HUMAN GENE THERAPY 14:1579–1585 (November 1, 2003) © Mary Ann Liebert, Inc. Brief Report A Comparison of Targeting Performance of Oncoretroviral Versus Lentiviral Vectors on Human Keratinocytes FERNANDO SERRANO, 3, * MARCELA DEL RIO, 1,2, * FERNANDO LARCHER, 1,2, * MARTA GARCIA, 1,2 EVANGELINA MUÑOZ, 1,2 MARÍA JOSÉ ESCAMEZ, 1 MARTA MUÑOZ, 1 ALVARO MEANA, 4 ANTONIO BERNAD, 3 and JOSÉ LUIS JORCANO, 1,2 ABSTRACT The epidermis, like other rapidly renewing tissues, relies on a stem cell compartment to undergo constant re- generation. In order to develop realistic and long-lasting therapeutic approaches for some skin disorders, gene transfer to these critical cells must be obtained. While efficient retroviral ex vivo targeting and transgene in- tegration in human keratinocytes is tightly dependent on proliferation, transferring genetic information to quiescent cells in culture also presents advantages, including the possibility of targeting putative dormant epi- dermal stem cells. In the present study we compared the efficiency of transduction achieved with a third-gen- eration of human immunodeficiency virus (HIV)-based lentiviral vector to that obtained with a Moloney murine leukemia oncoretroviral vector (MLV) on proliferating and quiescent human keratinocytes growing in vitro in standard Rheinwald and Green cultures as well as in confluent organotypic cultures. Each viral vector contained the enhanced green fluorescent protein (EGFP) as a reporter gene. The lentiviral vector, but not the MLV vector, led to EGFP expression both in nondividing and proliferating epidermal cell populations in vitro. This feature was clearly evident when direct targeting of human keratinocytes, forming part of the epidermal component of an organotypic skin culture, was attempted. Keratinocytes modified by both MLV and the lentiviral vector allowed long-term regeneration of genetically engineered human skin on the backs of immunodeficient nonobese diabetic/severe combined immunodeficiency disorders (NOD/SCID) mice. How- ever, EGFP transgene expression in the context of the MLV (long-terminal repeat [LTR]-driven) or lentivi- ral vector (cytomegalovirus [CMV]-driven) demonstrated clear differences both in quantitative terms and in the in vivo localization pattern. 1579 INTRODUCTION K ERATINOCYTE GENE THERAPY has a great theoretical ap- peal as a novel therapeutic approach for both skin dis- eases such as genodermatoses, and nonskin processes(Somani et al., 1999; Spirito et al., 2001; Del Rio et al., 2002a). Al- though the skin offers great advantages in terms of accessi- bility to in vivo gene therapy, gene transfer to large cell pop- ulations, including a stem cell compartment still requires a major effort in refinement to become a realistic possibility. Therefore, the ex vivo approach currently remains the method of choice to transfer genetic information efficiently to ker- atinocye progenitors, the required target for permanent gene therapy. In spite of efforts toward identification of the human epidermal stem cell compartment, its marking and isolation as a pure population from complex keratinocyte mixtures has not 1 Project of Cell and Molecular Biology and Gene Therapy, CIEMAT, 28040 Madrid, Spain. 2 Fundacion Marcelino Botín for Gene Therapy, 3 Department of Immunology and Oncology, Centro Nacional de Biotecnología, 28040 Madrid, Spain. 4 Centro de Transfusiones del Principado de Asturias, 33006 Oviedo, Spain. *These authors contributed equally to this study.