Structure of AAV-DJ, a Retargeted Gene Therapy Vector: Cryo-Electron Microscopy at 4.5 Å Resolution

AAV-DJ, a leading candidate vector for liver gene therapy, was created through random homologous recombination followed by directed evolution, selecting for in vivo liver tropism and resistance to in vitro immune neutralization. Here, the 4.5 Å resolution cryo-EM structure is determined for the engineered AAV vector, revealing structural features that illuminate its phenotype. The heparan sulfate receptor-binding site is little changed from AAV-2, and heparin-binding affinity is similar. A loop that is antigenic in other serotypes has a unique conformation in AAV-DJ that would conflict with the binding of an AAV-2 neutralizing monoclonal antibody. This is consistent with increased resistance to neutralization by human polyclonal sera, raising the possibility that changed tropism may be a secondary effect of altered immune interactions. The reconstruction exemplifies analysis of fine structural changes and the potential of cryo-EM, in favorable cases, to characterize mutant or ligand-bound complexes. [Display omitted] ► The 4.5 Åcryo-EM structure of AAV-DJ fully resolves the polypeptide backbone ► Liver tropism selected for in AAV-DJ has not changed the heparin binding site ► Changed conformation in an antigenic loop blocks binding of a neutralizing mAb ► Changed in vivo tropism may result from changed immune interactions AAV-DJ is an engineered adeno-associated virus (AAV) vector and a leading candidate for liver gene therapy. Here, Lerch et al. report a 4.5Å resolution cryo-EM structure of AAV-DJ, revealing structural features that underscore its unique phenotype. The reconstruction exemplifies potential of cryo-EM to analyze fine structural changes.