Currently, human skin equivalents (HSEs), used as 3D in vitro models, are a particularly attractive intermediate step between conventional cultures and in vivo experimentation. Our group is focused on the development of non-viral gene therapies, based on hyperbranched poly (β-amino esters) (HPAEs), for Recessive Dystrophic Epidermolysis Bullosa (RDEB). Traditionally, RDEB-HSEs have been used to evaluate ex vivo therapies by confirming that edited keratinocytes and fibroblasts are capable of forming a healthy skin with collagen VII (C7) restoration in the basement membrane zone (BMZ) and lack of blister formation. In this work, we have studied the use of RDEB-HSEs as a method to evaluate topical gene therapies efficacy for potential in vivo RDEB treatments. Transfected HSEs at different time points of their development with different treatments and topical formulations have shown recombinant C7 expression in the BMZ directly related with skin penetration and indirectly with stratum corneum development.
Nanoparticles, called polyplexes, where formed by combination of HPAEswith a full COL7A1 cDNA as described in Wang et al., 2020 (1). Efficacy of polyplexes was tested in conventional cultures of RDEB keratinocyte (RDEBK) and after 48 hours C7 was evaluated by Western-blotting and immunofluorescence. RDEB-HSEs were performed based on the ones described in Gache et al., 2004 and LLames et al. 2004, by increasing fibrinogen and aprotinin by 25%, doubling the ascorbic acid concentration and adding extra medium volume in the well when seeding the keratinocytes to avoid medium diffusion in the transwell (1,2). Test treatments applied topically after 4, 8, 12 and 16 days of maturation. Polyplexes were prepared in two buffer systems (H2O or NaOAc) and applied fresh or after lyophilization with jojoba oil. 48 hours post-treatment, HSEs were embedded in O.C.T. medium and snap frozen in liquid nitrogen, 7 μm sections were stained with anti-C7 antibody hLH 7:2pAb and mounted with mounting media with DAPI for immunofluorescence analysis (3). Microphotographs were taken on an Olympus CKX41 inverted microscope using a monochromatic camera (Q-Imaging) and CellSens imaging software (Olympus). Image post processing and analysis were performed by using ImageJ (NIH) open-source software.
Transfection in conventional in vitro culture of RDEBK induced 10-fold higher C7 formation compared with normal human keratinocytes. The generated HSEs showed a good microscopical structure, however, no significant differences were noted when using increased concentrations of fibrinogen and aprotinin compared to standard ones. Transfection was not successful at 16 days for any of the test treatments, most likely due to impaired cellular uptake of the treatment which is in contrast to in vitro transfection of keratinocytes at 48h after seeding, proving to be highly effective in producing C7. Decreased transfection could be a direct cause of keratinocyte terminal differentiation and stratum corneum formation, a well-known barrier for external agents despite our created defect.
RDEB-HSEs are a very good method to validate formation of restored skin when pre-treating the cells, however multiple challenges need to be overcome to use the matured skin equivalents as validated method to evaluate efficiency of topical gene therapies.