Long-Term Expression of Human Clotting Factor IX from Retrovirally Transduced Primary Human KeratinocytesIn Vivo

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Abstract
A persistent obstacle that has hampered gene transfer experiments is the short-term nature of transgene expression in vivo. In this article we present evidence for sustained expression from primary human keratinocytes, using the retroviral vector MFG. Primary keratinocytes were transduced in culture with the MFG retroviral vector containing the coding region from factor IX cDNA. Transduced keratinocytes, which secreted on average 830 ng of factor IX/106 cells/24 hr in tissue culture, were used to form a bilayered skin equivalent and grafted onto nude mice under a silicone transplantation chamber. Between 0.1 and 2.75 ng of human factor IX per milliliter was found in mouse plasma for more than 1 year, suggesting that keratinocyte stem cells were both transduced and grafted. The results show, for the first time, that long-term expression is obtainable in retrovirally transduced keratinocytes after transplantation. Factor IX retroviral vector (MFG-fIX) was used to transduce primary human keratinocytes in vitro, after which the cells were grafted as part of a bilayered skin equivalent onto nude mice. Silicone transplantation chambers protected the grafts while they became vascularized. The chambers fell off, generally within 6 weeks, allowing the site to heal and the grafted keratinocytes to become stably incorporated into mouse skin. Factor IX levels in mouse plasma stabilized on average between 40 and 50 days after transplantation and detectable levels of factor IX were present for more than 1 year. This result, in conjunction with immunohistochemical evidence, suggests that keratinocyte stem cells can be transduced, survive grafting, and express the transgene long term in vivo.