Oral Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

INTRODUCTION: The mammalian target of rapamycin (mTOR) is an intracellular serine/threonine kinase that has the two mTOR complexes 1 (mTORC1) containing the regulatory-associated protein of mTOR (RAPTOR) and 2 containing the rapamycin-insensitive companion of mTOR (RICTOR). The mTOR detects nutrient availability to coordinate cell growth and metabolism, and negatively regulate autophagy. We previously reported that in-vitro RNA interference (RNAi) targeting RAPTOR/mTORC1 could protect human disc cells against apoptosis, senescence, and matrix catabolism through autophagy and Akt induction, which however remains undetermined in vivo. Therefore, we designed an in-vivo study to clarify the effects of mTORC1 modulation by Raptor RNAi in a rat tail temporary static compression-induced disc degeneration model. METHODS: Twelve-week-old male Sprague–Dawley rats were used (n=60). (1) Contrast medium (1.0–5.0 µl) was injected into rat caudal discs and examined by computed tomography to determine the optimal volume. (2) Successful in-vivo intradiscal transfection of lipofection-mediated Alexa Fluor 555-labeled Raptor siRNA was assessed by immunofluorescence at 7–56 d post-injection. Transfection efficiency of Raptor RNAi, phosphorylation of mTOR signaling, and incidence of autophagy, apoptosis, and senescence were evaluated by Western blotting. (3) 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 24-h applied to induce disc disruption. Radiographic, histomorphological Safranin-O staining, and immunofluorescent assessments were performed to evaluate preventive effects of Raptor RNAi at 0–56 d post-compression. (4) To clarify treatment effects of Raptor RNAi, 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 0–28 d post-RNAi. RESULTS: (1) Contrast medium maximally filled the nucleus pulposus region up to 2.0 µl, which was used for following experiments (Figure 1). (2) Immunofluorescence detected intradiscal Raptor siRNA even at 56 d, surprisingly. Western blotting showed a prolonged reduction in RAPTOR protein expression at 7 (54.3%; p<0.01) and 56 d (60.4%; p<0.01) (n=6), autophagy induction with increased LC3-II and decreased p62/Sqstm1, and inhibition of apoptotic PARP cleavage and senescent p16/Ink4a expression (Figure 2). (3) Radiographic height analysis found no statistical differences between Raptor and control siRNA-injected loaded discs until 28 d, but disc height was significantly higher in 56-d Raptor siRNA-injected discs (p<0.01). Less degenerative histomorphological changes (p<0.01) with significantly lower percentages of apoptotic TUNEL-positive cells were observed in 56-d Raptor siRNA-injected loaded discs (p<0.01) (Figure 3). (4) Radiographic assessment showed no statistical differences between Raptor siRNA-injected and control discs. However, histomorphology presented a trend toward the reduced degeneration severity, e.g. preserved notochordal cells, in 28-d Raptor siRNA-injected discs (p=0.09). DISCUSSION: Successful in-vivo intradiscal lipofection-mediated Raptor RNAi demonstrated autophagy induction, apoptosis and senescence inhibition, and alleviated radiographic and histological degeneration. Its effects on degenerated discs were limited, however indicating the microenvironment improvement. Selective RAPTOR/mTORC1 RNAi is thus a potential gene therapeutic approach for disc degeneration, more effectively in early stages.