Gene transfer into human epidermis as an experimental model for somatic gene therapy

This review has been accepted as a thesis together with seven previously published papers, by the University of Aarhus, November 17, 2003, and defended on February 20, 2004.

Department of Human Genetics, Faculty of Health Sciences, University of Aarhus.

Correspondence: Thomas G. Jensen, Egeve 2, DK 8541 Skødstrup.

Official opponents: Finn Skou Pedersen, Lic.scient, and Finn Cilius Nielsen, consultant, MD.

Dan Med Bull 2004;51:155-66.

2. Gene transfer into keratinocytes

2.1. Initial studies of gene transfer
into keratinocytes

In 1987 Howard Green and co-workers reported retroviral mediated gene transfer of the human growth hormone (hGH) gene into primary human keratinocytes (19). Although the protein was synthesized in tissue culture, it could not be detected in the blood after transplantation onto mice. Two years later Taichman and co-workers reported non-viral transfection of primary human keratinocytes with a chloramphenicol acetyltransferase (CAT) reporter gene (20). Size fractionation of the transfected cells revealed that the transgene was expressed at highest levels in suprabasal cells, even though equal copy numbers of the transgenes were found in basal and suprabasal cells.

In 1990 we reported selective transfection of epidermal basal cells (I). We took advantage of the observation that after incubation of multilayered epidermal cultures in Ca 2+ - free media for three days all suprabasal layers can be removed (22, 23). These "calcium-stripped" cultures are able to regenerate the suprabasal layers, thus constituting an in vitro model for wound healing. We showed that after calcium stripping the remaining monolayer of basal cells can be efficiently transfected, and thereafter regenerate the multilayered tissue (I). Gene transfer with a β -galactosidase reporter gene into keratinocytes was used to label individual keratinocytes. The proportion of b-galactosidase positive cells was maximal one day after transfection, declined from day one to day two post-transfection, but then remained relatively stable (eight days post-transfection approximately 0.5% of the cells were still positive). After regeneration of the calcium-stripped cultures for 4 days, transfected cultures were found to be multilayered with approximately five layers. Both the histology of the transfected cultures and the size of the labeled cells compared to the unlabeled cells suggest that transfection of basal cells mainly results in marking of a cohort of cells entering the differentiation program.

In order to stimulate the proliferation of the undifferentiated, transfected cells, we investigated the use of the tumor promotor TPA, which in mouse skin has been described to stimulate proliferation of immature keratinocytes (24, 25). We found that incubation of transfected cultures with TPA increased the proportion of labeled cells, and size measurements showed that this was due preferentially to a relative expansion of small transfected cells (I). Still, it turned out that it was not possible to use TPA to further expand the population of cells expressing the transfected genes.

In order to increase the duration of the gene expression Epstein-Barr Virus (EBV) - based expression vectors were used in our initial transfection experiments. These vectors are relatively simple plasmids containing oriP sequences and the EBNA-1 gene from EBV. In human cells EBV-based expression vectors replicate as episomes and contain sequences conferring nuclear retention and thus stability (26-28). We modified the vectors from the original versions by inserting polylinker sequences and termination sequences (29) ( Figure 1 ). Although autonomous replication of EBV-based expression vectors in primary keratinocytes was difficult to show directly, we found that vectors containing EBV sequences lead to a more stable expression compared to vectors without EBV sequences (30).

2.2. Effects of the transfection
on primary human keratinocytes

Several investigators reported that differentiation of genetically labeled keratinocytes occurs in vitro (20, 21, 31) as well as in vivo (III, 33-34). One possibility is that the differentiation process is induced by the transgene itself; alternatively that a subpopulation of keratinocytes committed to differentiate is more susceptible to "productive transfection" (transgene expression after gene transfer). We found that transfection of primary human keratinocytes leads to cell cycle arrest of productively transfected cells, which is not due to the transgene expression per se, but probably due to an indirect effect of the exogeneous DNA (30).

2.3. Non-viral epidermal gene transfer

As seen in Table 1 and exemplified in Figure 2 numerous methods for efficient gene transfer into human epidermis have been developed during the last decade. For clinical applications direct in vivo gene transfer into the skin has several advantages due to the difficulties associated with cell culture and transplantation techniques. Surprisingly, Hengge et al discovered that simple injection of naked DNA intracutaneously leads to significant gene expression (35). Although the DNA is injected below the basement membrane, the expression is highest in the epidermal layer. The efficiency of DNA injection can be enhanced by electroporation. Use of a "gene gun" is another in vivo gene transfer method that has been used extensively for direct DNA transfer into the skin. This method is efficient for short time applications (33, 36, 37) and can be used for both mouse and human skin (Jensen UB, personal communication). Topical application of naked DNA or liposomes likewise leads to gene expression in the skin, but the expression is at low levels and of a short duration (36, 38).

A limitation of all the methods based on non-viral gene transfer into the skin is the absence of persistent and/or therapeutic gene expression because the vector genome does not integrate efficiently. To increase the integration frequency Khavari and co-workers have successfully used the C31 bacteriophage integrase which stably integrates large DNA sequences containing a specific 285-base-pair attB sequence into genomic "pseudo- attP sites" (39). This system was used to complement the genetic defect causing the skin disease dystrophic epidermolysis bullosa. Phenotypic correction was documented in vivo after grafting genetically modified keratinocytes onto immunodeficient mice.

2.4. Epidermal gene transfer using viral vectors

Several types of viral vectors have been used for keratinocyte gene transfer such as vectors based on murine retrovirus, lentivirus, adenovirus and AAV (Table 1). Keratinocyte stem cell targeting by retroviral vectors is indicated by prolonged gene expression, which is especially seen after grafting the cultures onto mice, and by the observation that individual retroviral transduced keratinocyte can yield more than 10 9 cells (40-44). Retroviral transduced keratinocytes have no apparent growth disadvantage in vitro (31), and as we have showed, efficient transduction is possible using several different retroviral envelopes (45).

Gene transfer using optimized murine retroviral vectors can lead to transduction frequencies approaching 100%, making it unnecessary to select for transduced cells in vitro (IV, 47). Using these highly efficient retroviral vectors the transgene expression and copy number in the keratinocytes can be even further increased by employing a centrifugation technique for the transduction ( Figure 3 ).

Taichman's group described that murine retroviral vectors can be used to obtain permanent expression in the skin on mice after in vivo gene transfer although this vector type only targets dividing cells (48). By creating a superficial skin wound keratinocyte proliferation was induced, and by using the wound crust to retain the virus suspension, conditions allowing transduction of proliferating epidermal cells were established. Since stem cells divide slowly in vivo and thus are difficult to target with murine retroviral vectors, several groups are currently investigating the use of lentiviral vectors for in vivo gene transfer to the skin (49-51).

2.5. Combined non-viral and viral epidermal
gene transfer

We have shown that after DNA-mediated gene transfer into primary human keratinocytes, it is possible to achieve production of retroviral vectors, leading to the transduction of co-cultured keratinocytes and prolonged green fluorescence protein (GFP) reporter gene expression (VI). The described method may be regarded as a first step in a strategy to generate retroviral producer cells in situ in the skin. Furthermore, the method can be used for rapid analysis of possible effects of transgenes in cultured human keratinocytes without preparatory retroviral vector production in packaging cell lines. We attempted to use the described technique in vivo after ballistic gene transfer or injection of naked DNA. Unfortunately, it was not possible to detect prolonged gene expression after gene transfer into mouse skin, which suggests that the efficiency of stem cell targeting in this model was too low. Preliminary results obtained by combining the method with topical selection for genetically modified cells in vivo in pig skin seem more promising (Pfützner W. & Jensen T.G., unpublished data).

2.6. Regulated expression

Regulatable gene expression can be achieved after DNA-mediated transfection by use of both the tetracyclin system and synthetic ligand-driven intracellular heterodimerization to activate a target gene (53, 54). Similar systems allowing regulatable expression have been used in a retroviral context (55, 56). Although gene delivery targeted to epidermis by either in vivo or ex vivo approaches can be enhanced by the availability of epidermis-specific promotors (57) encouraging evidence from experiments aiming at the correction of inherited skin diseases show that post-transcriptional mechanisms can result in correct protein localization in specific epidermal layers. Thus, using Moloney Murine Leukemia Virus Long Terminal Repeats (LTR) sequences known to be active in all layers of human epidermis, expression of the proteins transglutaminase-1 in lamellar ichthyosis (58), and BP180 (59) and laminin 5 b3 in junctional epidermolysis bullosa (60) was limited to the correct basal/suprabasal epidermal layers after grafting the gene modified tissue onto mice.

2.7. In vivo selection

Selection of stably transfected primary human keratinocytes in vitro is very difficult even though high levels of gene expression can be achieved (61), since the cells expressing the transgene cease to proliferate (30). Selection of keratinocytes retrovirally transduced with a resistance gene has been investigated for enhancing gene expression and to circumvent the problem of gene silencing. Ghazizadeh et al were able to demonstrate selection for neomycin transduced keratinocytes in raft cultures (62). More clinically relevant, colchicine treatment increased the percentage of multidrug resistance (MDR) expressing keratinocytes to almost 100% in raft cultures and in vivo on mice (63, 64). After topical selection MDR-transduced keratinocytes were able to proliferate normally and capable of forming a stratified differentiated epidermis.

