Chemical Chaperones Improve Protein Secretion and Rescue Mutant Factor VIII in Mice with Hemophilia A

Abstract

Inefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking.

Gene therapy for hemophilia

“…Incorporation of a point mutation (R1645H) from canine F.VIII into hF.VIII conferred many of these properties to the new transgene, and expression of this altered F.VIII via an AAV vector was more effective and comparably immunogenic to BDD-F.VIII (364). Other strategies for enhancing secretion include the incorporation of a fragment of the light chain into the heavy chain (whose secretion is the rate limiting step) or the use of chemical chaperones (365, 366). Alternatively, one can simply bypass F.VIII altogether.…”

A Thermolabile Aldolase A Mutant Causes Fever-Induced Recurrent Rhabdomyolysis without Hemolytic Anemia

“…Three other chaperons (sigma) were also tested, glycerol (100 mM) [25] for 10 days, betaine (10 and 50 mM) [26], [27] and benzylhydantoin (130 µM) for 3 days. Uniformly stable isotope labeled (U- 13 C 5 ) glutamine or (U- 13 C 6 ) arginine (Eurisotop, Saint-Aubin, France) were provided to myoblasts (1 mM) in the presence of glucose (2.5 mM) and incubated without serum for 6 hours.…”

Acute rhabdomyolysis

“…Ранее аргинин был опи-сан как защитный белок-шаперон, защищающий разные метаболические ферменты от термолабиль-ности [51][52][53]. …”