Intrinsic Differential Scanning Fluorimetry for Fast and Easy Identification of AAV Serotypes.

Rieser R, Penaud-Budloo M, Bouzelha M, Rossi A, Menzen T, Biel M, Büning H, Ayuso E, Winter G, Michalakis S.

Recombinant adeno-associated virus (rAAV) vectors have evolved as the most promising technology for gene therapy due to their good safety profile, high transduction efficacy and long-term gene expression in non-dividing cells. AAV-based gene therapy holds great promise for treating genetic disorders like inherited blindness, muscular atrophy or bleeding disorders. Multiple naturally occurring and engineered AAV serotypes exist, which differ in capsid sequence and as a consequence in cellular tropism. Individual AAV capsids differ in thermal stability and have a characteristic melting temperature (Tm), which enables serotype-specific discrimination of AAV vectors. Differential scanning fluorimetry (DSF) combined with a dye like SYPRO Orange (SO-DSF), which binds to hydrophobic regions of unfolded proteins, has been successfully applied to determine the Tm of AAV capsids. Here, we present DSF measurement of intrinsic fluorescence signal (iDSF) as a simple alternative method for determination of AAV capsid Tm. The study demonstrates that iDSF is a simple, accurate and rapid alternative to SO-DSF, which enables characterization of AAV capsid stability with excellent precision and without the need of SO or any other dye.

J Pharm Sci. 2020 Jan

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