2.8. Use of immortalized cells

Due to technical problems associated with the use of primary keratinocytes (caused by their limited lifespan), and to circumvent transcriptional downregulation in vivo, it has been investigated whether immortalized human keratinocytes can be used for transgene expression in vivo. Krueger et al showed that in vivo use of keratinocytes immortalized by Human Papilloma Virus (HPV) did result in persistent transgene expression (65). The spontaneous immortalized keratinocyte cell line HaCaT has likewise been used for transgene expression in vivo (66). Primary human keratinocytes can be immortalized by ectopic expression of viral oncogenes such as SV40 T antigen (67) or HPV E6 and/or E7 (68, 69). Another way to bypass senescence is ectopic expression of the human telomerase reverse transcriptase (hTert) gene. Ectopic expression of telomerase has been used to immortalize various human cell types such as skin fibroblasts, endothelial cells and, as we showed, cells from the bone marrow, while growth control and differentiation functions are maintained (70-72). We reported the establishment of Epidermolysis Bullosa Simplex (EBS) keratinocytes with an extended lifespan by telomerase expression (VII). After immortalization the cells retain normal growth and differentiation characteristics such as the ability to generate an epidermis with normal morphology in organ cultures and also a normal karyotype (VII). However, due to increased susceptibility of tumor formation associated with immortalization, the clinical use of these cells, e.g. by grafting, is not acceptable without extensive long term in vivo studies of the fate of the immortalized cells. It may be required to combine the immortalization procedure with a "safety switch" (e.g. based on the Cre-lox system) allowing conditional immortalization of the primary keratinocytes.

2.9. Genetic manipulation of epidermal stem cells

As mentioned above gene transfer into keratinocytes using murine retroviral vectors is very efficient (100% transduction can be achieved in vitro). Fiona Watt suggested that it may be possible not only to target epidermal stem cells but also to manipulate their differentiation program and fate in vivo (74). Molecules promoting exit from the stem cell compartment include c-Myc, β -integrin, MAPKK and a dominant negative β -catenin (55, 75, 76). Epidermal stem cells have higher β -catenin levels than transit amplifying cells, and expression of a stabilized truncated b-catenin increases the fraction of putative stem cells in vitro and cause keratinocytes to revert to a pluripotent state (77). Interestingly, Fuchs and co-workers have shown that a particular b-catenin mutant lacking the transcriptional domain promotes hair follicle morphogenesis when expressed in epidermis, but epidermal fate when expressed in hair cells, which demonstrates differences in the ways cells respond to β -catenin signalling (78). By using epidermal gene transfer it may be possible to stimulate lateral growth of epidermal stem cells or to specifically direct the formation of sebocytes, hair follicle and interfollicular epidermis and in this way enhance the function of epidermal grafts (74).

2.10. Gene repair

Novel strategies are under development for the specific repair of mutations. One strategy that has been used in the skin is based on the use of oligonucleotides composed of RNA and DNA residues that stimulate the correction of single-base mutations in mammalian cells (79, 80). Yoon and co-workers have shown that in cultured melanocytes it was possible to correct a point mutation in the mouse tyrosinase gene resulting in permanent restoration of tyrosinase enzymatic activity, melanin synthesis and pigmentation (81). Furthermore, topical application and intradermal injection of a RNA-DNA oligonucleotide to albino BALB/c mouse skin resulted in dark pigmentation of several hairs in a localized area, and the gene correction was maintained three months after the last application of the chimeric oligonucleotides (82). Gene repair in keratinocytes may be more difficult. Yoon and co-workers described that in contrast to HeLa cells, immortalized keratinocytes (HaCaT cells) or primary human keratinocytes did not show any detectable level of gene conversion (83). As we have discussed, a better understanding of the underlying mechanisms of the gene conversion process may help to increase the targeting frequency (80). Because evidence from mice with an inducible transgenic EBS phenotype (caused by keratin mutant proteins expressed in basal cells) shows that gene corrected keratinocytes from the basal layer have a growth advantage (84), gene therapy strategies aiming at correcting keratin mutations in keratinocytes are indeed worth pursuing. Immortalized patient keratinocytes will be useful as a research tool in the development of methods for the correction of disease-causing mutations in human keratinocytes (VII).

2.11. Topical gene delivery to the skin

Even though gene transfer to keratinocytes in vitro is efficient and allows long term expression, and genes can be expressed efficiently in the skin after direct transfer, there is still progress to be made in the development of topical delivery methods. Topical application of naked DNA or adenoviral vectors leads to cutaneous gene expression, though at low levels and of short duration (38, 85). Liposomes can also be used for topical gene delivery, but the resulting gene expression is mainly localized in hair follicles (86, 87). In contrast to genes, oligonucleotides and peptides can be efficiently transferred to all layers in epidermis after topical application (88-90). A gene delivery system based on peptides anchored to nucleic acids may be a viable strategy for efficient topical gene transfer into epidermis (91). Topical gene transfer combined with efficient integration, as for example catalysed by phage integrases, may fundamentally change the prospects of cutaneous gene therapy.

3. Applications

3.1. Gene therapy of inherited skin diseases

The final step in the differentiation pathway of a keratinocyte is the formation of the cornified envelope which is essential to the barrier function. The envelope consists of a rigid structure made of proteins cross-linked by the enzyme transglutaminase-1. Any defect in genes that regulate the assembly of the envelope can cause skin disorders (92).

In 1993, we reported experiments aiming at genetic complementation of keratinocytes from patients with X-linked ichthyosis (II). Thus we were the first research group to report progress towards gene therapy of inherited skin diseases. X-linked ichthyosis (RXLI), is a scaling disorder due to changes in the lipid composition of the stratum corneum (93). Patients with RXLI lack steroid sulfatase (STS), an enzyme capable of hydrolyzing sulfate esters of various sterols, including cholesterol sulfate (CS) (94, 95). The skin involvement, hyperkeratosis with the presence of dark scaly skin, is the main problem for the patients. However, RXLI is also a systemic metabolic disease associated with corneal opacities, insufficient estrogen synthesis in late pregnancy and an increased risk of development of testicular cancer (96).

In general, hyperkeratosis of the skin can be caused by two mechanisms: hyperproliferation or increased retention of the keratinocytes. It has been reported that keratinocyte proliferation in RXLI patients in vivo is normal (97, 98). In addition, evidence suggest that accumulation of CS in stratum corneum in RXLI patients leads to enhanced stickiness of the corneocytes followed by scaling of the surface of the skin (99). However, the pathological phenotype of patient keratinocytes is not well preserved in vitro. Thus, it was described that cultured keratinocytes from RXLI patients are in a hyperproliferative state and less differentiated than normal controls (100). We reported that initially in cultures of keratinocytes from RXLI patients extensive keratinization were prevailing (II). This phase was followed by shedding and degradation of suprabasal layers, resulting in relatively immature cultures. We hypothesized that the described hyperproliferation represent a secondary reaction to the increased shedding and, thus, to the lack of hyperkeratosis in vitro.

To explore the possibility of performing somatic gene therapy of RXLI we introduced and expressed the STS gene (inserted in EBV-based expression vectors) in keratinocytes from RXLI patients by using lipofection (II). Measurements of the STS protein by western blotting analysis and by enzyme activity assays showed that high STS expression could be achieved with enzyme activity levels exceeding those of normal keratinocytes, although only about 1% of the cells were productively transfected.

Since we were unable to select for the transfected cells, it was not possible to obtain homogeneous cultures of restored cells. Instead we performed a clonal analysis of differentiation by co-transfection with STS and b-galactosidase genes into RXLI keratinocytes. After four days in culture, we measured the cell size of b-galactosidase labeled and unlabeled keratinocytes. We observed that the population of small transfected keratinocytes had expanded, and that the size distribution of the total population had shifted toward higher cell sizes. In addition, we found that STS expression increased the amount of shedded, degradation resistant material (probably representing shedded corneocytes) in media from cultures. These data indicate that the differentiation program has been retarded and, consequently, that a more normal maturation was achieved, although both these types of analyses are indirect.

Later on Khavari's group used retroviral vectors for the delivery of STS genes into RXLI keratinocytes (102). The disease phenotype (both hyperkeratosis and impaired barrier function) was generated after grafting the uncorrected epidermal tissue onto mice. Retroviral mediated transfer of STS genes to the keratinocytes before grafting could restore the enzyme defect and complement the disease phenotype after grafting.

Numerous experiments aiming at the development of gene therapy of inherited skin diseases have been described during the last 10 years ( Table 2 ). Clinical trials are in preparation both in Italy and in USA involving ex vivo transfer of the β 3 gene of laminin-5 to patients with Junctional Epidermolysis Bullosa (JEB) (60, 103). This inherited skin disease is caused by genetic defects in proteins which are important in the adhesion of basal layer cells to the dermis, including the proteins laminin 5, BPAG2, and α 6/ β 4 integrin. Laminin-5 is a major basal lamina component formed by three distinct polypeptides α 3, β 3, and γ 2. Expression of β 3 from the constitutive LTR promoter in β 3 (-) JEB keratinocytes lead to appropriately localized protein expression in vivo after grafting to mice (60). Furthermore, although supra-normal quantities were expressed in culture, in vivo β 3 protein was detected at amounts indistinguishable from normal, probably due to the fact that β 3 is stabilized and associated with the limited quantities of the α 2 and γ 2 chains of laminin-5. Stable transduction of patient stem cells, long term expression in vivo and complete phenotypic correction of mutated keratinocytes combined with the facts that corrected cells are more adherent to the basal lamina and may have a selective advantage in vivo, lead to the proposed clinical trials.

