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Research Article

RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection

Wenhui Hu, Rafal Kaminski, Fan Yang, Yonggang Zhang, Laura Cosentino, Fang Li, Biao Luo, David Alvarez-Carbonell, Yoelvis Garcia-Mesa, Jonathan Karn, Xianming Mo, and Kamel Khalili
PNAS first published July 21, 2014; https://doi.org/10.1073/pnas.1405186111
Wenhui Hu
aDepartment of Neuroscience, Center for Neurovirology and The Comprehensive NeuroAIDS Center, Temple University School of Medicine, Philadelphia, PA 19140;
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  • For correspondence: kamel.khalili@temple.edu wenhui.hu@temple.edu
Rafal Kaminski
aDepartment of Neuroscience, Center for Neurovirology and The Comprehensive NeuroAIDS Center, Temple University School of Medicine, Philadelphia, PA 19140;
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Fan Yang
aDepartment of Neuroscience, Center for Neurovirology and The Comprehensive NeuroAIDS Center, Temple University School of Medicine, Philadelphia, PA 19140;
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Yonggang Zhang
aDepartment of Neuroscience, Center for Neurovirology and The Comprehensive NeuroAIDS Center, Temple University School of Medicine, Philadelphia, PA 19140;
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Laura Cosentino
aDepartment of Neuroscience, Center for Neurovirology and The Comprehensive NeuroAIDS Center, Temple University School of Medicine, Philadelphia, PA 19140;
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Fang Li
aDepartment of Neuroscience, Center for Neurovirology and The Comprehensive NeuroAIDS Center, Temple University School of Medicine, Philadelphia, PA 19140;
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Biao Luo
bCancer Genome Institute, Fox Chase Cancer Center, Temple University School of Medicine, Philadelphia, PA 19111;
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David Alvarez-Carbonell
cDepartment of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106; and
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Yoelvis Garcia-Mesa
cDepartment of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106; and
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Jonathan Karn
cDepartment of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106; and
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Xianming Mo
dLaboratory of Stem Cell Biology, State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China
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Kamel Khalili
aDepartment of Neuroscience, Center for Neurovirology and The Comprehensive NeuroAIDS Center, Temple University School of Medicine, Philadelphia, PA 19140;
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  • For correspondence: kamel.khalili@temple.edu wenhui.hu@temple.edu
  1. Edited by Anthony S. Fauci, National Institute of Allergy and Infectious Diseases, Bethesda, MD, and approved June 19, 2014 (received for review March 19, 2014)

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Significance

For more than three decades since the discovery of HIV-1, AIDS remains a major public health problem affecting greater than 35.3 million people worldwide. Current antiretroviral therapy has failed to eradicate HIV-1, partly due to the persistence of viral reservoirs. RNA-guided HIV-1 genome cleavage by the Cas9 technology has shown promising efficacy in disrupting the HIV-1 genome in latently infected cells, suppressing viral gene expression and replication, and immunizing uninfected cells against HIV-1 infection. These properties may provide a viable path toward a permanent cure for AIDS, and provide a means to vaccinate against other pathogenic viruses. Given the ease and rapidity of Cas9/guide RNA development, personalized therapies for individual patients with HIV-1 variants can be developed instantly.

Abstract

AIDS remains incurable due to the permanent integration of HIV-1 into the host genome, imparting risk of viral reactivation even after antiretroviral therapy. New strategies are needed to ablate the viral genome from latently infected cells, because current methods are too inefficient and prone to adverse off-target effects. To eliminate the integrated HIV-1 genome, we used the Cas9/guide RNA (gRNA) system, in single and multiplex configurations. We identified highly specific targets within the HIV-1 LTR U3 region that were efficiently edited by Cas9/gRNA, inactivating viral gene expression and replication in latently infected microglial, promonocytic, and T cells. Cas9/gRNAs caused neither genotoxicity nor off-target editing to the host cells, and completely excised a 9,709-bp fragment of integrated proviral DNA that spanned from its 5′ to 3′ LTRs. Furthermore, the presence of multiplex gRNAs within Cas9-expressing cells prevented HIV-1 infection. Our results suggest that Cas9/gRNA can be engineered to provide a specific, efficacious prophylactic and therapeutic approach against AIDS.

  • CRISPR/Cas9
  • genome editing
  • latency
  • retrovirus
  • reservoir

Footnotes

  • ↵1W.H. and R.K. contributed equally to this work.

  • ↵2To whom correspondence may be addressed. Email: kamel.khalili{at}temple.edu or wenhui.hu{at}temple.edu.
  • Author contributions: W.H., R.K., and K.K. designed research; W.H., R.K., F.Y., Y.Z., L.C., F.L., and B.L. performed research; D.A.-C., Y.G.-M., J.K., and X.M. contributed new reagents/analytic tools; W.H., B.L., and K.K. analyzed data; and W.H. and K.K. wrote the paper.

  • Conflict of interest statement: A patent application has been filed relating to this work.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1405186111/-/DCSupplemental.

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Genome editing eradicates HIV-1
Wenhui Hu, Rafal Kaminski, Fan Yang, Yonggang Zhang, Laura Cosentino, Fang Li, Biao Luo, David Alvarez-Carbonell, Yoelvis Garcia-Mesa, Jonathan Karn, Xianming Mo, Kamel Khalili
Proceedings of the National Academy of Sciences Jul 2014, 201405186; DOI: 10.1073/pnas.1405186111

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Genome editing eradicates HIV-1
Wenhui Hu, Rafal Kaminski, Fan Yang, Yonggang Zhang, Laura Cosentino, Fang Li, Biao Luo, David Alvarez-Carbonell, Yoelvis Garcia-Mesa, Jonathan Karn, Xianming Mo, Kamel Khalili
Proceedings of the National Academy of Sciences Jul 2014, 201405186; DOI: 10.1073/pnas.1405186111
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