Construction of Skin Equivalents for Gene Therapy of Recessive Dystrophic Epidermolysis Bullosa

Abstract

We have shown that retroviral vectors efficiently transfer the 9-kb collagen type VII cDNA into keratinocytes of dogs with recessive dystrophic epidermolysis bullosa (RDEB) and achieve correction of the RDEB phenotype in vitro. As a next step toward gene therapy applications, we have assessed the suitability of retroviral vectors to transduce human collagen type VII cDNA into primary human RDEB keratinocytes and generate transplantable autologous skin equivalents. The transduced RDEB keratinocytes permanently express high levels of recombinant collagen type VII that assembles into functional homotrimers readily secreted into the extracellular matrix. The recombinant collagen type VII reverts the migration and invasion potential of the transduced RDEB keratinocytes in vitro and is efficiently deposited at the dermal epidermal junction of artificial skin prepared with the reverted cells and artificial dermis made of biomaterial sponges embedded with dermal RDEB fibroblasts. Transplantable fibrin-based skin equivalents made with the transduced RDEB keratinocytes and grafted onto SCID mice either orthotopically or in accordance with the flap method generated cohesive and orderly stratified epithelia with all the characteristics of normal human epidermis, including rapid formation of anchoring fibrils. Because transplantable epithelia are routinely used to cure patients suffering from large skin or mucosal defects, the full phenotypic reversion of primary RDEB epidermal clonogenic cells mediated by recombinant retroviral vectors opens new perspectives in the long-term treatment of genodermatoses.

Current approaches and perspectives in human keratinocyte-based gene therapies

“…Recently, our labs have addressed the correction of RDEB in a clinically relevant manner. 13 In our setting, correction was achieved by using a conventional retroviral vector and a cultured skin equivalent to accomplish permanent epidermal regeneration in patients. 4 Our results evidenced that the size of the retroviral genome does not hamper its efficient packaging into the viral capsid and generation of infectious viral particles.…”

Long-Term Type VII Collagen Restoration to Human Epidermolysis Bullosa Skin Tissue

“…Subjects with RDEB develop painful blisters and wounds on the skin and mucous membranes, leading to a shortened life expectancy due to infection, organ failure, or squamous cell carcinoma (SCC). Preclinical RDEB studies have suggested various treatment strategies for the disease, such as direct virus-based (Woodley et al, 2004b), protein-based (Woodley et al, 2004a;Remington et al, 2009), and cell-based therapies including bone marrowderived cells (Tolar et al, 2009) and genetically modified keratinocytes (Chen et al, 2002;Ortiz-Urda et al, 2002;Gache et al, 2004) or fibroblasts (Ortiz-Urda et al, 2003;Woodley et al, 2003;Kern et al, 2009). The promise suggested by this body of preclinical research points to the emergence of new treatments for RDEB beyond currently available palliative wound care.…”

Nuevos modelos experimentales para el estudio de la homeostasis y la enfermedad cutánea

“…Por otro lado, estos modelos ofrecen la posibilidad de utilizar queratinocitos y/o fibroblastos humanos a los que previamente se les ha introducido una modificación genética. Esta estrategia ha permitido validar a nivel preclínico abordajes de terapia génica ex vivo 44,45,47 o conseguir la regeneración de la epidermis a partir de una única célula madre epidérmica 51 . El programa SkinModel-CM contempla, también, desarrollar estrategias de sobre-expresión y silenciamiento de los genes que codifican endoglina y podoplanina, para analizar sus efectos en la regeneración y arquitectura de la piel, así como en procesos neoplásicos y de cicatrización de heridas.…”