Recently, several research groups have focused on the development of gene therapy of Recessive Dystrophic Epidermolysis Bullosa (RDEB) caused by mutations in the COL7A1 gene that codes for the epidermal adhesion protein collagen VII. Since this large gene is difficult to fit into murine retroviral vectors, a truncated type VII collagen "minigene" was developed and used for retroviral-mediated transfer into patient keratinocytes (104). The entire COL7A1 locus has also been transferred to a RDEB cell line by microinjection (105), and a lentivirus-based minimal gene transfer vector has been developed and used to correct the disease phenotype (51). As mentioned above Khavari's group took advantage of a non-viral approach, based on the use of a bacteriophage integrase, to stably integrate the gene in primary patient keratinocytes, and was able to show normalization of the disease phenotype after grafting of the cells onto immunodeficient mice (39).

3.2. Secretion of proteins

Epidermis is an important source of secreted proteins with both local and systemic effects. In tissue culture keratinocytes secrete a wide variety of proteins encompassing a molecular weight range to over 180 kD including a variety of cytokines and lipoproteins (106). Taichman's group showed that human keratinocytes not only secrete apolipoprotein E in tissue culture, but the protein was also efficiently secreted into the circulation after grafting human keratinocytes onto nude mice (107).

In order to investigate the potential of epidermis to secrete medically relevant proteins, human growth hormone (hGH) has been widely used as a reporter protein (19, 61, 108). As previously mentioned, the first attempts by the group of Howard Green to secrete hGH from keratinocytes into the circulation were unsuccessful (19). It was found that the retroviral transduced primary human keratinocytes did secrete hGH in vitro, but after grafting no hGH could be detected in the circulation. The same group later showed that using a stably transfected human keratinocyte carcinoma cell line it was possible to demonstrate hGH in the circulation of grafted mice (61). However, the transfected cells were not grafted in a normal position, but underneath a mouse skin flap, perhaps facilitating transfer of the protein to the circulation.

We reported in 1994 that it was possible to detect hGH in the circulation from primary human keratinocytes, grafted in a normal position, on immunodeficient (nude) mice (III). We first showed that lipofection of primary human keratinocytes with an EBV-based expression vector containing the hGH gene resulted in a high hGH expression and secretion in vitro. The hormone was biologically active as judged by a receptor binding assay. Four days after grafting the transfected keratinocytes onto nude mice hGH could be detected in mouse serum in five out of five mice. Although the recombinant protein was present at low concentrations, the fact that the hGH half-life in mouse serum is less than 8 minutes implies that the protein was produced and secreted at steady levels. At day 10 after grafting hGH could not be detected in the serum, possible due to the fact that transfected keratinocytes undergoes differentiation after grafting, as observed in parallel experiments with the lacZ gene as reporter.

As shown in Table 3 , several groups have reported the production and secretion of medically relevant proteins from genetically modified epidermal tissue into the blood. These proteins include hGH (22 kD), apolipoprotein E (34 kD), α 1-antitrypsin (56 kD), factor IX (57 kD), factor VIII (170 kD) and various cytokines. Most of these proteins are relatively small. One exception is factor VIII, a large and heavily processed protein. It is cleaved into two chains of approximately 90 kD and 80 kD, both of which must access the vasculature to form a stable circulating protein (109). Factor VIII protein produced from genetically modified keratinocytes was even able to correct the coagulation defect in hemophilia A mice.

3.3. Wound healing

Non-healing wounds, a diverse group of diseases with different pathogenesis and manifestations, represent a major clinical problem. Advances in the understanding of the tissue repair process and its failure at the cellular and molecular levels are essential for the development of improved therapeutic strategies. Wound healing is a dynamic reponse to tissue injury involving interaction between various cell types, extracellular matrix molecules, soluble mediators, and cytokines. The development of recombinant growth factors raised the potential of manipulating the wound microenvironment giving hope for better healing of both acute and cronic wounds (110). In a wide variety of experimental models and clinical trials it has been shown that these peptides can accelerate the wound repair process. However, it has also become clear that delivery of the polypeptides can be a problematic task requiring repeated topical administration for optimal effects (111, 112).

In the first published study investigating the use of gene transfer to accelerate healing of wounds the gene gun was utilized for the gene transfer. In a porcine partial-thickness wound model it was found that expression of human epidermal growth factor accelerated wound repair (113). Since then, several strategies have been investigated with the purpose of promoting wound healing by use of gene transfer, aiming at stimulating the granulation process, increasing vascularisation, enhancing reepithelialisation or improving the scar quality ( Table 4 ).

One of the major regulators of tissue repair in the skin is platelet-derived growth factor (PDGF) (114). This protein (found in the three isoforms AA, AB, and BB dimers) is produced locally at the injury site by several cell types in the skin e.g. keratinocytes, endothelial cells, fibroblasts and smooth muscle cells. After release PDGF attracts monocytes, promotes neovascularization and stimulates dermal fibroblasts to proliferate, migrate and synthesize extracellular matrix components. Skin fibroblasts, unlike keratinocytes, express PDGF-receptors allowing both auto- and paracrine growth responses. Impaired skin repair is associated with reduced expression of both PDGF ligands and their corresponding receptors (115). In addition, topical administration of recombinant PDGF-BB protein promotes granulation tissue and vascular formation (116). Since optimal clinical efficiency of exogenous PDGF therapy requires repeated applications, gene transfer is investigated for the more prolonged delivery of PDGF. Gene transfer resulting in the production of both PDGF A and B has been described to enhance the wound healing process (117, 118). To potentiate the effect of PDGF-mediated skin repair it may be of advantage to make adjacent epidermis sensitive to PDGF stimuli. Thus, we introduced the PDGF-receptor gene to normal epidermal keratinocytes by retroviral mediated gene transfer and used the gene-modified keratinocytes to establish a receptor-positive skin equivalent (119). Our study demonstrated that transgene expression of human PDGF-receptor can be achieved in epidermal keratinocytes by retroviral transduction, and that ligand activation by recombinant PDGF-BB of such gene-modified skin equivalent enhances cell proliferation.

3.4. The skin as a "metabolic sink"

Accumulation of toxic substances can be involved in the pathogenesis of inborn errors of metabolism. One approach to perform somatic gene therapy of these diseases could be to genetically modify cells from the skin in such a way that the cells take up the toxic compound from the circulation and metabolize it (120).

3.4.1. Adenosine deaminase deficiency

In 1987 Palmer et al investigated the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy (121). The genetically modified cells produced 12 fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in blood in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. However, due to inactivation of the retroviral transferred genes the expression was of only a short duration.

Fenjves et al measured the capacity of transduced keratinocytes from ADA patients to metabolize adenosine (122). Patient keratinocytes transduced with an ADA containing retroviral vector deaminated dAdo 5.5 times more efficiently than normal controls. It was calculated that a graft occupying 2% of the total body area had the capacity to metabolize 337 mmol dAdo per day, corresponding to approximately 10% of the daily load. Calculations showed that the main rate-limiting factor for substrate conversion in vivo would be the amount of substrate reaching a skin graft from the blood circulation.

3.4.2. Ornithine aminotransferase deficiency

Another inborn error of metabolism, which could be a candidate disease for the "metabolic sink" approach, is ornithine aminotransferase (OAT) deficiency leading to atrophy of the choroid and retina and a slowly progressive loss of vision. Evidence suggests that it is of therapeutic benefit to decrease the concentration of ornithine in the circulation (123, 124). By using adenoviral-mediated overexpression of OAT in keratinocytes we showed that patient keratinocytes have a large capacity of OAT expression and that ornithine uptake and metabolism can be increased to levels exceeding normal (125). In order to achieve stable OAT expression in keratinocytes, which is required for clinical effect, we developed a retroviral vector containing the OAT gene (IV). We showed that retrovirally transduced patient keratinocytes contained OAT activity 25 to 75 times higher than that of normal keratinocytes, and the ability of transduced patient keratinocytes to eliminate ornithine from media was also higher than that of normal keratinocytes. Aiming at testing the approach on patients a Good Manufacturing Practice (GMP) grade retroviral vector was produced and a clinical trial was planned on the basis of these studies (126). However, due to various reasons, including the tragic death of a participant in another clinical gene therapy project and the subsequent general pause in the initiation of new gene therapy projects, our trial has still not been realized.

3.4.3. Phenylketonuria

Phenylketonuria, PKU, is yet another candidate disease for "metabolic sink" therapy. This disease is caused by deficiency of phenylalanine hydroxylase (PAH) resulting in increased levels of phenylalanine in body fluids. In the Western world, new-borns are screened for hyperphenylalaninemia (HPA) and identified patients are treated with a strict diet low in phenylalanine. Screening for hyperphenylalaninemia combined with a dietary treatment of PKU patients has essentially eliminated PKU as a cause of mental retardation. Despite recommandations of a life-long phenylalanine restricted diet, most adults end their diet since the effect of increased phenylalanine levels is most detrimental in the developing child. However, if a female PKU patient considers pregnancy, it is important that she returns to the diet before she becomes pregnant, since untreated PKU women have a very high risk of bearing offspring with microcephaly and mental retardation (127). Due to the difficulties in following the strict diet for adults, somatic gene therapy is worth investigating as a potential treatment modality for PKU.

In a transgenic mouse model it was shown that muscle specific expression of PAH led to decreased levels of serum phenylalanine when the mice were supplemented with BH 4 , the required co-factor for PAH (128). BH 4 is mainly synthesized in the liver and the rate limiting step in the synthesis is GTP cyclohydrolase I (GTP-CH).

