Here we show that supplementation with CC can improve FVIII secretion both in cell culture and in vivo
. In initial screening of CC candidates, the substance betaine showed the highest potency, in that it doubled the amount of secreted FVIII. Secreted FVIII was fully functional, which indicates no major limitation in other posttranslational processes required by the secreted protein. Other substances known to improve protein secretion either had no effect, or only a very mild effect, on secretion of FVIII. One possible explanation for this finding is the significant cellular toxicity observed with CC concentrations needed to enhance FVIII trafficking for these substances. On the other hand, experiments exploiting other CCs are derived from secretion studies of mis-folded mutant proteins. Here, we tested a system in which FVIII is already trapped in the ER as a result of an overload of the cell’s folding capacity, in turn due to high levels of recombinant protein over-expression. Betaine therefore appears to be an attractive option to overcome current limitations in pharmaceutical FVIII-production. In fact, a previous report described that betaine preserves cell viability at hyperosmolar conditions and therefore adds to increased productivity of thrombopoietin in CHO cells under these conditions 
. In addition, we observed that ectoine, thapsigargin and curcumin were able to further increase FVIII secretion when applied in combination with betaine. While ectoine is an osmolyte similar to betaine, the ER-ATPase inhibitors thapsigargin and curcumin reduce calcium levels in ER, which in turn alters interactions with calcium-dependent chaperones such calnexin and calreticulin 
, or affects the structure of MCFD2. Both of these scenarios might therefore produce additive effects.
In previous studies we observed that BDD FVIII concentrations in the ER are higher, and FVIII transport is more likely to occur by bulk flow, compared to FVIII FL 
. By contrast, the transport of the FL protein is primarily receptor mediated using LMAN1 and MCFD2 
. Different optimal betaine concentrations in FVIII-FL and –BDD expressing cell lines might therefore result in differences in protein trafficking. Supportingly as the administration of thapsigargin only improved FVIII-FL secretion, it seems to be more susceptive for alteration of the calcium level in the ER than FVIII-BDD. The B-domain contains most of the N-glycans which had to pass the quality control by the calnexin/calreticulin cycle. Both endoplasmic lectins are also known for prolonged binding of apparently misfolded glycoproteins. Possibly thapsigargin could influence these calcium-sensitive chaperone interactions with the B-domain and release FVIII-FL. An alternative hypothesis to consider could be differential betaine sensitivity of cell clones.
Betaine is proposed to stabilize proteins according to the “preferential exclusion“ model 
. The model hypothesizes that compatible solutes are excluded from the protein hydrate-sheath. The osmolytes are repelled form the peptide backbone due to osmophobic interactions, whereby water molecules are pushed to the protein surface, resulting in the protein adopting a more compact conformation 
. This process reduces inter-protein interactions by surface-exposed hydrophobic patches, leading to aggregate formation and could probably alter chaperone accessibility to proteins. In our study, betaine increases the proportion of correctly folded and processed FVIII in cell lysates. The total amount of detergent-soluble intracellular FVIII was higher, whereas the amount of FVIII in the insoluble fraction decreased. The improved solubility possibly points to an augmentation of transport-competent FVIII in the ER and a reduction of protein aggregation. Western blot analysis revealed that betaine also improved the maturity of FVIII, as more single-chain was cut into light and heavy chains, a process which requires transport from the ER to the Golgi. Likewise Santana et al. 
observed that betaine showed best results amongst various other CC in reversing the solubility and restoring the enzymatic activity of the mutant alanin:glyoxylate-aminotransferase protein (AGXT*LTM). Misfolded AGXT*LTM tends to build inactive aggregates in primary hyperoxaluria type 1 disease. The enzyme deficiency results in toxic oxalate accumulation in the kidney of homozygous patients. The authors could demonstrate that betaine induced an increase of soluble AGXT*LTM in COS-7 cell lysates accompanied by a reduction of insoluble protein. However the total protein amount remained unaffected. A similar effect was also shown for another methylamine, TMAO, in the correction of myocilin-causing glaucoma 
. In transfected cells TMAO improved the secretion of the aggregation-prone D384N mutant through increased solubility. Additionally, TMAO induced redistribution of the mutant protein from an ER-enriched to Golgi-enriched fraction in a density gradient system.
Our immunofluorescence staining suggested that the major portion of the translated FVIII protein is retained in the ER and does not reach the Golgi apparatus. These results are in accordance with our previous work in COS-7 cell lines 
. If FVIII passes ER quality control, it is packaged into COPII-coated carriers, mediated by LMAN1/MCFD2-receptors 
, prior to these carriers budding from the ER. These vesicles are transported to the ERGIC and further on to the Golgi apparatus 
. We observed in this study that betaine treatment partially changes the distribution of FVIII throughout the cell towards an increased perinuclear staining with punctate FVIII structures co-localizing with the cis-Golgi marker GM130. GM130 itself is known to shuttle between the ERGIC and Golgi 
, therefore one possibility is that the punctate structures of co-localization could be FVIII-vesicles in transit between the ERGIC and cis-Golgi. Live imaging analysis supported these findings, as betaine treatment led to a reduction in the number of FVIII-positive stationary elements, presumably representing accumulations of FVIII in ER exit sites or ER-retained FVIII aggregates, with the concomitant increase in active transport of FVIII.
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 (sugar beets, wheat germ, shrimps, spinach, etc.) 
, 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 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 (ClinicalTrials.gov Identifier NCT00283387). 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% (132 out of 170 with documented antigen levels) of missense mutations listed in the hemophilia A database (Dr. Geoffrey Kemball-Cook, The Haemophilia A Mutation, Structure, Test and Resource Site, http://hadb.org.uk
, accessed August 18, 2011) 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 two representative mutations, one located in the A1 domain (Q305P) and the other in the C2 domain (W2313A) of FVIII. Q305P lies within the binding site for immunoglobulin-binding-protein BiP (T291-F309) and disrupts the interaction with this chaperone 
. hFVIII-BDDW2313A is rapidly degraded intracellularly but has 90–100% of the specific activity of wild-type FVIII-BDD 
. For both mutations, betaine supplementation increased secretion of functional protein in cell culture. Q305P was further evaluated in FVIII deficient mice. For these experiments, human mutant coagulation factors were expressed in FVIII deficient mice using non-viral gene transfer. Addition of betaine to the drinking water resulted in higher FVIIIQ305P levels, but when betaine treatment was discontinued FVIII activity returned to baseline.
Similarly, betaine treatment also increased FVIII levels in mice expressing the human FVIII-BDD without missense mutation. In contrast, murine FVIII levels remained unchanged. We hypothesized that endogenous murine coagulation factors would be less affected by betaine treatment. Physiological expression might be more closely regulated and impaired folding as a result of ER capacity overload seems unlikely compared to hFVIII over-expression in limited numbers of liver cells in the gene transfer setting. To explore this difference, we used a similar gene transfer approach with the human coagulation FIX for non-viral gene transfer. Betaine increased recombinant FIX levels but did not influence endogenous murine FVIII. Although FIX is less affected by limitations of the ER to Golgi trafficking system, heterologous overexpression could also lead to impaired folding and processing of FIX in the ER.
Similar to FVIII, FIX mutations with impaired intracellular protein processing have also been described 
. The same is true for mutations affecting von Willebrand factor 
and combined FV/FVIII deficiencies, where an LMAN1/MCFD2 defect causes FVIII retention in the ER 
. All of these might therefore be targets for CC-improved protein secretion.