ined treatment. Post CC incubation HepG2 hFVIII-BDDQ305P cells were successively lysed in PBS/0.5% Triton X-100 and PBS/1% SDS. hFVIII antigen was determined in both fractions by indirect ELISA. hFVIII-BDDQ305P injected Hem A mice were treated with 2% betaine ad libitum per os in a crossover-study of two groups. After 3 days of treatment hFVIII antigen and activity was measured and treatment was switched between mouse groups. 3 days later, plasma levels were tested again. show hFVIII antigen levels, the related hFVIII activity levels in plasma of group I or II. represent the calculated overall effect of betaine on FVIII antigen levels or FVIII activity. square symbols indicate samples of the first measuring point, triangles the second one; clear: tap water-administration, filled: 2% betaine administration. Endogenous murine FIX levels in all injected FVIII knockout mice and murine FVIII levels of all used FIX knockout mice with and without betaine in the drinking water. Normal mouse levels were set to 100%. Values are presented as means 6 SEM. ANOVA; Student’s t-test; Wilcoxon signed rank test;P,.05, P,.005, P,.0005. 
doi:10.1371/journal.pone.0044505.g007 Following these promising results in cell culture we wanted to explore whether betaine treatment could also increase protein secretion in vivo. Betaine is a natural methyl group donor in homocysteine- and methionine-metabolism. The physiological source of betaine is food , or it is generated by oxidizing choline. It has approval as a dietary supplement in the USA and is commonly used for commercial feeding. Furthermore, betaine is easily administered by oral application and is already routinely used in patients suffering from hyperhomocysteinemia. Since 2001 betaine has been classified as orphan drug for treatment of homocystinuria by the EMA. Additional to its Neuromedin N web function as a methyl donor, betaine is also being evaluated for its function as a CC in a phase II study in patients with primary hyperoxaluria type 1. The disease is caused by a point mutation, which leads to a secretion defect of the liver enzyme alanine-glyoxylate aminotransferase. Betaine has also been tested in pre-clinical settings in cystic fibrosis. Several mutations leading to a trafficking defect are also known for FVIII. Although these described mutations are rare, 78% of missense mutations listed in the hemophilia A database have reduced antigen levels of below 50%, possibly as a consequence of impaired protein secretion. For this reason, a treatment strategy to improve protein trafficking could potentially benefit a wider patient population. Whether the improved processing and secretion of mutant FVIII protein might increase the risk for inhibitor formation has not been investigated at this point. In patients with missense mutations protein secretion is impaired due to intracellular retention of the mutant FVIII, but there is still residual protein in circulation. Therefore CC-rescued protein would not be foreign for the immune system. In theory, neo-antigens could occur due to release of protein that otherwise would never have reached the circulation. Although we cannot provide data addressing this concern, we believe that this risk is negligible since the effect of CC is relatively mild and aims on supporting correct protein folding rather than release of not correctly processed FVIII. Here, over-expression in single cells as observed in gene therapy might carry an even higher risk. In this study we investigated tw