We showed that co-overexpression of the two enzymes PAH and GTP-CH in primary human keratinocytes leads to high levels of phenylalanine clearance (V). Transfer of the two genes into the same cell is not necessary, since co-cultivation of cells transduced separately with PAH and GTP-CH also results in phenylalanine clearance indicating metabolic co-operation between cells overexpressing PAH and cells overexpressing GTP-CH (130). This metabolic co-operation requires direct cell to cell contact since growing the two types of cells in two separate layers with shared incubation medium failed to clear the medium for phenylalanine. Furthermore, adding BH 4 in excess to cells overexpressing PAH does not lead to high levels of phenylalanine clearance - which shows that only a small amount of the cofactor is taken up by the cells or that the cofactor is taken up in an oxidized form requiring further metabolism. Thus, BH 4 synthesized by keratinocytes overexpressing GTP-CH is probably transported through gap junctions. We showed that primary human keratinocytes overexpressing PAH and GTP-CH are capable of differentiating into a mulilayered tissue when grown in the interface between air and liquid (in raft cultures), and after differentiation the cells continue to clear phenylalanine at a high level (130). Direct comparison between skin fibroblasts and keratinocytes revealed that skin fibroblasts do not have the same high capacity for phenylalanine clearance as do keratinocytes (131).

By adding fresh medium to the keratinocytes every six hours we estimated that keratinocytes overexpressing both PAH and GTP-CH are capable of clearing more than 370 nmol phenylalanine/24 hours/10 6 cells (V). Calculating per cell basis, a graft of 20 cm × 20 cm, containing approximately 2 × 10 9 metabolically active cells (132) should remove more than 0.74 mmol phenylalanine/24 hours. In comparison, the minimal amount of phenylalanine in a diet, i.e. the amount necessary to maintain protein synthesis, is 1.2-3.0 mmol phenylalanine/24 hours (133). Thus, at the current level, the "metabolic sink" could only be supplementary to the diet relieving some of the strict dietary restrictions. Further optimization necessary for a potential clinical usefulness may include: 1) Use of PAH variants with supranormal PAH activity. PAH variants lacking the N-terminal regulatory domain have enhanced ability of forming active tetramers and display higher specific activity compared to the normal enzyme; 2) Stimulation of downstream processes. Tyrosine accumulation may be inhibiting phenylalanine conversion; 3) Stimulation of the transport of phenylalanine into cells by co-expression of phenylalanine transporters.

3.5. Gene transfer into the skin to alter
the immune reSponse

The skin is able to elicit immune responses to dangerous external agents and regulate the intensity of the response (134). Several cell types that are present in the skin microenvironment, including keratinocytes in epidermis and fibroblasts in dermis, have the capacity to secrete cytokines that initiate and control immune responses. Furthermore, the skin has resident epidermal and dermal dendritic cells; a very potent ("professional") antigen-presenting cell (135). Transfected dendritic cells have been shown to present transgenic peptides through both MHC class I- and II- restricted pathways (136, 137). In addition, this cell type is able to take up proteins from transfected neighboring cells in the case of secreted proteins or by phagocytosis of necrotic or apoptotic cells (138, 139). Both of these mechanisms can induce humoral and cellular immune responses. The contribution of non-professional antigen presenting cells to the extent and quality of the immune response is more unclear.

To produce an immune reaction against a foreign protein traditionally requires purification of the protein which is then injected into an animal. Such a response can also be elicited by introducing the gene encoding a protein directly into the skin of mice. Genetic immunization can be used to produce both polyclonal as well as monoclonal antibodies (140). Gene transfer into the skin has elicited effective immune responses against a large variety of different infectious agents and tumor antigens in animal models (reviewed in (141). The immune responses seen after DNA immunization are divided into the helper T-cell type 1 (Th1) and type 2 (Th2) responses. Although both of these can provide immunity to infectious organisms, a Th1 response may be superior against intracellular pathogens, while a Th2 response may be preferred when high titer neutralizing antibodies are required. DNA immunization by intradermal DNA injection generally yields Th1 responses, while the ballistic, topical and traditional protein vaccines produce a Th2 type immune response (reviewed in (142)). The presence of un-methylated cytosine guanine motifs (CpG) in plasmid DNA, known as immunostimulatory sequences (ISS), plays an important role in the initiation of Th1-biased responses (143, 144).

In animal models DNA transfer into the skin can lead to tumor regression by enhancing the tumor-directed immune response (145, 146). In a clinical study involving melanoma patients the gene encoding a foreign major histocompatibility complex protein, HLA-B7, was introduced into HLA-B7-negative patients by injection of DNA-liposome complexes (147, 148). Recombinant HLA-B7 protein was demonstrated in tumor biopsy tissue in all patients, and immune responses to HLA-B7 and autologous tumors could be detected. One patient demonstrated regression of injected nodules on two independent treatments and regression at distant sites. In another strategy, cytokine genes may be delivered directly to a tumor to stimulate destruction by the host immune system (145, 149).

3.6. Other applications

3.6.1. Alopecia

Hair follicles can be targeted by gene transfer creating the possibility of treating hair disorders such as alopecia or hair loss (86, 87). Embryonic development of hair follicles requires the protein Sonic hedgehog (Shh) (150). After adenoviral mediated expression of Shh in the skin of nineteen-day old mice, it was found that the hair follicle size increased and that there was a marked acceleration of the onset of new hair growth in the region of adenovirus administration (151). It was suggested that such a strategy may be beneficial in the treatment of some forms of alopecia associated with chemotherapy.

3.6.2. DNA repair

Xeroderma pigmentosum (XP) is an hereditary disease characterized by extreme sun sensitivity and an increased predisposition to develop skin cancer. Cultured cells from XP patients exhibit hypersensitivity to ultraviolet (UV) radiation due to defects in the nucleotide excision repair (NER) pathways and other cellular abnormalities. Gene transfer of NER genes into XP fibroblasts has been used to correct repair-defective cellular phenotypes by recovery of normal UV survival and RNA synthesis after UV irradiation, and also other cellular abnormalities resulting from NER defects (152, 153). Thus, it may be possible to genetically modify keratinocytes and fibroblasts to produce skin with normal DNA repair for XP patients.

3.6.3. Contact hypersensitivity

Inflammatory skin diseases like contact hypersensitivity may also be candidates for epidermal gene therapy. In a rat model it was showed that direct injection to the dorsal skin of rats of genes encoding IL-10 was able to suppress contact hypersensitivity to dinitrochlorobenzene applied at distant sites of the skin - at the skin on the ears (154). Interestingly, the efficiencies of transport to the blood circulation of cytokines produced in the skin after gene transfer differ a great deal. IL-4, 6, 10 and TGF- β 1 were readily transported into the circulation, whereas MCAF, GMCSF, TNF- α and IFN- γ could not be detected in the blood, although all eight cytokines were synthesized locally in the skin (155).

