In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors

Abstract

For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction. When lentiviral vectors expressing green fluorescent protein (GFP) were directly injected into the dermis of human skin grafted onto immunocompromised mice, transduction of dividing basal and nondividing suprabasal keratinocytes could be demonstrated, which was not the case when control retroviral vectors were used. However, flow cytometry analysis demonstrated low transduction efficiency, and histological analysis at later time points provided no evidence for progenitor cell targeting. In an alternative in vivo method, human keratinocytes were transduced in tissue culture (ex vivo) with either lentiviral or retroviral vectors and grafted as skin equivalents onto immunocompromised mice. GFP expression was analyzed in these human skin grafts after several cycles of epidermal turnover, and both the lentiviral and retroviral vector-transduced grafts had similar percentages of GFP-expressing keratinocytes. This ex vivo grafting study provides a good in vivo assessment of gene introduction into progenitor cells and suggests that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture. Achieving persistent and high-level gene expression in a significant percentage of target cells is important for establishing successful clinical applications of gene therapy. For gene therapy of the skin, a renewable tissue undergoing constant turnover, and for achieving long-term expression in a significant percentage of keratinocytes, gene targeting to keratinocyte stem cells (KSC) or long-lasting keratinocyte progenitor cells will be required (36). In ex vivo skin gene therapy, keratinocytes are removed from the donor and transduced with retroviral vectors in vitro without selectively targeting KSC. The genetically modified keratinocytes can then be grafted back onto the donor. Even though progress in skin gene therapy has enabled gene expression from retroviral vectors to be detected for sustained periods in grafted keratinocytes (9,24,32,56), transgene expression in vivo is frequently decreased over time, either in the duration of expression or in the percentage of keratinocytes expressing the gene (12,14,18,35,50). The factors contributing to this decreased expression may be similar to those for other tissues (14,27,41,51) and may include in...

Distinctive Roles for 2′,5′-Oligoadenylate Synthetases and Double-Stranded RNA-Dependent Protein Kinase R in the In Vivo Antiviral Effect of an Adenoviral Vector Expressing Murine IFN-β

“…This approach seems very plausible using lentiviruses as a potential delivery system for gene transfer (51). Several laboratories have demonstrated long-term expression associated with lentivirus transduction of epithelial stem cells (52)(53)(54). Alternatively, because of the highly innervated nature of the cornea, the possible use of viral vectors to deliver genes directly to the neurons of the sensory ganglion as demonstrated in the present study may have significant consequences to viral reactivation, for example, with HSV-1.…”

Gene transfer in human skin with different pseudotyped HIV-based vectors

“…14,15 Several types of viral vectors such as retrovirus, adenovirus, adeno-associated virus (AAV) and lentivirus have been evaluated for epidermal gene transfer in vivo. [16][17][18][19][20][21] Adenovirus vector can efficiently transduce keratinocytes, however, transgene expression is transient owing to the loss of unintegrated vector during epithelium differentiation as well as immunodestruction of target cells. 22 Retroviral vectors based on murine leukemia virus (MuLV) can lead to long-term expression of the transgene in keratinocytes both in in vitro 23 and in vivo, 24 probably owing to integration of the genetic material into the host genome.…”

Assessment of optimal transduction of primary human skin keratinocytes by viral vectors

“…Our results are in agreement with previous observations that human HIV-derived vectors efficiently infect sublethally irradiated or contact-inhibited cell cycle arrested keratinocytes. Moreover, long-term expression (16 weeks) of the transgene after grafting onto immunodeficient mice implies transduction of clonogenic primary keratinocytes [33,34]. Stem-cell-like keratinocytes are also efficiently transduced by oncoretroviruses and sustain permanent expression of the transgene in in vivo transplanted artificial skin [35].…”