Summary of immunological challenges and successes of liver-directed AAV gene therapy for hemophilia in humans. This is a representation of hepatocytes and LSEC-lined blood vessel within the liver. After intravenous delivery of an AAV vector, viral particles enter the hepatocyte, escape the endosome, and deliver their genetic payload to the nucleus. Preclinical animal models show that FVIII or FIX expression in the liver induces immunological tolerance through conversion of effector CD4⁺ T cells into Treg. Treg suppress activation of both B- and T-cell responses directed against FVIII or FIX. Preexisting NAB to the AAV capsid can block hepatocyte gene delivery and prevent therapeutic correction of the bleeding diathesis. On successful gene delivery to hepatocytes, primary or recall immune responses may lead to activation of capsid-specific cytotoxic CD8⁺ T cells and targeted elimination of hepatocytes presenting AAV capsid epitopes on MHC class I. Preclinical animal models of severe hemophilia show that suboptimal gene delivery may fail to induce tolerance to the transgene product because a threshold level of FVIII or FIX expression is needed for successful activation of Treg. In the absence of Treg, effector CD4⁺ T cells may become activated on recognition of FVIII or FIX epitopes presented on MHC class II, which is primarily expressed on professional antigen presenting cells. The resulting T help promotes B-cell maturation, leading to the production of high-affinity class-switched antibodies and terminal differentiation into antibody secreting plasma cells.