Ibba, Gabriele (2017) Eradication of persistent HBV genomes using SaCas9/3xgRNAs based gene therapy. Doctoral Thesis.
The existence of stable, drug-resistant, DNA intermediate in viral replication cycle allows many viruses to persist in infected cells and is the main obstacle for curative therapy. In the case of chronic hepatitis caused by HBV (hepatitis B virus), the viral genome persists in host cells as covalently closed, circular DNA (cccDNA). Here we demonstrate that the CRISPR/Cas9 gene editing technology cleaves HBV genome efficiently and specifically. To ensure successful disruption of cccDNA, we multiplexed three different gRNAs, targeting regions of viral polymerase gene (P) conserved among all ten HBV genotypes, in a single “all in” SaCas9/gRNAs AAV vector. Additionally, to achieve maximum gene editing efficiency, we added to our construct a shRNA-expressing cassette, targeting the viral trans-activator X mRNA, which should result in suppression of virus expression in treated cells. Transfecting our pX601-HBV3xgRNAs/shRNA plasmid into HepG2.2.15 cells (Vitro chronic hepatitis model for HBV) resulted in a significant reduction of viral DNA levels. Sanger sequencing of extracted PCR products confirmed canonical SaCas9 mediated cleavage of viral DNA. Disruption HBV cccDNA led to a significant reduction of intracellular viral DNA and RNA levels, as analyzed by qRT-PCRs. In summary, we designed and validated in vitro an application of SaCas9/gRNA gene editing platform, in a chronic hepatitis model, to directly target and cleave persistent episomal HBV genome. Our data set the stage for the next, pre-clinical phase of research in the animal model, toward developing a gene therapy-based treatment for patients with a chronic HBV hepatitis.
I documenti depositati in UnissResearch sono protetti dalle leggi che regolano il diritto d'autore
Repository Staff Only: item control page