Epidermal stem cells and ex vivo cutaneous gene therapy: application to xeroderma pigmentosum

Ex vivo cutaneous gene therapy is an alternative treatment for recessively inherited diseases with cutaneous traits. It relies on the transfer in cultured epidermal keratinocytes of the wild-type allele of the gene whose mutation is responsible for the disease. As for severely burnt patients, epithelial sheets developed from genetically corrected cells may then be grafted back to the patients. Long term correction and graft take depend on the genetic correction of stem cells. Success of such an approach has recently been reported in the case of one patient suffering from a severe case of junctional epidermolysis bullosae. Here we report a method for safely selecting keratinocytes populations after genetic manipulation. The method is non invasive and non immunogenic and allows high enrichment of genetically manipulated stem keratinocytes. This could perhaps contribute to ex vivo gene therapy approaches of cancer prone genodermatoses such as xeroderma pigmentosum.     

Cutaneous gene transfer and therapy: the present and the future

The easy accessibility of the skin as a therapeutic target provides an exciting potential for this organ for the development of gene therapy protocols for cutaneous diseases and a variety of metabolic disorders. Thus far, full phenotypic reversion of a diseased phenotype has been achieved in vivo for junctional epidermolysis bullosa and X-linked or lamellar ichthyosis and in vitro for xeroderma pigmentosum. These recessive skin diseases are characterized by skin blistering, abnormalities in epidermal differentiation and increased development of skin cancers, respectively. Corrective gene delivery at both molecular and functional levels was achieved by transduction of cultured skin cells using retroviral vectors carrying the specific curative cDNA. These positive results should prompt clinical trials based on transplantation of artificial epithelia reconstructed ex vivo using genetically modified keratinocytes. Promising results have also been obtained in phenotypic reversion of cells isolated from patients suffering from a number of metabolic diseases such as gyrate atrophy, familial hypercholesterolemia or phenylketonuria. In these diseases transplantation of autologous artificial epithelia expressing the transgenes of interest or direct transfer of the DNA to the skin represents a potential therapeutic approach for the systemic delivery of active molecules. Successful cutaneous gene therapy trials, however, require development of protocols for efficient gene transfer to epidermal stem cells, and information about the host immune response to the recombinant polypeptides produced by the implanted keratinocytes. The availability of spontaneous animal models for genodermatoses will validate the gene therapy approach in preclinical trials.     

Genetic reversion of inherited skin disorders

Human epidermis is a squamous stratified epithelium whose integrity relies on balanced processes of cell attachment, proliferation, and differentiation. In monogenic skin dermatoses, such as mecano-bullous diseases, or DNA repair deficiencies such as the xeroderma pigmentosum (XP), alterations of skin integrity may have devastating consequences as illustrated by the extremely high epidermal cancer proneness of XP patients. The lack of efficient pharmacological treatments, the easy accessibility of skin, and the possibility of long term culture and genetic manipulations ex vivo of epidermal keratinocytes, have encouraged approaches toward gene transfer and skin therapy prospects. We review here some of the human genetic disorders that exhibit major traits in skin, as well as requirements and difficulties inherent to approaches aimed at stable phenotypic correction.     

Enhanced DNA repair of cyclobutane pyrimidine dimers changes the biological response to UV-B radiation

The goal of DNA repair enzyme therapy is the same as that for gene therapy: to rescue a defective proteome/genome by introducing a substitute protein/DNA. The danger of inadequate DNA repair is highlighted in the genetic disease xeroderma pigmentosum. These patients are hypersensitive to sunlight and develop multiple cutaneous neoplasms very early in life. The bacterial DNA repair enzyme T4 endonuclease V was shown over 25 years ago to be capable of reversing the defective repair in xeroderma pigmentosum cells. This enzyme, packaged in an engineered delivery vehicle, has been shown to traverse the stratum corneum, reach the nuclei of living cells of the skin, and enhance the repair of UV-induced cyclobutane pyrimidine dimers (CPD). In such a system, changes in DNA repair, mutagenesis, and cell signaling can be studied without manipulation of the genome.     

Normal rate of DNA breakage in xeroderma pigmentosum complementation group E cells treated with 8-methoxypsoralen plus near-ultraviolet radiation

Treatment of normal and xeroderma pigmentosum complementation group E skin fibroblasts with 8-methoxypsoralen plus repeated doses of near-ultraviolet radiation elicited a marked increase in DNA strand breakage during a subsequent incubation. No such induction of breaks was noted with cells from xeroderma pigmentosum groups A and D. The results suggest that the gene product which is deficient in xeroderma pigmentosum group E cells is involved in a critical step of DNA repair of far-ultraviolet photoproducts but not so in the repair of psoralen cross-links.     

