title: Excision of HIV-1 DNA by gene editing: a proof-of-concept in vivo study.
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.
authors.affiliation: Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine at Temple University, Philadelphia, PA, USA.
authors.forename: R
authors.initials: R
authors.lastname: Kaminski
authors.affiliation: Microbiology and Clinical Microbiology, Department of Biomedical, Surgical and Dental Sciences, University of Milan, Milan, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Bella
authors.affiliation: Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine at Temple University, Philadelphia, PA, USA.
authors.forename: C
authors.initials: C
authors.lastname: Yin
authors.affiliation: Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine at Temple University, Philadelphia, PA, USA.
authors.forename: J
authors.initials: J
authors.lastname: Otte
authors.affiliation: Microbiology and Clinical Microbiology, Department of Biomedical, Surgical and Dental Sciences, University of Milan, Milan, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Ferrante
authors.affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Durham Research Center, Omaha, NE, USA.
authors.forename: H E
authors.initials: HE
authors.lastname: Gendelman
authors.affiliation: Behavioral Neuroscience, Department of Psychology, University of South Carolina, Columbia, SC, USA.
authors.forename: H
authors.initials: H
authors.lastname: Li
authors.affiliation: Behavioral Neuroscience, Department of Psychology, University of South Carolina, Columbia, SC, USA.
authors.forename: R
authors.initials: R
authors.lastname: Booze
authors.affiliation: Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine at Temple University, Philadelphia, PA, USA.
authors.forename: J
authors.initials: J
authors.lastname: Gordon
authors.affiliation: Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine at Temple University, Philadelphia, PA, USA.
authors.forename: W
authors.initials: W
authors.lastname: Hu
authors.affiliation: Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine at Temple University, Philadelphia, PA, USA.
authors.forename: K
authors.initials: K
authors.lastname: Khalili
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D064113:CRISPR-Cas Systems; D002478:Cells, Cultured; D004279:DNA, Viral / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics; D000072669:Gene Editing / Q000379:methods*; D015683:Gene Products, gag / Q000235:genetics; D018390:Gene Targeting / Q000379:methods; D015497:HIV-1 / Q000235:genetics*; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D051379:Mice; D009206:Myocardium / Q000378:metabolism; D051381:Rats
chemical_list: D004279:DNA, Viral; D015683:Gene Products, gag