Kim Van Vliet, 1Department of Biochemistry and Molecular Biology, University of Florida, College of Medicine, United States,
Carolyn S. Diaz, 2Interdisciplinary Center for Biotechnology Research, Proteomics Core, University of Florida, United States,
Robert Ng, 1Department of Biochemistry and Molecular Biology, University of Florida, College of Medicine, United States,
Brittney Gurda-Whittaker, 1Department of Biochemistry and Molecular Biology, University of Florida, College of Medicine, United States,
Richard O. Snyder, 3Department of Molecular Genetics and Microbiology, University of Florida, College of Medicine, United States,
Mavis Agbandje-McKenna 1Department of Biochemistry and Molecular Biology, University of Florida, College of Medicine, United States |
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Adeno-associated virus (AAV) is a small single-stranded DNA virus that has shown great promise for gene therapy applications. There are 12 known AAV serotypes (11) and approximately 100 genetic variants of AAV (1-3, 8-10) that have the potential to be developed into gene therapy vectors for delivering a therapeutic gene to specific cell types. Studies of virus assembly have shown that the AAV capsid proteins self-assemble into pre-formed empty capsids, into which the viral genomic DNA is inserted (4, 5). Viral replication and DNA packaging studies have shown that the minimal viral sequences required in cis for replication and packaging are the inverted terminal repeats (ITRs) (6, 13). To produce recombinant AAV vectors (rAAV), the viral genes, rep and cap, are supplied in trans, and the therapeutic transgene flanked by the ITRs is packaged into AAV capsids (4). For wt AAV, which contains viral genomic DNA, PCR-based methods have been developed for identifying the serotype of the wt AAV capsid, based on the DNA sequence of the cap gene (7). However, since AAV gene therapy vectors lack most of the viral DNA, these PCR-based assays are insufficient for determining the capsid identity of the recombinant AAV vector. For clinical vectors, it is desirable to have a diagnostic test to validate the serotype or protein composition of the AAV capsid that will be administered to patients. For this, we have developed a mass spectrometry-based assay for AAV capsid serotype identification (12). Using these methods, we have shown that we can distinguish AAV1 from AAV6, whose capsid proteins differ by only 6 amino acids. It is unlikely that antibody-based methodology would be useful for distinguishing these two serotypes from one another or for validating other AAV capsids which have been engineered for specific targeting of the therapeutic gene to certain tissues; therefore, protein identification by mass spectrometry is a preferred analytical technique for AAV capsid serotype identification.
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