Evaluation of BMP-2 Minicircle DNA for Enhanced Bone Engineering and Regeneration

Author(s): Alice Zimmermann, David Hercher, Benedikt Regner, Amelie Frischer, Simon Sperger, Heinz Redl, Ara Hacobian*

Journal Name: Current Gene Therapy

Volume 20 , Issue 1 , 2020


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Graphical Abstract:


Abstract:

Background: To date, the significant osteoinductive potential of bone morphogenetic protein 2 (BMP-2) non-viral gene therapy cannot be fully exploited therapeutically. This is mainly due to weak gene delivery and brief expression peaks restricting the therapeutic effect.

Objective: Our objective was to test the application of minicircle DNA, allowing prolonged expression potential. It offers notable advantages over conventional plasmid DNA. The lack of bacterial sequences and the resulting reduction in size, enables safe usage and improved performance for tissue regeneration.

Methods: We inserted an optimized BMP-2 gene cassette with minicircle plasmid technology. BMP-2 minicircle plasmids were produced in E. coli yielding plasmids lacking bacterial backbone elements. Comparative studies of these BMP-2 minicircles and conventional BMP-2 plasmids were performed in vitro in cell systems, including bone marrow derived stem cells. Tests performed included gene expression profiles and cell differentiation assays.

Results: A C2C12 cell line transfected with the BMP-2-Advanced minicircle showed significantly elevated expression of osteocalcin, alkaline phosphatase (ALP) activity, and BMP-2 protein amount when compared to cells transfected with conventional BMP-2-Advanced plasmid. Furthermore, the plasmids show suitability for stem cell approaches by showing significantly higher levels of ALP activity and mineralization when introduced into human bone marrow stem cells (BMSCs).

Conclusion: We have designed a highly bioactive BMP-2 minicircle plasmid with the potential to fulfil clinical requirements for non-viral gene therapy in the field of bone regeneration.

Keywords: C2C12, BMSCs, alkaline phosphatase, bone morphogenetic protein 2, DNA, bone engineering.

