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Optimization of human erythropoietin secretion from MLV‐infected human primary fibroblasts used for encapsulated cell therapy
Author(s) -
Schwenter F.,
Déglon N.,
Aebischer P.
Publication year - 2003
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.338
Subject(s) - biology , woodchuck hepatitis virus , vesicular stomatitis virus , transgene , viral vector , retrovirus , murine leukemia virus , multiplicity of infection , microbiology and biotechnology , virology , secretion , genetic enhancement , virus , recombinant dna , gene , hepadnaviridae , hepatitis b virus , biochemistry
Background The transplantation of encapsulated cells genetically engineered to secrete human erythropoietin (hEpo) represents an alternative to repeated injections of the recombinant hormone for the treatment of Epo‐responsive anemia. In the present study, the ability of primary human foreskin fibroblasts to secrete high levels of hEpo and the importance of cis ‐acting elements and infection conditions on transgene expression level were assessed. Methods The transduction efficiency was first evaluated with β‐galactosidase (LacZ)‐encoding retroviral vectors derived from the murine leukemia retrovirus (MLV) pseudotyped either with an amphotropic envelope or with the G glycoprotein of vesicular stomatitis virus (VSV‐G). Human fibroblasts were then infected with an amphotropic hEpo‐expressing retroviral vector, which was modified by insertion of a post‐transcriptional regulatory element from the woodchuck hepatitis virus (WPRE) and a Kozak consensus sequence (KZ). Human Epo production was further optimized by increasing the multiplicity of infection and by selecting high producer cells. The survival and the transgene expression of these fibroblasts were finally evaluated in vivo. The cells were encapsulated into microporous hollow fibers and subcutaneously implanted in nude mice. Results A secretion level of approximately 5 IU hEpo/10 6 cells/day was obtained with the basal vector. A 7.5‐fold increase in transgene expression was observed with the insertion of WPRE and KZ elements. Finally, according to the optimization of infection conditions, we obtained a 40‐fold increase in hEpo secretion, reaching approximately 200 IU hEpo/10 6 cells/day. The in vivo experiments showed an increase in the hematocrit during the first 2 weeks and elevated levels exceeding 60% were maintained over a 6‐week period. Conclusions These results indicate that primary human fibroblasts represent a promising source for encapsulated cell therapy. Copyright © 2003 John Wiley & Sons, Ltd.