Doctor of Philosophy, University of Georgia (2017)
Capsid engineering overcomes barriers toward Adeno-associated viral (AAV) vector-mediated transduction of endothelial cells.
Human gene therapy
Endothelial cells (EC) are targets in gene therapy and regenerative medicine, but are inefficiently transduced with Adeno-associated viral (AAV) vectors of various serotypes. To identify barriers hampering efficient transduction and to develop an optimized AAV variant for EC transduction, we screened an AAV serotype 2-based peptide display library on primary human macrovascular EC. Using a new high-throughput selection and monitoring protocol, we identified a capsid variant, AAV-VEC, which outperformed the parental serotype as well as first-generation targeting vectors in EC transduction. AAV vector uptake was improved resulting in significantly higher transgene expression levels from single-stranded vector genomes detectable already few hours post transduction. Notably, AAV-VEC transduced not only proliferating EC, but also quiescent EC although higher particle-per-cell ratios had to be applied. Also, induced pluripotent stem cell-derived endothelial progenitor cells, a novel tool in regenerative medicine and gene therapy, were highly susceptible toward AAV-VEC transduction. Thus, overcoming barriers by capsid-engineering significantly expands the AAV tool kit for a wide range of applications targeting EC.
View details for DOI 10.1089/hum.2019.027
View details for PubMedID 31407607
Genome-Wide CRISPR/Cas9 Screening Identifies GPR108 as a Highly Conserved AAV Entry Factor
CELL PRESS. 2019: 313–14
View details for Web of Science ID 000464381003125
Enterovirus pathogenesis requires the host methyltransferase SETD3.
Enteroviruses (EVs) comprise a large genus of positive-sense, single-stranded RNA viruses whose members cause a number of important and widespread human diseases, including poliomyelitis, myocarditis, acute flaccid myelitis and the common cold. How EVs co-opt cellular functions to promote replication and spread is incompletely understood. Here, using genome-scale CRISPR screens, we identify the actin histidine methyltransferase SET domain containing 3 (SETD3) as critically important for viral infection by a broad panel of EVs, including rhinoviruses and non-polio EVs increasingly linked to severe neurological disease such as acute flaccid myelitis (EV-D68) and viral encephalitis (EV-A71). We show that cytosolic SETD3, independent of its methylation activity, is required for the RNA replication step in the viral life cycle. Using quantitative affinity purification-mass spectrometry, we show that SETD3 specifically interacts with the viral 2A protease of multiple enteroviral species, and we map the residues in 2A that mediate this interaction. 2A mutants that retain protease activity but are unable to interact with SETD3 are severely compromised in RNA replication. These data suggest a role of the viral 2A protein in RNA replication beyond facilitating proteolytic cleavage. Finally, we show that SETD3 is essential for in vivo replication and pathogenesis in multiple mouse models for EV infection, including CV-A10, EV-A71 and EV-D68. Our results reveal a crucial role of a host protein in viral pathogenesis, and suggest targeting SETD3 as a potential mechanism for controlling viral infections.
View details for DOI 10.1038/s41564-019-0551-1
View details for PubMedID 31527793