Deimmunized AAV evades pre-existing human neutralizing antibodies and reduces antibody formation in vivo

Gene therapies have been limited by the presence of pre-existing neutralizing antibodies (NAbs) in human populations, excluding many patients from the therapeutic benefits. Here, we utilized PhIP-seq to comprehensively map linear IgG antibody-targeted sites for 14 adeno-associated virus (AAV) serotypes against pooled sera from 526 human donors and individual sera from 24 human donors, defining the first epitope map across entire AAV capsids. Our data reveals two patterns of human antibody recognition: binding sites within the conserved capsid N-terminal region and binding sites within the variable C-terminal region. Through rational engineering by substituting key amino acids in the N-terminal region, we developed AAV capsid variants that evade pre-existing human NAbs while maintaining bioproduction yield and transduction efficiency. These AAV variants exhibit up to ten-fold increase in resistance to neutralization to a NAb titer of 1:3.16, are directly portable to multiple serotypes used in the clinic, and potentially expand AAV therapies to previously ineligible patients. To further minimize the induction of primary humoral immune response in vivo, we incorporated a small immunomodulatory domain onto the AAV capsid, which simultaneously disrupts an additional epitope and reduces the generation of new anti-AAV antibodies in treated mice. The combination of these two immune-evading strategies generated deimmunized AAV vectors (AAV-DI) that overcome the pressing challenges of both pre-existing NAbs and primary antibody formation, offering a solution for safe and efficacious gene therapy applications.