TY  - GEN
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/28653/
A1  - Njenga, Robin Wanjiru
N2  - The Adeno-associated virus (AAV) presents itself as an attractive vector for gene therapy due to its broad tissue tropism, ease of manipulation and lack of pathogenicity. So far, three FDA approved AAV-based gene therapies are available, with more than 100 ongoing clinical trials. However, aspects of the AAV life cycle still limit its transduction capacities such as receptor binding, intracellular trafficking, uncoating, single-strand to double strand conversion and interaction with host cell factors. Although technologies have been developed to tackle most of these challenges, there is still a gap in knowledge on the restriction through the interaction with host cell factors. Bridging this gap in AAV biology shall additionally enable the generation of more efficient gene therapy vectors. In this study, the restriction of AAV through the ubiquitin-proteasome system was evaluated. The speckled-type POZ protein (SPOP) is a member of the E3-ubiquitin ligase complex with Cullin 3. Both Cullin 3 and SPOP were previously identified though a tandem-affinity purification as putative interaction partners of AAV capsid protein VP1. In addition, a yeast 2 hybrid screen also confirmed the interaction with SPOP. In this study, CRISPR-Cas9 technology was employed to knockout SPOP and Cullin 3; and evaluate their effects on AAV. SPOP was found to restrict AAV in both parts of the life cycle. During entry, SPOP had a 2-fold effect on AAV transduction and regulated the turnover of capsids (up to 3 fold more capsids were seen in the absence of SPOP). However, an increase of transduction through proteasome inhibition in the SPOP knockout cells indicated that it was not the sole E3 ubiquitin ligase complex involved in the restriction of AAV transduction. After de novo synthesis of capsid proteins, SPOP was able to regulate the turnover of both assembled capsids and unassembled capsid proteins. The latter could be visualized as distinct VP clusters in the nuclei of SPOP knockout cells. A restoration of SPOP in the knockout cells was able to destabilize the capsid proteins but wash not able to reach the levels seen in the parental condition, although the functionality of restored SPOP could not be confirmed as other endogenous SPOP substrates showed variable patterns. Proteasome inhibition was able to stabilize unassembled capsid proteins to comparable levels as the absence of SPOP but had no additive effect upon combination. Cullin 3 did not have a significant effect in either part of the AAV life cycle. The results of this study provide more concrete information about the involvement of the ubiquitin-proteasome system in the restriction of AAV.
ID  - heidok28653
Y1  - 2020///
TI  - Characterizing the Interaction of Adeno-Associated Virus (AAV) with Factors of the Ubiquitin-Proteasome System
AV  - public
CY  - Heidelberg
ER  -