Dihydroartemisinin Decreases Pre-existing Neutralizing Antibodies against Adeno-Associated Virus in Challenged Mice

Chen Ling^1,2*Jingjing Fang²Jinyan Xie¹Gao Xuxia¹Enze Cui²Yun He¹Ming Yang¹Zhengjun Zhou³Shaolai Zhou³Binbin Cheng²Chang-quan Ling^2*

• ¹Fudan University, Shanghai, China
• ²Department of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai, China
• ³Department of Liver Surgery and Transplantation, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China

The high prevalence of pre-existing neutralizing antibodies (NAb) against adeno-associated virus (AAV) presents a significant barrier to in vivo applications.Conventional immunosuppressive (IS) agents for overcoming anti-AAV NAb, such as corticosteroids, are often limited by their associated toxicity and side effects. In pursuit of a safer and more effective IS agent, we investigated dihydroartemisinin (DHA), a synthetic derivative of artemisinin, drawing on insights from traditional Chinese medicine (TCM) studies in our laboratory. Here, DHA was administered either at 30-or 210-day post-injection (PI) of rAAVDJ vectors in vivo. Our results demonstrated a notable reduction in anti-AAV NAb after DHA administration, without affecting transgene expression or vector genome biodistribution.Mechanistically, DHA treatment led to decreases in CD20 + B cells, splenic germinal center B cells, and plasma cells, accompanied by changes in splenic gene expression.Histological analysis and serum biochemical indicators confirmed that 125mg/kg per day DHA exhibited no hepatotoxicity. In vitro experiments showed that neither DHA nor its metabolites in blood inhibited infection of various rAAV serotypes in HEK293 cells. These studies offer proof-of-concept data for the use of TCM-derived drugs in addressing major challenges in gene therapy.