456. Gene Transfer to Skin Stem Cells by Early Gestational Intra_Amniotic Injection of Lentiviral Vector

Abstract

Early gestational gene transfer may provide developmental opportunities for transduction of accessible stem cell populations that are not available later in development. An example with potential clinical importance is skin_associated stem cells. During development, these cells become inaccessible for gene transfer due to formation of the periderm, stratification of epidermis, and involution of skin accessory structures. To test whether gene transfer to skin stem cells could be achieved early in gestation, and whether gene expression would be maintained long_term, we injected lentiviral vector carrying the green fluorescent protein (GFP) reporter gene into the fetal murine amniotic space from the late head fold/early somite stage (E8) to E18. From E8 to E12 amniotic space injections were performed under ultrasound image guidance using a [email protected] Vevo660 system with a 40 mHz scanhead. After E12, injections were performed under direct vision using a stereoscopic microscope. We observed the skin under fluorescence stereomicroscopy at sequential time points after birth and confirmed GFP expression by immunohistochemistry.GFP expression was observed in the skin of E8 to E12 groups after birth, but no expression was observed with injections after E12. In E8 to E10 injected groups, widespread expression over the entire skin surface, including hair and nails, was seen, whereas in the E11 and E12 groups, GFP expression was limited to the hair and skin of the scalp. Expression in these distributions persisted beyond 6 months of age in all animals. Histological analysis revealed that GFP was expressed in the skin accessory structures, including the bulge region of the hair follicle, the sebaceous glands, as well as the epidermis. After epidermal injury by punch biopsy, GFP positive cells from the surrounding skin and hair follicles were recruited into the epidermis and migrate in a linear manner toward the center of the wound, ultimately forming a distinctive radial pattern consistent with stem cell transduction. FACS analysis of the skin of E9 group revealed the existence of CD34+/GFP+ cells confirming gene transfer to the bulge stem cells.These finding confirm that gene transfer can be achieved to skin associated stem cell populations, including the bulge and epidermal stem cells, by early gestational intra_amniotic injection of lentiviral vectors. This gene transfer method may be useful for investigation of mechanisms of genetic and/or developmental disease and for the development of prenatal skin directed gene therapy for specific skin disorders. Early gestational gene transfer may provide developmental opportunities for transduction of accessible stem cell populations that are not available later in development. An example with potential clinical importance is skin_associated stem cells. During development, these cells become inaccessible for gene transfer due to formation of the periderm, stratification of epidermis, and involution of skin accessory structures. To test whether gene transfer to skin stem cells could be achieved early in gestation, and whether gene expression would be maintained long_term, we injected lentiviral vector carrying the green fluorescent protein (GFP) reporter gene into the fetal murine amniotic space from the late head fold/early somite stage (E8) to E18. From E8 to E12 amniotic space injections were performed under ultrasound image guidance using a [email protected] Vevo660 system with a 40 mHz scanhead. After E12, injections were performed under direct vision using a stereoscopic microscope. We observed the skin under fluorescence stereomicroscopy at sequential time points after birth and confirmed GFP expression by immunohistochemistry. GFP expression was observed in the skin of E8 to E12 groups after birth, but no expression was observed with injections after E12. In E8 to E10 injected groups, widespread expression over the entire skin surface, including hair and nails, was seen, whereas in the E11 and E12 groups, GFP expression was limited to the hair and skin of the scalp. Expression in these distributions persisted beyond 6 months of age in all animals. Histological analysis revealed that GFP was expressed in the skin accessory structures, including the bulge region of the hair follicle, the sebaceous glands, as well as the epidermis. After epidermal injury by punch biopsy, GFP positive cells from the surrounding skin and hair follicles were recruited into the epidermis and migrate in a linear manner toward the center of the wound, ultimately forming a distinctive radial pattern consistent with stem cell transduction. FACS analysis of the skin of E9 group revealed the existence of CD34+/GFP+ cells confirming gene transfer to the bulge stem cells. These finding confirm that gene transfer can be achieved to skin associated stem cell populations, including the bulge and epidermal stem cells, by early gestational intra_amniotic injection of lentiviral vectors. This gene transfer method may be useful for investigation of mechanisms of genetic and/or developmental disease and for the development of prenatal skin directed gene therapy for specific skin disorders.