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Journal article
Effects of Thermally Induced Configuration Changes on rAAV Genome's Enzymatic Accessibility
Molecular therapy. Methods & clinical development, v 18, pp 328-334
11 Sep 2020
PMID: 32671135
Featured in Collection : UN Sustainable Development Goals @ Drexel
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 degrees C-99 degrees C allowed only similar to 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.
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Details
- Title
- Effects of Thermally Induced Configuration Changes on rAAV Genome's Enzymatic Accessibility
- Creators
- Yinxia Xu - Huaqiao UniversityPing Guo - Huaqiao UniversityJunping Zhang - Indiana UniversityMatthew Chrzanowski - Temple UniversityHelen Chew - Temple UniversityJenni A. Firrman - Eastern Regional Research CenterNianli Sang - Drexel UniversityYong Diao - Huaqiao UniversityWeidong Xiao - Indiana University
- Publication Details
- Molecular therapy. Methods & clinical development, v 18, pp 328-334
- Publisher
- Elsevier
- Number of pages
- 7
- Grant note
- 81371672; 81371669 / National Natural Science Foundation of China; National Natural Science Foundation of China (NSFC) 2016N006; 2018C042R / Project of Science and Technology of Quanzhou of China HL114152; HL130871 / National Institutes of Health (NIH) of United States; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA 2018Y4009; 2019J05094 / Major Project of University and Industry Cooperation in Fujian Province of China
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Biology
- Web of Science ID
- WOS:000568405100031
- Scopus ID
- 2-s2.0-85087344731
- Other Identifier
- 991019167998304721
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- Collaboration types
- Domestic collaboration
- International collaboration
- Web of Science research areas
- Medicine, Research & Experimental