Genetic Manipulation of Skin Stem Cells: Success, Hope, and Challenges Ahead
Authors: Yann Barrandon
Journal: Molecular Therapy (2007)

Gene therapy has been suggested as a potential cure for patients with disabling genetic diseases. The possibility of using gene transfer to treat diseases of the skin was suggested nearly 20 years ago by a report demonstrating that human keratinocyte stem cells1Barrandon Y Green H Three clonal types of keratinocyte with different capacities for multiplication.Proc Natl Acad Sci USA. 1987; 84: 2302-2306Crossref PubMed Scopus (1103) Google Scholar could be genetically manipulated ex vivo using recombinant retroviruses.2Morgan JR Barrandon Y Green H Mulligan RC Expression of an exogenous growth hormone gene by transplantable human epidermal cells.Science. 1987; 237: 1476-1479Crossref PubMed Scopus (224) Google Scholar These early studies led to painstaking efforts by many groups, such as those led by De Luca, to develop gene transfer strategies with clinical potential to treat skin disease.3Dellambra E Vailly J Pellegrini G Bondanza S Golisano O Macchia C et al.Corrective transduction of human epidermal stem cells in laminin-5-dependent junctional epidermolysis bullosa.Hum Gene Ther. 1998; 9: 1359-1370Crossref PubMed Scopus (118) Google Scholar The successful transplantation of keratinocyte stem cells genetically corrected ex vivo to treat a rare form of junctional epidermolysis bullosa (JEB) by Mavilio et al.4Mavilio F Pellegrini G Ferrari S Di Nunzio F Di Iorio E Recchia A et al.Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells.Nat Med. 2006; 12: 1397-1402Crossref PubMed Scopus (522) Google Scholar unambiguously demonstrates the feasibility of gene therapy in the skin. The new work lends credence to the opinion that gene therapy may soon be part of our therapeutic arsenal and that it may eventually allow us to improve the lives of patients suffering from this horrendous skin disease. The epidermis (the outer epithelial layer of the skin) connects to the underlying stroma (the dermis) through the dermoepidermal junction that includes hemidesmosomes, a complex basement membrane, and anchoring fibers. Epidermolysis bullosa (EB) encompasses a number of skin diseases characterized by skin fragility and chronic blistering that results in the formation of painful wounds.5Uitto J Richard G Progress in epidermolysis bullosa: from eponyms to molecular genetic classification.Clin Dermatol. 2005; 23: 33-40Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar The formation of blisters and bullae in EB results from detachment of the epidermis from the dermis and is often the consequence of mechanical trauma. EB is classified into three major categories, each subdivided into several clinical forms based on the extent and location of the blistering and on the level of breakage within the dermoepidermal junction, which itself is closely linked to the nature of the genetic defect. In epidermolosysis bullosa simplex (EBS), breakage occurs above the dermoepidermal junction within the epidermis and results from mutations in keratin genes expressed in keratinocytes, which are the main lineage of the epidermis. In JEB, the deficit occurs within the dermoepidermal junction and results from a mutation in a gene encoding one of several proteins that participate in the formation of the hemidesmosomes or the basement membrane, such as the laminins and collagen XVII. In dystrophic epidermolysis bullosa (DEB), anchoring fibers are abnormal or absent, which results in breakage below the dermoepidermal junction. DEB results from mutations in the gene encoding collagen VII, a protein that participates in the formation of anchoring fibers. Some forms of JEB and DEB are particularly devastating, with patients living a life of permanent wounds and pain. Moreover, detachment may affect stratified epithelia other than the skin, for instance, the esophagus, leading to malnutrition, and the cornea, resulting in vision problems. Most important, skin carcinomas develop more frequently on chronic wounds of these devastating forms of EB, which results in poor prognosis. There is no cure for EB, and its treatment is only symptomatic. Patient associations exist worldwide that are strongly supportive of research, especially research aimed at developing gene therapy (see, for example, http://www.debra-international.org). Mavilio and colleagues' strategy was straightforward. The LAMB3 complementary DNA encoding the laminin 5 β3-chain, driven by a Moloney leukemia virus long terminal repeat, was transduced into autologous LAMB5 β3-deficient keratinocyte stem cells by means of an amphotropic retroviral vector. Importantly, the authors cultivated the stem cells using technology developed by Green and colleagues for the treatment of extensive burn wounds.6Gallico III, GG 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: 448-451Crossref PubMed Scopus (1121) Google Scholar Indeed, De Luca's group had thorough experience in transplanting cultivated keratinocyte stem cells to treat burns7Pellegrini G Ranno R Stracuzzi G Bondanza S Guerra L Zambruno G et al.The control of epidermal stem cells (holoclones) in the treatment of massive full-thickness burns with autologous keratinocytes cultured on fibrin.Transplantation. 