Marcela Del Rio

Human plasma as a dermal scaffold for the generation of a completely autologous bioengineered skin

Background. Keratinocyte cultures have been used for the treatment of severe burn patients. Here, we describe a new cultured bioengineered skin based on (1) keratinocytes and fibroblasts obtained from a single skin biopsy and (2) a dermal matrix based on human plasma. A high expansion capacity achieved by keratinocytes grown on this plasma-based matrix is reported. In addition, the results of successful preclinical and clinical tests are presented. Methods. Keratinocytes and fibroblasts were obtained by a double enzymatic digestion (trypsin and collagenase, respectively). In this setting, human fibroblasts are embedded in a clotted plasma-based matrix that serves as a three-dimensional scaffold. Human keratinocytes are seeded on the plasma-based scaffold to form the epidermal component of the skin construct. Regeneration performance of the plasma-based bioengineered skin was tested on immunodeficient mice as a preclinical approach. Finally, this skin equivalent was grafted on two severely burned patients. Results. Keratinocytes seeded on the plasma-based scaffold grew to confluence, allowing a 1,000-fold cultured-area expansion after 24 to 26 days of culture. Experimental transplantation of human keratinocytes expanded on the engineered plasma scaffold yielded optimum epidermal architecture and phenotype, including the expression of structural intracellular proteins and basement-membrane components. In addition, we report here the successful engraftment and stable skin regeneration in two severely burned patients at 1 and 2 years follow-up. Conclusions. Our data demonstrate that this new dermal equivalent allows for (1) generation of large bioengineered skin surfaces, (2) restoration of both the epidermal and dermal skin compartments, and (3) functional epidermal stem-cell preservation.

Inhibition of Xenografted Human Melanoma Growth and Prevention of Metastasis Development by Dual Antiangiogenic/Antitumor Activities of Pigment Epithelium-Derived Factor

Human melanoma mortality is associated with the growth of metastasis in selected organs including the lungs, liver, and brain. In this study, we examined the consequences of overexpression of pigment epithelium-derived factor (PEDF), a neurotrophic factor and potent angiogenesis inhibitor, on both melanoma primary tumor growth and metastasis development. PEDF overexpression by melanoma cells greatly inhibited subcutaneous tumor formation and completely prevented lung and liver metastasis in immunocompromised mice after tail vein injection of metastatic human melanoma cell lines. Whereas the effects of PEDF on primary tumor xenografts appear mostly associated with inhibition of the angiogenic tumor response, abrogation of melanoma metastasis appears to depend on direct PEDF effects on both migration and survival of melanoma cells. PEDF-mediated inhibition of melanoma metastases could thus have a major impact on existing therapies for melanoma.

A Preclinical Model for the Analysis of Genetically Modified Human Skin In Vivo

Although skin is perhaps the most accessible of all somatic tissues for therapeutic gene transfer, it is a challenging site when attempting gene delivery. In addition to the transience of gene expression, important obstacles to cutaneous gene therapy have included the inability to sustain gene expression in a large proportion of keratinocytes within a given skin compartment. In this study, we have developed a novel experimental strategy that allows long-term regeneration of entirely genetically engineered human skin on the backs of NOD/SCID mice. Primary human keratinocytes were infected with a retroviral vector encoding the enhanced green fluorescent protein (EGFP) produced by transient transfection of 293T cells. EGFP expression allowed cell-sorting selection of a polyclonal population of productively transduced keratinocytes that were assembled in a live fibroblast-containing fibrin dermal matrix and orthotopically grafted onto mice. Epifluorescent illumination of the transplanted zone allowed in vivo monitoring of the genetically modified graft. EGFP-positive human skin was present on mice for 22 weeks after grafting. In addition, frozen sections prepared from the grafts displayed consistently strong EGFP-based fluorescence in all epidermal strata at every time point examined. Persistence of transgene expression was further confirmed through EGFP protein immunodetection. Purified EGFP-positive keratinocytes grafted as part of the fibrin-based artificial skin were capable of generating multilayer human epidermis on mice, with well-developed granulosum and corneum strata, and clearly defined rete ridges. Finally, the large proportion of transduced keratinocytes in our grafts allowed us to study, for the first time, the long-term in vivo clonal reconstitution pattern of the regenerated skin. Analysis of the provirus insertion sites indicates that a discrete number of epidermal stem cell clones was responsible for the maintenance of human skin regenerated in NOD/SCID recipients.

Kindler syndrome: extension of FERMT1 mutational spectrum and natural history.

