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

• Published in: Scientific reports Vol. 7; no. 1; pp. 7401 - 11
• Main Authors: Scott, Tristan, Moyo, Buhle, Nicholson, Samantha, Maepa, Mohube Betty, Watashi, Koichi, Ely, Abdullah, Weinberg, Marc S., Arbuthnot, Patrick
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 07.08.2017; Nature Publishing Group; Nature Portfolio
• Subjects: 13; 42/41; 42/44; 631/154/152; 692/4017; Antiviral agents; Circular DNA; CRISPR; CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; Deoxyribonucleic acid; Dependovirus - genetics; DNA; DNA biosynthesis; Gene Editing; Genetic Vectors - pharmacology; Genome editing; Global health; Hep G2 Cells; Hepatitis; Hepatitis B; Hepatitis B - virology; Hepatitis B virus - drug effects; Hepatitis B virus - genetics; Hepatitis B virus - physiology; Hepatocytes; Humanities and Social Sciences; Humans; Inactivation; multidisciplinary; Mutagenesis; Mutagenesis, Site-Directed; Nucleotide sequence; Open Reading Frames; Replication; RNA, Guide, CRISPR-Cas Systems - chemical synthesis; RNA, Guide, CRISPR-Cas Systems - genetics; Science; Science (multidisciplinary); Staphylococcus aureus - metabolism; Streptococcus infections; Virus Replication - drug effects
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-017-07642-6

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.