FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by LARCHER, F.
Right arrow Articles by JORCANO, J. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by LARCHER, F.
Right arrow Articles by JORCANO, J. L.
(The FASEB Journal. 2001;15:1529-1538.)
© 2001 FASEB

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

FERNANDO LARCHER*,{dagger},12, MARCELA DEL RIO*,{dagger},1, FERNANDO SERRANO{ddagger}, JOSÉ CARLOS SEGOVIA*,{dagger}, ANGEL RAMÍREZ*, ALVARO MEANA§, ANGUSTIAS PAGE*, JOSÉ LUIS ABAD{ddagger}, MANUEL A. GONZÁLEZ{ddagger}, JUAN BUEREN*,{dagger}, ANTONIO BERNAD{ddagger} and JOSÉ LUIS JORCANO*,{dagger}

* Project of Cell and Molecular Biology and Gene Therapy. CIEMAT. Avenida Complutense 22, 28040 Madrid, Spain;
{dagger} Fundacion Marcelino Botín for Gene Therapy,
{ddagger} Department of Immunology and Oncology, Centro Nacional de Biotecnología, Madrid, Spain; and
§ Centro de Transfusiones del Principado de Asturias, Oviedo, Spain

2Correspondence: CIEMAT, Avenida Complutense 22 Edificio 7, 28040 Madrid, Spain. E-mail: Fernando.larcher{at}ciemat.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.—Larcher, F., Del Rio, M., Serrano, F., Segovia, J. C., Ramírez, A., Meana, A., Page, A., Abad, J. L., González, M. A., Bueren, J., Bernad, A., Jorcano, J. L. A cutaneous gene therapy approach to human leptin deficiencies: correction of the murine ob/ob phenotype using leptin-targeted keratinocyte grafts.


Key Words: skin • lipodistrophy • diabetes • leptin replacement


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
LEPTIN, THE 16 kDa product of the OB gene, is an essential hormone secreted mainly by adipocytes that regulates food intake and neuroendocrine function via specific receptors in the hypothalamus (1 , 2) . Experimental evidence suggests that leptin may also exert direct effects at the level of gene expression or cellular function on peripheral tissues including hematopoietic cells, T cells, the endocrine pancreas, pituitary, ovary, adipocytes, skeletal muscle, and hepatocytes (3 , 4) . In addition, a role for leptin has recently been reported in angiogenesis and bone formation (5 6 7) .

Congenital leptin deficiency has been identified in humans and is associated with a rare, severe early-onset obesity (8 , 9) . Recombinant leptin therapy is shown to lead to a sustained reduction in body weight in one of these patients, predominantly as a result of fat loss (10) .

Decreased circulating leptin levels are also reported in patients with different forms of generalized lipoatrophic diabetes (7) . Lipoatrophic diabetes designates a group of syndromes characterized by extreme paucity of adipose tissue, hyperglycemia, severe insulin resistance, hypoleptinemia, and voracious appetite (11 , 12) . Other serious clinical features in the congenital form of the disease include an anabolic syndrome with organomegaly and hypertrophic cardiomyopathy. Since no causal therapy has yet been identified, a prophylactic approach is used in these patients based on a rigid control of calorie consumption and anabolic syndrome in order to delay diabetic complications and progression of hypertrophic cardiomyopathy (11) .

Leptin administration has recently been shown to increase glucose metabolism and restore insulin sensitivity in two transgenic mouse models of congenital generalized lipodystrophy (13 14 15 16) , suggesting a critical role for leptin in the physiopathology of these disorders and its therapeutic potential for the treatment of human lipoatrophic diabetes.

Protein injection is frequently used to treat peptide hormone deficiencies. Although leptin has been delivered by this route with good results in a patient suffering from leptin-deficient inherited obesity (10) , the treatment may be compromised by its short duration of action and the need for repetitive daily dosing. Gene therapy is thus an attractive alternative approach for leptin delivery and has received recent experimental support (17 , 19)

On the basis of their proliferative capacity and the proven value of cultured keratinocytes for skin wound coverage, autologous grafts of genetically engineered keratinocytes have been proposed as a suitable vehicle to correct for deficiencies in circulating proteins (20) . Previous studies have shown that the product of exogenous genes such as GH, factor IX, interleukin 6 (IL-6), etc., reach the blood circulation after being synthesized, processed, and secreted by gene-transferred human epidermal cells (21 22 23 24) . In addition to these ex vivo approaches, transgenic mouse studies strongly support the concept of the epidermis as an efficient secretory bioreactor (25 26 27) .

A further boost to cutaneous gene therapy for systemic disorders has recently been provided. Partial correction of hemophilia A in factor VIII-deficient mice was achieved after grafting of factor VIII-expressing mouse skin from a loricrin factor VIII transgenic mouse (27) . Injection of plasmid DNA coding for IL-10 into rat skin also led to transient inhibition of contact hypersensitivity at a distant area of the skin (28) . To our knowledge, however, use of genetically modified human keratinocytes to correct for a systemic defect has not been described to date.

Here we report the successful correction of a leptin deficiency-derived metabolic disorder through cutaneous gene therapy using both murine and human keratinocytes. For this study, we used the obese ob/ob mutant mouse (29 , 30) as a model of leptin deficiency. In these animals, the absence of leptin accounts for a dramatic phenotype characterized by extreme obesity due to increased food intake and low energy expenditure. Other clinical features include decreased immune function, infertility, and impaired wound healing. At the biochemical level, the ob/ob mice present hyperglycemia, hyperinsulinemia, and reduced levels of gonadotrophins (30) .

Skin grafts from a leptin-overexpressing skin transgenic mouse proved highly efficient for the correction of the ob/ob mouse systemic defect. This result led us to the development of a human cutaneous gene therapy-relevant approach. Thus, ex vivo leptin-transduced human keratinocyte (HK) grafts comprising less than 10% of the body surface corrected the leptin deficiency of ob/ob mouse. This is also the first study demonstrating successful replacement of a missing circulating protein using genetically modified human keratinocyte grafts.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
BALB/c CB17-Prkdcscid and C57BL/6Job/ob mice were purchased from IFFA-Credo-Charles River (St. Aulin-les-Elbeuf, France) and maintained at the CIEMAT Laboratory Animals Facility (Spanish registration number 28079–21 A). Animals were housed in individually ventilated type II cages, maximum 4 mice each with 25 air changes per hour and 10 KGy gamma irradiated soft wood pellets as bedding. All handling was done under sterile conditions. All experimental procedures were carried out according to European and Spanish laws and regulations (European convention 123 about the use and protection of vertebrate mammals used in experimentation and other scientific purposes. Spanish R.D 223/88 and O.M. 13–10-89 of the Ministry of Agricultural, Food and Fisheries about the protection and use of animals in scientific research and internal biosafety and bioethics guidelines).

