Inhibiting periapical lesions through AAV-RNAi silencing of cathepsin K

Abstract

Dental caries, one of the most prevalent infectious diseases worldwide, affects approximately 80% of children and the majority of adults. Dental caries may result in endodontic disease, leading to dental pulp necrosis, periapical inflammation and bone resorption, severe pain, and tooth loss. Periapical inflammation may also increase inflammation in other parts of the body. Although many studies have attempted to develop therapies for this disease, there is still an urgent need for effective treatments. In this study, we applied a novel gene therapeutic approach using recombinant adeno-associated virus (AAV)-mediated RNAi knockdown of Cathepsin K (Ctsk) gene expression, to target osteoclasts and periapical bone resorption in a mouse model. We found that AAV-sh-Cathepsin K (AAV-sh-Ctsk) impaired osteoclast function in vivo and furthermore reduced bacterial infection-stimulated bone resorption by 88%. Reduced periapical lesion size was accompanied by decreases in mononuclear leukocyte infiltration and inflammatory cytokine expression. Our study shows that AAV-RNAi silencing of Cathepsin K in periapical tissues can significantly reduce endodontic disease development, bone destruction, and inflammation in the periapical lesion. This is the first demonstration that AAV-mediated RNAi knockdown gene therapy may significantly reduce the severity of endodontic disease.

Figure 1.

AAV-sh-Cathepsin K efficiently knocked down expression of Cathepsin K and impaired osteoclast-mediated bone resorption in vitro. (A) Immunofluorescent photomicrograph of AAV-sh-luc-YFP and AAV-sh-Cathepsin K treatment groups 7 days after transduction. The experiments were performed in triplicate. (B) Western blot of Cathepsin K expression in murine bone marrow cells stimulated with M-CSF/RANKL for 3 days to induce differentiation of osteoclasts, that were then transduced with no vector (mock), AAV-sh-luc-YFP (control vector), or AAV-sh-Cathepsin K. (C) Quantification of Western blot analysis demonstrates that the AAV-sh-Cathepsin K treatment group significantly reduced expression of Cathepsin K as compared with the AAV-sh-luc-YFP treatment group. (D) Untransduced osteoclasts (Mock) and osteoclasts transduced with AAV-sh-luc-YFP (control) or AAV-sh-luc-Cathepsin K stained with acridine orange to show extracellular acidification (top panel). Resorption pits on bone slices were visualized by WGA (middle panel) and scanning electron microscopy (SEM) (bottom panel). The experiment was performed in duplicate on 4 independent occasions. (E) Quantification of resorption pits on bone slices was significantly lower in the AAV-sh-Cathepsin K treatment group compared with the Mock and AAV-sh-luc-YFP groups. All assays were performed in triplicate; a representative photomicrograph from each assay is shown. N.S., Not Significant. **p < 0.01.

Figure 2.

AAV effectively transduced endodontic tissue in vivo and protected mice from bone loss due to endodontic infection. (A) Fluorescent micrographs of eGFP expression by AAV-infected cells in uninfected (Normal) mice and infected mice treated with AAV-sh-Cathepsin K. The experiments were performed in triplicate. (B) Radiographic imaging of the crown and distal root of the mandibular first molar in uninfected mice (Normal), infected mice treated with AAV-sh-luc-YFP, and infected mice treated with AAV-sh-luc-Cathepsin K. Severe periapical bone loss is visible in the AAV-sh-luc-YFP image compared with the minimal bone loss in the AAV-sh-Cathepsin K image (n = 21). (C) µCT analysis of the mandibular first molar in uninfected mice (Normal), infected mice treated with AAV-sh-luc-YFP, and infected mice treated with AAV-sh-Cathepsin K. Severe periapical bone loss is visible in the AAV-sh-luc-YFP image compared with the minimal bone loss in the AAV-sh-Cathepsin K image (n = 21). (D) Quantification of Bone Volume (BV)/Total Volume (TV) from uninfected (Normal) mice, infected mice treated with AAV-sh-luc-YFP, and infected mice treated with AAV-sh-Cathepsin K. N.S., Not Significant. **p < 0.01 (n = 21).

Figure 3.

AAV-sh-Cathepsin K significantly decreased cell infiltration and bone resorption in periapical tissues. (A) Hematoxylin and eosin (H&E) staining of sections from untreated, uninfected mice (Normal), infected mice treated with AAV-sh-luc-YFP (Control), and infected mice treated with AAV-sh-Cathepsin K. Bone resorption and mononuclear cell infiltration was significantly decreased in the AAV-sh-Cathepsin K treatment group compared with the control AAV-sh-luc-YFP group (n = 21). (B) Immunofluorescence staining of periapical lesion sections indicates that uninfected mice (Normal) and infected mice treated with AAV-sh-Cathepsin K have fewer CD3-positive (green) T-cells compared with infected mice treated with AAV-sh-luc-YFP. Cell nuclei were labeled with DAPI DNA stain (blue). The experiments were performed in triplicate.

Figure 4.

AAV-sh-Cathepsin K reduced the expression of inflammatory mediators in periapical tissues. (A) qRT-PCR results for osteoclast differentiation marker (CD115 Acp5) and inflammatory mediator (IL-1α, IL-1β, IL-17α) gene expression, in the periapical tissues of normal uninfected mice and infected mice treated with AAV-sh-luc-YFP or with AAV-sh-Cathepsin K. Expression levels were normalized to the housekeeping gene hypoxanthine-guanine phosphoribosyl transferase (Hprt) (n = 21). (B) Protein levels of IL-1α, IL-6, IL-12α, IL-17α, and TNF-α in periapical lesions as detected by ELISA. N.S., Not Significant. *p < 0.05; **p < 0.01; ***p < 0.005 (n = 21).