Evaluation of BMP2/miRNA co-expression systems for potent therapeutic efficacy in bone-tissue regeneration.

Reconstruction of bone defects and compensation of deficient repair mechanisms represent important goals within the field of regenerative medicine and require novel safe strategies for translation into the clinic. A non-viral osteogenic gene therapeutic vector system ('hybrid vectors') was generated, combining an improved bone morphogenetic protein 2 (BMP2) gene cassette and single pro-osteogenic microRNAs (miR-148b-3p, miR-20-5p, miR-590b-5p), driven by the U6 promoter. The vectors were tested in vitro for their osteogenic differentiation potential in C2C12 and C3H/10T1/2 cell lines, using BMP2 alone as control. After confirming BMP2 expression and miRNA transcription, increased osteogenic differentiation was observed by all hybrid vectors, but most consistently by BMP2/miR-590-5p, using alkaline phosphatase enzyme activity assays and osteogenic marker mRNA quantitation, including runt-related transcription factor 2 (Runx2), collagen type 1 (Col1a1) and osteocalcin. To visualise target mRNAs of the respective miRNAs, next generation sequencing was performed, confirming down-regulation of mRNA targets of the hybrid vectors. Since the hybrid vector consisting of BMP2 and miR-590-5p showed the largest increase in osteogenic differentiation in vitro, this was tested in a mouse ectopic-bone model. Mineralisation was more than with BMP2 alone. The present study showed hybrid vectors as a novel non-viral gene therapeutic plasmid system for combining therapeutic effects of recombinant protein expression and miRNA transcription that did not add to the burden of the translation machinery, while improving the therapeutic efficacies. In vivo proof-of-principle in the context of bone regeneration suggested that such hybrid vectors will be applicable in a wide array of gene therapeutic strategies.

Improved osteogenic vector for non-viral gene therapy.

Therapeutic compensation of deficient bone regeneration is a challenging task and a topic of on-going search for novel treatment strategies. One promising approach for improvement involves non-viral gene delivery using the bone morphogenetic protein-2 (BMP-2) gene to provide transient, local and sustained expression of the growth factor. However, since efficiency of non-viral gene delivery is low, this study focused on the improvement of a BMP-2 gene expression system, aiming for compensation of poor transfection efficiency. First, the native BMP-2 gene sequence was modified by codon optimisation and altered by inserting a highly truncated artificial intron (96 bp). Transfection of multiple cell lines and rat adipose-derived mesenchymal stem cells with plasmids harbouring the improved BMP-2 sequence led to a several fold increased expression rate and subsequent osteogenic differentiation. Additionally, comparing expression kinetics of elongation factor 1 alpha (EF1α) promoter with a state of the art CMV promoter revealed significantly higher BMP-2 expression when under the influence of the EF1α promoter. Results obtained by quantification of bone markers as well as osteogenic assays showed reduced sensitivity to promoter silencing effects of the EF1α promoter in rat adipose-derived mesenchymal stem cells. Finally, screening of several protein secretion signals using either luciferase or BMP-2 as reporter protein revealed no superior candidates for potential replacement of the native BMP-2 secretion signal. Taken together, by enhancing the exogenous BMP-2 expression system, low transfection efficiencies in therapeutic applications can be compensated, making safe non-viral systems even more suitable for tissue regeneration approaches.