Generation of K5-leptin transgenic mice
A SmaI/NotI fragment from plasmid pOB1 containing the mouse leptin cDNA (a generous gift of Dr. M. Reitman, NIDDK, NIH) was blunted and inserted into a cassette containing the 5.2 kb bovine regulatory sequences, the 5' rabbit ß-globin intron 2, and the 3' polyadenylation sequences (31 , 32) . The construct was designated pK5-leptin. The transgene was excised from the plasmid pK5-leptin with NotI, purified by low melting point agarose electrophoresis and Elutip columns (Schleicher and Schuell, Keene, NH), adjusted to a final concentration of ~2 µg/ml, and microinjected into (C57BL/6JxDBA/2)F2 mouse embryos as described (31 , 32) . Founder mice were identified by Southern blot analysis of tail DNA using the 5.2 kb fragment corresponding to the bovine keratin K5 promoter as a probe. A detailed description of the characterization of K5-leptin transgenic mice will be reported elsewhere (F. Larcher et al., unpublished results).

Generation of graft recipient immunodeficient ob/ob mice
Fertile heterozygous ob/+ males were first mated with homozygous severe combined immunodeficiency SCID/SCID female mice (BALB/c CB-17 strain) to obtain double heterozygous SCID/+, ob/+ animals. ob/+ mice were identified using the one-step PCR-based genotyping method described by Namae et al. (33) Double heterozygous SCID/+, ob/+ males were crossed with female homozygous SCID/SCID mice to obtain immunodeficient ob/+ breeding pairs. Immunodeficiency was assessed by the absence of B and T lymphocytes in blood by FACS analysis using FITC- and PE fluorochrome-conjugated antibodies specific for murine B cells (anti-B220; Caltag, Burlingame, CA) and T cells (anti-CD3; BD-PharMingen, San Diego, CA), respectively, as described (34) . Finally, male and female immunodeficient heterozygous ob/+ mice pairs were bred to obtain immunodeficient ob/ob graft recipient obese mice, which developed at normal mendelian rates. A similar breeding strategy was attempted to obtain immunodeficient ob/ob mice on a nu/nu background with low efficiency.

Mouse skin grafts
Dorsal full-thickness skin pieces of 2–2.5 cm2 were obtained from 5- to 7-wk-old K5-leptin transgenic mice or control littermates. Shaved donor skin pieces were grafted onto a wound created by removing a similar-sized piece of full-thickness back skin in female immunodeficient obese ob/ob recipient mice. Graft and host skin edges were joined using surgical silk suture and the grafted area was covered with a thin layer of NewSkin (Medtech, Jackson, WY) as the only protective dressing. This procedure allows graft-take monitoring and produced normal-haired donor skin.

Keratinocyte cell cultures and retroviral gene transfer
Human primary keratinocytes were obtained from newborn foreskins by repetitive trypsin incubation, then seeded on lethally irradiated 3T3-J2 cells (a gift from Dr. J. Garlick, SUNY) as described (35) . Retroviral infection was performed on small-sized cell colonies (8–16 cells) of first-passage primary keratinocytes. Cells were incubated with pLZRS-leptin-IRES-EGFP or pLZRS-IRES-EGFP vector supernatants at a titer of 1 to 5 x 106 CFU/ml for 4 h in the presence of polybrene (8 µg/ml) on 2 consecutive days. Keratinocytes at 60–80% confluence were trypsinized, resuspended in PBS/2% FCS, analyzed for EGFP expression and sorted by fluorescence-activated cell sorting on a FACStar PLUS flow cytometer (Becton Dickinson, San Jose, CA) as described (36) . Transduced, EGFP-sorted keratinocytes were seeded on live human fibroblast-containing fibrin gels at a density of 1 x 104 cells/cm2. Fibrin-fibroblast gels were prepared according to Del Rio et al. (36) .

Mouse primary keratinocyte cultures from newborn K5-leptin transgenic animals or control littermates were obtained as described (32) .

Human keratinocyte grafts and immunosuppression of ob/ob mice
After reaching confluence, transduced human keratinocyte cultures on fibrin-fibroblast gels were manually detached, divided into 2–2.5 cm2 squares, and grafted onto the dorsal region of female cyclosporin-A immunosuppressed ob/ob mice (one square/mouse) using the flap method described by Barrandon et al. (37) . Leptin- and control-transduced, EGFP-sorted human keratinocyte composite cultures were grafted in five immunosuppressed mice, respectively. Mice were housed in pathogen-free conditions for the duration of the experiment. Grafting experiments were also performed in ob/ob mice with no immunosuppressive treatment.

Human-to-mouse xenografts were maintained in cyclosporin-A-treated ob/ob mice according to Compton et al., with minor modifications (38) . Animals received daily intraperitoneal injections of 25 mg/kg of cyclosporin-A (Sandimmum 50 mg/ml, Novartis, Summit, NJ) in a sterile olive oil suspension (200 µl/mouse), starting 1 day before grafting and for the duration of the experiment (16 days).

Retroviral vector design and production
Retroviral expression vectors were constructed using the LZRS backbone vector (39 , 40) . To construct the pLZRS-IRES-EGFP vector, a XhoI/NotI fragment containing the IRES and the EGFP gene was obtained from plasmid pIRES2-EGFP (Clontech, Palo Alto, CA) and cloned into the XhoI/NotI sites of plasmid pL3 to capture a BamHI site. The resulting construct was digested with BamHI and NotI, and the fragment of interest cloned into the BamHI/NotI sites of the retroviral vector backbone pLZRS-CMV-EGFP (kindly provided by Dr. G.P. Nolan, Stanford University, CA). To construct the pLZRS-leptin-IRES-EGFP vector, a BamHI/EcoRI fragment containing the mouse leptin cDNA obtained from plasmid pK5-leptin was cloned into the BamHI/EcoRI sites of pLZRS-IRES-EGFP vector plasmid. Defective retroviruses were generated through transient transfection of 293-T cells with packaging and retroviral vector plasmids according to Yang et al. (40) .

