Thermal Stability as a Determinant of AAV Serotype Identity

Abstract

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 (Tm) for AAV serotype identification. This is a simple, rapid, cost effective, and robust method utilizing small amounts of purified AAV capsids (∼25 μL of ∼1011 particles). AAV1-9 and AAVrh.10 exhibit specific Tms in buffer formulations commonly used in clinical trials. Notably, AAV2 and AAV3, which are the least stable, have varied Tms, whereas AAV5, the most stable, has a narrow Tm 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.

Curating viscoelastic properties of icosahedral viruses, virus-based nanomaterials, and protein cages

“…This suggests that even while existing as a highly pressurized capsid, P22 can still be isovolumetrically distorted to a greater extent than the rigid AAV capsid by the shear forces of the liquid. Thermal and proteolytic stabilites of AAVs have shown that these capsids are extremely resistant to environmental changes [33,34] and our QCM-D results substantiate these results. The low rigidity of PBCV among compared to other viruses was expected due to the greater diameter and emphasizes that rigidity is not a simple outcome of subunit number.…”

“…Our experiments show that the rigidities of AAV capsids vary significantly with serotype. Thermal and proteolytic stabilities of empty and genome filled AAVs have also been well studied and have shown similar differences [33,34]. On the other hand, nanoindentation studies of AAV capsids using AFM pointed to a higher propensity for deformation of the empty forms [35].…”

“…In comparison, the AAV capsid has a higher shell thickness and the subunit protein is 3-4 times higher in molecular weight. It is important to note that CCMV (both closed and swollen forms) have native RNA in them and yet AAVs, both empty and genome-filled, have been shown to have significantly higher thermal stabilities [33,34,36] and stiffness [13,35,37] compared to CCMV. Our results corroborate with these previous observations.…”

Quantification of Adeno-Associated Virus with Safe Nucleic Acid Dyes

“…It has been demonstrated that AAV serotypes are different in their thermal stability, with rAAV2 being the least thermal stable and rAAV5 being the most thermal stable. In PBS buffer, the melting temperature (Tms) of different AAV serotypes range from 66.5 up to 89.5 °C 43 . Therefore we choose to heat rAAV samples at 95 °C.…”

Residues on Adeno-associated Virus Capsid Lumen Dictate Interactions and Compatibility with the Assembly-Activating Protein

“…Although AAV is the simplest form of an icosahedral capsid, the intermonomeric interactions occurring within the assembly are considerably complex and contribute to a remarkably stable virion that can withstand appreciably high temperatures (24)(25)(26), a wide range of pH (27), and long shelf-lives (27), while remaining infectious. There is a reasonably high energy barrier associated with assembling this highly stable product, corroborated by the inability of VP monomers to selfassemble (12) and the requirement for AAP, which promotes VP oligomerization to overcome such energy barriers.…”