Wenjun Li Junjiang Sun Susi Feng Ariana La Rosa Panli Zhang Eveline Wu Richard Loeser Chengwen Li ^*

• University of North Carolina at Chapel Hill, Chapel Hill, United States

Rheumatoid arthritis (RA) primarily affects the joints but can also affect multiple organs and profoundly impacts patients' ability to carry out daily activities, mental health, and life expectancy.Current treatments for RA are limited in terms of duration, efficacy, and adverse effects. PD-L1 is a checkpoint protein that plays important roles in immune regulation and has been implicated in the initiation and progression of multiple autoimmune diseases. In a previous study, we demonstrated that intra-articular injection with adeno-associated virus (AAV) vectors encoding wild type PD-L1 improved local inflammation in the joint in the collagen-induced arthritis (CIA) mouse model of RA. To further improve efficacy, we explored AAV-mediated delivery of the soluble PD-L1 (sPD-L1) to CIA mice. After intra-articular injection of AAV6 vectors expressing the optimal isoform of sPD-L1 (shPD-L1), more potency was observed when compared to wild type PD-L1, with a lower dose of AAV6/shPD-L1 needed for arthritis improvement. To study the therapeutic effect of systemic expression of sPD-L1, we administered AAV8/shPD-L1 gene therapy in CIA mice via retro-orbital injection and found significant improvements in joint inflammation and paw swelling, exhibiting similar phenotypes to that in naïve mice. The levels of total immunoglobulin and anti-collagen specific antibodies were lower in AAV8/shPD-L1 treated CIA mice than those in controls. The levels of pro-inflammatory cytokines in blood were also significantly decreased in shPD-L1 treated mice. Additionally, T cell apoptosis rates in the spleen showed a 2-fold increase in treated mice. Finally, we investigated the therapeutic effect of AAV/shPD-L1 via intramuscular injection. After injection of AAV6/shPD-L1, decreased paw swelling, reduced joint inflammation, and lower levels of pro-inflammatory cytokines in blood were achieved. The therapeutic effect of shPD-L1 was dose dependent via intramuscular treatment with AAV vectors. In conclusion, the findings in this study suggest that intra-articular injection of AAV vectors encoding sPD-L1 results in greater therapeutic benefit on arthritis, and systemic AAV/sPD-L1 is able to block the development of inflammatory arthritis with inhibition of the systemic immune response, underlining the potential of gene therapy with systemic delivery of shPD-L1 via AAV vectors in RA.