Glucose and leptin measurements
Blood samples were collected by tail incision. Blood glucose levels were determined between 9 and 10 AM on nonfasted mice using a Gluco-touch digital monitor (Lifescan, Johnson & Johnson, Burnaby, B.C., Canada). Both serum and cultured keratinocyte-derived mouse leptin were determined using the Quantikine murine leptin ELISA kit (R&D Systems, Abingdon, Oxon, UK) following the manufacturer’s instructions with minor modifications. Serum samples were assayed at 1:5–1:10 dilutions.

Animal weight and food intake
Body weight was measured every other day at the same time of day. For feeding studies, mice were placed in individual cages and acclimated to autoclaved standard rodent chow diet (A04, Panlab, Barcelona, Spain). A preweighed amount of diet was given per animal, and the difference in food weight after each time period provided a measure of food consumption.

Histology
Human keratinocyte graft recipient animals were killed at day 16 postgrafting. The graft-containing flap area was excised and tissue samples were formalin-fixed/paraffin-embedded for routine hematoxylin/eosin staining or frozen in OCT. Human involucrin immunostaining was performed on 5 µm paraffin sections using the human-specific mouse monoclonal anti-involucrin antibody SY5 (Sigma, St. Louis, MO) at a 1:100 dilution. The ABC elite peroxidase kit (Dako, Carpinteria, CA) was used for antibody detection.

EGFP fluorescence was analyzed in unfixed 7 µm frozen sections under the fluorescence microscope. Sections were mounted with Mowiol (Hoechst, Somerville, NJ) containing DAPI to stain nuclei.

Statistical analysis
Statistical significance was assessed by ANOVA with repeated measures analysis and Student’s t test. All values are expressed as the mean ± SE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Correction of the ob/ob phenotype using skin grafts from K5-leptin transgenic mice
To test the feasibility of using skin grafts containing genetically manipulated keratinocytes to correct the ob/ob mouse defect, we first developed transgenic mice overexpressing mouse leptin cDNA under the control of the bovine keratin K5 gene regulatory elements. As previously shown by our group and others, the 5.2 kb bovine K5 gene promoter enables high constitutive basal layer-specific expression of a wide variety of transgenes in the epidermis (31 , 32 , 41 , 42) . In this case, transgenic epidermal cells were able to efficiently express, process, and secrete leptin resulting in very high serum leptin levels (105±13 ng/ml), leading to a more marked lean phenotype than that of the transgenic skinny mouse described by Ogawa et al. (43) . A detailed description of K5-leptin mice will be reported elsewhere (F. Larcher et al., unpublished results). Transplantation of 2–2.5 cm2 shaved back skin pieces from K5-leptin mice to immunodeficient ob/ob mice induced complete correction of the obese phenotype (Figs. 1 2 3 4) . As early as 48 h after transplantation, a marked drop in blood glucose was observed. From day 5 postgrafting, all K5-leptin skin-grafted animals reached steady glucose levels comparable to those in lean (+/? genotype) immunodeficient mice (151±13 mg/dl). In contrast, glucose remained at diabetic values throughout the experiment in animals transplanted with skin from control nontransgenic littermates (Fig. 1a ). K5-leptin skin-grafted ob/ob mice also showed a rapid decrease in food intake. After a postsurgical recovery period that affected both control skin-grafted and K5-leptin skin-grafted mice, food intake of K5-leptin skin-grafted animals stabilized at values approximately half that of controls (Fig. 1b ).



View larger version (31K):
[in this window]
[in a new window]
  		Figure 1. Effect of K5-leptin skin graft on glucose levels and food consumption of immunodeficient ob/ob recipient mice. a) Glucose levels in K5-leptin skin graft recipients were significantly lower than those of controls at all times after 5 days of transplantation (P<0.01). The average glucose level for immunodeficient lean (+/? genotype) mice was 151 ± 13 mg/dl. Values are the mean ± SE. b) Mice were caged individually and food intake measured over 48 h periods. Food intake in K5-leptin skin graft recipients was significantly lower than that of controls at all times after 4 days of transplantation (P<0.01). The average food intake value for immunodeficient ob/ob mice was 7.4 ± 0.17 g per day. Values are the mean ± SE.



View larger version (45K):
[in this window]
[in a new window]
  		Figure 2. Effect of K5-leptin skin graft on body weight of immunodeficient ob/ob recipient mice. a) Body weight of K5-leptin skin graft recipient mice was significantly lower than that of controls at all times after 7 days of grafting (P<0.05 and P<0.01 from day 11 postgrafting). Values are the mean ± SE and represent the percentage of body weight on the day of transplant (day 0). The average body weight for ob/ob mice at day 0 was 45.3 ± 1.7 g. 34 days after the initial transplantation (arrow), K5-leptin skin grafts were excised and replaced by ob/ob mouse skin in two mice. b) 2 wk cumulative food intake in ob/ob recipient mice before (bars at left) and after K5-leptin skin graft removal (shaded bar in bars at right).



View larger version (19K):
[in this window]
[in a new window]
  		Figure 3. Circulating leptin levels in K5-leptin skin-grafted immunodeficient ob/ob recipient mice. Serum was collected from mice at the times indicated and leptin levels were measured using a commercial murine leptin ELISA kit. Values are the mean ± SE. Average serum leptin of age-matched lean mice (+/? genotype) was 3.4 ± 1.6 ng/ml. Serum leptin in control skin-grafted animals was undetectable at all time points measured.



View larger version (110K):
[in this window]
[in a new window]
  		Figure 4. K5-leptin skin graft-mediated correction of obesity in immunodeficient ob/ob mice. a) Physical appearance of a donor K5-leptin transgenic mouse (right) compared to a control littermate (left). The dashed square represents the donor skin piece to be grafted. b) Physical appearance of an immunodeficient ob/ob mouse immediately after transplantation (day 0) with K5-leptin skin graft (left). An immunodeficient lean mouse (+/? genotype) is shown on the right. c) Representative body shape of a corrected immunodeficient ob/ob mouse 35 days postgrafting (right). A comparison between age-matched immunodeficient ob/ob (middle) and immunocompetent C57BL/6J ob/ob mouse (left) is also shown.

