential AAs can be replaced with geneticin treatment. We systematically substituted PTCs with potential replacement AAs and found that activities of readthrough products are determined by the compatibility of replacement AA residues with FVIII secretion and activity. Our results suggest that DAP, with its high readthrough efficiency and low toxicity, is a promising drug candidate for HA patients carrying susceptible UGA mutations. This cell-based study identifies candidate patient mutations and provides a rationale for a future clinical study of DAP or one of its derivatives as a therapeutic for these patients.