Modifications of the fibroblast growth factor-2 gene led to a marked enhancement in secretion and stability of the recombinant fibroblast growth factor-2 protein.
Progress in FGF-2 gene therapy has been hampered by the difficulty in achieving therapeutic levels of FGF-2 secretion. This study tested whether the addition of BMP2/4 hybrid secretion signal to the FGF-2 gene and mutation of cys-70 and cys-88 to serine and asparagine, respectively, would increase the stability and secretion of active FGF-2 protein in mammalian cells using MLV-based vectors. Single or double mutations of cys-70 and cys-88 to ser-70 and asp-88, respectively, markedly increased the amounts of FGF-2 protein in conditioned media and cell lysates, which may be due to glycosylation, particularly at the mutated asp-88 residue. Addition of BMP2/4 secretion signal increased FGF-2 secretion, but also suppressed FGF-2 biosynthesis. The combination of BMP2/4 secretion signal and double cys-70 and cys-88 mutations increased the total amount of secreted FGF-2 protein >60-fold. The modifications did not alter its ability to stimulate cell proliferation and Erk1/2 phosphorylation in marrow stromal cells or its ability to bind heparin in vitro, suggesting that the modified FGF-2 protein was functionally as effective as the unmodified FGF-2. An ex vivo application of rat skin fibroblasts (RSF) transduced with the modified FGF-2 vector in a subcutaneous implant model showed that rats with implants containing cells transduced with the modified FGF-2 vector increased serum FGF-2 level >15-fold, increased growth of the implant, and increased vascularization within the implant, compared to rats that received implants containing beta-galactosidase- or wild-type FGF-2-transduced control cells. This modified vector may be useful in FGF-2 gene therapy investigations.
Chen
Shin-Tai
ST
The Gene Therapy Division, Musculoskeletal Disease Center (151), Jerry L. Pettis Memorial V.A. Medical Center, Loma Linda, California 92357, USA.
Gysin
Reinhard
R
Kapur
Sonia
S
Baylink
David J
DJ
Lau
K-H William
KH
D000818:Animals; D015153:Blotting, Western; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism* / Q000637:transplantation; D005822:Genetic Vectors / Q000235:genetics; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006493:Heparin / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism*; D012867:Skin / Q000166:cytology / Q000378:metabolism; D014162:Transfection
D019485:Bone Morphogenetic Proteins; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D006493:Heparin; D006026:Glycoside Hydrolases