REFERENCES

Potten CS. Cell replacement in epidermis (keratopoiesis) via discrete units of proliferation. Int Rev Cytol 1981; 69:271-318.
Potten CS, Wichmann HE, Loeffler M, Dobek K, Major D. Evidence for discrete cell kinetic subpopulations in mouse epidermis based on mathematical analysis. Cell Tissue Kinet 1982; 15:305-329.
Watt FM. Epidermal stem cells: markers, patterning and the control of stem cell fate. Philos Trans R Soc Lond B Biol Sci 1998; 353:831-837.
Jensen UB, Lowell S, Watt FM. The spatial relationship between stem cells and their progeny in the basal layer of human epidermis: a new view based on whole-mount labelling and lineage analysis. Development 1999; 126:2409-2418.
Panteleyev AA, Jahoda CA, Christiano AM. Hair follicle predetermination. J Cell Sci 2001; 114:3419-3431.
Taylor G, Lehrer MS, Jensen PJ, Sun TT, Lavker RM. Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 2000; 102:451-461.
Oshima H, Rochat A, Kedzia C, Kobayashi K, Barrandon Y. Morphogenesis and renewal of hair follicles from adult multipotent stem cells. Cell 2001; 104:233-245.
Reynolds AJ, Jahoda CA. Cultured dermal papilla cells induce follicle formation and hair growth by transdifferentiation of an adult epidermis. Development 1992; 115:587-593.
Ferraris C, Bernard BA, Dhouailly D. Adult epidermal keratinocytes are endowed with pilosebaceous forming abilities. Int J Dev Biol 1997; 41:491-498.
Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975; 6:331-343.
Boyce ST, Ham RG. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serumfree serial culture. J Invest Dermatol 1983; 81:33s-40s.
Flaxman BA. Replication and differentiation in vitro of epidermal cells from normal human skin and from benign (psoriasis) and malignant (basal cell cancer) hyperplasia. In Vitro 1972; 8:237-255.
Price FM, Camalier RF, Gantt R, Taylor WG, Smith GH, Sanford KK. A new culture medium for human skin epithelial cells. In Vitro 1980; 16:147-158.
Jensen PK, Norgard JO, Pedersen S, Bolund L. Morphological and functional differentiation in epithelial cultures obtained from human skin explants. Virchows Arch B Cell Pathol Incl Mol Pathol 1983; 44:305-322.
Asselineau D, Prunieras M. Reconstruction of "simplified" skin: control of fabrication. Br J Dermatol 1984; 111 Suppl 27:219-222.
Prunieras M, Regnier M, Woodley D. Methods for cultivation of keratinocytes with an air-liquid interface. J Invest Dermatol 1983; 81:28s-33s.
Gallico GG, 3rd, O'Connor NE, Compton CC, Kehinde O, Green H. Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med 1984; 311:448-451.
Guerra L, Capurro S, Melchi F, Primavera G, Bondanza S, Cancedda R, Luci A, De Luca M, Pellegrini G. Treatment of "stable" vitiligo by Timedsurgery and transplantation of cultured epidermal autografts. Arch Dermatol 2000; 136:1380-1389.
Morgan JR, Barrandon Y, Green H, Mulligan RC. Expression of an exogenous growth hormone gene by transplantable human epidermal cells. Science 1987; 237:1476-1479.
Lee JI, Taichman LB. Transient expression of a transfected gene in cultured epidermal keratinocytes: implications for future studies. J Invest Dermatol 1989; 92:267-271.
Jensen TG, Jensen PK, Norgard JO, Bolund L. High-frequency transfection of cultured human epidermal basal cells that differentiate to form a multilayered tissue. Exp Cell Res 1990; 189:163-168.
Read J, Watt FM. A model for in vitro studies of epidermal homeostasis: proliferation and involucrin synthesis by cultured human keratinocytes during recovery after stripping off the suprabasal layers. J Invest Dermatol 1988; 90:739-743.
Jensen PK, Bolund L. Low Ca2+ stripping of differentiating cell layers in human epidermal cultures: an in vitro model of epidermal regeneration. Exp Cell Res 1988; 175:63-73.
Yuspa SH, Ben T, Hennings H, Lichti U. Divergent responses in epidermal basal cells exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Cancer Res 1982; 42:2344-2349.
Yuspa SH, Kulesz-Martin M, Ben T, Hennings H. Transformation of epidermal cells in culture. J Invest Dermatol 1983; 81:162s-168s.
Yates JL, Warren N, Sugden B. Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature 1985; 313:812-815.
Belt PB, Groeneveld H, Teubel WJ, van de Putte P, Backendorf C. Construction and properties of an Epstein-Barr-virus-derived cDNA expression vector for human cells. Gene 1989; 84:407-417.
Krysan PJ, Haase SB, Calos MP. Isolation of human sequences that replicate autonomously in human cells. Mol Cell Biol 1989; 9:1026-1033.
Jensen TG, Andresen BS, Bross P, Jensen UB, Holme E, Kolvraa S, Gregersen N, Bolund L. Expression of wild-type and mutant medium-chain acyl-CoA dehydrogenase (MCAD) cDNA in eucaryotic cells. Biochim Biophys Acta 1992; 1180:65-72.
Jensen UB, Petersen MS, Lund TB, Jensen TG, Bolund L. Transgene expression in human epidermal keratinocytes: cell cycle arrest of productively transfected cells. Exp Dermatol 2000; 9:298-310.
Garlick JA, Katz AB, Fenjves ES, Taichman LB. Retrovirus-mediated transduction of cultured epidermal keratinocytes. J Invest Dermatol 1991; 97:824-829.
Jensen UB, Jensen TG, Jensen PK, Rygaard J, Hansen BS, Fogh J, Kolvraa S, Bolund L. Gene transfer into cultured human epidermis and its transplantation onto immunodeficient mice: an experimental model for somatic gene therapy. J Invest Dermatol 1994; 103:391-394.
Williams RS, Johnston SA, Riedy M, DeVit MJ, McElligott SG, Sanford JC. Introduction of foreign genes into tissues of living mice by DNA-coated microprojectiles. Proc Natl Acad Sci USA 1991; 88:2726-2730.
Vogt PM, Thompson S, Andree C, Liu P, Breuing K, Hatzis D, Brown H, Mulligan RC, Eriksson E. Genetically modified keratinocytes transplanted to wounds reconstitute the epidermis. Proc Natl Acad Sci USA 1994; 91:9307-9311.
Hengge UR, Chan EF, Foster RA, Walker PS, Vogel JC. Cytokine gene expression in epidermis with biological effects following injection of naked DNA. Nat Genet 1995; 10:161-166.
Tang DC, DeVit M, Johnston SA. Genetic immunization is a simple method for eliciting an immune response. Nature 1992; 356:152-154.
Johnston SA, Tang DC. The use of microparticle injection to introduce genes into animal cells in vitro and in vivo. Genet Eng 1993; 15:225-236.
Fan H, Lin Q, Morrissey GR, Khavari PA. Immunization via hair follicles by topical application of naked DNA to normal skin. Nat Biotechnol 1999; 17:870-872.
Ortiz-Urda S, Thyagarajan B, Keene DR, Lin Q, Fang M, Calos MP, Khavari PA. Stable nonviral genetic correction of inherited human skin disease. Nat Med 2002; 8:1166-1170.
Kolodka TM, Garlick JA, Taichman LB. Evidence for keratinocyte stem cells in vitro: long term engraftment and persistence of transgene expression from retrovirus-transduced keratinocytes. Proc Natl Acad Sci USA 1998; 95:4356-4361.
White SJ, Page SM, Margaritis P, Brownlee GG. Long-term expression of human clotting factor IX from retrovirally transduced primary human keratinocytes in vivo. Hum Gene Ther 1998; 9:1187-1195.
Deng H, Lin Q, Khavari PA. Sustainable cutaneous gene delivery. Nat Biotechnol 1997; 15:1388-1391.
Levy L, Broad S, Zhu AJ, Carroll JM, Khazaal I, Peault B, Watt FM. Optimised retroviral infection of human epidermal keratinocytes: long- term expression of transduced integrin gene following grafting on to SCID mice. Gene Ther 1998; 5:913-922.
Mathor MB, Ferrari G, Dellambra E, Cilli M, Mavilio F, Cancedda R, De Luca M. Clonal analysis of stably transduced human epidermal stem cells in culture. Proc Natl Acad Sci USA 1996; 93:10371-10376.
Christensen R, Jensen UB, Jensen TG. Cutaneous gene therapy-an update. Histochem Cell Biol 2001; 115:73-82.
Jensen TG, Sullivan DM, Morgan RA, Taichman LB, Nussenblatt RB, Blaese RM, Csaky KG. Retrovirus-mediated gene transfer of ornithine-delta-aminotransferase into keratinocytes from gyrate atrophy patients. Hum Gene Ther 1997; 8:2125-2132.
Deng H, Choate KA, Lin Q, Khavari PA. High-efficiency gene transfer and pharmacologic selection of genetically engineered human keratinocytes. Biotechniques 1998; 25:274-280.
Ghazizadeh S, Harrington R, Taichman L. In vivo transduction of mouse epidermis with recombinant retroviral vectors: implications for cutaneous gene therapy. Gene Ther 1999; 6:1267-1275.
Baek SC, Lin Q, Robbins PB, Fan H, Khavari PA. Sustainable systemic delivery via a single injection of lentivirus into human skin tissue. Hum Gene Ther 2001; 12:1551-1558.
Kuhn U, Terunuma A, Pfutzner W, Foster RA, Vogel JC. In vivo assessment of gene delivery to keratinocytes by lentiviral vectors. J Virol 2002; 76:1496-1504.
Chen M, Kasahara N, Keene DR, Chan L, Hoeffler WK, Finlay D, Barcova M, Cannon PM, Mazurek C, Woodley DT. Restoration of type VII collagen expression and function in dystrophic epidermolysis bullosa. Nat Genet 2002; 32:670-675.
Jensen TG, Jensen UB, Bolund L. Production of retroviral vectors in primary human keratinocytes after DNA-mediated gene transfer leads to prolonged gene expression. Acta Derm Venereol 2003; 83:83-7.