Enhanced expression of procollagenase in ataxia-telangiectasia and xeroderma pigmentosum fibroblasts

Summary Ataxia-telangiectasia and xeroderma pigmentosum are human hereditary diseases in which patients are cancer prone and demonstrate increased sensitivity to DNA damage by ionizing and ultraviolet radiation, respectively. In culture, both ataxia-telangiectasia and xeroderma pigmentosum skin fibroblasts show increased synthesis and secretion of the extracellular matrix proteins fibronectin and collagen. To determine whether these differences in protein production result from fundamental abnormalities in regulation of genes associated with cellular interactions, we compared the effects of trifluoperazine and 12-O-tetradecanoylphorbol-13-acetate on expression of the extracellular matrix-degrading metalloproteinases, procollagenase and prostromelysin, by normal, ataxia-telangiectasia, and xeroderma pigmentosum fibroblasts. After trifluoperazine treatment the overall levels of these metalloproteinases were much greater in three ataxia-telangiectasia cell strains and in cells from xeroderma pigmentosum complementation groups A and D than in normal cells. In contrast, cells from xeroderma pigmentosum complementation group C produced only slightly more procollagenase than normal cells. 12-O-tetradecanoylphorbol-13-acetate also induced higher than normal levels of procollagenase in some ataxia-telangiectasia and xeroderma pigmentosum strains, but less than that induced by trifluoperazine. Because increased extracellular accumulation of matrix-degrading enzymes has long been implicated in metastatic progression, this altered expression of procollagenase and prostromelysin in ataxia-telangiectasia and xeroderma pigmentosum cells could play an important role in the pathogenesis of various tumors in individuals with these genetic diseases. This work was supported by the Office of Health and Environmental Research, U. S. Department of Energy (contract DE-AC03-76-SF01012) (J. A., J. P. M.) and by a Fellowship in Medical Research from the A. P. Giannini/Bank of America Foundation (J. A.). A summary of these results has appeared previously in abstract form (1).     

Focal activation of a mutant allele defines the role of stem cells in mosaic skin disorders

Stem cells are crucial for the formation and maintenance of tissues and organs. The role of stem cells in the pathogenesis of mosaic skin disorders remains unclear. To study the molecular and cellular basis of mosaicism, we established a mouse model for the autosomal-dominant skin blistering disorder, epidermolytic hyperkeratosis (MIM 113800), which is caused by mutations in either keratin K1 or K10. This genetic model allows activation of a somatic K10 mutation in epidermal stem cells in a spatially and temporally controlled manner using an inducible Cre recombinase. Our results indicate that lack of selective pressure against certain mutations in epidermal stem cells leads to mosaic phenotypes. This finding has important implications for the development of new strategies for somatic gene therapy of dominant genodermatoses.     

In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors

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.     

Ex vivo gene therapy cures a blistering skin disease

A recent publication that describes gene therapy treatment of a patient with an inherited blistering skin disease, epidermolysis bullosa, demonstrates for the first time that gene therapy can cure a disease of solid tissue. The treatment relies on ex vivo transduction of autologous epidermal stem cells with a normal copy of the defective gene, followed by reconstitution of the patient's skin with epithelial sheets that are grown from these genetically corrected cells. This approach holds promise for treatment not only of inherited disorders of the skin but also of other solid tissues that are becoming amenable to tissue engineering.     

After sun reversal of DNA damage: enhancing skin repair

UV-induced DNA damage has been directly linked to skin cancer, and DNA repair is an important protection against this neoplasm. This is illustrated by the genetic disease xeroderma pigmentosum wherein a serious defect in DNA repair of cyclobutane pyrimidine dimers dramatically increases the rate of skin cancer. In other instances in which skin cancer rates are elevated, deficits in DNA repair may also be one of the causal factors. For example, solid organ transplant patients have elevated rates of skin cancer that are correlated with the dose and length of exposure to immunosuppressive drugs (predominantly cyclosporine A (CsA) and ascomycin (FK506)-related tacrolimus). We have found that treatment of cultured epidermal cells with CsA or ascomycin inhibits their removal of DNA damage by about 20% at 24 h. In a further example, people with a polymorphism in the DNA repair gene 8-oxo-guanine glycosylase (OGG1) have an increased risk of skin cancer. We have found that the cells with this variant polymorphism have an increased sensitivity of about 20% to a broad range of cytotoxic agents. The DNA deficits caused by immunosuppressive drugs or the OGG1 polymorphism can be overcome by the delivery of DNA repair enzymes in liposomes. The data suggests that deficits in DNA repair, even if they are not as severe as in the case of XP, may contribute to increased rates of cancer, and that topical therapy with DNA repair enzymes may be a promising avenue for after-sun protection.     

