Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient

Abstract

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Genome editing using CRISPR/Cas9 could provide new therapies because it can directly disrupt HBV genomes. However, because HBV genome sequences are highly diverse, the identical target sequence of guide RNA (gRNA), 20 nucleotides in length, is not necessarily present intact in the target HBV DNA in heterogeneous patients. Consequently, possible genome-editing drugs would be effective only for limited numbers of patients. Here, we show that an adenovirus vector (AdV) bearing eight multiplex gRNA expression units could be constructed in one step and amplified to a level sufficient for in vivo study with lack of deletion. Using this AdV, HBV X gene integrated in HepG2 cell chromosome derived from a heterogeneous patient was cleaved at multiple sites and disrupted. Indeed, four targets out of eight could not be cleaved due to sequence mismatches, but the remaining four targets were cleaved, producing irreversible deletions. Accordingly, the diverse X gene was disrupted at more than 90% efficiency. AdV containing eight multiplex gRNA units not only offers multiple knockouts of genes, but could also solve the problems of heterogeneous targets and escape mutants in genome-editing therapy.

Construction and application of adenoviral vectors

“…The Ad containing eight multiple gRNA units not only ensures that the target gene is knocked out multiple times, but also solves the problem of off-target effects and escape mutants in genome editing therapy. 208 …”

Development of therapeutic vaccines for the treatment of diseases

“…Additionally, some new strategies have been utilized to clear HBV with adenovirus vectors. For example, adenovirus was designed to deliver a CRISPR/Cas9 system and a single guide RNA (gRNA) system to degrade the HBV genome [96,97].…”

Adenoviral Vectors: Potential as Anti-HBV Vaccines and Therapeutics

“…These gene editors mediate sequence-specific deleterious mutations within the HBV DNA [ 22 ]. Although AdV-based anti-HBV TALEN delivery is not encouraging [ 59 ], promising outcomes when using AdVs to deliver anti-HBV CRISPR/Cas sequences have been reported [ 60 , 61 ].…”

“…A recent study by Kato et al successfully demonstrated the efficacy of an AdV expressing a CRISPR/Cas9 system with eight guide RNAs (gRNAs) targeting the HBV X gene ( HBx ) [ 60 ]. In this study, use of an AdV was ideal as the increased carrying capacity of the AdV allowed for inclusion of 8 gRNAs and a large Cas protein-encoding sequence isolated from Streptococcus pyogenes , spCas9.…”