Structurally Mapping Antigenic Epitopes of Adeno-Associated Virus 9: Development of Antibody Escape Variants.

Adeno-associated viruses (AAV) serve as vectors for therapeutic gene delivery. AAV9 vectors have been FDA approved, as Zolgensma®, for the treatment of spinal muscular atrophy and is being evaluated in clinical trials for the treatment of neurotropic and musculotropic diseases. A major hurdle for AAV-mediated gene delivery is the presence of pre-existing neutralizing antibodies in 40 to 80% of the general population. These pre-existing antibodies can reduce therapeutic efficacy through viral neutralization, and the size of the patient cohort eligible for treatment. In this study, cryo-electron microscopy and image reconstruction was used to define the epitopes of five anti-AAV9 monoclonal antibodies (MAbs); ADK9, HL2368, HL2370, HL2372, and HL2374, on the capsid surface. Three of these, ADK9, HL2370, and HL2374, bound on or near the icosahedral 3-fold axes, HL2368 to the 2/5-fold wall, and HL2372 to the region surrounding the 5-fold axes. Pseudo-atomic modeling enabled the mapping and identification of antibody contact amino acids on the capsid, including S454 and P659. These epitopes overlap with previously defined parvovirus antigenic sites. Capsid amino acids critical for the interactions were confirmed by mutagenesis followed by biochemical assays testing recombinant AAV9 (rAAV9) variants capable of escaping recognition and neutralization by the parental MAbs. These variants retained parental tropism and had similar or improved transduction efficiency compared to AAV9. These engineered rAAV9 variants could expand the patient cohort eligible for AAV9-mediated gene delivery by avoiding pre-existing circulating neutralizing antibodies. IMPORTANCE The use of recombinant AAVs (rAAVs) as delivery vectors for therapeutic genes is becoming increasingly popular, especially following the FDA approval of Luxturna® and Zolgensma®, based on serotypes AAV2 and AAV9, respectively. However, high titer anti-AAV neutralizing antibodies in the general population, exempts patients from treatment. The goal of this study is to circumvent this issue by creating AAV variant vectors not recognized by pre-existing neutralizing antibodies. The mapping of the antigenic epitopes of five different monoclonal antibodies (MAbs) on AAV9, to recapitulate a polyclonal response, enabled the rational design of escape variants with minimal disruption to cell tropism and gene expression. This study, which included four newly developed and now commercially available MAbs, provides a platform for the engineering of rAAV9 vectors that can be used to deliver genes to patients with pre-exiting AAV antibodies.