Exploration of CRISPR/Cas9-based gene editing as therapy for osteoarthritis

Abstract

Objectives: Osteoarthritis (OA) is a painful and debilitating disease and it is associated with aberrant upregulation of multiple factors, including matrix metalloproteinase 13 (MMP13), interleukin-1β (IL-1β) and nerve growth factor (NGF). In this study, we aimed to use the CRISPR/Cas9 technology, a highly efficient gene-editing tool, to study whether the ablation of OA-associated genes has OA-modifying effects.

Methods: We performed intra-articular injection of adeno-associated virus, which expressed CRISPR/Cas9 components to target each of the genes encoding MMP13, IL-1β and NGF, in a surgically induced OA mouse model. We also tested triple ablations of NGF, MMP13 and IL-1β.

Results: Loss-of-function of NGF palliates pain but worsens joint damage in the surgically induced OA model. Ablation of MMP13 or IL-1β reduces the expression of cartilage-degrading enzymes and attenuates structural deterioration. Targeting both MMP13 and IL-1β significantly mitigates the adverse effects of NGF blockade on the joints.

Conclusions: CRISPR-mediated ablation of NGF alleviates OA pain, and deletion of MMP13-1β or IL-1β attenuates structural damage in a post-traumatic OA model. Multiplex ablations of NGF, MMP13 and IL-1β provide benefits on both pain management and joint structure maintenance. Our results suggest that CRISPR-based gene editing is useful for the identification of promising drug targets and the development of feasible therapeutic strategies for OA treatment.

Keywords: knee osteoarthritis; osteoarthritis; treatment.

Figure 1

OA-modifying effects by CRISPR-mediated ablation of NGF, IL-1β and MMP13. (A) Results of von Frey test on the mice receiving the PMM surgery and administration of AAV that expresses control or NGF-targeting sgRNAs. n=9. Unpaired Student’s t-test. (B) Representative histology images of osteoarthritic knee joints, which were collected 3 months after injections of control or NGF-targeting AAV. Yellow arrowheads, loss of AC; red arrowheads, osteochondrophytes; black arrowheads, synovial hyperplasia; green arrowheads, subchondral sclerosis. n=9. Scale bar, 200 μm. (C) OARSI scoring of knee joint AC destruction in the mice receiving the PMM surgery and control or NGF-targeting AAV. Both medial femoral condyle and medial tibial plateau were analysed on three-level sections of the joints and summed OARSI scores for the entire joint were presented. Unpaired Student’s t-test. n=9. (D) Representative μCT images of osteoarthritic knee joints, which were collected 3 months after injections of control or NGF-targeting AAV. Red arrowheads, osteophytes. n=9. Scale bar, 1 mm. (E,F) Representative histological and μCT results of osteoarthritic knee joints, which were collected three months after injections of IL-1β-targeting (E) or MMP13-targeting AAV (F). (G,H) Results of von Frey tests on the mice receiving the PMM surgery and administration of AAV that expresses IL-1β- (G) or MMP13-targeting AAV (H). Unpaired Student’s t-test, n=9. AAV, adeno-associated virus; AC, articular cartilage; IL-1β, interleukin-1β; MMP13, matrix metalloproteinase 13; NGF, nerve growth factor; OA, osteoarthritis; PMM, partial meniscectomy.

Figure 2

CRISPR-mediated gene editing attenuated OA-associated downstream signalling. (A–C) Administration of gene-ablating AAV reduced the expression of the individual targets in osteoarthritic knee joints, such as NGF (A), IL-1β (B) and MMP13 (C). (D–F) NGF-targeting AAV downregulated the expression of βIII tubulin (D), MMP13 (E) and Adamts5 (F) and IL-1β-targeting AAV reduced the expression of MMP13 (E) and Adamts5 (F) in osteoarthritic knee joints. Arrowheads, IHC-positive cells. n=5. Scale bar, 50 μm. Unpaired Student’s t-test (A–D) or one-way ANOVA followed by the Tukey-Kramer test (E,F). **p<0.01, ***p<0.001, ****p<0.0001. AAV, adeno-associated virus; ANOVA, analysis of variance; IHC, immunohistochemistry; IL-1β, interleukin-1β; MMP13, matrix metalloproteinase 13; NGF, nerve growth factor; OA, osteoarthritis.

Figure 3

Concomitant loss-of-function of NGF, IL-1β and MMP13 relieves OA pain and mitigates OA progression. (A,B) Representative histology (A) and μCT (B) images of osteoarthritic knee joints, which were collected 6 months after injections of control, NGF-targeting or triple (NGF, IL-1β and MMP13)-targeting AAVs. Yellow arrowheads, loss of articular cartilage; red arrowheads, osteochondrophytes; black arrowheads, synovial hyperplasia; green arrowheads, subchondral sclerosis. n=9. Scale bar for histology, 200 μm. Scale bar for μCT, 1 mm. (C) Results of von Frey tests on the mice receiving the PMM surgery and control or triple-targeting AAV. n=9. Unpaired Student’s t-test. (D) OARSI scoring of AC destruction in osteoarthritic knee joints of the mice receiving control, NGF-targeting or tri-targeting AAVs. n=9. One-way ANOVA followed by the Tukey-Kramer test. (E–I) Simultaneous deletion of NGF, IL-1β and MMP13 attenuated OA-associated matrix proteases including MMP13 (F) and Adamts5 (G) and neural genes such as NGF (H) and βIII Tubulin (I), which were quantified and summarised (E). Scale bar, 50 μm. n=5, unpaired Student’s t-test. *p<0.05, ***p<0.001, ****p<0.0001. AAV, adeno-associated virus; ANOVA, analysis of variance; IL-1β, interleukin-1β; MMP13, matrix metalloproteinase 13; NGF, nerve growth factor; OA, osteoarthritis; PMM, partial meniscectomy.