Poor nuclear entry, especially into nondividing cells, is a limiting factor in nonviral gene delivery. We have engineered a novel chimeric vector relying on the controlled assembly of a TAT-tagged multisubunit DNA binding protein (EcoR124I) with expression plasmids containing the EcoR124I recognition site. Molecular interactions of this molecular assembly were studied by electrophoretic mobility shift assay and atomic force microscopy. Maintenance of nanocomplexes in an appropriate stoichiometric ratio was both necessary and sufficient to produce a significant (>8-fold) increase in the activity of the therapeutic -galactosidase A enzyme after intramuscular administration in the mouse model of Fabry disease. To our knowledge, this is the first molecular targeting system significantly enhancing plasmid-based expression in skeletal muscle. Coinjection with pluronic SP1017 produced further enhancement of gene expression, demonstrating cumulative effects of the increased nuclear delivery by TAT chimeras and transcription activation by the pluronic. Cell penetration peptides (CPP), such as TAT, have been shown to improve delivery of macromolecules, when linked directly. However, in our system TAT-enhanced targeting took place even though it was linked to the plasmid DNA molecule indirectly via two noncovalent bonds. Therefore, this proof-of principle result indicates that TAT (and potentially other CPP) can be used for targeting modular chimeric vectors and therapeutic nanodevices.—Lavigne, M. D., Yates, L., Coxhead, P., Górecki, D. C. Nuclear-targeted chimeric vector enhancing nonviral gene transfer into skeletal muscle of Fabry mice in vivo.