Cutaneous gene therapy: the graft takes

Prospects of ex vivo cutaneous gene therapy rely on stable corrective gene transfer in epidermal stem cells followed by engraftment of corrected cells in patients. In the case of cancer prone genodermatoses, such as xeroderma pigmentosum, cells that received the corrective gene must be selected. However, this step is potentially harmful and can increase risks of immune rejection of grafts. These obstacles have recently been overcome thanks to the labeling of genetically modified stem cells using a small epidermal protein naturally absent in stem cells. This approach was shown to be respectful of the fate of epidermal stem cells that retained full growth and differentiation capacities, as well as their potential to regenerate normal human skin when grafted in a mouse model in the long term. These progresses now open realistic avenues towards ex vivo cutaneous gene therapy of cancer prone genodermatoses such as xeroderma pigmentosum. However, major technical improvements are still necessary to preserve skin appendages which would contribute to aesthetic features and comfort of patients.     

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 correction of Huntington's disease phenotypes in induced pluripotent stem cells

Huntington's disease (HD) is caused by a CAG expansion in the huntingtin gene. Expansion of the polyglutamine tract in the huntingtin protein results in massive cell death in the striatum of HD patients. We report that human induced pluripotent stem cells (iPSCs) derived from HD patient fibroblasts can be corrected by the replacement of the expanded CAG repeat with a normal repeat using homologous recombination, and that the correction persists in iPSC differentiation into DARPP-32-positive neurons in vitro and in vivo. Further, correction of the HD-iPSCs normalized pathogenic HD signaling pathways (cadherin, TGF-β, BDNF, and caspase activation) and reversed disease phenotypes such as susceptibility to cell death and altered mitochondrial bioenergetics in neural stem cells. The ability to make patient-specific, genetically corrected iPSCs from HD patients will provide relevant disease models in identical genetic backgrounds and is a critical step for the eventual use of these cells in cell replacement therapy.     

Hematopoietic stem cell gene therapy for Niemann-Pick disease and other lysosomal storage diseases

Of the various gene therapy approaches under investigation for the treatment of genetic diseases, hematopoietic stem cell-mediated gene therapy has attracted the most interest. Enriched populations of hematopoietic stem cells can be obtained from diseased individuals, genetically modified to express normal gene products, and then transplanted back into these individuals without the risk of graft versus host disease. Following transplantation and engraftment, hematopoietically-derived cells can repopulate various sites of pathology and express the normal gene product in vivo. Such a procedure has been accomplished in several mouse models of human genetics diseases, leading to partial or complete correction of the disease phenotype, and current efforts are now focused on adapting the success of murine systems to larger animals, including man. This review will focus on the use of hematopoietic stem cell-mediated gene therapy for the treatment of lysosomal storage disorders, and discuss recent data obtained in the laboratory using a murine knock-out mouse model of Types A and B Niemann-Pick disease (NPD).     

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.     

Metabolic correction of congenital erythropoietic porphyria with iPSCs free of reprogramming factors

Congenital erythropoietic porphyria (CEP) is due to a deficiency in the enzymatic activity of uroporphyrinogen III synthase (UROS); such a deficiency leads to porphyrin accumulation and results in skin lesions and hemolytic anemia. CEP is a candidate for retrolentivirus-mediated gene therapy, but recent reports of insertional leukemogenesis underscore the need for safer methods. The discovery of induced pluripotent stem cells (iPSCs) has opened up new horizons in gene therapy because it might overcome the difficulty of obtaining sufficient amounts of autologous hematopoietic stem cells for transplantation and the risk of genotoxicity. In this study, we isolated keratinocytes from a CEP-affected individual and generated iPSCs with two excisable lentiviral vectors. Gene correction of CEP-derived iPSCs was obtained by lentiviral transduction of a therapeutic vector containing UROS cDNA under the control of an erythroid-specific promoter shielded by insulators. One iPSC clone, free of reprogramming genes, was obtained with a single proviral integration of the therapeutic vector in a genomic safe region. Metabolic correction of erythroblasts derived from iPSC clones was demonstrated by the disappearance of fluorocytes. This study reports the feasibility of porphyria gene therapy with the use of iPSCs.     

A cutaneous gene therapy approach to human leptin deficiencies: correction of the murine ob/ob phenotype using leptin-targeted keratinocyte grafts.

Leptin deficiency produces a phenotype of obesity, diabetes, and infertility in the ob/ob mouse. In humans, leptin deficiency occurs in some cases of congenital obesity and in lipodystrophic disorders characterized by reduced adipose tissue and insulin resistance. Cutaneous gene therapy is considered an attractive potential method to correct circulating protein deficiencies, since gene-transferred human keratinocytes can produce and secrete gene products with systemic action. However, no studies showing correction of a systemic defect have been reported. We report the successful correction of leptin deficiency using cutaneous gene therapy in the ob/ob mouse model. As a feasibility approach, skin explants from transgenic mice overexpressing leptin were grafted on immunodeficient ob/ob mice. One month later, recipient mice reached body weight values of lean animals. Other biochemical and clinical parameters were also normalized. In a second human gene therapy approach, a retroviral vector encoding both leptin and EGFP cDNAs was used to transduce HK and, epithelial grafts enriched in high leptin-producing HK were transplanted to immunosuppressed ob/ob mice. HK-derived leptin induced body weight reduction after a drop in blood glucose and food intake. Leptin replacement through genetically engineered HK grafts provides a valuable therapeutic alternative for permanent treatment of human leptin deficiency conditions.     

