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

Abstract

The combination of gene therapy and tissue engineering is one of the most promising strategies for the treatment of recessive dystrophic epidermolysis bullosa (RDEB). RDEB is a rare genetic disease characterised by mutations in the COL7A1 gene, encoding type VII collagen (COLVII), which forms anchoring fibrils at the dermal-epidermal junction of the skin. This disease causes severe blistering and only palliative treatments are offered. In this study, the base of a strategy combining gene therapy and a tissue-engineered skin substitute (TES), which would be suitable for the permanent closure of skin wounds, was set-up. As a high transduction efficiency into fibroblasts and/or keratinocytes seems to be a prerequisite for a robust and sustained correction of RDEB, different envelope pseudotyped retroviral vectors and the transduction enhancer EF-C were tested. When green fluorescent protein (GFP) was used as a reporter gene to evaluate the retroviral-mediated gene transfer, the fibroblast infection efficiency was 30 % higher with the Ampho pseudotyped vector as compared with the other pseudotypes. At least a 3.1-fold and a 1.3-fold increased transduction were obtained in fibroblasts and keratinocytes, respectively, with EF-C as compared with polybrene. A continuous and intense deposit of haemagglutinin (HA)-COLVII was observed at the dermal-epidermal junction of self-assembled TESs made of cells transduced with a HA-tagged COL7A1 vector. Furthermore, HA-tagged basal epidermal cells expressing keratin 19 were observed in TESs, suggesting stem cell transduction. This approach could be a valuable therapeutic option to further develop, in order to improve the long-term life quality of RDEB patients.

Unraveling the ECM-Immune Cell Crosstalk in Skin Diseases

“…Gene and stem cell therapy have proven highly beneficial for genetic skin disorders such as EB which continues to be a major health burden due to lack of effective treatment. The retroviral mediated transfer of COL7A1 into fibroblasts and keratinocytes have shown successful incorporation of collagen-VII at the dermal-epidermal junction of the engineered skin grafts (Ferrari et al, 2006; Piaceski et al, 2018). In a recent path-breaking paper, physicians were able to repair the skin of a 7-year old JEB patient who had lost 80% of his epidermis.…”

Generation of High-Titer Self-Inactivated γ-Retroviral Vector Producer Cells

“…The first one is the 8.9-kb COL7A1 cDNA that could be used to correct keratinocytes and fibroblasts of recessive dystrophic epidermolysis bullosa (RDEB) patients. A vector with such a long transgene would be less efficiently packaged and would lead to low titers 41, 42, 43. We hypothesized that the benefit of WPRE could be cancelled by its size (700 bp), which would further decrease the packaging ability of a COL7A1 vector.…”

“…WPRE was kept for the SIN GFP and PIGA vectors but not for the COL7A1 vector, as this sequence had no positive effect on titers (Figure 4). The blasticidin cassette was introduced in each of these retroviral plasmids that were transfected in the 293Vec-RD114 packaging cells for the GFP and the PIGA vectors and in the 293Vec-Ampho packaging cells for the COL7A1 vector, as amphotropic pseudotyped vectors are more efficient for transducing human fibroblasts 43 . All the 43 clones screened with the GFP vector had titers above 2 × 10 6 IU/mL, and for the PIGA vector, 90% had titers above 6 × 10 5 IU/mL.…”

“…Human RDEB keratinocytes and fibroblasts used in this study were isolated from a punch biopsy by the two-step thermolysine and trypsin method from one RDEB patient as described elsewhere 43 …”

“…Human primary keratinocytes were cultured with DMEM/Ham’s F12 (3:1) (Life Technologies) supplemented with 5% FetalClone II Serum (Hyclone, Logan, UT, USA), insulin (5 μg/mL; Sigma, St. Louis, MO, USA), hydrocortisone (0.4 μg/mL; Calbiochem, San Diego, CA, USA), isoproterenol hydrochloride (0.212 μg/mL; Sandoz Canada, Boucherville, QC, Canada), epidermal growth factor (10 ng/mL; Austral Biologicals, San Ramon, CA, USA), penicillin G (100 IU/mL), and gentamicin (25 μg/mL; Sigma). For all experiments, keratinocytes were co-cultured with irradiated human fibroblast feeder layers as previously described 43 …”