Thermal Stability as a Determinant of AAV Serotype Identity

• Published in: Molecular therapy. Methods & clinical development 2017-09, Vol.6 (C), p.171-182
• Main Authors: Bennett, Antonette, Patel, Saajan, Mietzsch, Mario, Jose, Ariana, Lins-Austin, Bridget, Yu, Jennifer C, Bothner, Brian, McKenna, Robert, Agbandje-McKenna, Mavis
• Format: Article
• Language: English
• Subjects: AAV; AAV buffer formulations; AAV capsid stability; AAV serotype identification; AAV vector buffers; adeno-associated virus; Amino acids; Capsids; Clinical trials; Congestive heart failure; Cystic fibrosis; differential scanning fluorimetry; Gene therapy; Genetic engineering; Genomes; Hemophilia; Identification; Methods; Neuronal ceroid lipofuscinosis; Original; parvovirus stability; Peptides; Rheumatoid arthritis; Thermal stability; viral vectors
• ISSN: 2329-0501; 2329-0501
• DOI: 10.1016/j.omtm.2017.07.003

Currently, there are over 150 ongoing clinical trials utilizing adeno-associated viruses (AAVs) to target various genetic diseases, including hemophilia (AAV2 and AAV8), congenital heart failure (AAV1 and AAV6), cystic fibrosis (AAV2), rheumatoid arthritis (AAV2), and Batten disease (AAVrh.10). Prior to patient administration, AAV vectors must have their serotype, concentration, purity, and stability confirmed. Here, we report the application of differential scanning fluorimetry (DSF) as a good manufacturing practice (GMP) capable of determining the melting temperature (T ) for AAV serotype identification. This is a simple, rapid, cost effective, and robust method utilizing small amounts of purified AAV capsids (∼25 μL of ∼10 particles). AAV1-9 and AAVrh.10 exhibit specific T s in buffer formulations commonly used in clinical trials. Notably, AAV2 and AAV3, which are the least stable, have varied T s, whereas AAV5, the most stable, has a narrow T range in the different buffers, respectively. Vector stability was dictated by VP3 only, specifically, the ratio of basic/acidic amino acids, and was independent of VP1 and VP2 content or the genome packaged. Furthermore, stability of recombinant AAVs differing by a single basic or acidic amino acid residue are distinguishable. Hence, AAV DSF profiles can serve as a robust method for serotype identification of clinical vectors.