Recent clinical trials have demonstrated the potential of adeno-associated virus 
(AAV)-based vectors for treating rare diseases. However, significant barriers 
remain for the translation of these vectors into widely available therapies. In 
particular, exposure to the AAV capsid can generate an immune response of 
neutralizing antibodies. One approach to overcome this response is to map the 
AAV-specific neutralizing epitopes and rationally design an AAV capsid able to 
evade neutralization. To accomplish this, we isolated a monoclonal antibody 
against AAV9 following immunization of BALB/c mice and hybridoma screening. This 
antibody, PAV9.1, is specific for intact AAV9 capsids and has a high 
neutralizing titer of >1:160,000. We used cryo-electron microscopy to 
reconstruct PAV9.1 in complex with AAV9. We then mapped its epitope to the 
3-fold axis of symmetry on the capsid, specifically to residues 496-NNN-498 and 
588-QAQAQT-592. Capsid mutagenesis demonstrated that even a single amino acid 
substitution within this epitope markedly reduced binding and neutralization by 
PAV9.1. In addition, in vivo studies showed that mutations in the PAV9.1 epitope 
conferred a "liver-detargeting" phenotype to the mutant vectors, unlike AAV9, 
indicating that the residues involved in PAV9.1 interactions are also 
responsible for AAV9 tropism. However, we observed minimal changes in binding 
and neutralizing titer when we tested these mutant vectors for evasion of 
polyclonal sera from mice, macaques, or humans previously exposed to AAV. Taken 
together, these studies demonstrate the complexity of incorporating mapped 
neutralizing epitopes and previously identified functional motifs into the 
design of novel capsids able to evade immune response.IMPORTANCE Gene therapy 
utilizing viral vectors has experienced recent success, culminating in U.S. Food 
and Drug Administration approval of the first adeno-associated virus vector gene 
therapy product in the United States: Luxturna for inherited retinal dystrophy. 
However, application of this approach to other tissues faces significant 
barriers. One challenge is the immune response to viral infection or vector 
administration, precluding patients from receiving an initial or readministered 
dose of vector, respectively. Here, we mapped the epitope of a novel 
neutralizing antibody generated in response to this viral vector to design a 
next-generation capsid to evade immune responses. Epitope-based mutations in the 
capsid interfered with the binding and neutralizing ability of the antibody but 
not when tested against polyclonal samples from various sources. Our results 
suggest that targeted mutation of a greater breadth of neutralizing epitopes 
will be required to evade the repertoire of neutralizing antibodies responsible 
for blocking viral vector transduction.