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In Vivo Delivery of MMP3-Shrna and Sox9 Lentivirus Cocktail Enhances Matrix Synthesis to Prevent Lumbar Disc Degeneration

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In Vivo Delivery of MMP3-Shrna and Sox9 Lentivirus Cocktail Enhances Matrix Synthesis to Prevent Lumbar Disc Degeneration Original papers In vivo delivery of MMP3-shRNA and Sox9 lentivirus cocktail enhances matrix synthesis to prevent lumbar disc degeneration Zheng Zhao1,A–F, Siyuan Li2,A–D,F, Hui Huang1,A,B,F, Jing Fang1,B,C,F, Huawei Wei1,B,C,F, Yongming Xi1,A–F 1 Department of Orthopedics, Affiliated Hospital of Qingdao University, China 2 Department of Orthopedics, Shandong Provincial Third Hospital, Jinan, China A – research concept and design; B – collection and/or assembly of data; C – data analysis and interpretation; D – writing the article; E – critical revision of the article; F – final approval of the article Advances in Clinical and Experimental Medicine, ISSN 1899–5276 (print), ISSN 2451–2680 (online) Adv Clin Exp Med. 2020;29(6):639–647 Address for correspondence Abstract Yongming Xi E-mail: [email protected] Background. Intervertebral disc degeneration (IDD) is characterized by increased proteolytic degradation of the extracellular matrix (ECM), leading to a loss of collagen II and proteoglycan in the nucleus pulposus (NP). Funding sources The National Natural Science Foundation of China Although MMP3 has been reported to play a central role in disc degeneration, it is still unknown whether (NSFC, grant No. 81470104). gene therapy targeting MMP3 can inhibit IDD. Objectives. To investigate whether lentivirus-mediated MMP3 knockdown is capable of attenuating IDD. Conflict of interest None declared More importantly, we also explored whether combined gene therapy that simultaneously antagonizes MMP3 and overexpresses Sox9 can synergistically inhibit IDD and induce augmented matrix reconstitution in the degenerative NP. Received on September 22, 2019 Reviewed on March 31, 2020 Material and methods. We performed direct injection of lentiviral vectors LV-MMP3-shRNA and/or LV- Accepted on April 29, 2020 Sox9 into rabbit lumbar discs. The animals were scanned using magnetic resonance imaging (MRI) at 8, 12 and 24 weeks after the operation. We also evaluated the gene expression and synthesis of NP matrix Published online on June 26, 2020 components, including collagen II, aggrecan and proteoglycan. Results. The MRI scans showed remarkable needle-puncture-induced progressive IDD in animals injected with PBS or 10^7 viral particles (VP) of the control virus. In contrast, injection of 10^7 VP of LV-MMP3-shRNA or LV-Sox9 substantially inhibited IDD. MMP3 knockdown or Sox9 overexpression stimulated collagen II and aggrecan expression, as well as proteoglycan synthesis. Notably, the injection of a cocktail of LV-MMP3-shRNA and LV-Sox9 (5 × 10^6 VP each) greatly delayed the development of IDD and induced the highest levels of collagen II and proteoglycan production, indicating a synergistic effect in ECM induction. Conclusions. Our results suggest that gene therapy targeting MMP3 is an efficient way to delay IDD. Combined gene therapy possesses a stronger capacity to induce matrix components in degenerative NP tissue than single-gene delivery. Key words: gene therapy, disc degeneration, SOX9, MMP3 Cite as Zhao Z, Li S, Huang H, Fang J, Wei H, Xi Y. In vivo delivery of MMP3-shRNA and Sox9 lentivirus cocktail enhances matrix synthesis to prevent lumbar disc degeneration. Adv Clin Exp Med. 2020;29(6):639–647. doi:10.17219/acem/121509 DOI 10.17219/acem/121509 Copyright © 2020 by Wroclaw Medical University This is an article distributed under the terms of the Creative Commons Attribution 3.0 Unported (CC BY 3.0) (https://creativecommons.org/licenses/by/3.0/) 640 Z. Zhao et al. Combined gene therapy for disc degeneration Introduction to prevent or delay IDD.11 Nishida et al. conducted an initial study showing that direct injection of adenoviral TGFβ1 Intervertebral disc degeneration (IDD) is a major cause vectors into the rabbit lumbar discs led to a 100% increase of most musculoskeletal disorders of the spine that re- in proteoglycan synthesis compared with intact control sults in lower back pain, morbidity and physical disability.1 tissues.12 Since then, gene therapy using several growth fac- Although the precise etiology and pathophysiology of disc tors, including BMP2, GDF5, IGF1, and interleukin (IL)-1 degeneration has not been fully elucidated, IDD is generally receptor antagonist (IL-1Ra) for disc degeneration has been believed to be a consequence of increased proteolytic deg- tested both in vitro and in vivo, as previously reported.11,13 radation of extracellular matrix (ECM) macromolecules. Co-transduction of adeno-associated virus (AAV)-based During the disc degenerative process, the nucleus pulposus Sox9 and OP1 vectors was reported to exhibit a stronger (NP) loses water and large amounts of aggregating pro- effect in the induction of proteoglycan and collagen II than teoglycans and type II collagen (COL2A1), which leads AAV-Sox9 or AAV-OP1 alone.14 to ECM breakdown and