Polymer-based transfection vectors are increasingly becoming the preferred alternative to viral vectors thanks to their safety and ease of production, but low transfection potency has limited their application. Many polycationic vectors show high efficiency in vitro, but their excessive charge density makes them toxic for in vivo applications. Herein, we demonstrate the synthesis of new and unique disulfide-reducible polymeric gene nanocarriers that exhibit significantly enhanced transfection potency and low cytotoxicity, particularly in skin cells, surpassing the efficiency of the well-known transfection reagents polyethylenimine (PEI) and Lipofectamine2000. The unique three-dimensional (3D) multiknot vectors were synthesized from in situ deactivation enhanced atom transfer radical (co)polymerization (DE-ATRP) of multivinyl monomers (MVMs). The high transfection levels and low toxicity of this multiknot structured polymer in vitro, combined with its ability to mediate collagen VII expression in 3D skin equivalents made from cells of recessive dystrophic epidermolysis bullosa patients, demonstrates its use as a platform nanotechnology which should be investigated further for dermatological disease therapies. Our findings suggest that the marked improvements stem from the dense multiknot architecture and degradable property, which facilitate both the binding and releasing process of the plasmid DNA.