[1]
Zura R, Xiong Z, Einhorn T, et al. Epidemiology of fracture nonunion in 18 human bones. JAMA Surg 2016; 151(11)e162775
[http://dx.doi.org/10.1001/jamasurg.2016.2775] [PMID: 27603155]
[2]
Pelled G, Ben-Arav A, Hock C, et al. Direct gene therapy for bone regeneration: gene delivery, animal models, and outcome measures. Tissue Eng Part B Rev 2010; 16(1): 13-20.
[http://dx.doi.org/10.1089/ten.teb.2009.0156] [PMID: 20143927]
[3]
Südkamp NP, Haas NP, Sinnig M, Sottmann G, Tscherne H. [Incidence of pseudarthroses in open fractures: analysis of 948 open fractures]. Aktuelle Traumatol 1993; 23(2): 59-67. [Incidence of pseudarthroses in open fractures: Analysis of 948 open fractures].
[PMID: 8098572]
[4]
Evans CH. Gene therapy for bone healing. Expert Rev Mol Med 2010; 12e18
[http://dx.doi.org/10.1017/S1462399410001493] [PMID: 20569532]
[5]
Sen MK, Miclau T. Autologous iliac crest bone graft: should it still be the gold standard for treating nonunions? Injury 2007; 38(Suppl. 1): S75-80.
[http://dx.doi.org/10.1016/j.injury.2007.02.012] [PMID: 17383488]
[6]
Calori GM, Mazza E, Colombo M, Ripamonti C, Tagliabue L. Treatment of long bone non-unions with polytherapy: indications and clinical results. Injury 2011; 42(6): 587-90.
[http://dx.doi.org/10.1016/j.injury.2011.03.046] [PMID: 21524745]
[7]
Schmidmaier G, Schwabe P, Wildemann B, Haas NP. Use of bone morphogenetic proteins for treatment of non-unions and future perspectives. Injury 2007; 38(Suppl. 4): S35-41.
[http://dx.doi.org/10.1016/S0020-1383(08)70007-X] [PMID: 18224735]
[8]
Hustedt JW, Blizzard DJ. The controversy surrounding bone morphogenetic proteins in the spine: a review of current research. Yale J Biol Med 2014; 87(4): 549-61.
[PMID: 25506287]
[9]
Tsuji K, Bandyopadhyay A, Harfe BD, et al. BMP2 activity, although dispensable for bone formation, is required for the initiation of fracture healing. Nat Genet 2006; 38(12): 1424-9.
[http://dx.doi.org/10.1038/ng1916] [PMID: 17099713]
[10]
Edgar CM, Chakravarthy V, Barnes G, Kakar S, Gerstenfeld LC, Einhorn TA. Autogenous regulation of a network of bone morphogenetic proteins (BMPs) mediates the osteogenic differentiation in murine marrow stromal cells. Bone 2007; 40(5): 1389-98.
[http://dx.doi.org/10.1016/j.bone.2007.01.001] [PMID: 17303481]
[11]
Kumar S, Nagy TR, Ponnazhagan S. Therapeutic potential of genetically modified adult stem cells for osteopenia. Gene Ther 2010; 17(1): 105-16.
[http://dx.doi.org/10.1038/gt.2009.116] [PMID: 19741731]
[12]
Kimelman N, Pelled G, Helm GA, Huard J, Schwarz EM, Gazit D. Review: gene- and stem cell-based therapeutics for bone regeneration and repair. Tissue Eng 2007; 13(6): 1135-50.
[http://dx.doi.org/10.1089/ten.2007.0096] [PMID: 17516852]
[13]
Evans CH. Gene delivery to bone. Adv Drug Deliv Rev 2012; 64(12): 1331-40.
[http://dx.doi.org/10.1016/j.addr.2012.03.013] [PMID: 22480730]
[14]
Brooks SA. Protein glycosylation in diverse cell systems: implications for modification and analysis of recombinant proteins. Expert Rev Proteomics 2006; 3(3): 345-59.
[http://dx.doi.org/10.1586/14789450.3.3.345] [PMID: 16771706]
[15]
Poynton AR, Lane JM. Safety profile for the clinical use of bone morphogenetic proteins in the spine. Spine 2002; 27(16)(Suppl. 1): S40-8.
[http://dx.doi.org/10.1097/00007632-200208151-00010] [PMID: 12205419]
[16]
