Identification and characterization of human nucleus pulposus cell specific serotypes of adeno-associated virus for gene therapeutic approaches of intervertebral disc disorders

BMC Musculoskeletal Disorders

Claudius Thomé¹  Demissew S. Mern¹ 

[1] Department of Neurosurgery, Innsbruck Medical University, Anichstrasse 35, Innsbruck A-6020, Austria

关键词: Maintenance of intervertebral disc matrix;    Targeted gene therapy;    Nucleus pulposus cell specific adeno-associated virus;    Nucleus pulposus cells;    Inflammatory and catabolic cytokines;    Intervertebral disc disorders;   

Others  :  1234034
DOI  :  10.1186/s12891-015-0799-4

 received in 2015-06-09, accepted in 2015-11-02,  发布年份 2015

【 摘 要 】

Background

Intervertebral disc (IVD) disorders are often accompanied by painful inflammatory and immunopathological processes. Nucleus pulposus (NP) cells play a pivotal role in maintenance of IVD by organizing the expression of anabolic, catabolic, anti-catabolic and inflammatory cytokines. Human NP cells have been targeted by gene therapeutic approaches using lentiviral or adenoviral systems that could be critical due to genome incorporation or immunological side effects. Adeno-associated viruses (AAVs), which do not express any viral gene and are not linked with any known disease in humans, are attractive gene delivery vectors. However, their lack of specific tissue tropism and preexisting immune response are main problems for therapeutic applications. Heretofore, AAVs have not been studied in human IVD research. Therefore, we attempted to identify NP cell specific AAV serotype by targeting human NP cells with different self-complementary AAV (scAAV) serotypes.

Identification and characterization of the proper serotype is crucial to establish less immunogenic and safer gene therapeutic approaches of IVD disorders.

Methods

Preoperative magnetic resonance imaging (MRI) was used for grading of IVD degeneration. NP cells were isolated, cultured with low-glucose and transduced with green fluorescent protein (GFP) packing scAAV serotypes (scAAV1-8) in a dose-dependent manner. scAAV titers were determined by quantitative polymerase chain reaction (qPCR). Transduction efficiencies were determined by fluorescence microscopy and fluorescence-activated cell sorting within 48 days of post-transduction. The 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine NP cell viability. Three-dimensional (3D) cell culture and enzyme-linked immunosorbant assay (ELISA) were performed to examine the expression levels of inflammatory, catabolic and matrix proteins in NP cells.

Results

scAAV6, scAAV2 and scAAV3 showed high and prolonged transgene GFP expressions with transdution efficiencies of 98.6 %, 91.5 % and 89.6 % respectively (p ≤ 0.002). Unlike scAAV6, the serotypes scAAV2 and scAAV3 declined the viability of NP cells by about 25 % and 10 % respectively (p ≤ 0.001). Moreover, scAAV6 did not affect the expression of the inflammatory, catabolic and matrix proteins.

Conclusions

As original primary research evaluating AAVs in degenerative human IVDs, this study identified scAAV6 as a proper serotype for high, stable and non-immunogenic target gene expression in human NP cells. The data could be very important to design efficient and safer gene therapeutic approaches of IVD disorders.

