摘要
背景: 到目前为止,骨形态发生蛋白2 (BMP-2)非病毒基因治疗的显著骨诱导潜能还不能完全用于治疗。这主要是由于基因传递弱,表达峰短,限制了治疗效果。 目的: 我们的目的是测试小圆DNA的应用,允许延长表达潜力。与传统的质粒DNA相比,它具有显著的优势。由于缺少细菌序列和由此导致的体积减小,使得安全使用和提高组织再生性能成为可能。 结果: 与转染传统BMP-2晚期质粒的细胞相比,转染BMP-2晚期小圆细胞的C2C12细胞骨钙素、碱性磷酸酶(ALP)活性和BMP-2蛋白含量均显著提高。此外,当质粒被引入人骨髓干细胞(BMSCs)时,显示出明显较高的碱性磷酸酶活性和矿化水平,显示出对干细胞方法的适应性。 结论:我们设计了一种具有高生物活性的BMP-2小圆质粒,有潜力满足骨再生领域非病毒基因治疗的临床需要。
关键词: C2C12
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[http://dx.doi.org/10.1038/nprot.2010.173] [PMID: 21212777]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[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]
[http://dx.doi.org/10.3892/mmr.2015.3954] [PMID: 26096280]
36
3