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Yin C , Zhang T , Qu X , Zhang Y , Putatunda R , Xiao X , Li F , Xiao W , Zhao H , Dai S , Qin X , Mo X , Young WB , Khalili K , Hu W
In Vivo Excision of HIV-1 Provirus by saCas9 and Multiplex Single-Guide RNAs in Animal Models
Mol Ther. 2017 May 3;25(5) :1168-1186
PMID: 28366764    PMCID: PMC5417847   
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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.
1525-0024 Yin, Chaoran Zhang, Ting Qu, Xiying Zhang, Yonggang Putatunda, Raj Xiao, Xiao Li, Fang Xiao, Weidong Zhao, Huaqing Dai, Shen Qin, Xuebin Mo, Xianming Young, Won-Bin Khalili, Kamel Hu, Wenhui T32 MH079785/MH/NIMH NIH HHS/United States P30 CA047904/CA/NCI NIH HHS/United States R01 NS087971/NS/NINDS NIH HHS/United States R21 NS094084/NS/NINDS NIH HHS/United States P30 MH092177/MH/NIMH NIH HHS/United States R21 MH100949/MH/NIMH NIH HHS/United States Journal Article United States Mol Ther. 2017 May 3;25(5):1168-1186. doi: 10.1016/j.ymthe.2017.03.012. Epub 2017 Mar 30.