Objective: Therapeutic potential of selective aggrecanase inhibition in osteoarthritis (OA) was previously demonstrated using a variant of endogenous tissue inhibitor of metalloproteinase-3 (TIMP-3); however, this relied on transgenic mice overexpressing TIMP-3. Here, we develop a translational approach for harnessing the aggrecanase-selective inhibitory activity of TIMP-3 using the latency associated peptide (LAP) technology. Methods: We successfully produced and purified recombinant LAP-TIMP-3 fusion proteins and determined the pharmacokinetics of these proteins in vivo following systemic injection. Surgical and non-surgical mouse models of OA were used to establish the therapeutic potential of these proteins in reducing ag- grecanase activity in mouse joints affected by OA. Results: The presence of the LAP conferred favourable TIMP-3 pharmacokinetics, with effective delivery of LAP-TIMP-3 to knee joints after systemic injection. We find that LAP-TIMP-3 also effectively reduced ag- grecanase activity in OA-affected joints, both in spontaneously-occurring OA and in the destabilisation of the medial meniscus (DMM) model of OA. We also found that reductions in aggrecanase activity in articular cartilage correlated with improved disease scores, but only in earlier stages of disease. Conclusions: This study describes the potential of LAP-TIMP-3 as a therapeutic agent in OA, showing de- livery to the cartilage of joints affected by OA after systemic administration and lower levels of the neoe- pitope of aggrecan in articular cartilage in mild disease (mean difference versus vehicle control for LAP- TIMP-3: 535 [95% CI: 336, 733] and for LAP-mutTIMP-3: 522 [95% CI: 323, 720] arbitrary units). These first in vivo data will inform further explorations into dose optimization and timing.