Efficient In Vivo Targeting of Epidermal Stem Cells by Early Gestational Intraamniotic Injection of Lentiviral Vector Driven by the Keratin 5 Promoter

Abstract

At the present time, no efficient in vivo method for gene transfer to skin stem cells exists. In this study, we hypothesized that early in gestation, specific epidermal stem cell populations may be accessible for gene transfer. To test this hypothesis, we injected lentiviral vectors encoding the green fluorescence protein marker gene driven by either the cytomegalovirus promoter or the keratin 5 (K5) promoter into the murine amniotic space at early developmental stages between embryonic days 8 and 12. This resulted in sustained green fluorescent protein (GFP) expression in both basal epidermal stem cells and bulge cells in the hair follicles of the skin. Transduction of stem cell populations was dependent on the developmental stage, and confirmed by the prolonged duration of GFP expression in all skin elements into adulthood. In addition, transduced stem cell populations responded to regenerative signals after wounding and actively participated in wound healing. Finally, we quantified the fraction of epidermal stem cells transduced, and the distribution of transduction related to the promoters utilized, confirming improved efficiency with the K5 promoter. This simple approach has possible biological applications in our study of gene functions in skin, and perhaps future clinical applications for treatment of skin based disorders.

The developmental stage determines the distribution and duration of gene expression after early intra-amniotic gene transfer using lentiviral vectors

“…We could detect GFP expression in organs derived from all three germ layers; however, most of the positive organs were derived from ectoderm. Stereomicroscopic images of representative positive organs, other than the skin and eye (which we have described in detail in our previous publications 28,29 ) are shown in Figure 2 and immunohistologic confirmation in Figure 3. The immunohistochemistry shown for specific tissues was derived from separate mice killed at similar, or for some tissues, later time points after IAGT to illustrate the longevity of gene expression.…”

“…5,12,19,24 However, in general, gene transfer after intra-amniotic vector injection has been inefficient and transient because of the relatively late developmental stage at which these experiments have been performed. 5,26,27 In contrast to previous studies, we have recently reported that early IAGT results in efficient transduction of skin stem cell populations 28 and the ectoderm-derived stem cell populations of the eye, 29 including stem cells in the neuroectoderm-derived retina. However, in the course of these studies we observed a broad range of transduction events involving multiple tissues of all three germ layers.…”

“…A typical example would be the expression in skin, which we have described in detail in our previous article that characterized the skin transduction after IAGT. 28 Although the purpose of this study was not quantitative comparison of the efficiency of transduction at different gestational ages for each tissue, there was a clear difference in the amount of GFP expression at early and late gestational IAGT, with greater expression in animals undergoing earlier IAGT, in all of the ectodermal tissues examined.…”

“…These mice are inclusive of the animals reported in our previous two reports of IAGT that focused on ocular and skin stem cell transduction. 28,29 From E8 to E12, we used an ultrasound-guided injection system to visualize and inject the amniotic space as previously described. 29 After E13, we injected under stereomicroscopic magnification.…”

Jaagsiekte sheep retrovirus pseudotyped lentiviral vector-mediated gene transfer to fetal ovine lung

“…In contrast, HIV-1-based lentiviral vectors (LVs) have increased packaging capacity (optimal transgene insert B5-7 Kb 23 ) and, following in utero delivery, are able to integrate into the host genome to provide life-long stable transgene expression. 16,[24][25][26][27] Fetal lung gene transfer has been investigated in non-human primates, 20,28 rabbits, 29 rats 30,31 and sheep 32 using vesicular stomatitis virus glycoprotein (VSVg) pseudotyped LV. Long-term luciferase transgene expression (that is 12-15 months after fetal gene transfer) has been reported 31,33 although there is a paucity of quantitative information on degree of epithelial transduction and cell types transduced.…”