【 授权许可】

   
2015 Mern and Thomé.

【 图 表 】

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【 参考文献 】

• [1]Freburger JK, Holmes GM, Agans RP, Jackman JD, Darter JD, Wallace AS et al.. The rising prevalence of chronic low back pain. Arch Intern Med. 2009; 169:251-8.
• [2]Wenig C, Schmidt CO, Kohlmann T, Schweikert B. Costs of back pain in Germany. Eur J Pain. 2009; 13:280-6.
• [3]Enthoven P, Skargren E, Oberg B. Clinical course in patients seeking primary care for back or neck pain: A prospective 5-year follow-up of outcome and health care consumption with subgroup analysis. Spine. 2004; 29:2458-65.
• [4]Schizas C, Kulik G, Kosmopoulos V. Disc degeneration: Current surgical options. Eur Cell Mater. 2010; 20:306-15.
• [5]Adams MA, Roughley PJ. What is intervertebral disc degeneration, and what causes it? Spine. 2006; 31:2151-61.
• [6]Kaneyama S, Nishida K, Takada T, Suzuki T, Shimomura T, Maeno K et al.. Fas ligand expression on human nucleus pulposus cells decreases with disc degeneration processes. J Orthop Sci. 2008; 13:130-5.
• [7]Hoyland JA, Le Maitre C, Freemont AJ. Investigation of the role of IL-1 and TNF in matrix degradation in the intervertebral disc. Rheumatology (Oxf). 2008; 47:809-14.
• [8]Burke JG, Watson RW, McCormack D, Dowling FE, Walsh MG et al.. Intervertebral discs which cause low back pain secrete high levels of proinflammatory mediators. J Bone Joint Surg Br. 2002; 84:196-201.
• [9]Mulleman D, Mammou S, Griffoul I, Watier H, Goupille P. Pathophysiology of disk-related sciatica. I. Evidence supporting a chemical component. Joint Bone Spine. 2006; 73:151-8.
• [10]Freemont AJ, Peacock TE, Goupille P, Hoyland JA, O'Brien J, Jayson MI. Nerve ingrowth into diseased intervertebral disc in chronic back pain. Lancet. 2007; 350(9072):178-81.
• [11]Peng B, Wu W, Hou S, Li P, Zhang C, Yang Y. The pathogenesis of discogenic low back pain. J Bone Joint Surg Br. 2005; 87(1):62-7.
• [12]Panjabi MM, Krag MH, Chung TQ. Effects of disc injury on mechanical behavior of the human spine. Spine. 1984; 9:707-13.
• [13]McNally DS, Shackleford IM, Goodship AE, Mulholland RC. In vivo stress measurement can predict pain on discography. Spine. 1996; 21:2580-7.
• [14]Panjabi MM. Clinical spinal instability and low back pain. J Electromyogr Kinesiol. 2003; 13:371-9.
• [15]Kandel R, Roberts S, Urban JP. Tissue engineering and the intervertebral disc: the challenges. Eur Spine J. 2008; 4:480-91.
• [16]Mern DS, Beierfuß A, Thomé C, Hegewald AA. Enhancing human nucleus pulposus cells for biological treatment approaches of degenerative intervertebral disc diseases: a systematic review. J Tissue Eng Regen Med. 2014; 12:925-36.
• [17]Mern DS, Fontana J, Beierfuß A, Thomé C, Hegewald AA. A combinatorial relative mass value evaluation of endogenously expressed bioactive proteins in three-dimensional cultured nucleus pulposus cells: Identification of potential target proteins for gene therapeutic approaches. PLoS One. 2013; 8(11): Article ID e81467
• [18]Mern DS, Fontana J, Beierfuß A, Thomé C, Hegewald AA. Imbalanced protein expression patterns of anabolic, catabolic, anti-catabolic and inflammatory cytokines in degenerative cervical disc cells: new indications for gene therapeutic treatments of cervical disc diseases. PLoS One. 2014; 9(5): Article ID e96870
• [19]Hegewald AA, Ringe J, Sittinger M, Thomé C. Regenerative treatment strategies in spinal surgery. Front Biosci. 2008; 13:1507-25.
• [20]An HS, Masuda K, Cs-Szabo G, Zhang Y, Chee A, Andersson GB et al.. Biologic repair and regeneration of the intervertebral disc. J Am Acad Orthop Surg. 2011; 19:450-2.
• [21]Leung VY, Tam V, Chan D, Chan BP, Cheung KM. Tissue engineering for intervertebral disk degeneration. Orthop Clin North Am. 2011; 42:575-83.
• [22]Lotz JC. Animal models of intervertebral disc degeneration: lessons learned. Spine. 2004; 29:2742-50.