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.     

Successful gene therapy of mice with congenital erythropoietic porphyria

Porphyrias are a group of disorders due to a genetic deficiency in one of the heme biosynthetic pathway enzymes. Congenital erythropoietic porphyria (CEP) is the most severe type characterized by a deficiency in uroporphyrinogen III synthase (UROS) activity. Bone marrow transplantation represents a curative treatment for patients, as long as human leucocyte antigen-compatible donor is available. We used a recently obtained murine model to check the feasibility of gene therapy in this disease. Lentivirus-mediated transfer of the human UROS cDNA into hematopoietic stem cells (HSCs) from Uros(mut 248) mice resulted in a complete and long-term enzymatic, metabolic and phenotypic correction of the disease, favored by a survival advantage of corrected red blood cells. These results demonstrate for the first time that the cure of this mouse model of CEP at moderate transduction level supports the proof of concept of a gene therapy in this disease by transplantation of genetically modified HSCs.     

Multiple classes of stem cells in cutaneous epithelium: a lineage analysis of adult mouse skin

Continuous renewal of the epidermis and its appendages throughout life depends on the proliferation of a distinct population of cells called stem cells. We have used in situ retrovirus-mediated gene transfer to genetically mark cutaneous epithelial stem cells of adolescent mice, and have followed the fate of the marked progeny after at least 37 epidermal turnovers and five cycles of depilation-induced hair growth. Histological examination of serial sections of labeled pilosebaceous units demonstrated a complex cell lineage. In most instances, labeled cells were confined to one or more follicular compartments or solely to sebaceous glands. Labeled keratinocytes in interfollicular epidermis were confined to distinct columnar units representing epidermal proliferative units. The contribution of hair follicles to the epidermis was limited to a small rim of epidermis at the margin of the follicle, indicating that long term maintenance of interfollicular epidermis was independent of follicle-derived cells. Our results indicate the presence of multiple stem cells in cutaneous epithelium, some with restricted lineages in the absence of major injury.     

Recent advances in gene therapy and future prospects

Gene therapy, which is treatment of diseases by introducing normal genes into the body, is becoming feasible as the result of advances in genetic engineering. The hematopoietic stem cells have been considered as the appropriate target for gene transfer in many genetic diseases for which allogeneic bone marrow transplantation has been employed successfully. However, there are still many problems to be solved. In particular, expression from retrovirally transduced genes in bone marrow cells has been transient and unstable. On the other hand, an alternative approach to somatic cell gene therapy using nonhematopoietic cells, including skin fibroblasts, endothelial cells, keratinocytes, and lymphocytes, has been shown to possess several advantages. This kind of approach is usually applied to supplementation therapy in not only hereditary disorders but also various acquired diseases, such as cancer or infectious diseases. Recently, clinical application of gene transfer into lymphocytes to treat cancer and immunodeficiency have been approved at NIH (USA). The trial could represent the start of a new era in molecular medicine.     

Efficient non-viral transfection of adult neural stem/progenitor cells, without affecting viability, proliferation or differentiation

BACKGROUND: Neurogenesis occurs in defined areas of the adult mammalian brain, including the dentate gyrus of the hippocampus. Rat neural stem/progenitor cells isolated from this region retain their multipotency in vitro and in vivo after grafting into the adult brain. Molecular signalling and lineage selection in these cells may be examined using genetic manipulation. However, valid analysis requires that this manipulation should not affect cellular viability, proliferation or differentiation. METHODS: We screened several transfection protocols to develop a method which met these criteria. We then tested the effects of transfection on viability, proliferation and differentiation into the three neural lineages: neurons, astrocytes and oligodendrocytes. RESULTS: In initial testing, ExGen500 and FuGene6 efficiently transfected adult neural stem/progenitor cells, in vitro. After optimisation, these agents transfected 16% and 11% of cells, respectively. FuGene6-treated cells did not differ from untransfected cells in their viability or rate of proliferation, whereas these characteristics were significantly reduced following ExGen500 transfection. Importantly, neither agent affected the pattern of differentiation following transfection. Both agents could be used to genetically label cells, and track their differentiation into the three neural lineages, after grafting onto ex vivo organotypic hippocampal slice cultures. CONCLUSIONS: These data demonstrate that non-viral transfection may be used to genetically manipulate neural stem/progenitor cells, without adversely affecting their growth or perturbing lineage selection. Such a method is valuable for examining the molecular mechanisms of cell fate determination in vitro. Furthermore, this protocol may be exploited in the development of cell-based gene therapy strategies.     

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.     

