Journal
ACS APPLIED MATERIALS & INTERFACES
Volume 11, Issue 34, Pages 30661-30672Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acsami.9b13135
Keywords
nonviral gene therapy; polymeric nanoparticles; highly branched poly(beta-amino esters)s; minicircle DNA; RDEB; keratinocyte transfection; type VII collagen; polyplex formulation
Funding
- Science Foundation Ireland (SFI) [13/IA/1962, 12/IP/1688]
- Health Research Board [HRA-POR2013-412]
- DEBRA Ireland
- Science Foundation Ireland (SFI) [13/IA/1962] Funding Source: Science Foundation Ireland (SFI)
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Recessive dystrophic epidermolysis bullosa (RDEB) is a severe congenital skin fragility disease caused by COL7A1 mutations that result in type VII collagen (C7) deficiency. Herein, we report a synergistic polyplex system that can efficiently restore C7 expression in RDEB keratinocytes. A highly branched multifunctional poly(beta-amino ester) (HPAE), termed as HC32-122, was optimized systematically as the high-performance gene delivery vector for keratinocytes, achieving much higher transfection capability than polyethylenimine, SuperFect, and Lipofectamine 2000 without inducing obvious cytotoxicity. Concurrently, a 12 kb length minicircle DNA encoding similar to 9 kb full-length COL7A1 (MCC7) devoid of bacterial sequence was biosynthesized as the therapeutic gene. Combining the highly potent polymer and the miniaturized gene structure, HC32-122/MCC7 polyplexes achieve 96.4% cellular uptake efficiency, 4019-fold COL7A1 mRNA enhancement, and robust recombinant C7 expression. Structure-property investigations reveal that HC32-122 can effectively condense MCC7 to form small, uniform, compact, and positively charged spherical nanoparticles with high DNA release flexibility. Moreover, formulation study shows that sucrose is conductive to lyophilized HC32-122/DNA polyplexes for maintaining the transfection capability. Direct frozen polyplexes can maintain full gene transfection capability after one-year storage. High efficiency, biocompatibility, facile manipulation, and long-term stability make the HC32-122/MCC7 system a promising bench-to-bed candidate for treating the debilitating RDEB.
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