Abstract: Objective To assess the toxicity of gene therapy drug AAV5-LPLS447X (GC304) in mice. Methods ①Acute toxicity study:40 C57BL/6N mice were randomly divided into 2 groups: the vehicle group and the test substance group. After a single tail vein injection, clinical symptoms, body weight and food intake were measured for 15 consecutive days, and necropsy was performed on the 15^th day.② Long-term toxicity study: 440 mice were randomly divided into 4 groups: the vehicle group, low-dose group(1×10¹³ vg·kg^-1), middle-dose group(5×10¹³ vg·kg^-1) and high-dose group(1.5×10¹⁴ vg·kg^-1). After a single tail vein injection, such indicators of toxicity as body weight, food intake, hematology, serum biochemistry, immunotoxicity and immunogenicity were measured. The mice were dissected at 4 weeks and 6 months after administration before being examined histopathologically. The distribution and expression of GC304 in various organs of the animals were detected by qPCR at 2 weeks, 4 weeks, 3 months, and 6 months. Results ①In the acute toxicity study: the clinical symptoms, body weight and food intake of the animals were normal, and no gross pathological changes related to GC304 were found.② In the long-term toxicity study: no obvious abnormalities related to GC304 were found in clinical symptoms, body weight, food intake, cytokines or T lymphocyte subsets. GC304 reduced plasma triglycerides and the albumin/globulin ratio associated with anti-drug antibodies, and induced the production of anti-AAV5-binding antibodies and neutralizing antibodies, which persisted up to 6 months after administration. After administration of GC304, no anti-LPL antibodies were detected. Histopathological results showed that drug-related inflammatory cell infiltration occurred at the injection site 4 weeks after administration in mice, and recovery was seen 6 months after administration. Drug-related changes were an active germinal center in the spleen with increased predisposition macrophages, and an active germinal center in the inguinal lymph nodes with increased predisposed macrophages. The results of qPCR showed that GC304 was widely distributed and expressed in the peripheral blood and organs of mice, especially in the liver. Conclusion After a single intravenous administration in C57BL/6N mice, GC304 is mainly expressed and distributed in the liver, and the animals are well tolerated without obvious toxic reactions, which provides reference for clinical trials of the drug.

Key words: gene therapy, GC304, toxicity study, mice