Introduction: Recessive dystrophic epidermolysis bullosa (RDEB) is a rare autosomal inherited skin blistering disorder caused by mutations in the type VII collagen gene (COL7A1). The large size of COL7A1 is a big delivery challenge for both viral and non-viral vectors and seriously hindered the development of therapeutic gene replacement therapy for RDEB. Our group has developed a series of hyper branched aminated polyesters (HAPE) as high-performance gene delivery vectors and combined them with a minicircle DNA coding full COL7A1 gene as a non-viral gene replacement strategy for therapeutic treatment of RDEB [1-2]. Here we further synthesized new HAPE with greater DNA delivery ability, higher gene expression efficiency and better biocompatibility.
Methodology: A new HAPE family was developed via an “A2+B3+C2” Michael addition strategy and were evaluated using green fluorescent protein (GFP) coding plasmid on human embryonic kidney 293 (HEK) cells and human recessive dystrophic epidermolysis bullosa keratinocyte (RDEBK) cells. The selected HAPE was further optimised with molecular weight and branch ratios for the best performance on DNA transfection on RDEBK cells. Then the optimised HAPE was formulated with minicircle DNA coding full human COL7A1 gene (MCC7) into nanoparticles, and its therapeutic effect was confirmed on RDEBK cells by quantitative polymerase chain reaction, immunocytochemistry staining and immunoblotting on both transcription and translation levels.
Results: The best HAPE (HAPE-B) had around 3 times higher GFP expression levels than other developed HAPEs on HEK cells. MCC7 delivered by further optimized HAPE-B (HAPE-BO) showed more efficient restoration of type VII collagen than the commercial reagents, Lipofectamine 3000 and JetPEI, on RDEBK cells.
Conclusion: The newly developed HAPE-BO can efficiently deliver the full COL7A1 gene into RDEBK cells in a cytocompatible manner and successfully restore the functional type VII collagen expression. The improved system developed here has a high potential for use as an efficient and safe non-viral topical treatment for RDEB patients in the clinic and can be adapted to other genetic diseases.
 Zhou, D. et al., Sci. Adv., 2(6), e1600102 (2016).
 Zeng, M., ACS Appl. Nano Mater., 11(34), 30661-30672 (2019).