SsAAVs containing cassettes encoding SaCas9 and guides targeting hepatitis B virus inactivate replication of the virus in cultured cells

Tristan Scott,Koichi Watashi,Marc S Weinberg,Abdullah Ely,Buhle Moyo,Samantha Nicholson,Patrick Arbuthnot,Mohube Betty Maepa

doi:10.1038/s41598-017-07642-6

Abstract

Management of infection with hepatitis B virus (HBV) remains a global health problem. Persistence of stable covalently closed circular DNA (cccDNA) during HBV replication is responsible for modest curative efficacy of currently licensed drugs. Novel gene editing technologies, such as those based on CRISPR/Cas9, provide the means for permanently disabling cccDNA. However, efficient delivery of antiviral sequences to infected hepatocytes is challenging. A limiting factor is the large size of sequences encoding Cas9 from Streptococcus pyogenes, and resultant incompatibility with the popular single stranded adeno-associated viral vectors (ssAAVs). We thus explored the utility of ssAAVs for delivery of engineered CRISPR/Cas9 of Staphylococcus aureus (Sa), which is encoded by shorter DNA sequences. Short guide RNAs (sgRNAs) were designed with cognates in the S open reading frame of HBV and incorporated into AAVs that also encoded SaCas9. Intended targeted mutation of HBV DNA was observed after transduction of cells with the all-in-one vectors. Efficacy against HBV-infected hNTCP-HepG2 cells indicated that inactivation of cccDNA was successful. Analysis of likely off-target mutagenesis revealed no unintended sequence changes. Use of ssAAVs to deliver all components required to disable cccDNA by SaCas9 is novel and the technology has curative potential for HBV infection.

Highlights

Chronic infection with hepatitis B virus (HBV) is responsible for a significant global disease burden[1]
Eight of the ten Short guide RNAs (sgRNAs) reduced concentrations of HBV S antigen (HBsAg) in the culture supernatants by 50–95% when compared to controls, which were a non-specific sgRNA or an sgRNA targeted to HIV (Fig. 1b)
This indicated that the inhibitory effect of sgRNA-8 on release of the HBV replication marker from cultured cells resulted from cleavage-mediated mutagenesis

Summary

Introduction

Chronic infection with hepatitis B virus (HBV) is responsible for a significant global disease burden[1]. Sequences encoding SaCas[9] as well as regulatory elements and the sgRNA cassette have been packaged into an ssAAV and used to edit endogenous genes in the liver This ‘all-in-one’ system, which overcomes the need for using more than one AAV, has potential for application to therapeutic gene editing and may be useful for inactivation of genes of HBV. To advance use of CRISPR/ Cas9-based technology for treatment of HBV infection, we incorporated cassettes encoding SaCas[9] and sgRNAs into ssAAVs. Targeting the HBV S open reading frame (ORF) resulted in efficient inactivation of HBV replication and mutation of cccDNA in cultured cells

Methods

Results

Conclusion