Normal development, wound healing, and adenovirus susceptibility in beta5-deficient mice

Integrins have been shown to play important roles in embryonic development, wound healing, metastasis, and other biological processes. alphavbeta5 is a receptor for RGD-containing extracellular matrix proteins that has been suggested to be important in cutaneous wound healing and adenovirus infection. To examine the in vivo function of this receptor, we have generated mice lacking beta5 expression, using homologous recombination in embryonic stem cells. Mice homozygous for a null mutation of the beta5 subunit gene develop, grow, and reproduce normally. Keratinocytes harvested from beta5(-/-) mice demonstrate impaired migration on and adhesion to the alphavbeta5 ligand, vitronectin. However, the rate of healing of cutaneous wounds is not different in beta5(-/-) and beta5(+/+) mice. Furthermore, keratinocytes and airway epithelial cells obtained from null mice show adenovirus infection efficiency equal to that from wild-type mice. These data suggest that alphavbeta5 is not essential for normal development, reproduction, adenovirus infection, or the healing of cutaneous wounds.     

Capturing epidermal stemness for regenerative medicine

The skin is privileged because several skin-derived stem cells (epithelial stem cells from epidermis and its appendages, mesenchymal stem cells from dermis and subcutis, melanocyte stem cells) can be efficiently captured for therapeutic use. Main indications remain the permanent coverage of extensive third degree burns and healing of chronic cutaneous wounds, but recent advances in gene therapy technology open the door to the treatment of disabling inherited skin diseases with genetically corrected keratinocyte stem cells. Therapeutic skin stem cells that were initially cultured in research or hospital laboratories must be produced according strict regulatory guidelines, which ensure patients and medical teams that the medicinal cell products are safe, of constant quality and manufactured according to state-of-the art technology. Nonetheless, it does not warrant clinical efficacy and permanent engraftment of autologous stem cells remains variable. There are many challenges ahead to improve efficacy among which to keep telomere-dependent senescence and telomere-independent senescence (clonal conversion) to a minimum in cell culture and to understand the cellular and molecular mechanisms implicated in engraftment. Finally, medicinal stem cells are expansive to produce and reimbursement of costs by health insurances is a major concern in many countries.     

Effective gene therapy of mice with congenital erythropoietic porphyria is facilitated by a survival advantage of corrected erythroid cells

Achieving long-term expression of a therapeutic gene in a given hematopoietic lineage remains an important goal of gene therapy. Congenital erythropoietic porphyria (CEP) is a severe autosomal-recessive disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. We used a recently obtained murine model to check the feasibility of gene therapy in this disease. Lentivirus-mediated transfer of the human UROS cDNA into hematopoietic stem cells (HSCs) from Uros^mut248 mice resulted in a complete and long-term enzymatic, metabolic, and phenotypic correction of the disease, favored by a survival advantage of corrected red blood cells. These results demonstrate that the cure of this mouse model of CEP at a moderate transduction level supports the proof of concept of a gene therapy in this disease by transplantation of genetically modified hematopoietic stem cells.     

Correction of Steroid Sulfatase Deficiency by Gene Transfer into Basal Cells of Tissue-Cultured Epidermis from Patients with Recessive X-Linked Ichthyosis

To develop an experimental model for somatic gene therapy we have tried to correct the steroid sulfatase (STS) deficiency in tissue-cultured primary epidermal keratinocytes from patients suffering from recessive X-linked ichthyosis. An efficient Epstein-Barr virus-based vector was constructed, in which full-length steroid sulfatase cDNA is located between an SV40 early promotor and processing signals. After STS gene transfer into cultured basal cells from ichthyotic skin, the cells produce large amounts of enzymatically active steroid sulfatase protein. The subpopulation of transfected cells can be made to produce approximately 100 times more STS activity than normal keratinocytes. Keratinocytes from patients suffering from recessive X-linked ichthyosis display an abnormal phenotype when developing a multilayered tissue in culture: Initially an extensive burst of keratinization is observed, followed by rapid, premature shedding and degradation of most suprabasal cell layers, leaving a culture with hyperproliferative relatively immature keratinocytes. Transfection of these immature ichthyotic cells with the functional STS construct led to an increase in the amount of retained cell material in the culture medium, indicating an increased cell maturation. It is possible to genetically label individual transfected epidermal cells with a reporter gene. Cotransfection experiments with STS and reporter gene vectors show that the cohort of transfected cells had a tendency to develop less rapidly since they became overrepresented in the smaller size classes at the same time the total population was somewhat shifted toward higher cell sizes. We interpret these results as an indication that restoration of the enzymatic activity induces a more normal maturation of the transfected keratinocytes.     

Long-term cure of the photosensitivity of murine erythropoietic protoporphyria by preselective gene therapy.

Definitive cure of an animal model of a human disease by gene transfer into hematopoietic stem cells has not yet been accomplished in the absence of spontaneous in vivo selection for transduced cells. Erythropoietic protoporphyria is a genetic disease in which ferrochelatase is defective. Protoporphyrin accumulates in erythrocytes, leaks into the plasma and results in severe skin photosensitivity. Using a mouse model of erythropoietic protoporphyria, we demonstrate here that ex vivo preselection of hematopoietic stem cells transduced with a polycistronic retrovirus expressing both human ferrochelatase and green fluorescent protein results in complete and long-term correction of skin photosensitivity in all transplanted mice.