Cellular immune responses to adeno-associated viral (AAV) vectors used for gene therapy have been linked to attenuated transgene expression and loss of efficacy. The impact of such cellular immune responses on the clinical efficacy of alipogene tiparvovec (Glybera; AAV1-LPL(S447X); uniQure), a gene therapy consisting of intramuscular administration of a recombinant AAV1 mediating muscle-directed expression of lipoprotein lipase (LPL), was investigated. Five subjects with LPL deficiency (LPLD) were administered intramuscularly with a dose of 1 × 10(12) gc/kg alipogene tiparvovec. All subjects were treated with immune suppression starting shortly before administration of alipogene tiparvovec and maintained until 12 weeks after administration. Systemic antibody and T cell responses against AAV1 and LPL(S447X), as well as local cellular immune responses in the injected muscle, were investigated in five LPLD subjects. Long-term transgene expression was demonstrated despite a transient systemic cellular response and a stable humoral immune response against the AAV1 capsid protein. Cellular infiltrates were found in four of the five subjects but were not associated with adverse clinical events or elevation of inflammation markers. Consistent herewith, CD8+ T cells in the infiltrates lacked cytotoxic potential. Furthermore, FoxP3+/CD4+ T cells were found in the infiltrates, suggesting that multiple mechanisms contribute to local tolerance. Systemic and local immune responses induced by intramuscular injection of alipogene tiparvovec did not appear to have an impact on safety and did not prevent LPL transgene expression. These findings support the use of alipogene tiparvovec in individuals with LPLD and indicate that muscle-directed AAV-based gene therapy remains a promising approach for the treatment of human diseases.