Cutaneous and mucosal cells can be genetically modified (by replication deficient retroviral vectors) to secrete therapeutic proteins at the local or systemic level. Fibroblasts, being secretory cells, are expected to secrete protein at higher rates compared to keratinocytes in this type of gene therapy. Objective: To compare protein secretion rates of genetically modified fibroblasts and keratinocytes in submerged and organotypic (raft) culture models. Methods: Bi-cistronic MMLV retroviral vectors constructed with IL-10/ApoE  (secretory protein) and GFP (reporter) genes were used to transduce human neonatal foreskin keratinocytes and fibroblasts at multiplicity of infection (M.O.I) of 0.3. In both cell types 30% of the cells were determined to be GFP positive by flow cytometric analysis. At this M.O.I each positive cell contains one provirus copy thus permitting comparative studies. Transduced cultures were grown in submerged and raft cultures.  Rafts were made with fibroblast-embedded collagen matrices over which keratinocytes were seeded. Media from culture supernatants were collected over time and analyzed by ELISA for IL10/ApoE levels. Secretory rates were calculated from the slope of lines thus generated.

Results:

Model system	Rates of secretion of ApoE (ng/hr/106cells)		Rates of secretion of IL10 (ng/hr/106cells)
Submerged culture	KC=12.33+/-2.3	HF=9.58+/-1.01	KC=3.04+/-0.31	HF=1.62+/-0.57
Raft cultures	KC=0.4260+/-0.24	HF=0.195+/-0.14	KC=0.101+/-0.060	HF=0.113+/-0.0631

Conclusions:  The results suggest that keratinocytes in submerged cultures are slightly more efficient than fibroblasts in secreting the transgene product but when organized into rafts, rates are similar in the two cell types. In-vivo the rates are likely to be similar as well. However since there are more keratinocytes than fibroblasts per square millimeter of cutaneous/mucosal tissue, the bulk of secretion is likely to originate from the epithelial compartment. Interestingly there is a drop in secretory rate for both cells when shifted from submerged cultures to rafts suggesting that secretion is altered by cell microenvironment.   Supported by NIH-DEO4511 (to LBT)