Freiberg RA, Ho SN, Khavari PA. Transcriptional control in keratinocytes and fibroblasts using synthetic ligands. J Clin Invest 1997; 99:2610-2615.
Yao F, Eriksson E. A novel tetracycline-inducible viral replication switch. Hum Gene Ther 1999; 10:419-427.
Gandarillas A, Watt FM. c-Myc promotes differentiation of human epidermal stem cells. Genes Dev 1997; 11:2869-2882.
Gal EM, Nelson JM, Sherman AD. Biopterin. III. Purification and characterization of enzymes involved in the cerebral synthesis of 7,8-dihydrobiopterin. Neurochem Res 1978; 3:69-88.
Ghazizadeh S, Doumeng C, Taichman LB. Durable and stratum-specific gene expression in epidermis. Gene Ther 2002; 9:1278-1285.
Choate KA, Medalie DA, Morgan JR, Khavari PA. Corrective gene transfer in the human skin disorder lamellar ichthyosis. Nat Med 1996; 2:1263-1267.
Seitz CS, Giudice GJ, Balding SD, Marinkovich MP, Khavari PA. BP180 gene delivery in junctional epidermolysis bullosa. Gene Ther 1999; 6:42-47.
Robbins PB, Lin Q, Goodnough JB, Tian H, Chen X, Khavari PA. In vivo restoration of laminin 5 beta 3 expression and function in junctional epidermolysis bullosa. Proc Natl Acad Sci USA 2001; 98:5193-5198.
Teumer J, Lindahl A, Green H. Human growth hormone in the blood of athymic mice grafted with cultures of hormone-secreting human keratinocytes. Faseb J 1990; 4:3245-3250.
Ghazizadeh S, Harrington R, Garfield J, Taichman LB. Retrovirus-mediated transduction of porcine keratinocytes in organ culture. J Invest Dermatol 1998; 111:492-496.
Pfutzner W, Hengge UR, Joari MA, Foster RA, Vogel JC. Selection of keratinocytes transduced with the multidrug resistance gene in an in vitro skin model presents a strategy for enhancing gene expression in vivo. Hum Gene Ther 1999; 10:2811-2821.
Pfutzner W, Terunuma A, Tock CL, Snead EK, Kolodka TM, Gottesman MM, Taichman L, Vogel JC. Topical colchicine selection of keratinocytes transduced with the multidrug resistance gene (MDR1) can sustain and enhance transgene expression in vivo. Proc Natl Acad Sci USA 2002; 99:13096-13101.
Krueger GG, Jorgensen CM, Matsunami N, Morgan JR, Liimatta A, Meloni-Ehrig A, Shepard R, Petersen MJ. Persistent transgene expression and normal differentiation of immortalized human keratinocytes in vivo. J Invest Dermatol 1999; 112:233-239.
Page SM, Brownlee GG. Differentiation-specific enhancer activity in transduced keratinocytes: a model for epidermal gene therapy. Gene Ther 1998; 5:394-402.
Morley SM, Dundas SR, James JL, Gupta T, Brown RA, Sexton CJ, Navsaria HA, Leigh IM, Lane EB. Temperature sensitivity of the keratin cytoskeleton and delayed spreading of keratinocyte lines derived from EBS patients. J Cell Sci 1995; 108:3463-3471.
Pirisi L, Creek KE, Doniger J, DiPaolo JA. Continuous cell lines with altered growth and differentiation properties originate after transfection of human keratinocytes with human papillomavirus type 16 DNA. Carcinogenesis 1988; 9:1573-1579.
Durst M, Dzarlieva-Petrusevska RT, Boukamp P, Fusenig NE, Gissmann L. Molecular and cytogenetic analysis of immortalized human primary keratinocytes obtained after transfection with human papillomavirus type 16 DNA. Oncogene 1987; 1:251-256.
Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB, Shay JW, Lichtsteiner S, Wright WE. Extension of life-span by introduction of telomerase into normal human cells. Science 1998; 279:349-352.
Yang J, Chang E, Cherry AM, Bangs CD, Oei Y, Bodnar A, Bronstein A, Chiu CP, Herron GS. Human endothelial cell life extension by telomerase expression. J Biol Chem 1999; 274:26141-26148.
Simonsen JL, Rosada C, Serakinci N, Justesen J, Stenderup K, Rattan SI, Jensen TG, Kassem M. Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells. Nat Biotechnol 2002; 20:592-596.
Jensen TG, Sorensen CB, Jensen UB, Bolund L. Epidermolysis Bullosa Simplex Keratinocytes with Extended Lifespan established by ectopic Expression of Telomerase. Exp Dermatol 2003; in press.
Watt FM. Epidermal stem cells as targets for gene transfer. Hum Gene Ther 2000; 11:2261-2266.
Zhu AJ, Haase I, Watt FM. Signaling via beta1 integrins and mitogen-activated protein kinase determines human epidermal stem cell fate in vitro. Proc Natl Acad Sci USA 1999; 96:6728-6733.
Zhu AJ, Watt FM. beta-catenin signalling modulates proliferative potential of human epidermal keratinocytes independently of intercellular adhesion. Development 1999; 126:2285-2298.
Gat U, DasGupta R, Degenstein L, Fuchs E. De Novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin. Cell 1998; 95:605-614.
DasGupta R, Rhee H, Fuchs E. A developmental conundrum: a stabilized form of beta-catenin lacking the transcriptional activation domain triggers features of hair cell fate in epidermal cells and epidermal cell fate in hair follicle cells. J Cell Biol 2002; 158:331-344.
Yoon K, Cole-Strauss A, Kmiec EB. Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA.DNA oligonucleotide. Proc Natl Acad Sci USA 1996; 93:2071-2076.
Andersen MS, Sorensen CB, Bolund L, Jensen TG. Mechanisms underlying targeted gene correction using chimeric RNA/DNA and single-stranded DNA oligonucleotides. J Mol Med 2002; 80:770-781.
Alexeev V, Yoon K. Stable and inheritable changes in genotype and phenotype of albino melanocytes induced by an RNA-DNA oligonucleotide. Nat Biotechnol 1998; 16:1343-1346.
Alexeev V, Igoucheva O, Domashenko A, Cotsarelis G, Yoon K. Localized in vivo genotypic and phenotypic correction of the albino mutation in skin by RNA-DNA oligonucleotide. Nat Biotechnol 2000; 18:43-47.
Santana E, Peritz AE, Iyer S, Uitto J, Yoon K. Different frequency of gene targeting events by the RNA-DNA oligonucleotide among epithelial cells. J Invest Dermatol 1998; 111:1172-1177.
Cao T, Longley MA, Wang XJ, Roop DR. An inducible mouse model for epidermolysis bullosa simplex: implications for gene therapy. J Cell Biol 2001; 152:651-656.
Tang DC, Shi Z, Curiel DT. Vaccination onto bare skin. Nature 1997; 388:729-730.
Domashenko A, Gupta S, Cotsarelis G. Efficient delivery of transgenes to human hair follicle progenitor cells using topical lipoplex. Nat Biotechnol 2000; 18:420-423.
Alexander MY, Akhurst RJ. Liposome-medicated gene transfer and expression via the skin. Hum Mol Genet 1995; 4:2279-2285.
Robbins PB, Oliver SF, Sheu SM, Goodnough JB, Wender P, Khavari PA. Peptide delivery to tissues via reversibly linked protein transduction sequences. Biotechniques 2002; 33:190-192, 194.
White PJ, Gray AC, Fogarty RD, Sinclair RD, Thumiger SP, Werther GA, Wraight CJ. C-5 propyne-modified oligonucleotides penetrate the epidermis in psoriatic and not normal human skin after topical application. J Invest Dermatol 2002; 118:1003-1007.
Mehta RC, Stecker KK, Cooper SR, Templin MV, Tsai YJ, Condon TP, Bennett CF, Hardee GE. Intercellular adhesion molecule-1 suppression in skin by topical delivery of anti-sense oligonucleotides. J Invest Dermatol 2000; 115:805-812.
Branden LJ, Smith CI. Bioplex technology: novel synthetic gene delivery system based on peptides anchored to nucleic acids. Methods Enzymol 2002; 346:106-124.
Huber M, Rettler I, Bernasconi K, Frenk E, Lavrijsen SP, Ponec M, Bon A, Lautenschlager S, Schorderet DF, Hohl D. Mutations of keratinocyte transglutaminase in lamellar ichthyosis. Science 1995; 267:525-528.
Shapiro LJ, Yen P, Pomerantz D, Martin E, Rolewic L, Mohandas T. Molecular studies of deletions at the human steroid sulfatase locus. Proc Natl Acad Sci USA 1989; 86:8477-8481.
Shapiro LJ, Weiss R, Buxman MM, Vidgoff J, Dimond RL, Roller JA, Wells RS. Enzymatic basis of typical X-linked icthyosis. Lancet 1978; 2:756-757.
Koppe G, Marinkovic-Ilsen A, Rijken Y, De Groot WP, Jobsis AC. X-linked icthyosis. A sulphatase deficiency. Arch Dis Child 1978; 53:803-806.
Lykkesfeldt G, Bennett P, Lykkesfeldt AE, Micic S, Rorth M, Skakkebaek NE, Svenstrup B. Testis cancer. Ichthyosis constitutes a significant risk factor. Cancer 1991; 67:730-734.
Frost P, Weinstein GD, Van Scott EJ. The ichthyosiform dermatoses. II. Autoradiographic studies of epidermal proliferation. J Invest Dermatol 1966; 47:561-567.
Frost P, Van Scott EJ. Ichthyosiform dermatoses. Classification based on anatomic and biometric observations. Arch Dermatol 1966; 94:113-126.
Epstein EH, Williams ML, Elias PM. The epidermal cholesterol sulfate cycle. J Am Acad Dermatol 1984; 10:866-868.
Jensen PK, Herrmann FH, Hadlich J, Bolund L. Proliferation and differentiation of cultured epidermal cells from patients with X-linked ichthyosis and ichthyosis vulgaris. Acta Derm Venereol 1990; 70:99-104.
Jensen TG, Jensen UB, Jensen PK, Ibsen HH, Brandrup F, Ballabio A, Bolund L. Correction of steroid sulfatase deficiency by gene transfer into basal cells of tissue-cultured epidermis from patients with recessive X- linked ichthyosis. Exp Cell Res 1993; 209:392-397.
Freiberg RA, Choate KA, Deng H, Alperin ES, Shapiro LJ, Khavari PA. A model of corrective gene transfer in X-linked ichthyosis. Hum Mol Genet 1997; 6:927-933.
