Effects of Thermally Induced Configuration Changes on rAAV Genome’s Enzymatic Accessibility

Abstract

Physical titers for recombinant adeno-associated viral (rAAV) vectors are measured by quantifying viral genomes. It is generally perceived that AAV virions disassemble and release DNA upon thermal treatment. Here, we present data on enzymatic accessibility of rAAV genomes when AAV virions were subjected to thermal treatment. For rAAV vectors with a normal genome size (≤4.7 kb), thermal treatment at 75°C–99°C allowed only ∼10% of genomes to be detectable by quantitative real-time PCR. In contrast, greater than 70% of AAV genomes can be detected under similar conditions for AAV vectors with an oversized genome (≥5.0 kb). The permeability of virions, as measured by ethidium bromide (EB) staining, was enhanced by thermal stimulation. These results suggest that in rAAV virions with standard-sized genomes, the capsid and DNA are close enough in proximity for heat-induced “crosslinking,” which results in inaccessibility of vector DNA to enzymatic reactions. In contrast, rAAV vectors with oversized genomes release their DNA readily upon thermal treatment. These findings suggested that the spatial arrangement of capsid protein and DNA in AAV virions is genome-size dependent. These results provide a foundation for future improvement of vector assays, design, and applications.

Quantitative analysis of genome packaging in recombinant AAV vectors by charge detection mass spectrometry

“…A number of previous studies have reported on temperature-induced changes in AAV vectors. [69][70][71][72] A more detailed accounting of the CDMS measurements will be reported elsewhere. We mention these results here because we use the masses from the heat-treated samples below.…”

Reactive oxygen species enhance rAAV transduction by promoting its escape from late endosomes

“…Afterwards, the TIANamp Virus DNA/RNA Kit (Tiangen, Beijing, China) was employed for extracting total cellular DNA as described in the manufacturer's guidance. qPCR was conducted with the AceQ qPCR SYBR Green Master Mix kit (SYBR Green I, Vazyme Biotechnology, Nanjing, China) using transgene-specific primers to measure the infection efficiency of rAAV as described previously [ 20 ]. For internalization assays, cells were infected for 1 h at 4 °C and cultured for 1 h at 37 °C with 5% CO 2 , followed by trypsin treatment to remove those non-internalized surface-bound virions.…”

Reversed Phase-Liquid Chromatography for Recombinant AAV Genome Integrity Assessment

“…The copurified DNA is, in most of the cases, removed by a Benzonase or DNase I treatment during downstream processing. , These species should be monitored carefully due to their potential genotoxicity . Another source of external DNA can be encapsidated DNA, which can be ejected from the capsid when samples are stressed. , Next to the external, also internal DNA can contain selection markers but also fragments or truncated forms of the intended genome. − All of these species are potentially immunogenic and/or genotoxic for patients . For this reason, profound characterization of the rAAV genome is of the utmost importance to ensure a safe and effective product.…”

“… 17 Another source of external DNA can be encapsidated DNA, which can be ejected from the capsid when samples are stressed. 18 , 19 Next to the external, also internal DNA can contain selection markers but also fragments or truncated forms of the intended genome. 20 − 23 All of these species are potentially immunogenic and/or genotoxic for patients.…”