We describe the 2-year follow-up of an open-label trial (CT-AMT-011–01) of AAV1-LPL S447X gene therapy for lipoprotein lipase (LPL) deficiency (LPLD), an orphan disease associated with chylomicronemia, severe hypertriglyceridemia, metabolic complications and potentially life-threatening pancreatitis. The LPL S447X gene variant, in an adeno-associated viral vector of serotype 1 (alipogene tiparvovec), was administered to 14 adult LPLD patients with a prior history of pancreatitis. Primary objectives were to assess the long-term safety of alipogene tiparvovec and achieve a⩾ 40% reduction in fasting median plasma triglyceride (TG) at 3–12 weeks compared with baseline. Cohorts 1 (n= 2) and 2 (n= 4) received 3× 10 11 gc kg− 1, and cohort 3 (n= 8) received 1× 10 12 gc kg− 1. Cohorts 2 and 3 also received immunosuppressants from the time of alipogene tiparvovec administration and continued for 12 weeks. Alipogene tiparvovec was well tolerated, without emerging safety concerns for 2 years. Half of the patients demonstrated a⩾ 40% reduction in fasting TG between 3 and 12 weeks. TG subsequently returned to baseline, although sustained LPL S447X expression and long-term changes in TG-rich lipoprotein characteristics were noted independently of the effect on fasting plasma TG.