Dellambra E, Pellegrini G, Guerra L, Ferrari G, Zambruno G, Mavilio F, De Luca M. Toward epidermal stem cell-mediated ex vivo gene therapy of junctional epidermolysis bullosa. Hum Gene Ther 2000; 11:2283-2287.
Chen M, O'Toole EA, Muellenhoff M, Medina E, Kasahara N, Woodley DT. Development and characterization of A recombinant truncated type VII collagen "Minigene". Implication for gene therapy of dystrophic epidermolysis bullosa. J Biol Chem 2000; 275:24429-24435.
Mecklenbeck S, Compton SH, Mejia JE, Cervini R, Hovnanian A, Bruckner-Tuderman L, Barrandon Y. A microinjected COL7A1-PAC vector restores synthesis of intact procollagen VII in a dystrophic epidermolysis bullosa keratinocyte cell line. Hum Gene Ther 2002; 13:1655-1662.
Katz AB, Taichman LB. Epidermis as a secretory tissue: an in vitro tissue model to study keratinocyte secretion. J Invest Dermatol 1994; 102:55-60.
Fenjves ES, Gordon DA, Pershing LK, Williams DL, Taichman LB. Systemic distribution of apolipoprotein E secreted by grafts of epidermal keratinocytes: implications for epidermal function and gene therapy. Proc Natl Acad Sci USA 1989; 86:8803-8807.
Wang X, Zinkel S, Polonsky K, Fuchs E. Transgenic studies with a keratin promoter-driven growth hormone transgene: prospects for gene therapy. Proc Natl Acad Sci USA 1997; 94:219-226.
Fakharzadeh SS, Zhang Y, Sarkar R, Kazazian HH, Jr. Correction of the coagulation defect in hemophilia A mice through factor VIII expression in skin. Blood 2000; 95:2799-2805.
Lawrence WT, Diegelmann RF. Growth factors in wound healing. Clin Dermatol 1994; 12:157-169.
Falanga V. Chronic wounds: pathophysiologic and experimental considerations. J Invest Dermatol 1993; 100:721-725.
Lauer G, Sollberg S, Cole M, Flamme I, Sturzebecher J, Mann K, Krieg T, Eming SA. Expression and proteolysis of vascular endothelial growth factor is increased in chronic wounds. J Invest Dermatol 2000; 115:12-18.
Andree C, Swain WF, Page CP, Macklin MD, Slama J, Hatzis D, Eriksson E. In vivo transfer and expression of a human epidermal growth factor gene accelerates wound repair. Proc Natl Acad Sci USA 1994; 91:12188-12192.
Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev 1999; 79:1283-1316.
Beer HD, Longaker MT, Werner S. Reduced expression of PDGF and PDGF receptors during impaired wound healing. J Invest Dermatol 1997; 109:132-138.
Robson MC, Phillips LG, Thomason A, Robson LE, Pierce GF. Platelet-derived growth factor BB for the treatment of chronic pressure ulcers. Lancet 1992; 339:23-25.
Eming SA, Lee J, Snow RG, Tompkins RG, Yarmush ML, Morgan JR. Genetically modified human epidermis overexpressing PDGF-A directs the development of a cellular and vascular connective tissue stroma when transplanted to athymic mice - implications for the use of genetically modified keratinocytes to modulate dermal regeneration. J Invest Dermatol 1995; 105:756-763.
Eming SA, Whitsitt JS, He L, Krieg T, Morgan JR, Davidson JM. Particle-mediated gene transfer of PDGF isoforms promotes wound repair. J Invest Dermatol 1999; 112:297-302.
Rollman O, Jensen UB, Ostman A, Bolund L, Gústafsdóttir SM, Jensen TG. Platelet-derived growth factor-responsive epidermis formed from human keratinocytes transduced with the PDGF beta-receptor gene. 2003; submitted.
Taichman LB. Epithelial gene therapy. In: Leigh IM, Lane EB, Watt FM, editors. The Keratinocyte Handbook. Cambridge: Cambridge University Press; 1994. p. 543-551.
Palmer TD, Hock RA, Osborne WR, Miller AD. Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human. Proc Natl Acad Sci USA 1987; 84:1055-1059.
Fenjves ES, Schwartz PM, Blaese RM, Taichman LB. Keratinocyte gene therapy for adenosine deaminase deficiency: a model approach for inherited metabolic disorders. Hum Gene Ther 1997; 8:911-917.
Kaiser-Kupfer MI, de Monasterio F, Valle D, Walser M, Brusilow S. Visual results of a long-term trial of a low-arginine diet in gyrate atrophy of choroid and retina. Ophthalmology 1981; 88:307-310.
Kaiser-Kupfer MI, Caruso RC, Valle D. Gyrate atrophy of the choroid and retina. Long-term reduction of ornithine slows retinal degeneration. Arch Ophthalmol 1991; 109:1539-1548.
Sullivan DM, Jensen TG, Taichman LB, Csaky KG. Ornithine-delta-aminotransferase expression and ornithine metabolism in cultured epidermal keratinocytes: toward metabolic sink therapy for gyrate atrophy. Gene Ther 1997; 4:1036-1044.
Phase 1 study in the safety and efficacy of transduced keratinocytes for possible treatment of gyrate atrophy. NIH/ORDA receipt date: 3-4-99. Principal investigator: Robert B. Nussenblatt, National Institutes of Health, Bethesda, Maryland. FDA protocol number: 9903-295.
Lenke RR, Levy HL. Maternal phenylketonuria and hyperphenylalaninemia. An international survey of the outcome of untreated and treated pregnancies. N Engl J Med 1980; 303:1202-1208.
Harding CO, Wild K, Chang D, Messing A, Wolff JA. Metabolic engineering as therapy for inborn errors of metabolism - development of mice with phenylalanine hydroxylase expression in muscle. Gene Ther 1998; 5:677-683.
Christensen R, Kolvraa S, Blaese RM, Jensen TG. Development of a skin-based metabolic sink for phenylalanine by overexpression of phenylalanine hydroxylase and GTP cyclohydrolase in primary human keratinocytes. Gene Ther 2000; 7:1971-1978.
Christensen R, Jensen UB, Jensen TG. Skin genetically engineered as a bioreactor or a "Metabolic Sink". Cells Tissues Organs 2002; 172:96-104.
Christensen R, Guttler F, Jensen T. Comparison of epidermal keratinocytes and dermal fibroblasts as potential target cells for somatic gene therapy of phenylketonuria. Mol Genet Metab 2002; 76:313.
Weinstein GD, McCullough JL, Ross P. Cell proliferation in normal epidermis. J Invest Dermatol 1984; 82:623-628.
Scriver CR, Kaufman S, Eisensmith RC, Woo SLC. The Hyperphenylalaninemias. In: The metabolic and molecular bases of inherited disease. McGraw-Hill; 1995. p. 1015-1075.
Bos JD, Kapsenberg ML. The skin immune system: progress in cutaneous biology. Immunol Today 1993; 14:75-78.
Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 1991; 9:271-296.
Condon C, Watkins SC, Celluzzi CM, Thompson K, Falo LD, Jr. DNA-based immunization by in vivo transfection of dendritic cells. Nat Med 1996; 2:1122-1128.
Porgador A, Irvine KR, Iwasaki A, Barber BH, Restifo NP, Germain RN. Predominant role for directly transfected dendritic cells in antigen presentation to CD8+ T cells after gene gun immunization. J Exp Med 1998; 188:1075-1082.
Celluzzi CM, Falo LD, Jr. Physical interaction between dendritic cells and tumor cells results in an immunogen that induces protective and therapeutic tumor rejection. J Immunol 1998; 160:3081-3085.
Albert ML, Sauter B, Bhardwaj N. Dendritic cells acquire antigen from apoptotic cells and induce class I- restricted CTLs. Nature 1998; 392:86-89.
Barry MA, Johnston SA. Biological features of genetic immunization. Vaccine 1997; 15:788-791.
Larregina AT, Falo LD, Jr. Generating and regulating immune responses through cutaneous gene delivery. Hum Gene Ther 2000; 11:2301-2305.
Vogel JC, Udey MC. Activation of skin dendritic cells by immunostimulatory DNA. Springer Semin Immunopathol 2000; 22:45-54.
Jakob T, Walker PS, Krieg AM, Udey MC, Vogel JC. Activation of cutaneous dendritic cells by CpG-containing oligodeoxynucleotides: a role for dendritic cells in the augmentation of Th1 responses by immunostimulatory DNA. J Immunol 1998; 161:3042-3049.
Sato Y, Roman M, Tighe H, Lee D, Corr M, Nguyen MD, Silverman GJ, Lotz M, Carson DA, Raz E. Immunostimulatory DNA sequences necessary for effective intradermal gene immunization. Science 1996; 273:352-354.
Rakhmilevich AL, Turner J, Ford MJ, McCabe D, Sun WH, Sondel PM, Grota K, Yang NS. Gene gun-mediated skin transfection with interleukin 12 gene results in regression of established primary and metastatic murine tumors. Proc Natl Acad Sci USA 1996; 93:6291-6296.
Sun WH, Burkholder JK, Sun J, Culp J, Turner J, Lu XG, Pugh TD, Ershler WB, Yang NS. In vivo cytokine gene transfer by gene gun reduces tumor growth in mice. Proc Natl Acad Sci USA 1995; 92:2889-2893.
Nabel GJ, Nabel EG, Yang ZY, Fox BA, Plautz GE, Gao X, Huang L, Shu S, Gordon D, Chang AE. Direct gene transfer with DNA-liposome complexes in melanoma: expression, biologic activity, and lack of toxicity in humans. Proc Natl Acad Sci USA 1993; 90:11307-11311.
Nabel GJ, Gordon D, Bishop DK, Nickoloff BJ, Yang ZY, Aruga A, Cameron MJ, Nabel EG, Chang AE. Immune response in human melanoma after transfer of an allogeneic class I major histocompatibility complex gene with DNA-liposome complexes. Proc Natl Acad Sci USA 1996; 93:15388-15393.
Quintin-Colonna F, Devauchelle P, Fradelizi D, Mourot B, Faure T, Kourilsky P, Roth C, Mehtali M. Gene therapy of spontaneous canine melanoma and feline fibrosarcoma by intratumoral administration of histoincompatible cells expressing human interleukin-2. Gene Ther 1996; 3:1104-1112.
