Structural Study of Aavrh.10 Receptor and Antibody Interactions

Abstract

Recombinant Adeno-associated virus (rAAV) vectors are one of the leading tools for the delivery of therapeutic genes in human gene therapy applications. For a successful transfer of their payload, the AAV vectors have to circumvent potential pre-existing neutralizing host antibodies and bind to the receptor of the target cells. Both these aspects have not been structurally analyzed for AAVrh.10. Here, cryo-electron microscopy (cryo-EM) and three-dimensional image reconstruction were used to map the binding site of sulfated N -Acetyllactosamine (LacNAc, previously shown to bind AAVrh.10) and a series of four monoclonal antibodies (MAbs). LacNAc was found to bind to a pocket located on the side of the 3-fold capsid protrusion, that is mostly conserved to AAV9 and equivalent to its galactose-binding site. As a result, AAVrh.10 was also shown to be able to bind to cell surface glycans with terminal galactose. For the antigenic characterization, it was observed that several anti-AAV8 MAbs cross-react with AAVrh.10. The binding sites of these antibodies were mapped to the 3-fold capsid protrusions. Based on these observations, the AAVrh.10 capsid surface was engineered to create variant capsids that escape these antibodies while maintaining infectivity. Importance Gene therapy vectors based on Adeno-associated virus rhesus isolate 10 (AAVrh.10) have been used in several clinical trials to treat monogenetic diseases. However, compared to other AAV serotypes little is known about receptor binding and antigenicity of the AAVrh.10 capsid. Particularly, pre-existing neutralizing antibodies against capsids are an important challenge that can hamper treatment efficiency. This study addresses both topics and identifies critical regions of the AAVrh.10 capsid for receptor and antibody binding. The insights gained were utilized to generate AAVrh.10 variants capable of evading known neutralizing antibodies. The findings of this study could further aid the utilization of AAVrh.10 vectors in clinical trials and help the approval of the subsequent biologics.

Natural Adeno-Associated Virus Serotypes and Engineered Adeno-Associated Virus Capsid Variants: Tropism Differences and Mechanistic Insights

“…Five amino acid residues (N470, D271, N272, Y446, and W503) that form a pocket at the base of the three-fold axis of symmetry are key to AAV9–galactose binding and tropism [ 115 , 116 ]. Finally, AAVrh.10 has been shown to bind to sulfated N -acetyllactosamine (LacNAc), a glycan with terminal galactose, via a pocket located on the three-fold capsid protrusions [ 118 , 132 ].…”

Structural characterization of antibody-responses from Zolgensma treatment provides the blueprint for the engineering of an AAV capsid suitable for redosing

“…These engineered capsids will not prevent future immune responses upon vector administration but will expand the patient cohort treatable with AAV vectors that previously were precluded due to their pre-existing antibodies. In the past 12 years, the antibody responses against the AAV capsids were primarily simulated with mouse mAbs 13,20,33,34 . However, our recent study characterizing 21 anti-AAV9 capsid mAbs from human patients indicated that human and mouse antibodies may exhibit differential binding characteristics to the AAV capsids 24 .…”

“…The visualization of asymmetric features in otherwise icosahedral structures, such as the Fabs binding near or at the capsids' icosahedral symmetry axes, using traditional cryo-EM reconstruction procedures has been challenging as their information is lost due to averaging of the particle. However, standard asymmetric reconstruction is unfeasible here as the Fab binding modes are independent at each axis resulting in a vast number of structural classes (~3 20 possible permutations for the 3-fold binder) 26 . Thus, the localized reconstruction method was used in combination with symmetry relaxation to structurally characterize the 2-, 3-, or 5-fold region to reconstruct the Fabs not conforming to the icosahedral symmetry of the capsid [25][26][27] .…”

“…The identified interacting residues form the basis for AAV capsid engineering efforts by rational design or by directed evolution 22 . Modifications of capsid aa have been shown to result in capsid variants capable of escaping the characterized antibodies in vitro and in vivo [19][20][21]23 . To date, this structural mapping approach has relied primarily on mouse monoclonal antibodies (mAbs) to simulate the human antibody response against the AAV capsids.…”

“…Initially, the resolutions of AAV capsid-antibody cryo-EM maps were low (∼5–20 Å) 8,19,20 . However, recent advancements of the cryo-EM technology have enabled the analysis of the interaction between the antibody and the capsid surface at atomic resolution 21 .…”