Key Inclusion Criteria: * Diagnosis of Hemophilia B with known severe or moderately severe FIX deficiency (≤2% normal circulating FIX activity) for which the subject is on continuous, stable and adequate FIX prophylaxis * Have acceptable laboratory values of a) Hemoglobin ≥11g/dL; b) Platelets ≥100,000 cells/µL; c) AST, ALT and alkaline phosphatase (ALP) ≤ upper limit of normal (ULN); d) Serum albumin \> lower limit of normal (LLN); e) Total bilirubin ≤1.5 x ULN (except if caused by Gilbert's disease); f) Serum creatinine ≤2.0mg/dL. * Level of neutralizing anti-AAV-S3 antibodies below the limit of the pre-established clinical cutoff using an in vitro transduction inhibition assay within the 4 weeks prior to FLT180a administration * Has demonstrated ability to accurately, independently and in a timely manner enter bleed diary data during the lead-in study, as judged by the investigator * At least 150 exposure days to FIX concentrates * At least 6 months of satisfactory controlled prospective baseline data for bleeding events and FIX consumption data from the FLT-01 lead-in study (ECLIPSE) Key Exclusion Criteria: * Any history of alcohol or drug dependence * Presence of neutralizing anti human FIX antibodies (inhibitor; determined by the Nijmegen modified Bethesda inhibitor assay) at the time of enrolment or a previous history of FIX inhibitor * Subjects at high risk of thromboembolic events * Evidence of advanced liver fibrosis * Prior treatment with a gene transfer medicinal product * Subjects with active hepatitis B or C * Serological evidence of HIV-1, not controlled with anti-viral therapy and as evidenced by cluster of differentiation 4 (CD4)+ counts ≤200 μL * Cytomegalovirus (CMV) immunoglobulin G positive subjects who are CMV polymerase chain reaction (PCR) positive at screening * Known coagulation disorder other than hemophilia B * High sensitivity (hs) troponin-T ≥14 pg/mL during screening * History of uncontrolled cardiac failure, unstable angina, or myocardial infarction or other acute cardiac conditions requiring clinical management in the past 6 months * Planned surgical procedure within the next 12 months requiring prophylactic FIX treatment * Known active severe infection (including documented coronavirus (COVID)-19 infection), or any other significant concurrent, uncontrolled medical condition