Determining the concentration of recombinant adeno-associated virus (AAV) productions, also known as titering, is crucial not only for quality control purposes but also for comparative studies of preclinical and clinical gene therapy trials. Recently, several AAVs were engineered by inserting seven amino acids in a protruding loop of the AAV capsid structure: variable region VIII (VR-VIII) loop. These variants have demonstrated increased transduction capabilities over naturally occurring AAV serotypes in several studies. However, they have also been shown to produce lower yields when titered using standard techniques, raising questions about their adequacy for clinical development and use. Here, we investigated why peptide insertion onto AAV capsids reduces their titer by examining viral stocks using electron microscopy and PCR-based titering. We reveal that the DNase digestion step, performed to eliminate free-floating DNA prior to qPCR or ddPCR, adversely impacts engineered capsid stability due to exposure to heat, artificially lowering viral titers of engineered serotypes. Titering without heating yields significantly higher titers for these variants which have melting temperatures (Tm) close to the DNase inactivation temperature, while titers for parental serotypes with higher Tm remain unchanged. Our findings provide an important perspective for titering engineered variants with lower thermostability, especially when comparing their effectiveness with their parental serotypes.
Keywords: AAV; AAV titering; engineered variants; gene therapy; molecular biology; thermostability.
© 2025 The Authors. Published by Elsevier Inc. on behalf of The American Society of Gene and Cell Therapy.