Sustained weight loss was apparent early after transplantation in K5-leptin skin-grafted mice, although weight loss kinetics changed with time. Whereas moderate weight decline occurred during the first days after transplantation, a more abrupt weight loss began around day 7, concomitant with hair regrowth in the graft (Fig. 2a ). This may be because the number of K5/leptin-expressing cells per area of skin increase during the anagen phase of hair follicle growth. On the contrary, control skin-grafted ob/ob mice started to regain weight after the slight weight loss, during the first 24–48 h postgrafting. Recipient animals showed an average initial weight at grafting of 45.3 ± 1.4 g. Five to six weeks after grafting, K5-leptin skin-transplanted mice lost 40% of body weight, reaching values within the range of age- and sex-matched lean immunodeficient littermates (+/? genotype) (Fig. 2a ).

Complete reversion of leptin action after graft removal
One advantage of skin gene therapy lies in the possibility of graft excision in case of adverse reactions (44) . To determine the extent of reversibility of the graft-driven systemic response, we tested the effect of K5-leptin skin graft removal on ‘slimmed’ immunodeficient ob/ob mice. The K5-leptin skin graft was replaced with a size-matched skin piece from an ob/ob mouse donor. A sudden drop in circulating leptin after graft excision appeared to act as a strong starvation signal, and rapid weight gain was observed concomitant with the recovery of food intake that reached control skin-grafted immunodeficient ob/ob values (Fig. 2a , b ). In contrast, immunodeficient ob/ob animals that conserved the K5-leptin skin graft remained within lean animal values (90 days after grafting, corresponding to the latest records; data not shown).

K5-leptin skin grafts produce stable serum leptin levels
Serum leptin was determined at different times postgrafting. K5-leptin skin-grafted mice showed values ranging from 3.1 to 4.5 ng/ml, a range within the physiological levels found in age-matched lean mice (Fig. 3 ). The presence of stable serum leptin throughout the experiment indicates that the K5 promoter remains active in the graft. Serum leptin was undetectable in control skin-grafted mice. Thus, a contribution of the subcutaneous (s.c.) adipose tissue from the control skin graft to circulating leptin appears negligible. As predicted, animals that underwent K5-leptin skin graft removal showed an absence of serum leptin, as determined 5 days after graft excision (data not shown). Both a long-lasting corrective effect and a reversible effect on graft removal were the hallmarks of this transgenic approach. A representative sequence of experimental steps and changes in physical appearance is illustrated in Fig. 4 .

Correction of the ob/ob phenotype through ex vivo leptin-transduced human keratinocyte grafts
Having achieved efficient systemic leptin delivery from transgenic mouse keratinocytes, we sought a relevant ex vivo approach for human gene therapy.

Efficient leptin gene transfer into primary HKs was carried out with high-titer replication-defective retroviruses obtained through transient transfection in 293T cells (39 , 40) . The retroviral construct (Fig. 5a ) contained an internal ribosome entry site (IRES) to coexpress leptin with EGFP as a selectable marker. The same construct lacking leptin cDNA was used to transduce control keratinocytes. Use of the IRES sequence in retroviral vectors for keratinocyte targeting has been described (45 , 46) . To produce populations of entirely genetically engineered human keratinocytes before grafting, transduced cells were selected by EGFP sorting. This technique, previously used to select EGFP gene-transferred pig keratinocytes (36) , renders viable HK cells without affecting their capacity for growth and differentiation (M. Del Rio et al. unpublished observations). Sorted cells were plated either on 3T3 feeder layer or on live fibroblast-containing fibrin gels (35 , 36) , and their conditioned medium was assayed for murine leptin. Cells cultured on either substrate produced similar amounts of leptin (Fig. 5b ).



View larger version (37K):
[in this window]
[in a new window]
  		Figure 5. Retroviral constructs and in vitro leptin levels. a) The retroviral constructs are based on LZRS vector backbone. Shaded boxes indicate regulatory sequences (viral LTR and IRES). b) In vitro mouse leptin produced by cultured leptin-transduced human (HK) or K5-leptin primary newborn mouse (MK) keratinocytes. Values are the mean ± SE of two independent experiments performed in duplicate.

To predict the possible outcome of the experiment using leptin-transduced human epidermal cells, we compared in vitro leptin levels of transduced HKs with those of primary mouse keratinocyte cultures (MK) obtained from K5-leptin transgenic mouse. Leptin-transduced HKs produced 30% more leptin than newborn K5-leptin mouse keratinocytes (357±31 vs. 255±35 ng/106 cells/24 h), suggesting that leptin-transduced HKs should perform adequately as a leptin delivery source (Fig. 5b ).