Carragee EJ, Chu G, Rohatgi R, et al. Cancer risk after use of recombinant bone morphogenetic protein-2 for spinal arthrodesis. J Bone Joint Surg Am 2013; 95(17): 1537-45.
[http://dx.doi.org/10.2106/JBJS.L.01483] [PMID: 24005193]
[17]
Garrison KR, Donell S, Ryder J, et al. Clinical effectiveness and cost-effectiveness of bone morphogenetic proteins in the non-healing of fractures and spinal fusion: a systematic review. Health Technol Assess 2007; 11(30): 1-150, iii-iv.
[http://dx.doi.org/10.3310/hta11300] [PMID: 17669279]
[18]
Gaspar V, de Melo-Diogo D, Costa E, et al. Minicircle DNA vectors for gene therapy: advances and applications. Expert Opin Biol Ther 2015; 15(3): 353-79.
[http://dx.doi.org/10.1517/14712598.2015.996544] [PMID: 25539147]
[19]
Hacobian AR, Posa-Markaryan K, Sperger S, et al. Improved osteogenic vector for non-viral gene therapy. Eur Cell Mater 2016; 31: 191-204.
[http://dx.doi.org/10.22203/eCM.v031a13] [PMID: 26995192]
[20]
Hacobian A, Hercher D. Pushing the right buttons: Improving efficacy of therapeutic DNA vectors. Tissue Eng Part B Rev 2017; •••
[PMID: 29264951]
[21]
Bleiziffer O, Eriksson E, Yao F, Horch RE, Kneser U. Gene transfer strategies in tissue engineering. J Cell Mol Med 2007; 11(2): 206-23.
[http://dx.doi.org/10.1111/j.1582-4934.2007.00027.x] [PMID: 17488473]
[22]
Glover DJ, Lipps HJ, Jans DA. Towards safe, non-viral therapeutic gene expression in humans. Nat Rev Genet 2005; 6(4): 299-310.
[http://dx.doi.org/10.1038/nrg1577] [PMID: 15761468]
[23]
Al-Dosari MS, Gao X. Nonviral gene delivery: principle, limitations, and recent progress. AAPS J 2009; 11(4): 671-81.
[http://dx.doi.org/10.1208/s12248-009-9143-y] [PMID: 19834816]
[24]
Kay MA. State-of-the-art gene-based therapies: the road ahead. Nat Rev Genet 2011; 12(5): 316-28.
[http://dx.doi.org/10.1038/nrg2971] [PMID: 21468099]
[25]
Mingozzi F, High KA. Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat Rev Genet 2011; 12(5): 341-55.
[http://dx.doi.org/10.1038/nrg2988] [PMID: 21499295]
[26]
Wang W, Li W, Ma N, Steinhoff G. Non-viral gene delivery methods. Curr Pharm Biotechnol 2013; 14(1): 46-60.
[PMID: 23437936]
[27]
Mayrhofer P, Schleef M, Jechlinger W. Use of minicircle plasmids for gene therapy. Methods Mol Biol 2009; 542: 87-104.
[http://dx.doi.org/10.1007/978-1-59745-561-9_4] [PMID: 19565897]
[28]
Jechlinger W. Optimization and delivery of plasmid DNA for vaccination. Expert Rev Vaccines 2006; 5(6): 803-25.
[http://dx.doi.org/10.1586/14760584.5.6.803] [PMID: 17184219]
[29]
Stenler S, Blomberg P, Smith CI. Safety and efficacy of DNA vaccines: plasmids vs. minicircles. Hum Vaccin Immunother 2014; 10(5): 1306-8.
[http://dx.doi.org/10.4161/hv.28077] [PMID: 24553064]
[30]
Klinman DM. Immunotherapeutic uses of CpG oligodeoxynucleotides. Nat Rev Immunol 2004; 4(4): 249-58.
[http://dx.doi.org/10.1038/nri1329] [PMID: 15057783]
[31]
Krieg AM, Yi AK, Matson S, et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 1995; 374(6522): 546-9.
[http://dx.doi.org/10.1038/374546a0] [PMID: 7700380]
[32]
Schwartz DA, Quinn TJ, Thorne PS, Sayeed S, Yi AK, Krieg AM. CpG motifs in bacterial DNA cause inflammation in the lower respiratory tract. J Clin Invest 1997; 100(1): 68-73.
[http://dx.doi.org/10.1172/JCI119523] [PMID: 9202058]
[33]
Darquet AM, Rangara R, Kreiss P, et al. Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer. Gene Ther 1999; 6(2): 209-18.
[http://dx.doi.org/10.1038/sj.gt.3300816] [PMID: 10435105]
[34]
