Genetic diseases can result from nonsense mutations that cause premature translation termination. Nonsense mutations account for ~10–15% of all cases of hemophilia A and B and this population may benefit from small molecule-induced readthrough of nonsense codons. Recent studies document the ability of an orally bioavailable small molecule, PTC124, to facilitate dose-dependent readthrough of nonsense codons in a variety of in vitro and in vivo systems, including reporter gene constructs, mdx mice (a murine model of Duchenne muscular dystrophy;

Nature
447
:
87
–91,
2007
), and in a mouse model of cystic fibrosis (
PNAS
105
:
2064
–2069,
2008
). PTC124 was well-tolerated in healthy human volunteers (
J. Clin. Pharmacol.
47
:
430
–444,
2007
) and in patients with cystic fibrosis (The Lancet, online August 21, 2008). We previously constructed murine models of severe hemophilia B with nonsense mutations in a transgenic human factor IX (hF.IX) gene resulting in nonsense codons at amino acid positions 29 or 338 (R29X and R338X;
Blood
104
:
2767
–2774,
2004
). These mice have no circulating hF.IX detectable by ELISA. To evaluate the effect of small molecules on readthrough in the hemophilia B model, we administered PTC124 to mice carrying the R338X mutation by subcutaneous injection for three days. Measurable plasma levels of hF.IX were detected by ELISA (n=3) and high levels of hF.IX were detected in the liver by immunohistochemistry in the single mouse that was sacrificed. Following oral administration of PTC124 to R338X mice for three days, 20% of mice demonstrated detectable circulating hF.IX levels in the range of 3–5 ng/mL. In a separate experiment, we administered another small molecule, PTC-EMK, by IP injection for three days to R338X mice. With this regimen, 40% of mice showed detectable circulating hF.IX levels. To examine the effect of PTC124 or PTC-EMK on other nonsense mutations, we prepared ten of the most frequently reported nonsense mutations in the hF.IX gene (R29X, R116X, W194X, R248X, R252X, Y266X, W310X, R333X, R338X, and W407X) and transiently transfected these constructs into HEK293 cells. The cells were treated with PTC124 or PTCEMK at various concentrations for 72 hrs and the level of hF.IX protein in the medium was quantified using the ELISA. Of the nonsense containing constructs analyzed, 6/10 mutants showed positive responses with the largest response noted for cells containing the R29X mutation treated with PTC-EMK. In summary, our results demonstrate the ability of PTC124 and PTC-EMK to read through nonsense codons in the hF.IX mRNA and support the potential use of orally bioavailable small molecules as a therapeutic option for patients with hemophilia due to nonsense mutations.

Topics:
hemophilia a, psychological suppression, heart failure, ataluren, molecule, cystic fibrosis, hemophilia b, administration, oral, amino acids, duchenne's muscular dystrophy

Disclosures: Sheedy:PTC Therapeutics Inc.: Employment. Welch:PTC Therapeutics Inc.: Employment. Weetall:PTC Therapeutics Inc.: Employment.

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2008, The American Society of Hematology
2008
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