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Excision of HIV-1 DNA by gene editing: a proof-of-concept in vivo study

Gene Therapy volume 23, pages 690–695 (2016)Cite this article

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A Corrigendum to this article was published on 04 August 2016

Abstract

A CRISPR/Cas9 gene editing strategy has been remarkable in excising segments of integrated HIV-1 DNA sequences from the genome of latently infected human cell lines and by introducing InDel mutations, suppressing HIV-1 replication in patient-derived CD4+ T-cells, ex vivo. Here, we employed a short version of the Cas9 endonuclease, saCas9, together with a multiplex of guide RNAs (gRNAs) for targeting the viral DNA sequences within the 5′-LTR and the Gag gene for removing critically important segments of the viral DNA in transgenic mice and rats encompassing the HIV-1 genome. Tail-vein injection of transgenic mice with a recombinant Adeno-associated virus 9 (rAAV9) vector expressing saCas9 and the gRNAs, rAAV:saCas9/gRNA, resulted in the cleavage of integrated HIV-1 DNA and excision of a 978 bp DNA fragment spanning between the LTR and Gag gene in the spleen, liver, heart, lung and kidney as well as in the circulating lymphocytes. Retro-orbital inoculation of rAAV9:saCas9/gRNA in transgenic rats eliminated a targeted segment of viral DNA and substantially decreased the level of viral gene expression in circulating blood lymphocytes. The results from the proof-of-concept studies, for the first time, demonstrate the in vivo eradication of HIV-1 DNA by CRISPR/Cas9 on delivery by an rAAV9 vector in a range of cells and tissues that harbor integrated copies of viral DNA.

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Acknowledgements

We wish to thank past and present members of the Department of Neuroscience and Center for Neurovirology for sharing the ideas and reagents. We thank Fang Li and Ting Zhang for technical assistance in cloning of the vector. Preparation of the rAAV9 vector was done by the Penn Vector Core, Gene Therapy Program at the University of Pennsylvania. This work was supported by: P30MH092177 (KK), R01MH106392 (RB), R01MH092371 (KK), R01DA013137 (RB), and R01NS087971 (KK, WH), R01HD043680 (RB), P01DA037830 (KK).

Author contributions

KK, JG, WH, RB conceived experiments; RK, RB, CY, JO, RB, HL performed experiments; KK, JG, WH, HEG, PF, RB analyzed data and KK, RK, JG, RB helped in preparing manuscript.

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Authors and Affiliations

  1. Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine at Temple University, Philadelphia, PA, USA

    R Kaminski, C Yin, J Otte, J Gordon, W Hu & K Khalili

  2. Department of Biomedical, Microbiology and Clinical Microbiology, Surgical and Dental Sciences, University of Milan, Milan, Italy

    R Bella & P Ferrante

  3. Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Durham Research Center, Omaha, NE, USA

    H E Gendelman

  4. Department of Psychology, Behavioral Neuroscience, University of South Carolina, Columbia, SC, USA

    H Li & R Booze

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  1. R Kaminski
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  2. R Bella
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  10. W Hu
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Corresponding authors

Correspondence to J Gordon, W Hu or K Khalili.

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Kaminski, R., Bella, R., Yin, C. et al. Excision of HIV-1 DNA by gene editing: a proof-of-concept in vivo study. Gene Ther 23, 690–695 (2016). https://doi.org/10.1038/gt.2016.41

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