Regeneration of the entire human epidermis using transgenic stem cells

Abstract

Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, life-threatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.

Translating the combination of gene therapy and tissue engineering for treating recessive dystrophic epidermolysis bullosa

“…A recent paper from De Luca's group shows that the grafting of transfected holoclones over 80 % of total body surface area results in long-term correction in a patient suffering from JEB (Hirsch et al, 2017). As described in our study for RDEB cells (Fig.…”

“…After expansion of the corrected cells, they successfully transplanted cultured epidermal sheets onto small areas containing wounds. Very recently, the same team was able to successfully graft 80 % of the body surface of a JEB patient with a transgenic epidermis, a procedure that was life-saving (Hirsch et al, 2017). The transgene was expressed during the 21-month follow-up and the epidermis was fully functional and did not form blisters.…”

“…This percentage might have been insufficient to target stem cells and the harsh infection condition could have led to cellular differentiation. In comparison, more than 90 % of keratinocytes are transduced in the JEB studies with a gentle transduction procedure (De Rosa et al, 2014;Hirsch et al, 2017;Mavilio et al, 2006). This important difference in viral transduction is due to the extended length of the COL7A1 cDNA that prevents the generation of high-titer vector batches.…”

“…Codon optimisation is used to enhance the stability of the transcript, aiming at obtaining high viral titers and transgene expression in transduced cells Moreno-Carranza et al, 2009). Also, SIN retroviral vectors without viral enhancers are safer and less prone to transformation upon integration when compared to vectors with intact LTRs (Cavazza et al, 2013;Hacein-Bey-Abina et al, 2008;Howe et al, 2008;Modlich et al, 2006). Fibronectin and retronectin enhance retroviral gene transfer in keratinocytes (Bajaj et al, 2005;Goto et al, 2006), but the cost of the good manufacturing practices (GMP)-grade fibronectin or retronectin and the numerous manipulation steps limit the clinical development of a gene therapy strategy for RDEB.…”

“…The transgene was expressed during the 21-month follow-up and the epidermis was fully functional and did not form blisters. This proof of principle suggests that this strategy could be applied to other EB types (De Rosa et al, 2014;Hirsch et al, 2017;Mavilio et al, 2006).…”