Journal Article

Self-complementary adeno-associated virus serotype 6 mediated knockdown of ADAMTS4 induces long-term and effective enhancement of aggrecan in degenerative human nucleus pulposus cells: A new therapeutic approach for intervertebral disc disorders

Shenegelegn Mern, Demissew, Tschugg, Anja, Thomé, Claudius, Hartmann, Sebastian

2017

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: ADAMTS4 Protein - antagonists & inhibitors / ADAMTS4 Protein - genetics / Adult / Aggrecan / Aggrecans - metabolism / Analysis / Biology and Life Sciences / Care and treatment / Cells, Cultured / Cytokines / Degeneration / Delivery systems / Deoxyribonucleic acid / Dependovirus - genetics / Disorders / DNA / Expression vectors / Female / Flow cytometry / Fluorescence / Fluorescence microscopy / Gene expression / Gene transfer / Genes / Genetic aspects / Genetic Therapy / Genetic Vectors - administration & dosage / Growth factors / Humans / Immune response / Immunoassays / Immunogenicity / Immunology / Inflammation / Intervertebral Disc Degeneration - genetics / Intervertebral Disc Degeneration - pathology / Intervertebral Disc Degeneration - therapy / Intervertebral discs / Intervertebral disk / Laboratories / Magnetic resonance / Magnetic resonance imaging / Male / Medicine and Health Sciences / Middle Aged / Neurological diseases / Neurosurgery / Nuclei (cytology) / Nucleus pulposus / Nucleus Pulposus - metabolism / Nucleus Pulposus - pathology / Proteins / Research and Analysis Methods / Reverse transcription / Serogroup / Tissues / Toxicity / Tumor necrosis factor-TNF / Viral proteins / Viruses