The skin as a biofactory for systemic secretion of erythropoietin: potential of genetically modified keratinocytes and fibroblasts.
BACKGROUND
The skin is an interesting target tissue for gene therapy applications because of its ready accessibility. One possibility would be to utilize the genetically modified skin as a biofactory secreting a systemically needed product, such as erythropoietin (EPO).


METHODS
Keratinocytes (KC) and fibroblasts (FB) were transduced with a retroviral vector encoding human EPO. Gene transfer efficiency was assessed by real-time PCR analysis and flow cytometry of transduced cells. In addition, EPO synthesis and secretion were analysed by quantifying the amount of RNA and secreted protein in both monolayer cultures and skin equivalents (SE).


RESULTS
When cultured as a monolayer, EPO-KC synthesized significantly more EPO than EPO-FB, as shown by quantitatively measuring the amount of secreted protein and RNA. This correlated with an increased EPO-vector incorporation in KC compared with FB, demonstrated by determining both the percentage of transduced cells and the average transgene copy number per cell. In addition, in transduced cell cultures enriched to equally high percentages of EPO+ cells, KC showed a higher activity of EPO secretion than FB. Finally, when assembled in a SE, EPO-KC secreted significantly higher amounts of EPO than EPO-FB, although reduced secretory activity of EPO-KC monolayers grown in high calcium concentrations suggested that in stratified epidermis differentiated KC secrete less EPO than non-differentiated KC.


CONCLUSIONS
In summary, while both transduced KC and FB are able to synthesize and secrete human EPO, KC show higher potential in serving as possible target cells for therapeutic substitution with EPO, probably because of improved transduction rates and increased secretory activity.
Scheidemann
Frank
F
Department of Dermatology and Allergology, University of Munich, Munich, Germany.
Löser
Maria
M
Niedermeier
Andrea
A
Kromminga
Arno
A
Therrien
Jean-Philippe
JP
Vogel
Jon
J
Pfützner
Wolfgang
W
D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D003078:Colchicine / Q000008:administration & dosage; D004247:DNA / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D004921:Erythropoietin / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D005434:Flow Cytometry; D019450:Genes, MDR; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D012313:RNA / Q000032:analysis; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000378:metabolism*; D017768:Skin, Artificial; D014161:Transduction, Genetic; D019076:Transgenes
D004921:Erythropoietin; D012313:RNA; D004247:DNA; D003078:Colchicine