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EVALUATION of BMP2/Mirna CO-EXPRESSION SYSTEMS for POTENT THERAPEUTIC EFFICACY in BONE-TISSUE REGENERATION

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EVALUATION of BMP2/Mirna CO-EXPRESSION SYSTEMS for POTENT THERAPEUTIC EFFICACY in BONE-TISSUE REGENERATION EuropeanTK Brenner Cells et al. and Materials Vol. 41 2021 (pages 245-268) DOI: Non-viral 10.22203/eCM.v041a18 BMP2/miRNA co-expression ISSN 1473-2262 systems EVALUATION OF BMP2/miRNA CO-EXPRESSION SYSTEMS FOR POTENT THERAPEUTIC EFFICACY IN BONE-TISSUE REGENERATION T.K. Brenner1,§, K. Posa-Markaryan1,§, D. Hercher1,4, S. Sperger1,4, P. Heimel1,4, C. Keibl1, S. Nürnberger1,2,3,4, J. Grillari1,4,5, H. Redl1,4 and A. Hacobian1,4,* 1 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology/AUVA Research Centre, The Austrian Cluster for Tissue Regeneration, European Institute of Excellence on Tissue Engineering and Regenerative Medicine Research (Expertissues EEIG), Donaueschingenstrasse 13, 1200 Vienna, Austria 2 Department of Orthopaedics and Trauma-Surgery, Division of Trauma-Surgery, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria 3 University Clinic of Dentistry, Medical University of Vienna, Sensengasse 2a, 1090 Vienna, Austria 4 Austrian Cluster for Tissue Engineering 5 Institute for Molecular Biotechnology, Department of Biotechnology, BOKU – University of Natural Resources and Life Sciences, Vienna, Austria § These authors contributed equally to this work Abstract 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, usingBMP2 alone as a 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. Keywords: Gene therapy, bone morphogenetic protein 2, microRNA-590, microRNA-148b, microRNA-20a, microRNA, ectopic bone, hybrid vector. *Address for correspondence: Ara Hacobian, Donaueschingenstrasse 13, 1200 Vienna, Austria. Telephone number: +43 6801348411 Email: [email protected] Copyright policy: This article is distributed in accordance with Creative Commons Attribution Licence (http://creativecommons.org/licenses/by-sa/4.0/). List of Abbreviations BAMBI BMP and activin membrane bound inhibitor ACTR5 actin-related protein 5 BMP2 bone morphogenetic protein 2 Alk-2 activin receptor-like kinase-2 BMPR2 BMP receptor 2 ALP alkaline phosphatase cDNA complementary DNA ALPL liver/bone/kidney alkaline COL1A1 collagen type 1 phosphatase CRIM1 cysteine-rich transmembrane BMP APC anaphase-promoting complex regulator 1 245 www.ecmjournal.org TK Brenner et al. Non-viral BMP2/miRNA co-expression systems ΔCT delta threshold cycle plasmids represent a suitable strategy for clinical DMEM Dulbecco’s modified Eagle’s medium translation within tissue regenerative gene therapy EF1α translation elongation factor 1 alpha (Bleiziffer et al., 2007; Glover et al., 2005). ELISA enzyme-linked immunosorbent An improved osteogenic vector system encoding assay BMP2 was recently introduced (Hacobian et al., 2016). FCS foetal calf serum The main bottle-neck of non-viral gene therapy – low FGF fibroblast growth factor target-gene expression – was compensated for by FOP fibrodysplasia ossificans progressiva use of a strong promoter, codon optimisation and hASCs human adipose-tissue-derived insertion of an artificial largely truncated intron stromal cells sequence, resulting in an improved therapeutic HE haematoxylin and eosin BMP2 vector (BMP2-Advanced, in the following hMSCs human mesenchymal stem cells referred to as pBMP2ADV) (Hacobian et al., 2016). HPRT hypoxanthine-guanine Aside from the need to increase therapeutic protein phosphoribosyl transferase production, further improvement of therapeutic hsa-miR homo sapiens microRNA vector bio-efficacy, such as the simultaneous µCT micro-computed tomography knockdown of transgene inhibitors, is considered MetLuc Metridia longa luciferase to support successful translation of non-viral miRNA microRNA vectors to the clinic (Al-Dosari and Gao, 2009; Kay, mRNA messenger RNA 2011; Mingozzi and High, 2011; Wang et al., 2013). MSCs mesenchymal stem cells Recently, the application of miRNAs in tissue NGS next generation sequencing