Location: Dairy and Functional Foods ResearchTitle: Effects of thermally induced configuration changes on rAAV genome’s enzymatic accessibility
|XU, YINXIA - School Of Biomedical Sciences, Huaqiao University|
|GUO, PING - School Of Biomedical Sciences, Huaqiao University|
|CHRZANOWSKI, MATTHEW - Temple University|
|CHEW, HELEN - Temple University|
|SANG, NIANGLI - Drexel University|
|DIAO, YONG - School Of Biomedical Sciences, Huaqiao University|
|XIAO, WEIDONG - Indiana University|
|ZHANG, JUNPING - Indiana University|
Submitted to: Molecular Therapy Methods and Clinical Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/3/2020
Publication Date: 6/5/2020
Citation: Xu, Y., Guo, P., Chrzanowski, M., Chew, H., Firrman, J., Sang, N., Diao, Y., Xiao, W., Zhang, J. 2020. Effects of thermally induced configuration changes on rAAV genome’s enzymatic accessibility. Molecular Therapy. 18: 328-334. https://doi.org/10.1016/j.omtm.2020.06.005.
Interpretive Summary: Gene therapy is a type of treatment that is able to ease symptoms of disease by giving a human cell a correct version of a dysfunctional gene. The Adeno-Associated Virus (AAV) is a safe virus used in gene therapy treatment to deliver the correct gene. One important part of using AAV for gene therapy is knowing how much virus is being given to each person. Currently, the amount of virus is determined using a method that can count how much virus DNA is present in a sample. However, in this study we found that during experiments to count AAV, sometimes the outside capsid layer of the virus interacted with the DNA, making it impossible to detect and count the DNA. Whether or not the capsid interacted with the DNA depended on how much DNA was inside the virus. This means that the methods currently used to count AAV may not always give exact information on how much virus is present in a sample. These results provide a foundation for future improvement of vector assays, design, and applications.
Technical 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 (=4.7kb), thermal treatment at 75-99°C allowed only ~10% of genomes to be detectable by qPCR. In contrast, greater than 70% of AAV genomes can be detected under similar condition for AAV vectors with an oversized genome (=5.0kb). 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 “cross-linking”, 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.