Mutations in the FERMT1 gene (also known as KIND1), encoding the focal adhesion protein kindlin‐1, underlie the Kindler syndrome (KS), an autosomal recessive skin disorder with an intriguing progressive phenotype comprising skin blistering, photosensitivity, progressive poikiloderma with extensive skin atrophy, and propensity to skin cancer. Herein we review the clinical and genetic data of 62 patients, and delineate the natural history of the disorder, for example, age at onset of symptoms, or risk of malignancy. Although most mutations are predicted to lead to premature termination of translation, and to loss of kindlin‐1 function, significant clinical variability is observed among patients. There is an association of FERMT1 missense and in‐frame deletion mutations with milder disease phenotypes, and later onset of complications. Nevertheless, the clinical variability is not fully explained by genotype–phenotype correlations. Environmental factors and yet unidentified modifiers may play a role. Better understanding of the molecular pathogenesis of KS should enable the development of prevention strategies for disease complications. Hum Mutat 32:1204–1212, 2011. ©2011 Wiley Periodicals, Inc.

1α,25-Dihydroxyvitamin D3 regulates the expression of Id1 and Id2 genes and the angiogenic phenotype of human colon carcinoma cells

1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3) has antitumor activity in addition to its classical action on calcium metabolism and bone tissue biology. It is thought to regulate the expression of multiple target genes and thus modulate processes critical for tumor growth and metastases. Here we show that 1α,25(OH)2D3 differentially regulates the expression of Id1 and Id2 genes, members of a family of transcriptional regulators of cell proliferation and differentiation. 1α,25(OH)2D3 induced epithelial differentiation in SW480-ADH human colon carcinoma cell line by promoting expression of the proteins implicated in adherent junction formation, including E-cadherin, and by inhibiting β-catenin transcriptional activity. 1α,25(OH)2D3 activated the human Id1 gene promoter and rapidly induced Id1 RNA and protein. Ectopic overexpression of Id1 was not sufficient to induce E-cadherin, which was critical for the morphological changes induced by 1α,25(OH)2D3 in SW480-ADH cells. Conversely, Id2 transcription rate, RNA and protein levels were decreased by 1α,25(OH)2D3. Id2 downregulation by 1α,25(OH)2D3 mediated the antiproliferative effect of 1α,25(OH)2D3 on SW480-ADH cells. In addition, we showed that 1α,25(OH)2D3 changed the levels of the inducer of angiogenesis, vascular endothelial growth factor and the potent antiangiogenic factor thrombospondin-1, leading to a balanced change in the angiogenic potential of SW480-ADH human colon carcinoma cells.

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

Leptin deficiency produces a phenotype of obesity, diabetes, and infertility in the ob/ob mouse. In humans, leptin deficiency occurs in some cases of congenital obesity and in lipodystrophic disorders characterized by reduced adipose tissue and insulin resistance. Cutaneous gene therapy is considered an attractive potential method to correct circulating protein deficiencies, since gene‐transferred human keratinocytes can produce and secrete gene products with systemic action. However, no studies showing correction of a systemic defect have been reported. We report the successful correction of leptin deficiency using cutaneous gene therapy in the ob/ob mouse model. As a feasibility approach, skin explants from transgenic mice overexpressing leptin were grafted on immunodeficient ob/ob mice. One month later, recipient mice reached body weight values of lean animals. Other biochemical and clinical parameters were also normalized. In a second human gene therapy approach, a retroviral vector encoding both leptin and EGFP cDNAs was used to transduce HK and, epithelial grafts enriched in high leptin‐producing HK were transplanted to immunosuppressed ob/ob mice. HK‐derived leptin induced body weight reduction after a drop in blood glucose and food intake. Leptin replacement through genetically engineered HK grafts provides a valuable therapeutic alternative for permanent treatment of human leptin deficiency conditions.—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‐tar‐geted keratinocyte grafts. FASEB J. 15, 1529–1538 (2001)

Correction of Laminin-5 Deficiency in Human Epidermal Stem Cells by Transcriptionally Targeted Lentiviral Vectors

Deficiency of the basement membrane component laminin-5 (LAM5) causes junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. Autologous transplantation of epidermal stem cells genetically corrected with a Moloney leukemia virus (MLV)-derived retroviral vector reconstitutes LAM5 synthesis, and corrects the adhesion defect in JEB patients. However, MLV-derived vectors have genotoxic characteristics, and are unable to reproduce the physiological, basal layer-restricted expression of LAM5 chains. We have developed an alternative gene transfer strategy based on self-inactivating (SIN) or long terminal repeat (LTR)-modified lentiviral vectors, in which transgene expression is under the control of different combinations of promoter-enhancer elements derived from the keratin-14 (K14) gene. Analysis in human keratinocyte cultures and in fully differentiated skin regenerated onto immunodeficient mice showed that gene expression directed by K14 enhancers is tissue-specific and restricted to the basal layer of the epidermis. Transcriptionally targeted lentiviral vectors efficiently transduced clonogenic stem/progenitor cells derived from a skin biopsy of a JEB patient, restored normal synthesis of LAM5 in cultured keratinocytes, and reconstituted normal adhesion properties in human skin equivalents transplanted onto immunodeficient mice. These vectors are therefore an effective, and potentially more safe, alternative to MLV-based retroviral vectors in gene therapy of JEB.Molecular Therapy (2008) 16 12, 1977-1985 doi:10.1038/mt.2008.204.