Generation of immunodeficient ob/ob mice involves a long-term breeding process, since several rounds of heterozygote matings were required to obtain enough double mutant mice for use in these experiments (see Materials and Methods). To obtain an appropriate number of readily available animals, we performed the human xenotransplants on cyclosporin-A immunosuppressed ob/ob mice. Previous studies showed that cyclosporin-treated C57BL/6J mice accepted human keratinocyte xenografts for an average of 16 days (38) , a period when a clear corrective response was observed with the K5-leptin skin grafts. Both control (IRES-EGFP) and leptin (leptin-IRES-EGFP)-transduced keratinocytes grown to confluence on fibrin-fibroblast gels were transplanted using an s.c. flap technique (36 , 37) in cyclosporin-A immunosuppressed C57BL/6J ob/ob mice. A study of serum glucose levels and food intake showed a rapid corrective effect in the leptin-HK transplanted mice (Fig. 6a , b ). A drop in glucose within 48 h of transplantation was the first sign of keratinocyte-derived leptin action (Fig. 6a ). Food intake was markedly reduced in leptin-HK transplanted mice, and food intake measurements showed a 50% reduction compared with control-HK transplanted mice (Fig. 6b ). The rate of weight loss was rapid and sustained. At the end of the experiment (16 days after grafting), leptin-HK transplanted mice had lost an average of 30% of starting weight. In contrast, control HK-transplanted mice showed a moderate weight gain (Fig. 6c ). Serum leptin was determined at three different times throughout the experiment. Leptin levels were stable and ranged from 1.9 to 2.5 ng/ml (mean 2.20±0.24 ng/ml, n=5 at day five; 2.11±0.16 ng/ml at day 10, n=4; 2.10±0.21 ng/ml at day 15, n=4; Table 1 ). At day 16 postgrafting, animals were killed to analyze graft histopathology. Histological evaluation revealed a well-developed, healthy stratified epithelium of human origin, as determined by hematoxylin/eosin staining and specific human involucrin immunostaining (Fig. 7a , b ). Active gene expression was confirmed by EGFP fluorescence in the graft (Fig. 7c ). One of the leptin-HK-grafted animals showed a progressive loss of the phenotypic corrective effect starting around day 8 postgrafting (data not shown). Histological examination of the grafted area in this animal was consistent with T cell-mediated rejection (Fig. 7d ), indicating premature loss of the immunosuppressive treatment efficacy.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 6. Effect of leptin-transduced human keratinocyte grafts on blood glucose, food consumption and body weight on immunosuppressed ob/ob recipient mice. a) Glucose levels in leptin-transduced human keratinocyte graft recipients were significantly lower than controls at all times after 48 h of transplantation (P<0.01). b) Mice were caged individually and food intake was measured over a 48 h period. Values are the mean ± SE. P < 0.01 for ob/ob transplanted with leptin-transduced HK grafts compared to ob/ob transplanted with control HK grafts 4 days after transplantation. c) Body weight of leptin-transduced human keratinocyte graft recipient mice was significantly lower than that of controls at all times after 2 days of grafting (P < 0.05 and P<0.01 after 6 days). Body weight is expressed as the percent of body weight on the day of transplant (day 0). The average body weight for immunosuppressed ob/ob mice at day 0 was 46.1 ± 1.5 g.


View this table:
[in this window]
[in a new window]
  		Table 1. In vivo leptin levelsa



View larger version (100K):
[in this window]
[in a new window]
  		Figure 7. Histological appearance of leptin-transduced human skin graft in immunodeficient ob/ob mice. a) Human involucrin immunostaining of a section of the grafted area on the inner surface of the recipient mouse dorsal region demonstrating positive staining in the leptin-transduced human epithelium (arrowhead). b) Hematoxylin/eosin staining of leptin-HK graft, showing the presence of a well-differentiated epithelium. c) Detection of transduced gene expression through EGFP-fluorescence in an unfixed frozen section of the leptin-HK graft. Nuclei were visualized by DAPI staining (blue fluorescence). d) Hematoxylin/eosin staining of a graft section from a leptin-HK-grafted mouse that lost the leptin-corrective effect. Note the presence of lymphocyte infiltrate consistent with T cell-mediated rejection of the graft. x40 (a) or x400 (b–d).

As also found for the K5-leptin grafts, a 2–2.5 cm2 ex vivo genetically engineered HK graft sufficed to replace physiological leptin levels, which in turn reversed the ob/ob syndrome in a short-term experiment.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The majority of systemic protein deficiencies (i.e., type I diabetes, hemophilia, dwarfism) are currently treated by multiple injections of the deficient polypeptide. Safe administration procedures that would eliminate this unpleasant duty on the part of the patient would be highly desirable (19) . Somatic gene therapy offers an attractive alternative for permanent protein delivery.

Numerous ex vivo, in vivo, and transgenic studies have established the potential therapeutic use of genetically manipulated keratinocytes as a noninvasive vehicle of circulatory systemic proteins (20 21 22 23 24 25 26 27) .

Most studies of systemic keratinocyte gene therapy have emphasized critical issues such as duration of transgene expression and serum levels of the modified keratinocyte-derived proteins (47 48 49) . Less work has addressed the correction of circulating protein deficiencies. To date, only a partial corrective effect, in a mouse model of hemophilia A, has been achieved through grafts of involucrin factor VIII transgenic mouse skin (27) .

In the present study, we have been able to fully correct the phenotype of the leptin-deficient ob/ob mouse. When K5-leptin transgenic skin patches were grafted onto immunodeficient ob/ob mice, a rapid, lasting corrective effect was achieved as the clinical and biochemical parameters related to the obese phenotype returned to normal values. High serum leptin protein levels resulted from the basal cell-specific keratin K5 gene promoter activity. High hGH protein levels were similarly found in the blood of transgenic mice in which an hGH cDNA was under the control of the keratin K14 gene promoter (26) . The long-lasting effect achieved with the K5-leptin transgenic skin grafts reinforces the idea that the use of strong epidermal promoters would be a major asset to skin gene therapy. To be able to use gene constructs similar to those used in the generation of transgenic mice, however, improved nonviral gene transfer methods for human keratinocytes are still required (36) .

Transgenic approaches, although not of direct clinical relevance, allow us to address issues critical to modeling a therapeutic human ex vivo system, such as 1) whether the product of transferred genes can be delivered from epidermis to blood circulation, 2) to compare therapeutic protein concentrations in epidermal cell culture, 3) to make a rough estimate of the graft size needed, and 4) to assess potential undesired effects.

We used the K5-leptin transgenic skin grafting studies to predict whether the ex vivo approach using genetically engineered human keratinocytes would be feasible. Differences in promoter specificity (viral LTRs vs. K5) and/or the presence of epidermal appendages, such as hair follicles in the transgenic mouse skin grafts, may lead to substantial variations in keratinocyte-derived leptin per area unit of grafted skin in vivo. In fact, although transduced and selected human keratinocytes produced 30% more bioactive leptin than keratinocytes derived from K5-leptin transgenic mice in vitro, serum leptin levels obtained with leptin HKs in vivo were slightly lower than predicted. Nevertheless, when grafts of leptin-producing human keratinocytes of similar size to those from K5-leptin mice were used, a rapid corrective effect was also achieved. Both the transgenic and ex vivo cutaneous gene therapy approaches reported here appear to be as efficient as another long-term delivery system in which a leptin-carrying adeno-associated virus is targeted to muscle in ob/ob mice (18) . Skin accessibility, ex vivo keratinocyte manipulation, and the possibility of graft removal highlight the feasibility as well as the safety advantages of this procedure over other gene therapy methods for leptin delivery. Both the immunosuppressive protocol and the grafting technique used allowed only a short-term study. We have nonetheless been able to show that ex vivo, genetically modified human keratinocyte grafts comprising less than 10% of total body surface were suitable for correction of leptin deficiency in mice. Some clinical and biochemical parameters of the ob/ob phenotype were not monitored during our corrective study. Based on previous studies of recombinant leptin administration and the fact that the serum leptin levels reached either after transgenic mouse skin or human keratinocyte grafts were within values found in lean animals, glucose, weight, and food intake appear to be representative markers of the complete ob/ob syndrome (50) . Experiments are under way to determine whether orthotopic grafting of genetically engineered leptin-expressing human keratinocytes are able to produce long-term correction in immunodeficient ob/ob mice. This goal appears within reach, considering the recent success in achieving sustainable gene expression in genetically engineered human keratinocytes (24 , 49 ; M. Del Rio et al., unpublished results).

