Effects of Thermally Induced Configuration Changes on rAAV Genome's Enzymatic Accessibility.
Mol Ther Methods Clin Dev
; 18: 328-334, 2020 Sep 11.
Article
en En
| MEDLINE
| ID: mdl-32671135
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 â¼10% 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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Idioma:
En
Revista:
Mol Ther Methods Clin Dev
Año:
2020
Tipo del documento:
Article
País de afiliación:
China