Extended Data Fig. 1: K9 LNP enables therapeutic genome editing in a DMM-induced osteoarthritis model.

a, Schematic of the dosing schedule in the destabilization of the medial meniscus (DMM) surgical model. Mice received either a single dose or two weekly intra-articular injections of K9 LNPs (total RNA dose: 5 μg/joint; mCas9:sgMMP-13 = 3:1), starting two months after DMM surgery. Tissues were collected for analysis four months after the final dose for analysis. b, Editing efficiency at the MMP13 locus in knee joints and liver, assessed by sequencing (n = 3 biologically independent mice per group; mean ± SD; P values were calculated by ordinary one-way ANOVA). c-d, Quantification of MMP-13 expression in the knee joint by (c) ELISA (n = 4 biologically independent mice per group; mean ± SD; P values were calculated by ordinary one-way ANOVA). and (d) RT-qPCR (n = 3 biologically independent mice per group; each sample was measured in duplicate; mean ± SD; P values were calculated by ordinary one-way ANOVA). e-f, Mechanism of the MMPSense probe and representative in vivo images showing total MMP activity with varying LNP doses (e), and the quantification result (f). Blue circles represent the area of quantified fluorescence. (n = 3 biologically independent mice per group; mean ± SD; P values were calculated by ordinary one-way ANOVA). g-h, Detection of MMP-13 protein in cartilage by (g) fluorescence imaging and (h) corresponding quantification. (n = 6 biologically independent mice per group; mean ± SD; P values were calculated by ordinary one-way ANOVA). Scale bar, 100 μm. i-j, (i) SHG imaging of articular cartilage and (j) quantification of cartilage thickness and organization. (n = 3 biologically independent mice per group; mean ± SD; P values were calculated by ordinary one-way ANOVA). k, Representative histological images of knee joints from sham control, untreated, and K9-treated DMM mice stained with Safranin O and TRAP; magnified insets highlight cartilage and osteoclast features. Healthy cartilage appears deep red due to Safranin O staining of proteoglycans, while TRAP staining highlights osteoclasts in red. Scale bar, 100 μm. l-m, Osteoarthritis severity assessed using OARSI scoring (l) and quantification of osteoclast numbers from TRAP-stained sections (m) (n = 5 biologically independent mice per group; mean ± SD; P values were calculated by ordinary one-way ANOVA). n, Representative microCT 3D reconstructions of knee joints from sham, untreated, and K9-treated DMM mice, shown in frontal (left) and posterior (right) views with magnified insets below. Red arrows highlight areas of disrupted joint architecture, ectopic ossification, and osteophyte formation. o, Quantification of trabecular bone volume fraction (BV/TV, %) from microCT scans, indicating changes in subchondral bone density across treatment groups. (n = 3 biologically independent mice per group; mean ± SD; P values were calculated by ordinary one-way ANOVA).

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