Assessment of Optimal Virus-Mediated Growth Factor Gene Delivery for Human Cutaneous Wound Healing Enhancement

Using a recently described skin-humanized model based on the engraftment of human bioengineered skin equivalents onto immunodeficient mice, we compared the efficacy of different in vivo gene transfer strategies aimed at delivering growth factors to promote skin wound healing. The approaches involving transient delivery of keratinocyte growth factor (KGF) to wounds performed in the engrafted human skin included (1) KGF gene transfer by intradermal adenoviral injection; (2) KGF gene transfer by adenoviral vector immobilized in a fibrin carrier; and (3) KGF-adenoviral gene-transferred human fibroblasts embedded in a fibrin matrix. All delivery systems achieved KGF protein overproduction at the wound site, with a concomitant re-epithelialization enhancement. However, although direct gene delivery strategies exhibited variability in terms of the number of successfully transduced humanized mice, the use of genetically modified fibroblast-containing matrix as an in situ protein bioreactor was highly reproducible, leading to a significant improvement of the overall healing process. This latter approach appeared to be the most reliable means to deliver growth factors to wounds and also avoided the potential danger of scoring cases of faulty administration as therapeutic failures and direct exposure to viral vectors. The combined use of cell and gene therapy appears a robust tool to aid healing in a clinical context.

Acetylsalicylic Acid Inhibits Cell Proliferation by Involving Transforming Growth Factor-β

Background—Acetylsalicylic acid (ASA) inhibits cell proliferation. This may be mediated by transforming growth factor-&bgr; (TGF-&bgr;). TGF-&bgr; directly stops cell proliferation, restrains cells in G0, and inhibits the uptake of platelet-derived growth factor and insulin-like growth factor. These effects are identical to those observed with ASA treatment. Methods and Results—We cultured rat thoracic aorta vascular smooth muscle cells and measured cytotoxicity, cell proliferation, cell cycle, transcription of TGF-&bgr;1, and concentration of TGF-&bgr;1 in supernatant medium. ASA dose-dependently restrained cells in G0 phase with no cytotoxic effect and inhibited cell proliferation by 30.86%. Anti–TGF-&bgr;1 reversed this inhibition by 30.21%. However, ASA treatment decreased TGF-&bgr;1 transcription and had no significant effect on TGF-&bgr;1 concentration. Conclusions—TGF-&bgr; seems to play an important role in ASA-mediated inhibition of cell proliferation. Therefore, treatment with ASA prevents coronary disease not only by means of its antiplatelet properties but also by an important inhibition of plaque growth. This relationship between ASA and TGF-&bgr; explains many other effects, such as cancer chemoprevention, immunomodulation, and wound healing. The aim of this study was to demonstrate this link.

Ex-vivo Gene Therapy Restores LEKTI Activity and Corrects the Architecture of Netherton Syndrome-derived Skin Grafts

Netherton syndrome (NS) is a debilitating congenital skin disorder caused by mutations in the SPINK5 gene encoding the lymphoepithelial Kazal-type-related inhibitor (LEKTI). It is characterized by defective keratinization, recurrent infections, and hypernatraemic dehydration with a mortality rate of about 10% in the first year of life. Currently, there are no curative treatments for NS. We have developed a HIV-1 based, self-inactivating lentiviral vector to express SPINK5 in keratinocytes as part of an ex-vivo gene therapy strategy for NS. High transduction efficiency was achieved in NS keratinocytes and reconstitution of LEKTI expression was confirmed in previously deficient cells. These genetically corrected keratinocytes were further tested in an in vitro organotypic culture (OTC) system and in vivo mouse/human skin engraftment model. Results showed correction of epidermal architecture in both OTCs and regenerated skin grafts. Importantly, the results from corrected skin grafts indicated that even where detectable LEKTI expression was restored to a limited numbers of cells, a wider bystander benefit occurred around these small populations. As LEKTI is a secreted protein, the genetically modified graft may provide not only an immediate local protective barrier, but also act as a source of secreted LEKTI providing a generalized benefit following ex-vivo gene therapy.