Obesity in humans is a major chronic disorder with a high incidence of morbidity; its molecular pathogenesis is the subject of intense study. Most obese individuals appear to be resistant to leptin, showing higher than normal levels of the hormone. In fact, inherited leptin deficiency appears to be a rare cause of extreme obesity in humans (8 , 9) . Recombinant human leptin has been tested successfully on one such leptin-deficient patient (10) . Nonetheless, since leptin is a short half-life protein susceptible to breakdown and inactivation by the gastrointestinal system, daily s.c. injections need to be administered. It has become apparent that a group of patients suffering from some forms of lipodystrophy show reduced leptin levels (12) . Alleviation of severe insulin resistance after leptin administration has recently been shown in two transgenic mouse models of congenital generalized lipodystrophy (CGL) (13 14 15 16) . CGL patients would thus probably benefit from leptin replacement. It is conceivable that, under experimental conditions equivalent to those described here, a single operation replacing less than 10% of the epidermal surface with leptin-producing keratinocyte sheets would suffice to restore normal leptin levels in patients suffering from leptin deficiency.

Epidermal regeneration obtained with cultured keratinocytes has been shown to be long-lasting as well as life-saving in cases of severe wounds (51 , 52) . In the last few years, cultured epidermal sheets bearing melanocytes have also been used to treat stable vitiligo with encouraging results; more important, new surgical procedures have been demonstrated that allow grafting without scarring (53) . These technical advantages and the feasibility of stable transduction of epidermal stem cells (24 , 47 , 48) lead us to predict a promising future for keratinocyte-mediated gene therapy for leptin and perhaps for other protein deficiencies.


   ACKNOWLEDGMENTS
 
This work was supported by grants PM98–0039 and SAF 98–0047 from the Spanish Ministry for Science and Technology. We wish to thank Dr. Alberto Alvarez and Israel Orman for FACS analysis, Cathy Mark for editorial revision of the manuscript, Isabel de los Santos for technical help, Dr. Clara Martin for surgical assistance, Jesus Martinez for animal care, and Dr. Fernando Benavidez for helpful comments on animal breeding strategies.


