Efficient retroviral transduction of human epidermal stem cells using recombinant fibronectin
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The epidermis is an attractive target for gene therapy because it is easily accessible and shows great potential as an ectopic site for protein delivery in vivo. Genetically modified epidermal keratinocytes are used to prepare three-dimensional skin equivalents, which when transplanted into animals act as in vivo "bioreactors" that produce and deliver the desired therapeutic proteins either systematically or locally. Although retroviral transduction results in permanent genetic modification, differentiation and loss of transduced cells from the epidermis results in temporary transgene expression. To ensure permanent genetic modification epidermal stem cells must be transduced with high efficiency. To this end, we used recombinant fibronectin (rFN) to immobilize retroviral particles. We show that retrovirus binds to the heparin-binding domain of rFN and that retrovirus associated heparan sulfate is important for this interaction. Our results show that cells transduced on rFN exhibit significantly higher transduction efficiency, which correlates with the levels of expression of integrin subunits β1 and α5. These integrins have been previously shown to correlate with stem cell phenotype. Notably, cells that adhere rapidly to rFN are transduced more efficiently than slowly adherent cells. Most importantly, the efficiency of gene transfer increases with cell passage. An increase in the fraction of transduced cells over time suggests selective transduction of progenitor cells, as the untransduced differentiated cells exhaust their proliferative potential and stop propagating. We were also able to demonstrate that rFN, besides preferentially promoting gene transfer to these progenitor cells, was also instrumental in maintenance of their undifferentiated state. Next, we demonstrated that the JNK MAPK signaling pathway is important for retroviral gene transfer to keratinocytes. Chemical inhibitors of this pathway starkly reduced the efficiency of gene transfer. These results may have important implications for gene therapy of the skin.