OBJECTIVE: Joint pain is the major clinical symptom of arthritis that affects millions of people. Controlling the excitability of knee-innervating dorsal root ganglion (DRG) neurons (knee neurons) could potentially provide pain relief. We undertook this study to evaluate whether the newly engineered adeno-associated virus (AAV) serotype, AAV-PHP.S, can deliver functional artificial receptors to control knee neuron excitability following intraarticular knee injection. METHODS: The AAV-PHP.S virus, packaged with dTomato fluorescent protein and either excitatory (Gq ) or inhibitory (Gi ) designer receptors exclusively activated by designer drugs (DREADDs), was injected into the knee joints of adult mice. Labeling of DRG neurons with AAV-PHP.S from the knee was evaluated using immunohistochemistry. The functionality of Gq - and Gi -DREADDs was evaluated using whole-cell patch clamp electrophysiology on acutely cultured DRG neurons. Pain behavior in mice was assessed using a digging assay, dynamic weight bearing, and rotarod performance, before and after intraperitoneal administration of the DREADD activator, Compound 21. RESULTS: We showed that AAV-PHP.S can deliver functional genes into ~7% of lumbar DRG neurons when injected into the knee joint in a similar manner to the well-established retrograde tracer, fast blue. Short-term activation of AAV-PHP.S-delivered Gq -DREADD increased excitability of knee neurons in vitro (P = 0.02 by unpaired t-test), without inducing overt pain in mice when activated in vivo. By contrast, in vivo Gi -DREADD activation alleviated digging deficits induced by Freund's complete adjuvant-mediated knee inflammation (P = 0.0002 by repeated-measures analysis of variance [ANOVA] followed by Holm-Sidak multiple comparisons test). A concomitant decrease in knee neuron excitability was observed in vitro (P = 0.005 by ANOVA followed by Holm-Sidak multiple comparisons test). CONCLUSION: We describe an AAV-mediated chemogenetic approach to specifically control joint pain, which may be utilized in translational arthritic pain research.