Poster Presentation 51st International Society for the Study of the Lumbar Spine Annual Meeting 2025

INTRODUCTION: The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that has mTOR complex 1 (mTORC1) containing RAPTOR and mTORC2 containing RICTOR. We previously reported that in-vitro RNA interference (RNAi) targeting RAPTOR/mTORC1 protected human disc cells against apoptosis, senescence, and matrix catabolism through autophagy and Akt induction, however in-vivo effects remain undetermined. This is an in-vivo study to clarify effects of mTORC1 modulation by Raptor RNAi and temsirolimus, a selective mTORC1 inhibitor, in a rat tail temporary static compression model.

METHODS: Twelve-week-old male Sprague–Dawley rats were used (n=60). (1) To confirm in-vivo transfection, Alexa Fluor 555-labeled Raptor siRNA into discs was assessed by immunofluorescence at 7–56 d post-injection. Transfection efficiency of Raptor RNAi, expression of mTOR signaling components, and incidence of autophagy, apoptosis, and senescence were evaluated by Western blotting. (2) Rat tails were affixed between the 8^th and 10^th coccygeal (C) vertebrae with an Ilizarov-type apparatus with springs. Non-specific siRNA was injected into C9–C10 (loaded-control) and C12–C13 (unloaded-control) discs, while Raptor siRNA was into C8–C9 (loaded-experimental) and C11–C12 (unloaded-experimental) discs. Subsequently, 1.3-MPa axial force was applied for 24 h to induce degeneration. Radiographic, histomorphological, immunofluorescent, and immunohistochemistry for extracellular matrix degradation by ADAMTS4, MMP3 and aggrecan neoepitopes were performed to evaluate preventive effects of Raptor RNAi at 0–56 d post-compression. (3) To clarify treatment effects, Raptor and non-specific siRNAs were injected into already degenerated discs at 28 d after 24-h compression. Radiographic, histomorphological, and immunofluorescent assessments were performed at 0–56 d post-RNAi. (4) To clarify the difference between RNAi and an inhibitor drug, we performed the similar experiments using temsirolimus to (2) and (3).

RESULTS: (1) Immunofluorescence detected Raptor siRNA in discs even at 56 d. Western blotting showed prolonged RAPTOR suppression at 7 (54.3%, p=0.001) and 56 d (60.4%, p=0.002), autophagy induction with increased LC3-II and decreased p62/SQSTM, and inhibition of apoptotic PARP cleavage and senescent p16/INK4a (Figure 1). (2) Radiographic height found no significant differences between Raptor and no-specific siRNA-injected loaded discs until 28 d (p=0.082), but disc height was higher in Raptor siRNA-injected discs at 56 d (61.1% vs. 46.0%, p=0.002) (Figure 2). Reduced histomorphological degeneration (6.3 vs. 10.7 points, p=0.001) with a lower percentage of TUNEL-positive cells and reduced aggrecan degradation were observed in Raptor siRNA-injected loaded discs at 56 d (Figure 3). (3) Although radiographic height showed no significant difference, histology showed the trend toward reduced degeneration in Raptor siRNA-injected loaded discs at 56 d post-RNAi (8.5 vs. 9.5 points, p=0.082). (4) Western blotting showed RAPTOR reduction at 7 d (59.5%, p=0.003); however, there was no difference at 28 d. Radiography and histology showed no difference at 28–56 d post-injection (Figure 4).

DISCUSSION: In-vivo Raptor RNAi induced autophagy, inhibited apoptosis and senescence, and mitigated radiographic and histological degeneration while its effects on degenerated discs were limited. Although temsirolimus temporarily inhibited RAPTOR/mTORC1, it showed no marked changes at later time points. In summary, RNAi of RAPTOR/mTORC1 is a potential gene therapy for early-stage disc degeneration.