Transgenic studies with a keratin promoter-driven growth hormone transgene: Prospects for gene therapy

Abstract

Keratinocytes are potentially appealing vehicles for the delivery of secreted gene products because they can be transferred to human skin by the relatively simple procedure of grafting. Adult human keratinocytes can be efficiently propagated in culture with sufficient proliferative capacity to produce enough epidermis to cover the body surface of an average adult. However, the feasibility of delivering secreted proteins through skin grafting rests upon (i) the strength of the promoter in keratinocytes and (ii) the efficiency of protein transport through the basement membrane of the stratified epithelium and into the bloodstream. In this paper, we use transgenic technology to demonstrate that the activity of the human keratin 14 promoter remains high in adult skin and that keratinocyte-derived human growth hormone (hGH) can be produced, secreted, and transported to the bloodstream of mice with efficiency that is sufficient to exceed by an order of magnitude the circulating hGH concentration in growing children. Transgenic skin grafts from these adults continue to produce and secrete hGH stably, at Ϸ1͞10 physiological levels in the bloodstream of nontransgenic recipient mice. These studies underscore the utility of the keratin 14 promoter for expressing foreign transgenes in keratinocytes and demonstrate that keratinocytes can be used as effective vehicles for transporting factors to the bloodstream and for eliciting metabolic changes. These findings have important implications for considering the keratinocyte as a possible vehicle for gene therapy.

EpithelialBmpr1aregulates differentiation and proliferation in postnatal hair follicles and is essential for tooth development

“…Transgenic founders were tested for Cre activity by breeding to ROSA26R mice (B6.129S4-Gt(ROSA)26Sor tm1Sor ) (Soriano, 1999) (Jackson Laboratories). Detection of β-galactosidase in embryo wholemounts and 20 µm frozen sections of P1 tissue were as described previously (Phippard et al, 1999;Wang et al, 1997).…”

“…To determine the requirement for Bmpr1a in development of the epidermis and its appendages, we generated three lines of transgenic mice that constitutively express Cre recombinase in epidermal cells under the control of a well-characterized keratin 14 (K14) promoter (Wang et al, 1997). This promoter is active in surface ectoderm and basal epidermis from embryonic day (E) 9.5, and drives expression in developing hair follicle and tooth epithelia (Byrne et al, 1994;Dassule et al, 2000).…”

Efficient gene transfer into human keratinocytes with recombinant adeno-associated virus vectors

“…2 Skin grafting procedures have been established for the clinical treatment of burned patients. 3 These achievements provide the tools to manipulate large amounts of skin cells ex vivo and prepare the ground for skin gene therapy ex vivo.…”

Seeing the gene therapy: application of gene gun technique to transfect and decolour pigmented rat skin with human agouti signalling protein cDNA

“…21 High concentrations of transcription products in both the dermis and epidermis have been achieved using cytomegalovirus (CMV) promoters. 22 We found that gene gun administration of pCMV-hASP caused the local skin melanin index of rats to decrease by 70% (Figure 2).…”