• [23]Alini M, Eisenstein SM, Ito K, Little C, Kettler AA, Masuda K et al.. Are animal models useful for studying human disc disorders/degeneration? Eur Spine J. 2008; 17:2-19.
• [24]Robbins PD, Ghivizzani SC. Viral vectors for gene therapy. Pharmacol Ther. 1998; 80:35-47.
• [25]Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P et al.. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science. 2003; 302(5644):415-19.
• [26]Wallach CJ, Sobajima S, Watanabe Y, Kim JS, Georgescu HI, Robbins P et al.. Gene transfer of the catabolic inhibitor TIMP-1 increases measured proteoglycans in cells from degenerated human intervertebral discs. Spine. 2003; 28:2331-37.
• [27]Moon SH, Nishida K, Gilbertson LG, Lee HM, Kim H, Hall RA et al.. Biologic response of human intervertebral disc cells to gene therapy cocktail. Spine. 2008; 33:1850-55.
• [28]Gilbertson L, Ahn SH, Teng PN, Studer RK, Niyibizi C, Kang JD. The effects of recombinant human bone morphogenetic protein-2, recombinant human bone morphogenetic protein-12, and adenoviral bone morphogenetic protein-12 on matrix synthesis in human annulus fibrosis and nucleus pulposus cells. Spine J. 2008; 8:449-56.
• [29]Lee JY, Hall R, Pelinkovic D, Cassinelli E, Usas A, Gilbertson L et al.. New use of a three-dimensional pellet culture system for human intervertebral disc cells: initial characterization and potential use for tissue engineering. Spine. 2001; 26:2316-22.
• [30]Yang Y, Nunes FA, Berencsi K, Furth EE, Gönczöl E, Wilson JM. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc Natl Acad Sci U S A. 1994; 91(10):4407-11.
• [31]Mazzolini G, Narvaiza I, Pérez-Diez A, Rodriguez-Calvillo M, Qian C, Sangro B et al.. Genetic heterogeneity in the toxicity to systemic adenoviral gene transfer of interleukin-12. Gene Ther. 2001; 8:259-67.
• [32]Nemunaitis J, Cunningham C, Buchanan A, Blackburn A, Edelman G, Maples P et al.. Intravenous infusion of a replication-selective adenovirus (ONYX-015) in cancer patients: safety, feasibility and biological activity. Gene Ther. 2001; 8:746-59.
• [33]Lozier JN, Csako G, Mondoro TH, Krizek DM, Metzger ME, Costello R et al.. Toxicity of a first generation adenoviral vector in rhesus macaques. Hum Gene Ther. 2002; 13:113-24.
• [34]Grieger JC, Samulski RJ. Adeno-associated virus as a gene therapy vector: vector development, production and clinical applications. Adv Biochem Eng Biotechnol. 2005; 99:119-45.
• [35]Samulski RJ, Zhu X, Xiao X, Brook JD, Housman DE, Epstein N et al.. Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J. 1991; 10:3941-50.
• [36]Wang Z, Ma HI, Li J, Sun L, Zhang J, Xiao X. Rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo. Gene Ther. 2003; 10:2105-11.
• [37]Mori S, Wang L, Takeuchi T, Kanda T. Two novel adeno-associated viruses from cynomolgus monkey: pseudotyping characterization of capsid protein. Virology. 2004; 330(2):375-83.
• [38]Pfirrmann CW, Metzdorf A, Zanetti M, Hodler J, Boos N. Magnetic resonance classification of lumbar intervertebral disc degeneration. Spine. 2001; 26:1873-1878.
• [39]Landis RJ, Koch GG. The measurement of observer agreement for categorical data. Biometrics. 1977; 33:159-74.
• [40]Fechner H, Sipo I, Westermann D, Pinkert S, Wang X, Suckau L et al.. Cardiac-targeted RNA interference mediated by an AAV9 vector improves cardiac function in coxsackievirus B3 cardiomyopathy. J Mol Med. 2008; 86:987-97.
• [41]Rothe D, Wajant G, Grunert HP, Zeichhardt H, Fechner H, Kurreck J. Rapid construction of adeno-associated virus vectors expressing multiple short hairpin RNAs with high antiviral activity against echovirus 30. Oligonucleotides. 2010; 20:191-8.
