Tissue regenerative gene therapy requires expression strategies that deliver therapeutic effective amounts of transgenes. As physiological expression patterns are more complex than high-level expression of a singular therapeutic gene, we aimed at constitutive or inducible co-expression of 2 transgenes simultaneously. Co-expression of human bone morphogenetic protein 2 and 7 (BMP2/7) from constitutively expressing and doxycycline inducible plasmids was evaluated in vitro in C2C12 cells with osteocalcin reporter gene assays and standard assays for osteogenic differentiation. The constitutive systems were additionally tested in an in vivo pilot for ectopic bone formation after repeated naked DNA injection to murine muscle tissue. Inductor controlled differentiation was demonstrated in vitro for inducible co-expression. Both co-expression systems, inducible and constitutive, achieved significantly better osteogenic differentiation than single factor expression. The potency of the constitutive co-expression systems was dependent on relative expression cassette topology. In vivo, ectopic bone formation was demonstrated in 6/13 animals (46% bone formation efficacy) at days 14 and 28 in hind limb muscles as proven by in vivo µCT and histological evaluation. In vitro findings demonstrated that the devised single vector BMP2/7 co-expression strategy mediates superior osteoinduction, can be applied in an inductor controlled fashion and that its efficiency is dependent on expression cassette topology. In vivo results indicatethatco-expression of BMP2/7 applied by non-viral naked DNA gene transfer effectively mediates bone formation without the application of biomaterials, cells or recombinant growth factors, offering a promising alternative to current treatment strategies with potential for clinical translation in the future.