Epidermal stem cells - role in normal, wounded and pathological psoriatic and cancer skin

In this review we focus on epidermal stem cells in the normal regeneration of the skin as well as in wounded and psoriatic skin. Furthermore, we discuss current data supporting the idea of cancer stem cells in the pathogenesis of skin carcinoma and malignant melanoma. Epidermal stem cells present in the basal layer of the interfollicular epidermis and in the bulge region of the hair follicle play a critical role for normal tissue maintenance. In wound healing, multipotent epidermal stem cells contribute to re-epithelization. It is possible that defects in growth control of either epidermal stem cells or transit amplifying cells constitute a primary pathogenetic factor in the epidermal hyperproliferation seen in psoriasis. In cutaneous malignancies mounting evidence supports a stem cell origin in skin carcinoma and malignant melanoma and a possible existence of cancer stem cells.     

Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells

The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles. Cultured keratinocyte stem cells, known as holoclones, generate sheets of epithelium used to restore severe skin, mucosal and corneal defects. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder. Epidermal stem cells from an adult patient affected by LAM5-beta3-deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-beta3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up (1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease.     

Retrovirus-mediated DNA repair gene transfer into xeroderma pigmentosum cells: Perspectives for a gene therapy

The rare hereditary disease xeroderma pigmentosum (XP) is clinically characterized by extreme sun sensitivity and an increased predisposition for developing skin cancer. Cultured cells from XP patients exhibit hypersensitivity to ultraviolet (UV) radiation due to the defect in nucleotide excision repair (NER), and other cellular abnormalities. Seven genes identified in the classical XP forms, XPA to XPG, are involved in the NER pathway. In view of developing a strategy of gene therapy for XP, we devised recombinant retrovirus-carrying DNA repair genes for transfer and stable expression of these genes in cells from XP patients. Results showed that these retroviruses are efficient tools for transducing XP fibroblasts and correcting repair-defective cellular phenotypes by recovering normal UV survival, unscheduled DNA synthesis, and RNA synthesis after UV irradiation, and also other cellular abnormalities resulting from NER defects. These results imply that the first step of cellular gene therapy might be accomplished successfully.     

Nucleotide excision repair and cancer

Nucleotide excision repair (NER) is the most versatile and best studied DNA repair system in humans. NER can repair a variety of bulky DNA damages including UV-light induced DNA photoproducts. NER consists of a multistep process in which the DNA lesion is recognized and demarcated by DNA unwinding. Then, a ~28 bp DNA damage containing oligonucleotide is excised followed by gap filling using the undamaged DNA strand as a template. The consequences of defective NER are demonstrated by three rare autosomal-rezessive NER-defective syndromes: xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). XP patients show severe sun sensitivity, freckling in sun exposed skin, and develop skin cancers already during childhood. CS patients exhibit sun sensitivity, severe neurologic abnormalities, and cachectic dwarfism. Clinical symptoms of TTD patients include sun sensitivity, freckling in sun exposed skin areas, and brittle sulfur-deficient hair. In contrast to XP patients, CS and TTD patients are not skin cancer prone. Studying these syndromes can increase the knowledge of skin cancer development including cutaneous melanoma as well as basal and squamous cell carcinoma in general that may lead to new preventional and therapeutic anticancer strategies in the normal population.     

Poly(ADP-ribose)-synthesis and excision repair in light sensitive skin disorders

Several data suggest a relationship of poly(ADP-ribose) (PAR) synthesis to DNA repair and the influence of some trace elements on the semiconservative and unscheduled DNA synthesis (UDS). Previously we found certain alterations in the UV-light induced UDS and in the contents of trace elements in the lymphocytes of patients with light sensitive skin disorders. In the recent study in polymorphic light eruption, cutaneous porphyrias and xeroderma pigmentosum the PAR synthesis and zinc, copper and manganese contents in the chromatin of the lymphocytes (measured by neutron activation analysis) were investigated. UV induced PAR synthesis was generally lower in the cells of polymorphic light eruption and especially in xeroderma pigmentosum with a reduced repair capacity whereas in cutaneous porphyrias no difference was observed. Some correlations occurred between the contents of trace elements studied and UDS as well in each group tested. It seems that PAR investigations throw new light upon our understanding of the pathomechanism of photodermatoses.     

