title: Sustained long-term RNA interference in nucleus pulposus cells in vivo mediated by unmodified small interfering RNA.
abstract: RNA interference (RNAi) technology has recently emerged as an important biological strategy for gene silencing. Previously, the efficacies of RNAi in cultured nucleus pulposus cells in vitro have been reported. However, RNAi in the disc in vivo has never been reported. Therefore, the aims of the present study were to establish a method for RNAi in the disc in vivo and to evaluate the applicability of this technique for endogenous genes in the intervertebral discs using Fas Ligand (FasL) as a representative endogenous gene. To evaluate the efficacy of RNAi in vivo, two reporter luciferase plasmids (Firefly and Renilla) were used. These plasmids and unmodified short interference RNA (siRNA) duplex for targeting Firefly luciferase were co-transfected into coccygeal intervertebral disc of Sprague-Dawley rats in vivo using the ultrasound gene transfer technique. To evaluate the RNAi of the endogenous gene in vivo, siRNAs targeting rat FasL were transfected with the same technique. Non-specific siRNA was used as the negative control. The discs receiving no siRNAs were used as the control. The inhibitory effect of Firefly luciferase against Renilla luciferase was obtained using the results of dual-luciferase assay. Down-regulation of endogenous FasL was calculated by the data from real-time PCR. Our results showed that siRNA for Firefly luciferase can dramatically down-regulate the Firefly luciferase gene expression in vivo compared with Renilla luciferase. The inhibitory effects were maintained for at least 24 weeks and at 24 weeks post transfection, the inhibitory rate was 80% compared with the control group. Furthermore, the siRNA co-transfection group inhibited endogenous FasL expression by 53% compared with the control group. The present study demonstrates long-term down-regulation mediated by unmodified siRNA is possible not only for the exogenous reporter gene, but also for endogenous FasL expression in rat discs in vivo. This application of RNAi might be promising as a local therapy for disc degeneration and associated disorders by down-regulating some of the genes that are harmful for the normal physiology of the disc and may cause disc degeneration.
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
authors.forename: Teppei
authors.initials: T
authors.lastname: Suzuki
authors.forename: Kotaro
authors.initials: K
authors.lastname: Nishida
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Kakutani
authors.forename: Koichiro
authors.initials: K
authors.lastname: Maeno
authors.forename: Takashi
authors.initials: T
authors.lastname: Yurube
authors.forename: Toru
authors.initials: T
authors.lastname: Takada
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kurosaka
authors.forename: Minoru
authors.initials: M
authors.lastname: Doita
mesh_terms: D000818:Animals; D019902:Chondrocytes / Q000502:physiology*; D053222:Fas Ligand Protein / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D007403:Intervertebral Disc / Q000166:cytology*; D008297:Male; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D053222:Fas Ligand Protein; C505662:Faslg protein, rat; D034741:RNA, Small Interfering