Mutant macaque factor IX T262A: a tool for hemophilia B gene therapy studies in macaques.
BACKGROUND
Gene therapy is expected to be the next generation therapy for hemophilia, and a good animal model is required for hemophilia gene therapy preclinical studies.


METHODS
Taking advantage of the human factor IX (FIX) specificity of monoclonal antibody 3A6, the epitope of which resides in the amino acid polypeptide segment including Ala 262 of human FIX, mutant macaque FIX with an amino acid substitution of Thr 262 to Ala (macaque FIX T262A) was generated and its reactivity to monoclonal antibody 3A6, biological activity and expression in vivo were studied.


RESULTS
Enzyme-linked immunosorbent assays (ELISAs) and Western blot analyses showed that monoclonal antibody 3A6 bound to human FIX and macaque FIX T262A but not to wild-type macaque FIX. Recombinant macaque FIX T262A exhibited a comparable coagulation activity to wild-type macaque FIX and human FIX. High expression of macaque FIX T262A was achieved in mice by injection of AAV8 vectors carrying the macaque FIX T262A gene and reached levels of up to 31.5microg/mL (1050% of the normal human FIX concentration). Macaque FIX T262A expressed in the liver of mice was as biologically active as that expressed in vitro. In addition, the macaque FIX T262A concentrations determined by a 3A6-based ELISA were not influenced by the presence of normal macaque plasma.


CONCLUSIONS
The results of the present study suggest that macaque FIX T262A may be processed appropriately in vivo and that the macaque FIX T262A concentration in the macaque circulation can be quantified precisely by a monoclonal antibody 3A6-based ELISA.
Ishiwata
Akira
A
Divisions of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi-ken 329-0498, Japan.
Mimuro
Jun
J
Mizukami
Hiroaki
H
Kashiwakura
Yuji
Y
Yasumoto
Atsushi
A
Sakata
Asuka
A
Ohmori
Tsukasa
T
Madoiwa
Seiji
S
Ono
Fumiko
F
Shima
Midori
M
Yoshioka
Akira
A
Ozawa
Keiya
K
Sakata
Yoichi
Y
D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005164:Factor IX / Q000032:analysis / Q000235:genetics* / Q000276:immunology*; D015316:Genetic Therapy; D002836:Hemophilia B / Q000628:therapy; D006801:Humans; D008251:Macaca; D051379:Mice; D023421:Models, Animal; D016297:Mutagenesis, Site-Directed
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005164:Factor IX