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

Molecular Therapy. Methods & Clinical Development
Yinxia XuWei-dong Xiao

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 v...Continue Reading

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Citations

Jul 31, 2021·Human Gene Therapy·David Nathan Quan, Joseph Shiloach

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Methods Mentioned

BETA
electron
atomic force microscopy
thermal
PCR
thermal treatment
electrophoresis
DNA assay
Assay

Software Mentioned

SPSS
GelAnalyzer

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