Inhibition of p38 MAPK facilitates ex vivo expansion of skin epithelial progenitor cells

Ex vivo expansion of skin epithelial stem cells has long attracted great interest because of the potential utilization in transplantation and gene therapy. The use of cultured stem or progenitor cells was limited by the lack of applicable culturing system with both satisfactory expansion efficacy and well suppressed differentiation ex vivo. The p38 mitogen-activated protein kinase (MAPK) pathways are responsible for cell growth and differentiation process. We investigated the function of p38 inhibitor SB203580 in the ex vivo expansion of skin epithelial progenitor cells by comparing media with or without addition of this inhibitor. Our results showed that the culturing medium with murine 3T3 feeder layers added with 10 μM SB203580 was more effective in promoting clonal growth of human skin epithelial progenitors or stem cells than the conventional medium without SB203580. The clone initial day in cells treated with 10 μM SB203580 came 2 d earlier with higher colony formation efficiency. The skin epithelial progenitor cells treated with 10 μM SB203580 formed clones that were uniformly smaller in size, longer in sustained proliferation, shorter in clone doubling time, higher in S-phase cells percentage, and lower in levels of differentiation markers such as K10 along with higher levels of stem-cell-associated markers such as p63, K15, and ABCG2 than those cultured in the conventional medium. Collectively, these results indicate that the p38 MAPK pathways inhibitor SB203580 can be used as a culture medium additive that helps to achieve more effective ex vivo expansion of skin epithelial progenitor cells.     

Inhibition of protein synthesis does not antagonize induction of UV-induced sister-chromatid exchange in xeroderma pigmentosum cells

Cycloheximide strongly antagonizes the induction of sister-chromatid exchanges by ethyl methanesulfonate or mitomycin C in human skin fibroblast and xeroderma pigmentosum cells (group A). Analogous behavior has been observed in several other species including Chinese hamster and plant cells. This report documents an exception to that pattern: cycloheximide fails to antagonize UV-induced sister chromatid exchange in xeroderma pigmentosum cells, whereas it does in normal human skin fibroblast cells. A genetic defect in these cells is postulated to alter the UV-mediated DNA recombination process.     

Dangerous entanglements

The promise of stem-cell therapy is long-term repair of damaged organs by transplantation. Inoculum size might be a determinant of successful repair, and efforts are being made to improve conditions for ex vivo expansion of stem cells prior to transplantation. A recent article by Damelin and colleagues raised concerns about this approach, demonstrating that stem cells lack the decatenation G2 checkpoint that preserves genetic stability. Cells that fail to disentangle chromatids prior to mitosis are prone to the types of chromosomal aberrations that are observed in cancer cells. Ex vivo expansion of stem cells might have the unintended consequence of encouraging malignant progression.     

Low synthesis of retinoic acid due to impaired cytochrome P450 1a1 expression in mouse xeroderma pigmentosum fibroblasts

New tumor formation was suppressed by retinoic acid (RA) administration in xeroderma pigmentosum (XP) patients who have a defect in nuclear excision repair. However, the inhibition is not due to enhanced removal of UV-damaged DNA. These results prompted us to investigate whether or not RA metabolism is abnormal in XP fibroblasts and what the underlying mechanism is. Compared with wild type fibroblasts, low activities of RA synthesis were determined on HPLC in mouse fibroblasts lacking XP group A (XPA) gene and UV-induced XPA deficient cancer cells. Moreover, we observed an impaired expression of cytochrome P450 1a1 in XPA deficient fibroblasts by RT-PCR and a decreased expression of retinoic acid receptor gamma in XPA deficient cancer cells by Western blotting. Finally, pre-treatment of RA isoforms significantly protected the XPA deficient fibroblasts from UV-induced death. These results suggest that decreased structure activity of RA synthesis, resulting from impaired mRNA expression of cytochrome P450 1a1 may, at least together with UV irradiation, involve in skin carcinogenesis in XP patients.     

Hypersensitivity of xeroderma pigmentosum cells to dietary carcinogens

Xeroderma pigmentosum patients, in addition to ultraviolet-induced skin cancers, have an increased prevalence of neoplasms occurring in sites shielded from ultraviolet radiation. We postulated that these internal neoplasms might be related to ingestion of dietary carcinogens. As model dietary carcinogens, we studied the tryptophan pyrolysis products, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). These dietary compounds bind to DNA and are highly mutagenic and carcinogenic. Cytotoxicity of these compounds was examined in cultured lymphoblastoid cell lines from xeroderma pigmentosum patients in complementation groups A, B, C, D and E and the variant form and from normal donors. All xeroderma pigmentosum lymphoblastoid cell lines showed a greater reduction in viable cell concentration than the 2 normal lymphoblastoid cell lines following addition of Trp-P-1 or Trp-P-2 (5 micrograms/ml) to the culture medium. Possible differences in cellular activation of these compounds were overcome by treating the cells with rat-liver microsome-activated Trp-P-2. There was a greater reduction in viable cell concentration in the xeroderma pigmentosum group A and D cells than in the normal lymphoblastoid cell lines after treatment with activated Trp-P-2. These data suggest that the xeroderma pigmentosum DNA-repair system is defective in repairing Trp-P-1 and Trp-P-2 induced DNA damage in addition to being defective in repairing ultraviolet-induced DNA damage. Thus xeroderma pigmentosum patients may be at increased risk of toxicity from some dietary carcinogens.