structural failure.2 So far, gene therapy which targets an individual MMP A variety of studies have focused on the mechanisms molecule (particularly MMP3) against disc degeneration underlying ECM degradation in IDD pathogenesis. An in- has not yet been reported. In our study, we performed direct creasing amount of evidence has shown that matrix metal- injection of lentiviral vectors expressing MMP3-shRNA loproteinases (MMPs) play important roles in the degrada- into NP tissues of lumbar discs in rabbits and evaluated tion of matrix components during the disc degenerative the reconstitution of matrix components, specifically process. Several MMP family members, such as MMP1, collagen type II and proteoglycan. We also investigated MMP2, MMP3, MMP7, MMP8, MMP9, MMP12, and whether a cocktail of both MMP3-shRNA and Sox9 lenti- MMP26, have been reported to be increasingly expressed viruses could trigger a synergistic effect to further augment in degenerated disc tissues, suggesting an association matrix synthesis. between MMP and IDD.3–6 Notably, Bachmeier et al. reported that among all MMPs tested (MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, and MMP13), the expres- Material and methods sion of MMP3 was consistently and substantially upregu- lated in degenerated disc samples, and this process was Lentiviral vectors accompanied with increased enzymatic activity of MMP3,7 implying that MMP3 plays a central role in disc degenera- The lentiviral vectors harboring MMP3-shRNA (LV- tion. Besides elevated matrix-degrading MMPs, the loss MMP3-shRNA), Sox9 (LV-Sox9), or empty vectors were of matrix-producing Sox9 also plays a role in ECM deg- provided by Biowit Technologies (Shenzhen, China). radation in IDD. Being a key transcription factor in chon- All lentivirus vectors were stored at −80°C and diluted drogenic differentiation, Sox9 can directly stimulate gene with phosphate-buffered saline (PBS) to a concentration expression of collagen type II and aggrecan,8 the major core of 5 × 105 viral particles (vp)/μL right before use. protein in proteoglycan. Sive et al. detected a strong signal of Sox9 in normal NP tissues using in situ hybridization, Animal study which was accompanied by high-level expression of col- lagen II and aggrecan.9 However, the expression of Sox9 This is an experimental study in a rabbit model. The pro- decreases with aging and disc degeneration.10 These stud- tocol for the animal study was reviewed and approved ies suggest that MMP-induced excessive ECM degrada- by the Animal Care Committee of Qingdao University, tion or loss of Sox9-reduced ECM synthesis is critically China. A total of 25 New Zealand white rabbits were used implicated in the pathogenesis of IDD. Hence, molecular in this study, with an average age of 4 months and an av- therapy which targets MMP or restores Sox9 can be used erage body weight of 2.5 kg. Before surgery, the animals to reconstitute matrix components in a degenerative NP. underwent magnetic resonance imaging (MRI) to exclude Although spine disorders resulting from disc degen- congenital spine deformity and disc disease. eration can be treated with surgery, including discectomy For direct gene transfer, the animals were anesthetized or spinal fusion, they are not curative and are associated with an intramuscular injection of 1.5% pentobarbital so- with various complications. Non-surgical treatments dium at a dosage of 2 mL/kg. Following anesthesia, each – such as drugs, massage or physical therapy – however, animal was placed in a lateral position with all 4 limbs fixed. can only relieve the clinical symptoms; they cannot delay A vertical incision of about 3 cm was made from the iliac or reverse the disc degenerative process. As a result, gene crest to the lower edge of the 12th rib via a right extraperito- therapy has received considerable interest, as this approach neal approach. The peritoneum was bluntly dissected to ex- can transfer the genes of therapeutic proteins to the disc pose the transverse processes from L3 to L7. A 24-gauge cells, enabling them to manufacture the proteins endog- needle was used to pierce into the center of the exposed enously, on a continuous or regular basis, allowing for L3/4, L4/5, L5/6, and L6/7 lumbar discs (~5 mm) parallel long-term regulation of matrix synthesis with the potential to the upper and lower endplates. Then, a volume of 20 μL Adv Clin Exp Med. 2020;29(6):639–647 641 Fig. 1. In vivo gene delivery. Lentiviral vectors are directly injected into the rabbit lumbar discs (L3/4, L4/5, L5/6, and L6/7) through a surgical approach containing PBS or 107 vp of lentivirus was injected directly of the scanner were as follows: T2-weighted sequence rep- into the NP tissue. All 25 rabbits were randomly divided into etition time – 2,000–6,000 ms; echo time – 80–120 ms; 5 groups and injected with a total volume of 20 μL of PBS bandwidth – 25 Hz; microwave – 16; matrix – 384 × 224 px; or lentivirus, as follows: group I – PBS; group II – 107 vp nex – 4; field of vision – 16 × 16 cm; thickness – 3 mm; of empty virus (control); group III – 107 vp of LV-MMP3- and spacing – 0.2 mm.
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