St-Jacques B, Dassule HR, Karavanova I, Botchkarev VA, Li J, Danielian PS, McMahon JA, Lewis PM, Paus R, McMahon AP. Sonic hedgehog signaling is essential for hair development. Curr Biol 1998; 8:1058-1068.
Sato N, Leopold PL, Crystal RG. Induction of the hair growth phase in postnatal mice by localized transient expression of Sonic hedgehog. J Clin Invest 1999; 104:855-864.
Dumaz N, Drougard C, Quilliet X, Mezzina M, Sarasin A, Daya-Grosjean L. Recovery of the normal p53 response after UV treatment in DNA repair- deficient fibroblasts by retroviral-mediated correction with the XPD gene. Carcinogenesis 1998; 19:1701-1704.
Zeng L, Sarasin A, Mezzina M. Retrovirus-mediated DNA repair gene transfer into xeroderma pigmentosum cells: perspectives for a gene therapy. Cell Biol Toxicol 1998; 14:105-110.
Meng X, Sawamura D, Tamai K, Hanada K, Ishida H, Hashimoto I. Keratinocyte gene therapy for systemic diseases. Circulating interleukin 10 released from gene-transferred keratinocytes inhibits contact hypersensitivity at distant areas of the skin. J Clin Invest 1998; 101:1462-1467.
Meng X, Sawamura D, Ina S, Tamai K, Hanada K, Hashimoto I. Keratinocyte gene therapy: cytokine gene expression in local keratinocytes and in circulation by introducing cytokine genes into skin. Exp Dermatol 2002; 11:456-461.
Harper JR, Greenhalgh DA, Yuspa SH. Expression of transfected DNA by primary murine keratinocytes. J Invest Dermatol 1988; 91:150-153.
Jiang CK, Connolly D, Blumenberg M. Comparison of methods for transfection of human epidermal keratinocytes. J Invest Dermatol 1991; 97:969-973.
Rhim JS, Park JB, Jay G. Neoplastic transformation of human keratinocytes by polybrene-induced DNA-mediated transfer of an activated oncogene. Oncogene 1989; 4:1403-1409.
Staedel C, Remy JS, Hua Z, Broker TR, Chow LT, Behr JP. High-efficiency transfection of primary human keratinocytes with positively charged lipopolyamine:DNA complexes. J Invest Dermatol 1994; 102: 768-772.
Sawamura D, Meng X, Ina S, Ishikawa H, Tamai K, Nomura K, Hanada K, Hashimoto I, Kaneda Y. In vivo transfer of a foreign gene to keratinocytes using the hemagglutinating virus of Japan-liposome method. J Invest Dermatol 1997; 108:195-199.
Huber M, Limat A, Wagner E, Hohl D. Efficient in vitro transfection of human keratinocytes with an adenovirus-enhanced receptor-mediated system. J Invest Dermatol 2000; 114:661-666.
Reiss M, Jastreboff MM, Bertino JR, Narayanan R. DNA-mediated gene transfer into epidermal cells using electroporation. Biochem Biophys Res Commun 1986; 137:244-249.
Rorth P, Nerlov C, Blasi F, Johnsen M. Transcription factor PEA3 participates in the induction of urokinase plasminogen activator transcription in murine keratinocytes stimulated with epidermal growth factor or phorbol-ester. Nucleic Acids Res 1990; 18:5009-5017.
Glasspool-Malone J, Somiari S, Drabick JJ, Malone RW. Efficient nonviral cutaneous transfection. Mol Ther 2000; 2:140-146.
Eriksson E, Yao F, Svensjo T, Winkler T, Slama J, Macklin MD, Andree C, McGregor M, Hinshaw V, Swain WF. In vivo gene transfer to skin and wound by microseeding. J Surg Res 1998; 78:85-91.
Cartier R, Ren SV, Walther W, Stein U, Lewis A, Schlag PM, Li M, Furth PA. In vivo gene transfer by low-volume jet injection. Anal Biochem 2000; 282:262-265.
Setoguchi Y, Jaffe HA, Danel C, Crystal RG. Ex vivo and in vivo gene transfer to the skin using replication- deficient recombinant adenovirus vectors. J Invest Dermatol 1994; 102:415-421.
Braun-Falco M, Doenecke A, Smola H, Hallek M. Efficient gene transfer into human keratinocytes with recombinant adeno-associated virus vectors. Gene Ther 1999; 6:432-441.
Hengge UR, Mirmohammadsadegh A. Adeno-associated virus expresses transgenes in hair follicles and epidermis. Mol Ther 2000; 2:188-194.
Caplen NJ, Higginbotham JN, Scheel JR, Vahanian N, Yoshida Y, Hamada H, Blaese RM, Ramsey WJ. Adeno-retroviral chimeric viruses as in vivo transducing agents. Gene Ther 1999; 6:454-459.
Dellambra E, Vailly J, Pellegrini G, Bondanza S, Golisano O, Macchia C, Zambruno G, Meneguzzi G, De Luca M. Corrective transduction of human epidermal stem cells in laminin-5- dependent junctional epidermolysis bullosa. Hum Gene Ther 1998; 9:1359-1370.
Vailly J, Gagnoux-Palacios L, Dell'Ambra E, Romero C, Pinola M, Zambruno G, De Luca M, Ortonne JP, Meneguzzi G. Corrective gene transfer of keratinocytes from patients with junctional epidermolysis bullosa restores assembly of hemidesmosomes in reconstructed epithelia. Gene Ther 1998; 5:1322-1332.
Medin JA, Tudor M, Simovitch R, Quirk JM, Jacobson S, Murray GJ, Brady RO. Correction in trans for Fabry disease: expression, secretion and uptake of alpha-galactosidase A in patient-derived cells driven by a high- titer recombinant retroviral vector. Proc Natl Acad Sci USA 1996; 93:7917-7922.
Moullier P, Bohl D, Heard JM, Danos O. Correction of lysosomal storage in the liver and spleen of MPS VII mice by implantation of genetically modified skin fibroblasts. Nat Genet 1993; 4:154-159.
Krueger GG, Morgan JR, Jorgensen CM, Schmidt L, Li HL, Kwan MK, Boyce ST, Wiley HS, Kaplan J, Petersen MJ. Genetically modified skin to treat disease: potential and limitations. J Invest Dermatol 1994; 103:76S-84S.
Gerrard AJ, Hudson DL, Brownlee GG, Watt FM. Towards gene therapy for haemophilia B using primary human keratinocytes. Nat Genet 1993; 3:180-183.
Da Costa TH, Williamson DH, Ward A, Bates P, Fisher R, Richardson L, Hill DJ, Robinson IC, Graham CF. High plasma insulin-like growth factor-II and low lipid content in transgenic mice: measurements of lipid metabolism. J Endocrinol 1994; 143:433-439.
Larcher F, Del Rio M, Serrano F, Segovia JC, Ramirez A, Meana A, Page A, Abad JL, Gonzalez MA, Bueren J, Bernad A, Jorcano JL. A cutaneous gene therapy approach to human leptin deficiencies: correction of the murine ob/ob phenotype using leptin-targeted keratinocyte grafts. Faseb J 2001; 15:1529-1538.
Cheng J, Turksen K, Yu QC, Schreiber H, Teng M, Fuchs E. Cachexia and graft-vs.-host-disease-type skin changes in keratin promoter-driven TNF alpha transgenic mice. Genes Dev 1992; 6:1444-1456.
Petersen MJ, Kaplan J, Jorgensen CM, Schmidt LA, Li L, Morgan JR, Kwan MK, Krueger GG. Sustained production of human transferrin by transduced fibroblasts implanted into athymic mice: a model for somatic gene therapy. J Invest Dermatol 1995; 104:171-176.
Rio MD, Larcher F, Meana A, Segovia J, Alvarez A, Jorcano J. Nonviral transfer of genes to pig primary keratinocytes. Induction of angiogenesis by composite grafts of modified keratinocytes overexpressing VEGF driven by a keratin promoter. Gene Ther 1999; 6:1734-1741.
Rosenthal FM, Cao L, Tanczos E, Kopp J, Andree C, Stark GB, Mertelsmann R, Kulmburg P. Paracrine stimulation of keratinocytes in vitro and continuous delivery of epidermal growth factor to wounds in vivo by genetically modified fibroblasts transfected with a novel chimeric construct. In Vivo 1997; 11:201-208.
Bryant M, Drew GM, Houston P, Hissey P, Campbell CJ, Braddock M. Tissue repair with a therapeutic transcription factor. Hum Gene Ther 2000; 11:2143-2158.
Sun L, Xu L, Chang H, Henry FA, Miller RM, Harmon JM, Nielsen TB. Transfection with aFGF cDNA improves wound healing. J Invest Dermatol 1997; 108:313-318.
Jeschke MG, Richter G, Herndon DN, Geissler EK, Hartl M, Hofstatter F, Jauch KW, Perez-Polo JR. Therapeutic success and efficacy of nonviral liposomal cDNA gene transfer to the skin in vivo is dose dependent. Gene Ther 2001; 8:1777-1784.
Yamasaki K, Edington HD, McClosky C, Tzeng E, Lizonova A, Kovesdi I, Steed DL, Billiar TR. Reversal of impaired wound repair in iNOS-deficient mice by topical adenoviral-mediated iNOS gene transfer. J Clin Invest 1998; 101:967-971.
Eming SA, Medalie DA, Tompkins RG, Yarmush ML, Morgan JR. Genetically modified human keratinocytes overexpressing PDGF-A enhance the performance of a composite skin graft. Hum Gene Ther 1998; 9:529-539.
Tyrone JW, Mogford JE, Chandler LA, Ma C, Xia Y, Pierce GF, Mustoe TA. Collagen-embedded platelet-derived growth factor DNA plasmid promotes wound healing in a dermal ulcer model. J Surg Res 2000; 93:230-236.
Breitbart AS, Grande DA, Laser J, Barcia M, Porti D, Malhotra S, Kogon A, Grant RT, Mason JM. Treatment of ischemic wounds using cultured dermal fibroblasts transduced retrovirally with PDGF-B and VEGF121 genes. Ann Plast Surg 2001; 46:555-561; discussion 561-552.
Liechty KW, Nesbit M, Herlyn M, Radu A, Adzick NS, Crombleholme TM. Adenoviral-mediated overexpression of platelet-derived growth factor-B corrects ischemic impaired wound healing. J Invest Dermatol 1999; 113:375-383.
Taub PJ, Marmur JD, Zhang WX, Senderoff D, Nhat PD, Phelps R, Urken ML, Silver L, Weinberg H. Locally administered vascular endothelial growth factor cDNA increases survival of ischemic experimental skin flaps. Plast Reconstr Surg 1998; 102:2033-2039.