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

Abstract

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 indicate that co-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.

Evaluation of fibrin-based gene-activated matrices for BMP2/7 plasmid codelivery in a rat nonunion model

“…Preparation and in vitro testing of the BMP2 and BMP7 plasmids are described elsewhere [23]. The plasmids were amplified in E.coli TOP10 (Life Technologies Ltd, Paisely, UK, ♯C404003) and isolated as endotoxin-free maxipreps using the EndoFree Plasmid Maxi or Giga kits (Quiagen GmbH, Hilden, Germany, ♯12362).…”

Delivery of the improved BMP-2-Advanced plasmid DNA within a gene-activated scaffold accelerates mesenchymal stem cell osteogenesis and critical size defect repair

“…They can exist as homodimers, where both sub-units are identical, such as BMP-2, and also as heterodimers, where the sub-units are different, for example BMP-2/4 and BMP-2/7. While both types are active, some studies have demonstrated that heterodimers such as BMP-2/4 and BMP-2/7 are more active than homodimers [18][19][20]. For this reason, we sought to investigate the use of a multi-cistronic plasmid encoding BMP-2 and BMP-7 ( Figure 1B) which may induce expression of BMP-2/7 heterodimers and enhance the efficiency of the geneactivated scaffold [20].…”

Ultrasound-responsive gene-activated matrices for osteogenic gene therapy using matrix-assisted sonoporation

“…The data essentially demonstrate that it is possible to control gene transfer‐dependent osteogenic differentiation within GAMs in vitro using an external stimulus. The data obtained with the TetON system also suggest that additional control of transgene expression and subsequent osteogenic differentiation can be incorporated into the novel GAM by judiciously manipulating control of expression of therapeutic nucleic acid constructs that would respond to inducers in vivo at a molecular genetic level as shown in previous work (Feichtinger et al ., ).…”