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Degenerative disk disease (DDD) that aggravates structural deterioration of intervertebral disks (IVDs) can be accompanied by painful inflammation and immunopathological progressions. Current surgical or pharmacological therapies cannot repair the structure and function of IVDs. Nucleus pulposus (NP) cells are crucial for the preservation or restoration of IVDs by balancing the anabolic and catabolic factors affecting the extracellular matrix. Imbalanced anabolic and catabolic factors cause increased degradation of aggrecan. Aggrecanases A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS)4 and ADAMTS5 are the main degrading enzymes of aggrecan. Previously, we characterized adeno-associated virus (AAV6) as the most suitable serotype with marked NP cellular tropism and demonstrated that ADAMTS4 could be silenced by self-complementary adeno-associated virus grade 6 small helix ribonucleic acid (scAAV6-shRNA) in NP cells of degeneration grade III, which resulted in enrichment of aggrecan. Nonetheless, neither scAAV6-shRNA-mediated inhibition of ADAMTS5 nor joint inhibitions of ADAMTS4 and ADAMTS5 have been investigated, although both enzymes are regulated by analogous proinflammatory cytokines and have the same cleavage sites in aggrecan. Therefore, we attempted scAAV6-shRNA-mediated inhibitions of both enzymes in NP cells of degeneration grade IV to increase efficacies in treatments of DDD. The degeneration grade of IVDs in patients was determined by magnetic resonance imaging (MRI) before surgical operations. After isolation and culturing of NP cells, cells were transduced with scAAV6-shRNAs targeting ADAMTS4 or ADAMTS5. Transduced cells were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR), fluorescence microscopy, flow cytometry-assisted cell sorting (FACS), MTT assay (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay), immunoblotting, and enzyme-linked immunosorbent assay (ELISA). Joint transduction of NP cells exhibited high transduction efficacies (98.1%), high transduction units (TU) (1381 TU/Cell), and no effect on cell viability or proliferation. Above all joint treatments resulted in effective knockdown of ADAMTS4 (92.8%) and ADAMTS5 (93.4%) along with additive enrichment of aggrecan (113.9%). Treatment effects were significant for more than 56 days after transduction ( P < 0.001). In conclusion, scAAV6-shRNA-mediated combined molecular therapy could be very valuable for more effective, durable, and less immunogenic treatment approaches in DDD., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Inhibition of intervertebral disc (IVD) degeneration, which is often accompanied by painful inflammatory and immunopathological processes, is challenging. Current IVD gene therapeutic approaches are based on adenoviral gene delivery systems, which are limited by immune reactions to their viral proteins. Their applications in IVDs near to sensitive neural structure could provoke toxicity and immunological side-effects with neurological deficits. Self-complementary adeno-associated virus (scAAV) vectors, which do not express any viral gene and are not linked with any known disease in humans, are attractive therapeutic gene delivery vectors in degenerative IVDs. However, scAAV-based silencing of catabolic or inflammatory factor has not yet been investigated in human IVD cells. Therefore, we used scAAV6, the most suitable serotype for transduction of human nucleus pulposus (NP) cells, to knockdown the major catabolic gene (ADAMTS4) of IVD degeneration. IVD degeneration grades were determined by preoperative magnetic resonance imaging. Lumbar NP tissues of degeneration grade III were removed from 12 patients by nucleotomy. NP cells were isolated and cultured with low-glucose. Titre of recombinant scAAV6 vectors targeting ADAMTS4, transduction efficiencies, transduction units, cell viabilities and expression levels of target genes were analysed using quantitative PCR, fluorescence microscopy, fluorescence-activated cell sorting, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assays, quantitative reverse transcription PCR, western blot and enzyme-linked immunosorbent assays during 48 days of post-transduction. Transduction efficiencies between 98.2% and 37.4% and transduction units between 611 and 245 TU/cell were verified during 48 days of post-transduction (p<0.001). scAAV6-mediated knockdown of ADAMTS4 with maximum 87.7% and minimum 40.1% was confirmed on day 8 and 48 with enhanced the level of aggrecan 48.5% and 30.2% respectively (p<0.001). scAAV6-mediated knockdown of ADAMTS4 showed no impact on cell viability and expression levels of other inflammatory catabolic proteins. Thus, our results are promising and may help to design long-term and less immunogenic gene therapeutic approaches in IVD disorders, which usually need prolonged therapeutic period between weeks and months.