title: In Vivo Excision of HIV-1 Provirus by saCas9 and Multiplex Single-Guide RNAs in Animal Models.
abstract: CRISPR-associated protein 9 (Cas9)-mediated genome editing provides a promising cure for HIV-1/AIDS; however, gene delivery efficiency in vivo remains an obstacle to overcome. Here, we demonstrate the feasibility and efficiency of excising the HIV-1 provirus in three different animal models using an all-in-one adeno-associated virus (AAV) vector to deliver multiplex single-guide RNAs (sgRNAs) plus Staphylococcus aureus Cas9 (saCas9). The quadruplex sgRNAs/saCas9 vector outperformed the duplex vector in excising the integrated HIV-1 genome in cultured neural stem/progenitor cells from HIV-1 Tg26 transgenic mice. Intravenously injected quadruplex sgRNAs/saCas9 AAV-DJ/8 excised HIV-1 proviral DNA and significantly reduced viral RNA expression in several organs/tissues of Tg26 mice. In EcoHIV acutely infected mice, intravenously injected quadruplex sgRNAs/saCas9 AAV-DJ/8 reduced systemic EcoHIV infection, as determined by live bioluminescence imaging. Additionally, this quadruplex vector induced efficient proviral excision, as determined by PCR genotyping in the liver, lungs, brain, and spleen. Finally, in humanized bone marrow/liver/thymus (BLT) mice with chronic HIV-1 infection, successful proviral excision was detected by PCR genotyping in the spleen, lungs, heart, colon, and brain after a single intravenous injection of quadruplex sgRNAs/saCas9 AAV-DJ/8. In conclusion, in vivo excision of HIV-1 proviral DNA by sgRNAs/saCas9 in solid tissues/organs can be achieved via AAV delivery, a significant step toward human clinical trials.
authors.affiliation: Department of Neuroscience, Center for Neurovirology and the Comprehensive NeuroAIDS Center, Temple University Lewis Katz School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA.
authors.forename: Chaoran
authors.initials: C
authors.lastname: Yin
authors.affiliation: Department of Neuroscience, Center for Neurovirology and the Comprehensive NeuroAIDS Center, Temple University Lewis Katz School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA.
authors.forename: Ting
authors.initials: T
authors.lastname: Zhang
authors.affiliation: Department of Radiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA.
authors.forename: Xiying
authors.initials: X
authors.lastname: Qu
authors.affiliation: Department of Neuroscience, Center for Neurovirology and the Comprehensive NeuroAIDS Center, Temple University Lewis Katz School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA.
authors.forename: Yonggang
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Neuroscience, Center for Neurovirology and the Comprehensive NeuroAIDS Center, Temple University Lewis Katz School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA.
authors.forename: Raj
authors.initials: R
authors.lastname: Putatunda
authors.affiliation: Department of Neuroscience, Center for Neurovirology and the Comprehensive NeuroAIDS Center, Temple University Lewis Katz School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA.
authors.forename: Xiao
authors.initials: X
authors.lastname: Xiao
authors.affiliation: Department of Neuroscience, Center for Neurovirology and the Comprehensive NeuroAIDS Center, Temple University Lewis Katz School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA.
authors.forename: Fang
authors.initials: F
authors.lastname: Li
authors.affiliation: Department of Microbiology and Immunology, Temple University Lewis Katz School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA.
authors.forename: Weidong
authors.initials: W
authors.lastname: Xiao
authors.affiliation: Department of Clinical Science, Temple University Lewis Katz School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA.
authors.forename: Huaqing
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Department of Neuroscience, Center for Neurovirology and the Comprehensive NeuroAIDS Center, Temple University Lewis Katz School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA.
authors.forename: Shen
authors.initials: S
authors.lastname: Dai
authors.affiliation: Department of Neuroscience, Center for Neurovirology and the Comprehensive NeuroAIDS Center, Temple University Lewis Katz School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA.
authors.forename: Xuebin
authors.initials: X
authors.lastname: Qin
authors.affiliation: Laboratory of Stem Cell Biology, State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China.
authors.forename: Xianming
authors.initials: X
authors.lastname: Mo
authors.affiliation: Department of Radiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA. Electronic address: bioimaging@mail.com.
authors.forename: Won-Bin
authors.initials: WB
authors.lastname: Young
authors.affiliation: Department of Neuroscience, Center for Neurovirology and the Comprehensive NeuroAIDS Center, Temple University Lewis Katz School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA. Electronic address: kamel.khalili@temple.edu.
authors.forename: Kamel
authors.initials: K
authors.lastname: Khalili
authors.affiliation: Department of Neuroscience, Center for Neurovirology and the Comprehensive NeuroAIDS Center, Temple University Lewis Katz School of Medicine, 3500 N. Broad Street, Philadelphia, PA 19140, USA. Electronic address: whu@temple.edu.
authors.forename: Wenhui
authors.initials: W
authors.lastname: Hu
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D016679:Genome, Viral; D015658:HIV Infections / Q000473:pathology / Q000628:therapy* / Q000821:virology; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011533:Proviruses / Q000235:genetics* / Q000378:metabolism; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics* / Q000378:metabolism; D013211:Staphylococcus aureus / Q000737:chemistry / Q000201:enzymology; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism; D054302:pol Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D009841:Oligonucleotides; D000094704:RNA, Guide, CRISPR-Cas Systems; D054301:gag Gene Products, Human Immunodeficiency Virus; D054302:pol Gene Products, Human Immunodeficiency Virus; D004720:Endonucleases
keywords: AAV; Cas9; HIV; Tg26 mice; animal models; gRNA; gene therapy; genome editing; humanized BLT mice; viral latency