In this murine study, we demonstrate that IAGT using lentiviral vectors can result in efficient, diffuse and sustained gene transfer to cutaneous epithelial lineages. With appropriate timing of vector administration, gene transfer to stem cell populations that generate and maintain the skin and skin appendages can be achieved. To our knowledge, this is the first report that demonstrates widespread transduction of skin stem cell/progenitor populations by any in vivo gene transfer strategy.
Our results are best interpreted in the context of normal skin development.23
During early murine development until E9, the skin is comprised of only a single cell layer. Focal thickening of the epidermis next occurs, creating the epidermal placodes.24
The epidermal placodes then signal the mesenchyme to aggregate beneath the placodes.25
Once aggregated, mesenchymal–epidermal interaction induces the development of the skin appendages.24
In our study, we achieved gene transfer to all of the skin appendages by early IAGT. This finding suggests that early IAGT results in gene transfer to epidermal stem cells prior to the differentiation of the epidermal placodes. A second event in fetal skin development is formation of the periderm, an epithelial layer covering the emerging epidermis in early embryogenesis, around E9.5.23,26
The periderm is a transient layer of cells, interfacing between the developing epidermis and amniotic fluid. The periderm differentiates itself in tandem with epidermal development, completes its formation by E12, and is sloughed into amniotic fluid by E17 as the skin becomes fully keratinized.27
The epidermis starts to stratify at the end of E9 and is fully differentiated by E18.5. Thus, our inability to transduce skin stem cells after E9, and inability to transduce any skin epidermal cell population after E12, corresponds with the formation of the periderm and stratification of the epidermis that presumably acts as a physical barrier. Similarly, the pattern of loss of transduction with gestational age corresponds with the pattern of periderm formation. The periderm completes its formation on the limb buds first, followed by the dorsal and ventral trunk regions and scalp.28,29
These observations support our hypothesis of a unique developmental “window of opportunity” during which skin associated stem cells are accessible for gene transfer.
The transduction of stem cells is of obvious importance with respect to the experimental and therapeutic potential of this approach. There have been two types of stem cells/progenitors identified in the skin; the epidermal basal cell and the bulge stem cell.30–34
These populations play important, but distinct roles in skin maintenance, regeneration, and repair.30,31
Recently, using transgenic mice with an inducible reporter gene under control of the K15 promoter, Ito et al
. demonstrated that bulge stem cells contribute to early stages of wound healing but not to homeostasis of the epidermis, and that stem cells in the basal layers contribute to maintain the homeostasis of the epidermis.30
Several observations in our study indicate that we transduced both populations of stem cells. First, the longevity of epidermal GFP expression argues strongly for stem cell transduction. The maintenance of epidermal GFP expression beyond 6 months is clearly derived from transduction of nascent epidermal basal stem/progenitor cells. Second, after making a full-thickness wound in the skin, GFP-positive cells migrated from single hair follicles at the wound edge towards the center of the wound. This pattern is identical to that described by Ito et al
. in observing the bulge cell response to a similar wound. Finally, we found significant levels of gene transduction in sorted cell populations highly enriched for each stem/progenitor cell type.
Our initial observations using a lentiviral vector driven by the CMV promoter demonstrated diffuse expression in the suprabasal layers of the epithelium, and a robust contribution of transduced stem cells to wound healing. This was inconsistent with our quantitative finding of only 0.41 and 1.23% of isolated bulge and basal stem cells respectively, expressing GFP. A possible explanation for this discrepancy could be that the stem cell populations were efficiently targeted but that promoter specificity limited transgene expression to the superficial epithelial strata. This is consistent with previous data that examined transduction of keratinocytes in regenerated human skin with a lentiviral vector driven by the CMV promoter; here, GFP expression was seen only in the suprabasal layers, which arose from transduced basal stem cells.14
In the hopes of achieving greater basal layer gene expression, we compared vectors driven by the CMV and the K5 promoters. In contrast to CMV, the K5 promoter is expressed only in ectodermal tissues with high expression in the basal layer of the epidermis.21
In this study, there were distinct differences in the distribution of epidermal expression between the vectors. Expression with the K5 promoter driven vector was very high in the basal epithelial cells of the epidermis, whereas expression driven by the CMV promoter was primarily in the superficial epidermal layer. Our FACS data quantitatively confirms this difference, as most of the collected cells are α6-integrin positive basal layer cells (63–71% of total cells).
Another factor that may result in underestimation of the true transduction efficiency is in vivo
silencing. Mechanisms of silencing for retroviral vectors are thought to be both genetic and epigenetic. Epigenetic mechanisms may silence gene expression after expression is established by de novo
DNA methylation within the promoter region or other mechanisms resulting in a repressive chromatin configuration.35–37
In contrast to epigenetic silencing, genetic mechanisms result in primary failure of expression and relate to the integration site but not to epigenetic changes of the integrated element.38,39
It has been recently documented in a study designed to ensure only single round infection kinetics and the expression of only integrated transgene, that only a minority of human immunodeficiency virus-1-based vector integrations express transgene40
but that once transgene expression is established it is likely to remain stable favoring a predominantly genetic mechanism of silencing, at least in this defined system. This would support the concept that the likelihood of gene expression as well as the strength of expression would depend upon the number of copies integrated. Consistent with these concepts we found the EGFP
transgene in all of the cell populations to be independent of GFP expression, suggesting better transduction efficiency than represented by the histology and FACS analysis, and significant levels of in vivo
gene silencing with both promoter constructs. Higher levels of expression in stem cell populations using the K5 promoter reflect the relative specificity of the K5 promoter for expression in basal layer cells. Therefore the choice of promoter for therapeutic application to congenital skin diseases would depend upon which level of the epidermis one wished to target. For EB, that affects the basal layer, the K5 promoter would appear to be a better choice.41,42
Our demonstration of high efficiency transduction of skin stem cell compartments using the IAGT approach addresses the need for an in vivo
methodology for manipulating skin gene expression. From an experimental and biological perspective, combination of this approach with technologies such as RNAi, Cre/loxP, and drug inducible On/Off systems will provide versatile systems for elucidating mechanisms of skin differentiation, regeneration, wound healing, and scarring. From a therapeutic perspective, this approach may allow prenatal treatment of genetic skin disorders such as EB in which defective protein synthesis occurs at the dermal, epidermal interface. However, there are a number of hurdles that would need to be overcome prior to any clinical application of this approach. First, although we have chosen to focus on skin gene transfer in this study, IAGT is not specific for skin. While some selectivity occurs due to the limited interface between amniotic fluid and other fetal tissues, we have documented multiple other epidermal tissues that are transduced by IAGT.20
This raises obvious concerns about the potential for insertional mutagenesis, developmental effects, and the potential for germ line alteration that exists for lentiviral vector based approaches. These dangers are only heightened by early gestational transduction.43
While greater tissue specificity and safety can probably be accomplished by the use of tissue specific promoters, or regulated transgene expression, safer gene transfer techniques will need to be developed to alleviate these concerns. The second major impediment is that stage for stage, the timing of our early gestational injections between E8 and E11 in the mouse, corresponds to the 21st to 55th day of gestation in human fetal development,23
a time in pregnancy that precedes current capabilities for prenatal diagnosis. Nevertheless, in the foreseeable future, prenatal diagnosis may allow for diagnosis of genetic disorders during this period of gestation. Finally, because of the early and relatively high efficiency of gene transfer to stem cell populations using this approach, it may offer a relatively rigorous assay for safety studies on vector based gene transfer.