   FOOTNOTES
 
1 These authors contributed equally to these studies.

Received for publication February 12, 2001. Accepted for publication March 26, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Friedman, J. M., Halaas, J. L. (1998) Leptin and the regulation of body weight in mammals. Nature (London) 395,763-770[Medline]
  2. Ahima, R. S., Flier, J. S. (2000) Leptin. Annu. Rev. Physiol. 62,413-437[Medline]
  3. Wang, M. Y., Koyama, K., Shimabukuro, M., Newgard, C. B., Unger, R. H. (1998) OB-Rb gene transfer to leptin-resistant islets reverses diabetogenic phenotype. Proc. Natl. Acad. Sci. USA 95,714-718[Abstract/Free Full Text]
  4. Kim, Y. B., Uotani, S., Pierroz, D. D., Flier, J. S., Kahn, B. B. (2000) In vivo administration of leptin activates signal transduction directly in insulin-sensitive tissues: overlapping but distinct pathways from insulin. Endocrinology 141,2328-2339[Abstract/Free Full Text]
  5. Ducy, P., Amling, M., Takeda, S., Priemel, M., Schilling, A. F., Beil, F. T., Shen, J., Vinson, C., Rueger, J. M., Karsenty, G. (2000) Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass. Cell 100,197-207[Medline]
  6. Bouloumie, A., Drexler, H. C., Lafontan, M., Busse, R. (1998) Leptin, the product of Ob gene, promotes angiogenesis. Circ. Res. 83,1059-1066[Abstract/Free Full Text]
  7. Sierra-Honigmann, M. R., Nath, A. K., Murakami, C., Garcia-Cardena, G., Papapetropoulos, A., Sessa, W. C., Madge, L. A., Schechner, J. S., Schwabb, M. B., Polverini, P., et al (1998) Biological action of leptin as an angiogenic factor. Science 281,1683-1686[Abstract/Free Full Text]
  8. Montague, C. T., Farooqi, I. S., Whitehead, J. P., Soos, M. A., Rau, H., Wareham, N. J., Sewter, C. P., Digby, J. E., Mohammed, S. N., Hurst, J. A., Cheetham, C., Earley, A., Barnett, A., Prins, J., O’Rahilly, S. (1997) Congenital leptin deficiency is associated with severe early-onset obesity in humans. Nature (London) 387,903-908[Medline]
  9. Strobel, A., Issad, T., Camoin, L., Ozata, M., Strosberg, A. D. (1998) A leptin missense mutation associated with hypogonadism and morbid obesity. Nat. Genet. 3,213-215
  10. Farooqi, I. S., Jebb, S. A., Langmack, G., Lawrence, E., Cheetham, C. H., Prentice, A. M., Hughes, I. A., McCamish, M. A., O’Rahilly, S. (1999) Effects of recombinant leptin therapy in a child with congenital leptin deficiency. N. Engl. J. Med. 341,879-884[Free Full Text]
  11. Seip, M., Trygstad, O. (1996) Generalized lipodystrophy, congenital and acquired (lipoatrophy). Acta Paediatr. Suppl. 13,2-28
  12. Pardini, V. C., Victoria, I. M., Rocha, S. M., Andrade, D. G., Rocha, A. M., Pieroni, F. B., Milagres, G., Purisch, S., Velho, G. (1998) Leptin levels, beta-cell function, and insulin sensitivity in families with congenital and acquired generalized lipoatropic diabetes. J. Clin. Endocrinol. Metab. 83,503-508[Abstract/Free Full Text]
  13. Shimomura, I., Hammer, R. E., Richardson, J. A., Ikemoto, S., Bashmakov, Y., Goldstein, J. L., Brown, M. S. (1998) Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissue: model for congenital generalized lipodystrophy. Genes Dev 12,3182-3194[Abstract/Free Full Text]
  14. Moitra, J., Mason, M. M., Olive, M., Krylov, D., Gavrilova, O., Marcus-Samuels, B., Feigenbaum, L., Lee, E., Aoyama, T., Eckhaus, M., Reitman, M., Vinson, C. (1998) Life without white fat: a transgenic mouse. Genes Dev 12,3168-3181[Abstract/Free Full Text]
  15. Shimomura, I., Hammer, R. E., Ikemoto, S., Brown, M. S., Goldstein, J. L. (1999) Leptin reverses insulin resistance and diabetes mellitus in mice with congenital lipodystrophy. Nature (London) 401,73-76[Medline]
  16. Ebihara, K., Ogawa, Y., Masuzaki, H., Miyanaga, F., Shintani, M., Abe, M., Hayashi, T., Hosoda, K., Inoue, G., Yoshimasa, Y., Reitman, M., Nakao, K. (2000) Increased glucose metabolism and insulin sensitivity in a mouse model of lipoatrophic diabetes by transgenic overexpression of leptin. Diabetes 49(Suppl.),A210(abstr.)
  17. Muzzin, P., Eisensmith, R. C., Copeland, K. C., Woo, S. L. (1996) Correction of obesity and diabetes in genetically obese mice by leptin gene therapy. Proc. Natl. Acad. Sci. USA 93,14804-14808[Abstract/Free Full Text]
  18. Murphy, J. E., Zhou, S., Giese, K., Williams, L. T., Escobedo, J. A., Dwarki, V. J. (1997) Long-term correction of obesity and diabetes in genetically obese mice by a single intramuscular injection of recombinant adeno-associated virus encoding mouse leptin. Proc. Natl. Acad. Sci. USA 94,13921-13926[Abstract/Free Full Text]
  19. Morsy, M. A., Gu, M. C., Zhao, J. Z., Holder, D. J., Rogers, I. T., Pouch, W. J., Motzel, S. L., Klein, H. J., Gupta, S. K., Liang, X., et al (1998) Leptin gene therapy and daily protein administration: a comparative study in the ob/ob mouse. Gene Ther 5,8-18[Medline]
  20. Fenjves, E. S., Smith, J., Zaradic, S., Taichman, L. B. (1994) Systemic delivery of secreted protein by grafts of epidermal keratinocytes: prospects for keratinocyte gene therapy. Hum. Gene Ther. 5,1241-1248[Medline]
  21. Morgan, J. R., Barrandon, Y., Green, H., Mulligan, R. C. (1987) Expression of an exogenous growth hormone gene by transplantable human epidermal cells. Science 237,1476-1479[Abstract/Free Full Text]
  22. Teumer, J., Lindahl, A., Green, H. (1990) Human growth hormone in the blood of athymic mice grafted with cultures of hormone-secreting human keratinocytes. FASEB J 4,3245-3250[Abstract]
  23. Gerrard, A. J., Hudson, D. L., Brownlee, G. G., Watt, F. M. (1993) Towards gene therapy for haemophilia B using primary human keratinocytes. Nat. Genet. 3,180-183[Medline]
  24. Mathor, M. B., Ferrari, G., Dellambra, E., Cilli, M., Mavilio, F., Cancedda, R., De Luca, M. (1996) Clonal analysis of stably transduced human epidermal stem cells in culture. Proc. Natl. Acad. Sci. USA 93,10371-10376[Abstract/Free Full Text]
  25. Alexander, M. Y., Bidichandani, S. I., Cousins, F. M., Robinson, C. J., Duffie, E., Akhurst, R. J. (1995) Circulating human factor IX produced in keratin-promoter transgenic mice: a feasibility study for gene therapy of haemophilia B. Hum. Mol. Genet. 4,993-999[Abstract/Free Full Text]
  26. Wang, X., Zinkel, S., Polonsky, K., Fuchs, E. (1997) Transgenic studies with a keratin promoter-driven growth hormone transgene: prospects for gene therapy. Proc. Natl. Acad. Sci. USA 94,219-226[Abstract/Free Full Text]
  27. Fakharzadeh, S. S., Zhang, Y., Sarkar, R., Kazazian, H. H., Jr (2000) Correction of the coagulation defect in hemophilia A mice through factor VIII expression in skin. Blood 95,2799-2805[Abstract/Free Full Text]
  28. Meng, X., Sawamura, D., Tamai, K., Hanada, K., Ishida, H., Hashimoto, I. (1998) 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. 101,1462-1467[Medline]
