I. Mirones

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.

Modeling normal and pathological processes through skin tissue engineering.

Skin tissue engineering emerged as an experimental regenerative therapy motivated primarily by the critical need for early permanent coverage of extensive burn injuries in patients with insufficient sources of autologous skin for grafting. With time, the approach evolved toward a wider range of applications including disease modeling. We have established a skin‐humanized mouse model system consisting in bioengineered human‐skin‐engrafted immunodeficient mice. This new model allows to performing regenerative medicine, gene therapy, genomics, and pathology studies in a human context on homogeneous samples. Starting from skin cells (keratinocytes and fibroblasts) isolated from normal donor skin or patients biopsies, we have been able to deconstruct‐reconstruct several inherited skin disorders including genodermatoses and cancer‐prone diseases in a large number of skin humanized mice. In addition, the model allows conducting studies in normal human skin to gain further insight into physiological processes such as wound healing or UV‐responses.

Structural characterization of two CD1A allelic variants

CD1 molecules are specialized in presenting lipidic antigens to T lymphocytes. They are structurally and evolutionary related to MHC molecules and show very limited polymorphism. We have previously described and partially characterized a new human CD1A allele differing from the wild type CD1A by a substitution of Cysteine by Tryptophan at position 52 in the alpha1 domain of the CD1A molecule. The frequency of this allele varies from 10% in individuals of Caucasian origin to 56% in Chinese people. The aim of the present work was to structurally characterize this CD1A allele. To do this we have cloned and sequenced the full-length cDNA encoding the new CD1A allele. The cDNA sequence of this allele encodes a protein differing the wild type in two amino acids at positions 14 (Threonine versus Isoleucine) and 52 (Cysteine versus Tryptophan). The cDNAs encoding both wild type and mutant CD1A were cloned in the expression vector pSRalphaNeo and transfected into C1R and L721.221 cells. Cell surface expression of the protein products in transfected cell lines were analyzed by flow cytometry and immunoprecipitation using CD1a-specific monoclonal antibodies. Our results indicate that both allelic products are efficiently expressed on the cell surface.

Complexity of VEGF Responses in Skin Carcinogenesis Revealed through Ex Vivo Assays Based on a VEGF-A Null Mouse Keratinocyte Cell Line

Vascular endothelial growth factor (VEGF-A) is a critical player in cutaneous angiogenesis. However, the relative contribution of VEGF-A from different sources including epithelial and mesenchymal cells has not been fully characterized during skin repair and tumorigenesis. Moreover, the actual involvement of other vascular-specific acting molecules has remained elusive in part due to the masking and/or overlapping effects of VEGF-A. To shed light on these uncertainties we generated and characterized a clonal VEGF-null mouse keratinocyte cell line, through in vitro adenoviral gene transfer of Cre recombinase to VEGF-LoxP primary keratinocytes followed by repeated cell passaging under controlled conditions and cloning. In vitro and in vivo assays demonstrated that VEGF-null keratinocytes were nontumorigenic and expressed normal differentiation markers after calcium switch. Hras-induced tumorigenesis of immortalized VEGF-null keratinocytes upon subcutaneous injection was markedly reduced but not fully suppressed. However, the metastatic ability of Hras-transformed VEGF-null keratinocytes was abolished. These ex vivo approaches suggest the existence of VEGF-dependent and independent angiogenic stimuli in skin carcinogenesis. The VEGF-null mouse keratinocyte cell line arises as an important tool to assess the actual contribution of keratinocyte-derived VEGF with respect to other angiogenic factors in skin homeostasis and malignancy.

148 In Vivo Adenoviral Gene Transfer of SPARC in a Skin‐Humanized Mouse Wound Healing Model

SPARC (secreted protein, acidic and rich in cysteine), a matricellular glycoprotein, modulates the interactions of cells with the extracellular matrix. Studies in null-mice revealed a role of SPARC in wound healing. Here we examined the effect of SPARC in a skin-humanized mouse wound healing model. This model is based in the regeneration of human skin onto the back of nude mice by transplantation of a dermo-epidermal equivalent. The regenerated human skin was excisionally wounded with biopsy punches. At the moment of wounding an adenoviral vector encoding the cDNA for SPARC was intradermally injected. We are currently assessing the effects of in vivo gene transfer of SPARC at the wound site in the healing process. Criticals events of wound healing including reepithelialization, regeneration of dermoepidermal junction and dermal remodeling were studied at different time points postwounding.