• [42]Wagner A, Röhrs V, Kedzierski R, Fechner H, Kurreck J. A novel method for the quantification of adeno-associated virus vectors for RNA interference applications using quantitative polymerase chain reaction and purified genomic adeno-associated virus DNA as a standard. Hum Gene Ther Methods. 2013; 24(6):355-63.
• [43]Liebscher T, Haefeli M, Wuertz K, Nerlich AG, Boos N. Age-related variation in cell density of human lumbar intervertebral discs. Spine. 2011; 36:153-59.
• [44]Nishida K, Kang JD, Gilbertson LG, Moon SH, Suh JK, Vogt MT et al.. Modulation of the biologic activity of the rabbit intervertebral disc by gene therapy: an in vivo study of adenovirus-mediated transfer of the human transforming growth factor beta 1 encoding gene. Spine. 1999; 24:2419-25.
• [45]Nishida K, Doita M, Takada T, Kakutani K, Miyamoto H, Shimomura T et al.. Sustained transgene expression in intervertebral disc cells in vivo mediated by microbubble-enhanced ultrasound gene therapy. Spine. 2006; 31:1415-19.
• [46]Okuma M, Mochida J, Nishimura K, Sakabe K, Seiki K. Reinsertion of stimulated nucleus pulposus cells retards intervertebral disc degeneration: an in vitro and in vivo experimental study. J Orthop Res. 2000; 18:988-97.
• [47]Driesse MJ, Esandi MC, Kros JM, Avezaat CJ, Vecht C, Zurcher C et al.. Intra-CSF administered recombinant adenovirus causes an immune response-mediated toxicity. Gene Ther. 2000; 7:1401-09.
• [48]Wallach CJ, Kim JS, Sobajima S, Lattermann C, Oxner WM, McFadden K et al.. Safety assessment of intradiscal gene transfer: a pilot study. Spine J. 2006; 6:107-112.
• [49]Ren XF, Diao ZZ, Xi YM, Qi ZH, Ren S, Liu YJ et al.. Adeno-associated virus-mediated BMP-7 and SOX9 in vitro co-transfection of human degenerative intervertebral disc cells. Genet Mol Res. 2015; 14(2):3736-44.
• [50]Ren S, Liu Y, Ma J, Liu Y, Diao Z, Yang D et al.. Treatment of rabbit intervertebral disc degeneration with co-transfection by adeno-associated virus-mediated SOX9 and osteogenic protein-1 double genes in vivo. Int J Mol Med. 2013; 32(5):1063-8.
• [51]Xi YM, Dong YF, Wang ZJ, Liu Y, Diao ZZ, Hu YG. Co-transfection of adeno-associated virus-mediated human vascular endothelial growth factor165 and transforming growth factor-β1 into annulus fibrosus cells of rabbit degenerative intervertebral discs. Genet Mol Res. 2013; 12(4):4895-908.
• [52]Lattermann C, Oxner WM, Xiao X, Li J, Gilbertson LG, Robbins PD et al.. The adeno associated viral vector as a strategy for intradiscal gene transfer in immune competent and pre-exposed rabbits. Spine. 2005; 30(5):497-504.
• [53]Sowa G, Westrick E, Pacek C, Coelho P, Patel D, Vadala G et al.. In vitro and in vivo testing of a novel regulatory system for gene therapy for intervertebral disc degeneration. Spine. 2011; 36(10):E623-8.
• [54]Levicoff EA, Kim JS, Sobajima S, Wallach CJ, Larson JW, Robbins PD et al.. Safety assessment of intradiscal gene therapy II: effect of dosing and vector choice. Spine. 2008; 33(14):1509-16.
• [55]McCarty DM. Self-complementary AAV Vectors; Advances and Applications. Mol Ther. 2008; 16(10):1648-56.
• [56]Bartlett JS, Wilcher R, Samulski RJ. Infectious entry pathway of adeno-associated virus and adeno-associated virus vectors. J Virol. 2000; 74:2777-85.
• [57]Wu Z, Asokan A, Samulski RJ. Adeno-associated virus serotypes: Vector toolkit for human gene therapy. Mol Ther. 2006; 14:316-27.
• [58]Le Maitre CL, Hoyland JA, Freemont AJ. Catabolic cytokine expression in degenerate and herniated human intervertebral discs: IL-1beta and TNF-alpha expression profile. Arthritis Res Ther. 2007; 9:R77. BioMed Central Full Text
• [59]Malfait AM, Liu RQ, Ijiri K, Komiya S, Tortorella MD. Inhibition of ADAM-TS4 and ADAM-TS5 prevents aggrecan degradation in osteoarthritic cartilage. J Biol Chem. 2002; 277:22201-8.
• [60]Naito S, Shiomi T, Okada A, Kimura T, Chijiiwa M, Fujita Y et al.. Expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic cartilage. Pathol Int. 2007; 57:703-11.