Kreiss P, Cameron B, Rangara R, et al. Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency. Nucleic Acids Res 1999; 27(19): 3792-8.
[http://dx.doi.org/10.1093/nar/27.19.3792] [PMID: 10481017]
[35]
Yin W, Xiang P, Li Q. Investigations of the effect of DNA size in transient transfection assay using dual luciferase system. Anal Biochem 2005; 346(2): 289-94.
[http://dx.doi.org/10.1016/j.ab.2005.08.029] [PMID: 16213455]
[36]
Chabot S, Orio J, Schmeer M, Schleef M, Golzio M, Teissié J. Minicircle DNA electrotransfer for efficient tissue-targeted gene delivery. Gene Ther 2013; 20(1): 62-8.
[http://dx.doi.org/10.1038/gt.2011.215] [PMID: 22257936]
[37]
Keeney M, Chung MT, Zielins ER, et al. Scaffold-mediated BMP-2 minicircle DNA delivery accelerated bone repair in a mouse critical-size calvarial defect model. J Biomed Mater Res A 2016; 104(8): 2099-107.
[http://dx.doi.org/10.1002/jbm.a.35735] [PMID: 27059085]
[38]
Yoon CS, Jung HS, Kwon MJ, et al. Sonoporation of the minicircle-VEGF(165) for wound healing of diabetic mice. Pharm Res 2009; 26(4): 794-801.
[http://dx.doi.org/10.1007/s11095-008-9778-x] [PMID: 18998201]
[39]
Chang CW, Christensen LV, Lee M, Kim SW. Efficient expression of vascular endothelial growth factor using minicircle DNA for angiogenic gene therapy. J Control Release 2008; 125(2): 155-63.
[http://dx.doi.org/10.1016/j.jconrel.2007.10.014] [PMID: 18063165]
[40]
Viecelli HM, Harbottle RP, Wong SP, et al. Treatment of phenylketonuria using minicircle-based naked-DNA gene transfer to murine liver. Hepatology 2014; 60(3): 1035-43.
[http://dx.doi.org/10.1002/hep.27104] [PMID: 24585515]
[41]
Huang M, Chen Z, Hu S, et al. Novel minicircle vector for gene therapy in murine myocardial infarction. Circulation 2009; 120(11)(Suppl.): S230-7.
[http://dx.doi.org/10.1161/CIRCULATIONAHA.108.841155] [PMID: 19752373]
[42]
Wu J, Xiao X, Zhao P, et al. Minicircle-IFNgamma induces antiproliferative and antitumoral effects in human nasopharyngeal carcinoma. Clin Cancer Res 2006; 12(15): 4702-13.
[http://dx.doi.org/10.1158/1078-0432.CCR-06-0520] [PMID: 16899621]
[43]
Narsinh KH, Jia F, Robbins RC, Kay MA, Longaker MT, Wu JC. Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors. Nat Protoc 2011; 6(1): 78-88.
[http://dx.doi.org/10.1038/nprot.2010.173] [PMID: 21212777]
[44]
Mayrhofer P, Jug H, Štrancar A, et al. https://www.biorxiv.org/ content/10.1101/300236v1
[45]
Hartikka J, Sawdey M, Cornefert-Jensen F, et al. An improved plasmid DNA expression vector for direct injection into skeletal muscle. Hum Gene Ther 1996; 7(10): 1205-17.
[http://dx.doi.org/10.1089/hum.1996.7.10-1205] [PMID: 8793545]
[46]
Valera A, Perales JC, Hatzoglou M, Bosch F. Expression of the neomycin-resistance (neo) gene induces alterations in gene expression and metabolism. Hum Gene Ther 1994; 5(4): 449-56.
[http://dx.doi.org/10.1089/hum.1994.5.4-449] [PMID: 7914094]
[47]
Chen ZY, Riu E, He CY, Xu H, Kay MA. Silencing of episomal transgene expression in liver by plasmid bacterial backbone DNA is independent of CpG methylation. Mol Ther 2008; 16(3): 548-56.
[http://dx.doi.org/10.1038/sj.mt.6300399] [PMID: 18253155]
[48]
Gracey Maniar LE, Maniar JM, Chen ZY, Lu J, Fire AZ, Kay MA. Minicircle DNA vectors achieve sustained expression reflected by active chromatin and transcriptional level. Mol Ther 2013; 21(1): 131-8.
[http://dx.doi.org/10.1038/mt.2012.244] [PMID: 23183534]
[49]
