SUSTAINABILITY OF KERATINOCYTE GENE-TRANSFER AND CELL-SURVIVAL IN-VIVO

The epidermis is an attractive site for therapeutic gene delivery beca use it is accessible and capable of delivering polypeptides to the sys temic circulation. A number of difficulties, however, have emerged in attempts at cutaneous gene delivery, and central among these is an ina bility to sustain therapeutic gene production. We have examined two ma jor potential contributing factors, viral vector stamina and involveme nt of long-lived epidermal progenitor cells. Human keratinocytes were either untreated or transduced with a retroviral vector for beta-galac tosidase (beta-Gal) at >99% efficiency and then grafted onto immunodef icient mice to regenerate human epidermis. Human epidermis was monitor ed in vivo after grafting for clinical and histologic appearance as we ll as for gene expression. Although integrated vector sequences persis ted unchanged in engineered epidermis at 10 weeks post-grafting, retro viral long terminal repeat (LTR)-driven beta-Gal expression ceased in vivo after approximately 4 weeks. Endogenous cellular promoters, howev er, maintained consistently normal gene expression levels without evid ence of time-dependent decline, as determined by immunostaining with s pecies-specific antibodies for human involucrin, filaggrin, keratinocy te transglutaminase, keratin 10, type VII collagen, and Laminin 5 prot eins out to week 14 post-grafting. Transduced human keratinocytes gene rated multilayer epidermis sustained through multiple epidermal turnov er cycles; this epidermis demonstrated retention of a spatially approp riate pattern of basal and suprabasal epidermal marker gene expression . These results confirm previous findings suggesting that viral promot er-driven gene expression is not durable and demonstrate that keratino cytes passaged in vitro can regenerate and sustain normal epidermis fo r prolonged periods.