Constitutive and inducible co-expression systems for non-viral osteoinductive gene therapy

• DOI: 10.22203/ecm.v027a13
• Publication Date: 2016-11-28T05:27:46Z
• Authors: GA Feichtinger; A Hacobian; AT Hofmann; K Wassermann; A Zimmermann; M van Griensven; H Redl
• Keywords: Transcriptional Activation; 0301 basic medicine; Bone Morphogenetic Protein 7; Genetic Vectors; Osteocalcin; Bone Morphogenetic Protein 2; Diseases of the musculoskeletal system; Cell Line; Mice; 03 medical and health sciences; Osteogenesis; Animals; Humans; Bone Morphogenetic Protein 7/genetics; Orthopedic surgery; 0303 health sciences; Osteoblasts; Mesenchymal Stem Cells/cytology; Osteoblasts/cytology; Mesenchymal Stem Cells; Genetic Therapy; Bone Morphogenetic Protein 2/genetics; 3. Good health; Genetic Vectors/genetics; RC925-935; Osteocalcin/genetics; RD701-811

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.