regeneration has attracted much interest (Penget al., OCN osteocalcin 2015). At the same time, scientific knowledge of the PPARG peroxisome-proliferator-activated involvement of miRNAs throughout the osteogenic receptor γ differentiation cascade (Lian et al., 2012; Zhai et al., rhBMP2 recombinant human BMP2 protein 2017) and in bone formation and osteoporosis ( et al., rRNA ribosomal RNA 2015a) has increased. In general, miRNA-function ROCK1 rho-associated coiled-coil containing can be divided into two categories depending on protein kinase 1 the degree of sequence complementarity: specific ROI region of interest downregulation of target mRNA transcription RT-qPCR reverse transcription quantitative and/or translation. One mRNA can be regulated polymerase chain reaction by multiple miRNAs just as a single miRNA can RUNX2 runt-related transcription factor 2 recognise a multitude of different targets at once SD standard deviation (Lim et al., 2005). Utilisation of miRNAs for in vivo shRNA short hairpin RNA bone tissue engineering has recently emerged to siRNA short interfering RNA specifically enhance or suppress differentiation and SMAD7 SMAD family member 7 target-gene expression (Deng et al., 2014; Eskildsen et snRNA small nuclear RNA al., 2011; Li et al., 2017; Liao et al., 2014). Therefore, a TGFβ transforming growth factor β combinatorial approach of miRNA-dependent gene TGFBR2 TGFβ receptor 2 regulation and overexpression of a therapeutic gene WNT3A Wnt family member 3A encoded by a single-plasmid-system (hybrid vector) has the potential to increase biological efficacy of therapeutic plasmids and drive cellular fate in a Introduction desired direction. Following a literature search, multiple miRNAs Reconstruction of bone defects and compensation potentially involved in the osteogenic pathway were for deficient repair mechanisms represent important selected for integration into the non-viral pBMP2ADV goals within the field of regenerative medicine. Large vector. In preliminary tests (data not shown), the bone regeneration is required in complex clinical selection was reduced to the three most potent conditions, such as reconstruction of large bone- candidates, human miR-148b-3p, hsa-miR-590-5p and defects caused by trauma, infection and tumour hsa-miR-20a-5p. These miRNAs were individually resection, or cases in which the regenerative process incorporated into pBMP2ADV, under the control of is compromised, including non-union bone defects a human U6 promoter, a well-studied competent and osteoporosis (Audigé et al., 2005; Dimitriou et RNA polymerase III promoter that constitutively al., 2011). Current strategies for stimulating bone transcribes small RNAs (Duvoisin et al., 2012). growth range from application of recombinant Combination of these elements resulted in the protein to viral as well as non-viral gene therapeutic generation the hybrid plasmids. Then, these plasmids approaches (Bleiziffer et al., 2007; Feichtinger et al., were tested for their osteoinductive potential, in vitro 2014a; Kempen et al., 2010; Koh et al., 2008; Sood et as well as in vivo, using an ectopic-bone mouse model. al., 2012; Southwood et al., 2004). Due to transient The study hypothesis was that these newly designed expression profiles and clinical safety, non-viral miRNA-BMP2 co-expression constructs possessed a www.ecmjournal.org 246 TK Brenner et al. Non-viral BMP2/miRNA co-expression systems greater osteoinductive potency than the previously A schematic illustration of control plasmid introduced BMP2 overexpression plasmid (Hacobian pHybrid_MetLuc, specifically designed to investigate et al., 2016). miRNA influence on differentiation processes uncoupled from BMP2ADV expression, is also depicted in Fig. 1a. BMP2ADV was replaced by secreted MetLuc Materials and Methods gene. To investigate successful knockdown of a reporter Plasmid construction gene via the incorporated shRNA/U6 promoter The BMP2ADV-Hybrid vectors (Fig. 1a) include a system, an artificial hairpin structure (anti-Luc) was codon-optimised human BMP2
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