  29. Coleman, D. L. (1978) Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice. Diabetologia 14,141-148[Medline]
  30. Leiter, E. H. (1992) Variation in the severity and duration of hyperglycemia in the C57BL/6-ob/ob (obese) mouse. JAX Notes ,451
  31. Ramirez, A., Bravo, A., Jorcano, J. L., Vidal, M. (1994) Sequences 5' of the bovine keratin 5 gene direct tissue- and cell type-specific expression of a lacZ gene in the adult and during development. Differentiation 58,53-64[Medline]
  32. Murillas, R., Larcher, F., Conti, C. J., Santos, M., Ullrich, A., Jorcano, J. L. (1995) Expression of a dominant negative mutant of epidermal growth factor receptor in the epidermis of transgenic mice elicits striking alterations in hair follicle development and skin structure. EMBO J 14,5216-5223[Medline]
  33. Namae, M., Mori, Y., Yasuda, K., Kadowaki, T., Kanazawa, Y., Komeda, K. (1998) New method for genotyping the mouse Lep(ob) mutation, using a polymerase chain reaction assay. Lab. Anim. Sci. 48,103-105[Medline]
  34. Albella, B., Segovia, J. C., Güenechea, G., Pragnell, I. B., Bueren, J. A. (1999) Preserved long-term repopulation and differentiation properties of hematopoietic grafts subjected to ex vivo expansion with stem cell factor and interleukin 11. Transplantation 67,1348-1357[Medline]
  35. Meana, A., Iglesia, J., Del Rio, M., Larcher, F., Madrigal, B., Fresno, M. F., Martin, C., San Roman, F., Tevar, F. (1998) Large surface of cultured human epithelium obtained on a dermal matrix based on live fibroblast-containing fibrin gels. Burns 7,621-630
  36. Del Rio, M., Larcher, F., Meana, A., Segovia, J., Jorcano, J. (1999) Non-viral 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 6,1734-1741[Medline]
  37. Barrandon, Y., Li, V., Green, H. (1988) New techniques for the grafting of cultured human epidermal cells onto athymic animals. J. Invest. Dermatol. 91,315-318[Medline]
  38. Compton, C. C., Rizk, I., Regauer, S., Burd, E., Holaday, J., Kenner, J. (1994) The effect of bromocryptine-induced hypoprolactinemia on xenogeneic and allogeneic skin graft survival in a mouse model. J. Burn Care Rehabil. 15,393-400[Medline]
  39. Kinsella, T. M., Nolan, G. P. (1996) Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum. Gene Ther. 7,1405-1413[Medline]
  40. Yang, S., Delgado, R., King, S., Woffendin, C., Barker, C., Yang, Z., Xu, L., Nolan, G., Nabel, G. (1999) Generation of retroviral vector for clinical studies using transient transfection. Hum. Gene Ther. 10,123-132[Medline]
  41. Robles, A. I., Larcher, F., Whalin, R. B., Murillas, R., Richie, E., Gimenez-Conti, I. B., Jorcano, J. L., Conti, C. J. (1996) Expression of cyclin D1 in epithelial tissues of transgenic mice results in epidermal hyperproliferation and severe thymic hyperplasia. Proc. Natl. Acad. Sci. USA 93,7634-7638[Abstract/Free Full Text]
  42. DiGiovanni, J., Bol, D. K., Wilker, E., Beltran, L., Carbajal, S., Moats, S., Ramirez, A., Jorcano, J., Kiguchi, K. (2000) Constitutive expression of insulin-like growth factor-1 in epidermal basal cells of transgenic mice leads to spontaneous tumor promotion. Cancer Res 60,1561-1570[Abstract/Free Full Text]
  43. Ogawa, Y., Masuzaki, H., Hosoda, K., Aizawa-Abe, M., Suga, J., Suda, M., Ebihara, K., Iwai, H., Matsuoka, N., Satoh, N., et al (1999) Increased glucose metabolism and insulin sensitivity in transgenic skinny mice overexpressing leptin. Diabetes 48,1822-1829[Abstract]
  44. Taichman, L. (1994) Epithelial gene therapy. Leight, I. Lane, B. Watt, F. eds. The Keratinocyte Handbook ,543-551 Cambridge Univ. Press
  45. Deng, H., Choate, K. A., Lin, Q., Khavari, P. A. (1998) High-efficiency gene transfer and pharmacologic selection of genetically engineered human keratinocytes. BioTechniques 25,274-280[Medline]
  46. Pfutzner, W., Hengge, U. R., Joari, M. A., Foster, R. A., Vogel, J. C. (1999) 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. 10,2811-2821[Medline]
  47. Deng, H., Lin, Q., Khavari, P. A. (1997) Sustainable cutaneous gene delivery. Nat. Biotechnol. 15,1388-1391[Medline]
  48. De Luca, M., Pellegrini, G. (1997) The importance of epidermal stem cells in keratinocyte-mediated gene therapy. Gene Ther 4,381-383[Medline]
  49. Kolodka, T., Garlick, J., Taichman, L. (1998) Evidence for keratinocyte stem cells in vitro: long term engraftment and persistence of transgene expression from retrovirus-transduced keratinocytes. Proc. Natl. Acad. Sci. USA 95,4356-4361[Abstract/Free Full Text]
  50. Ioffe, E., Moon, B., Connolly, E., Friedman, J. M. (1998) Abnormal regulation of the leptin gene in the pathogenesis of obesity. Proc. Natl. Acad. Sci. USA 95,11852-11857[Abstract/Free Full Text]
  51. Gallico, G. G., O’Connor, N. E., Compton, C. C., Kehinde, O., Green, H. (1984) Permanent coverage of large burn wounds with autologous cultured human epithelium. N. Engl. J. Med. 311,448-451[Medline]
  52. Pellegrini, G., Bondanza, S., Guerra, L., De Luca, M. (1998) Cultivation of human keratinocyte stem cells: current and future clinical applications. Med. Biol. Eng. Comput. 36,778-790[Medline]
  53. Guerra, L., Capurro, S., Melchi, F., Primavera, G., Bondanza, S., Cancedda, R., Luci, A., De Luca, M., Pellegrini, G. (2000) Treatment of ‘stable’ vitiligo by timed surgery and transplantation of cultured epidermal autografts. Arch. Dermatol. 136,1380-1389[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Hum Mol GenetHome page
B. D. Yu, A. Mukhopadhyay, and C. Wong
Skin and hair: models for exploring organ regeneration
Hum. Mol. Genet., April 15, 2008; 17(R1): R54 - R59.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
M. J. Jurczak, A. M. Danos, V. R. Rehrmann, M. B. Allison, C. C. Greenberg, and M. J. Brady
Transgenic overexpression of protein targeting to glycogen markedly increases adipocytic glycogen storage in mice
Am J Physiol Endocrinol Metab, March 1, 2007; 292(3): E952 - E963.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. Garcia, N. I. Fernandez-Garcia, V. Rivas, M. Carretero, M. J. Escamez, A. Gonzalez-Martin, E. E. Medrano, O. Volpert, J. L. Jorcano, B. Jimenez, et al.
Inhibition of Xenografted Human Melanoma Growth and Prevention of Metastasis Development by Dual Antiangiogenic/Antitumor Activities of Pigment Epithelium-Derived Factor
Cancer Res., August 15, 2004; 64(16): 5632 - 5642.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
R. B. CEDDIA, H. A. KOISTINEN, J. R. ZIERATH, and G. SWEENEY
Analysis of paradoxical observations on the association between leptin and insulin resistance
FASEB J, August 1, 2002; 16(10): 1163 - 1176.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by LARCHER, F.