1999; 68: 868-879Crossref PubMed Scopus (317) Google Scholar and limbal stem cells to repair corneal defects.8Pellegrini G Traverso CE Franzi AT Zingirian M Cancedda R De Luca M Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium.Lancet. 1997; 349: 990-993Abstract Full Text Full Text PDF PubMed Scopus (1124) Google Scholar Mastering the cultivation and the transplantation of keratinocyte stem cells was key to success. This involved first infecting LAMB5 β3-deficient keratinocyte stem cells by cocultivating them onto a feeder layer of a lethally irradiated producer line, which helped maintain stemness and resulted in high transduction efficiency. Consequently, there was no need for drug selection, thus sparing another source of stress to cultivated stem cells. Finally, the choice to transplant chronic nonhealing wounds depleted in resident stem cells was crucial, because it probably removed competition between stem cells present in the skin appendages (i.e., in hair follicles and sweat glands) and the transplanted stem cells. Although autologous transplantation of cultivated keratinocyte stem cells has saved thousands of lives worldwide, some are questioning the origin of the healing cells, and consequently the stemness of the transplanted keratinocytes. For example, it has been suggested that epidermal reconstitution may originate not from skin stem cells but from stem cells from another source, such as bone marrow. Importantly, Mavilio and colleagues demonstrated unambiguously that the transduced keratinocyte stem cells transplanted into the JEB wounds provided long-term, if not permanent, engraftment. These new data, along with recent evidence obtained by clonal analysis of genetically labeled stem cells transplanted in rodents,9Claudinot S Nicolas M Oshima H Rochat A Barrandon Y Long-term renewal of hair follicles from clonogenic multipotent stem cells.Proc Natl Acad Sci USA. 2005; 102: 14677-14682Crossref PubMed Scopus (258) Google Scholar should finally end this debate. Another important finding is that few stem cells were necessary to reconstruct and renew the recombinant epidermis. Obviously long-term follow-up, together with clonal analysis, should provide more information on how the transplanted stem cells participated in epidermal renewal. The road to successful skin gene therapy is still long—and paved with ambushes. Nevertheless, Mavilio and colleagues fuel us with hope; they have provided a proof of principle that skin gene therapy can work in human patients. Nevertheless, several important questions remain that span all aspects of the procedure, including safety. It is not clear that retroviruses provide the best and safest shuttle vectors for skin gene therapy. Self-inactivating lentiviruses deserve investigation in this regard. An interesting question is what would happen if transduced stem cells were transplanted on wounds that still contained resident stem cells. Competition between the stem cells might complicate the therapy, in that it is not clear whether the transduced stem cells would have time to engraft permanently. Indeed, it is possible that the transduced stem cells would only act as a biological dressing that favored the migration of resident stem cells out of skin appendages. It is also unclear how easy it will be to transplant large areas of skin. Many other questions remain. Are keratinocyte stem cells the best cells for gene therapy of JEB and DEB? Can systemic gene therapy be envisioned to treat lesions not affecting the skin? Continuing research is focusing on these questions. It is also important to minimize risk factors, as for any medical procedure. It remains to be seen whether regulatory officials would authorize cocultivation of human stem cells onto an amphotropic producer cell line or stem cell expansion onto a mouse feeder layer. This seems unlikely. Would they allow fetal bovine serum to remain part of the procedure? Again, unlikely. This could be problematic because, to our knowledge, no successful permanent engraftment of keratinocyte stem cells expanded in a defined medium has been achieved. Nevertheless, xenotransplantation of irradiated mouse feeder cells is a global concern, and the move to ban xenocells is irreversible, even if they originated from an approved master bank and no adverse reaction has been reported so far. It is then crucial to search for technological alternatives to expand human keratinocyte stem cells. Otherwise, ex vivo gene therapy of the skin will remain at the stage of proof of principle and unavailable to our therapeutic arsenal for the foreseeable future. Nevertheless, for the time being, one might consider allowing the current procedure to treat these horrendous genetic skin diseases. On this particular issue, patients who are suffering these painful wounds should definitely have their say.