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

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...

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Autores principales:	Tristan Scott, Buhle Moyo, Samantha Nicholson, Mohube Betty Maepa, Koichi Watashi, Abdullah Ely, Marc S. Weinberg, Patrick Arbuthnot
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a6a126a42f17449db43d6d0992861f02
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spelling	oai:doaj.org-article:a6a126a42f17449db43d6d0992861f022021-12-02T11:52:39ZssAAVs containing cassettes encoding SaCas9 and guides targeting hepatitis B virus inactivate replication of the virus in cultured cells10.1038/s41598-017-07642-62045-2322https://doaj.org/article/a6a126a42f17449db43d6d0992861f022017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-07642-6https://doaj.org/toc/2045-2322Abstract 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.Tristan ScottBuhle MoyoSamantha NicholsonMohube Betty MaepaKoichi WatashiAbdullah ElyMarc S. WeinbergPatrick ArbuthnotNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-11 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Tristan Scott Buhle Moyo Samantha Nicholson Mohube Betty Maepa Koichi Watashi Abdullah Ely Marc S. Weinberg Patrick Arbuthnot ssAAVs containing cassettes encoding SaCas9 and guides targeting hepatitis B virus inactivate replication of the virus in cultured cells
description	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.
format	article
author	Tristan Scott Buhle Moyo Samantha Nicholson Mohube Betty Maepa Koichi Watashi Abdullah Ely Marc S. Weinberg Patrick Arbuthnot
author_facet	Tristan Scott Buhle Moyo Samantha Nicholson Mohube Betty Maepa Koichi Watashi Abdullah Ely Marc S. Weinberg Patrick Arbuthnot
author_sort	Tristan Scott
title	ssAAVs containing cassettes encoding SaCas9 and guides targeting hepatitis B virus inactivate replication of the virus in cultured cells
title_short	ssAAVs containing cassettes encoding SaCas9 and guides targeting hepatitis B virus inactivate replication of the virus in cultured cells
title_full	ssAAVs containing cassettes encoding SaCas9 and guides targeting hepatitis B virus inactivate replication of the virus in cultured cells
title_fullStr	ssAAVs containing cassettes encoding SaCas9 and guides targeting hepatitis B virus inactivate replication of the virus in cultured cells
title_full_unstemmed	ssAAVs containing cassettes encoding SaCas9 and guides targeting hepatitis B virus inactivate replication of the virus in cultured cells
title_sort	ssaavs containing cassettes encoding sacas9 and guides targeting hepatitis b virus inactivate replication of the virus in cultured cells
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/a6a126a42f17449db43d6d0992861f02
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ssAAVs containing cassettes encoding SaCas9 and guides targeting hepatitis B virus inactivate replication of the virus in cultured cells

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