Sustainability of Keratinocyte Gene Transfer and Cell SurvivalIn Vivo

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Abstract
The epidermis is an attractive site for therapeutic gene delivery because it is accessible and capable of delivering polypeptides to the systemic circulation. A number of difficulties, however, have emerged in attempts at cutaneous gene delivery, and central among these is an inability to sustain therapeutic gene production. We have examined two major potential contributing factors, viral vector stamina and involvement of long-lived epidermal progenitor cells. Human keratinocytes were either untreated or transduced with a retroviral vector for β-galactosidase (β-Gal) at >99% efficiency and then grafted onto immunodeficient mice to regenerate human epidermis. Human epidermis was monitored in vivo after grafting for clinical and histologic appearance as well as for gene expression. Although integrated vector sequences persisted unchanged in engineered epidermis at 10 weeks post-grafting, retroviral long terminal repeat (LTR)-driven β-Gal expression ceased in vivo after approximately 4 weeks. Endogenous cellular promoters, however, maintained consistently normal gene expression levels without evidence of time-dependent decline, as determined by immunostaining with species-specific antibodies for human involucrin, filaggrin, keratinocyte transglutaminase, keratin 10, type VH collagen, and Laminin 5 proteins out to week 14 post-grafting. Transduced human keratinocytes generated multilayer epidermis sustained through multiple epidermal turnover cycles; this epidermis demonstrated retention of a spatially appropriate pattern of basal and suprabasal epidermal marker gene expression. These results confirm previous findings suggesting that viral promoter-driven gene expression is not durable and demonstrate that keratinocytes passaged in vitro can regenerate and sustain normal epidermis for prolonged periods. Loss of retroviral transgene expression has frustrated efforts at durable gene delivery in a number of tissues, including the skin. The degree to which factors such as cell loss and promoter inactivation contribute to this phenomenon, however, is not clearly known. In this study, we regenerated human skin on immune-deficient mice using primary human keratinocytes transduced at high efficiency with a retroviral expression vector for β-galactosidase. Long terminal repeat (LTR)-driven transgene expression in vivo was lost after 4 weeks, in spite of genomic retention of vector sequences. In contrast, engineered keratinocytes continued to produce normal levels of endogenous genes in vivo. These results indicate that long-lived human keratinocyte progenitor cells survive ex vivo retroviral transduction but that LTR-driven transgene production is not sustained in engineered epidermis in vivo.