Jostins L, Ripke S, Weersma RK, et al. Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature 2012; 491(7422): 119-24.
[http://dx.doi.org/10.1038/nature11582] [PMID: 23128233]
[50]
Oliveira PH, Mairhofer J. Marker-free plasmids for biotechnological applications - implications and perspectives. Trends Biotechnol 2013; 31(9): 539-47.
[http://dx.doi.org/10.1016/j.tibtech.2013.06.001] [PMID: 23830144]
[51]
Ribeiro S, Mairhofer J, Madeira C, et al. Plasmid DNA size does affect nonviral gene delivery efficiency in stem cells. Cell Reprogram 2012; 14(2): 130-7.
[http://dx.doi.org/10.1089/cell.2011.0093] [PMID: 22339198]
[52]
Lukacs GL, Haggie P, Seksek O, Lechardeur D, Freedman N, Verkman AS. Size-dependent DNA mobility in cytoplasm and nucleus. J Biol Chem 2000; 275(3): 1625-9.
[http://dx.doi.org/10.1074/jbc.275.3.1625] [PMID: 10636854]
[53]
Darquet AM, Cameron B, Wils P, Scherman D, Crouzet J. A new DNA vehicle for nonviral gene delivery: supercoiled minicircle. Gene Ther 1997; 4(12): 1341-9.
[http://dx.doi.org/10.1038/sj.gt.3300540] [PMID: 9472558]
[54]
Kay MA, He CY, Chen ZY. A robust system for production of minicircle DNA vectors. Nat Biotechnol 2010; 28(12): 1287-9.
[http://dx.doi.org/10.1038/nbt.1708] [PMID: 21102455]
[55]
Zhang X, Epperly MW, Kay MA, et al. Radioprotection in vitro and in vivo by minicircle plasmid carrying the human manganese superoxide dismutase transgene. Hum Gene Ther 2008; 19(8): 820-6.
[http://dx.doi.org/10.1089/hum.2007.141] [PMID: 18699723]
[56]
Chen ZY, He CY, Ehrhardt A, Kay MA. Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo. Mol Ther 2003; 8(3): 495-500.
[http://dx.doi.org/10.1016/S1525-0016(03)00168-0] [PMID: 12946323]
[57]
Tsiridis E, Upadhyay N, Giannoudis P. Molecular aspects of fracture healing: which are the important molecules? Injury 2007; 38(Suppl. 1): S11-25.
[http://dx.doi.org/10.1016/j.injury.2007.02.006] [PMID: 17383481]
[58]
Stacey KJ, Sweet MJ, Hume DA. Macrophages ingest and are activated by bacterial DNA. J Immunol 1996; 157(5): 2116-22.
[PMID: 8757335]
[59]
Hartmann G, Weiner GJ, Krieg AM. CpG DNA: a potent signal for growth, activation, and maturation of human dendritic cells. Proc Natl Acad Sci USA 1999; 96(16): 9305-10.
[http://dx.doi.org/10.1073/pnas.96.16.9305] [PMID: 10430938]
[60]
Ballas ZK, Rasmussen WL, Krieg AM. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. J Immunol 1996; 157(5): 1840-5.
[PMID: 8757300]
[61]
Liu S, Liu Y, Jiang L, et al. Recombinant human BMP-2 accelerates the migration of bone marrow mesenchymal stem cells via the CDC42/PAK1/LIMK1 pathway in vitro and in vivo. Biomater Sci 2018; 7(1): 362-72.
[http://dx.doi.org/10.1039/C8BM00846A] [PMID: 30484785]
[62]
Sharma S, Sapkota D, Xue Y, et al. Adenoviral mediated expression of BMP2 by bone marrow stromal cells cultured in 3D Copolymer scaffolds enhances bone formation. PLoS One 2016; 11(1)e0147507
[http://dx.doi.org/10.1371/journal.pone.0147507] [PMID: 26808122]
[63]
Sun J, Li J, Li C, Yu Y. Role of bone morphogenetic protein-2 in osteogenic differentiation of mesenchymal stem cells. Mol Med Rep 2015; 12(3): 4230-7.
[http://dx.doi.org/10.3892/mmr.2015.3954] [PMID: 26096280]


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VOLUME: 20
ISSUE: 1
Year: 2020
Published on: 23 June, 2020
Page: [55 - 63]
Pages: 9
DOI: 10.2174/1566523220666200427121350
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