Journal Article

In Vivo Excision of HIV-1 Provirus by saCas9 and Multiplex Single-Guide RNAs in Animal Models

Qin, Xuebin, Zhao, Huaqing, Zhang, Ting, Li, Fang, Xiao, Xiao, Young, Won-Bin, Mo, Xianming, Yin, Chaoran, Xiao, Weidong, Dai, Shen, Zhang, Yonggang, Putatunda, Raj, Khalili, Kamel, Qu, Xiying, Hu, Wenhui

2017

Overview

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. [Display omitted] Yin et al. use multiplex CRISPR/Cas9 genome editing technology to excise the HIV-1 provirus in a precise manner in three different HIV-1 animal models via in vivo AAV gene delivery. The feasibility of HIV excision in infected cells in vivo paves the way toward human clinical trials to cure HIV-1 infection.

• Publisher: Elsevier Inc,Elsevier Limited,American Society of Gene & Cell Therapy
• Subject: AAV / Acquired immune deficiency syndrome / Advisors / AIDS / Animal models / Animals / Bacterial Proteins - genetics / Bacterial Proteins - metabolism / Base Sequence / Bioluminescence / Bone marrow / Cas9 / Chronic infection / Clinical trials / Clustered Regularly Interspaced Short Palindromic Repeats / Colon / CRISPR / CRISPR-Cas Systems / Deoxyribonucleic acid / Dependovirus - genetics / Dependovirus - metabolism / Disease Models, Animal / DNA / Efficiency / Endonucleases - genetics / Endonucleases - metabolism / gag Gene Products, Human Immunodeficiency Virus - genetics / gag Gene Products, Human Immunodeficiency Virus - metabolism / Gene Editing - methods / gene therapy / Gene transfer / Genes / Genetic Therapy - methods / Genetic Vectors - chemistry / Genetic Vectors - metabolism / Genome editing / Genome, Viral / Genomes / Genotyping / gRNA / HIV / HIV Infections - pathology / HIV Infections - therapy / HIV Infections - virology / HIV Long Terminal Repeat / HIV-1 - genetics / HIV-1 - metabolism / Human immunodeficiency virus / humanized BLT mice / Humans / Infections / Intravenous administration / Liver / Mice / Mice, Transgenic / Neural stem cells / Neuroimaging / Oligonucleotides - genetics / Oligonucleotides - metabolism / Original / pol Gene Products, Human Immunodeficiency Virus - genetics / pol Gene Products, Human Immunodeficiency Virus - metabolism / Polymerase chain reaction / Progenitor cells / Proviruses - genetics / Proviruses - metabolism / Ribonucleic acid / RNA / RNA, Guide, CRISPR-Cas Systems - genetics / RNA, Guide, CRISPR-Cas Systems - metabolism / Spleen / Staphylococcus aureus - chemistry / Staphylococcus aureus - enzymology / Studies / Tg26 mice / Thymus / Transgenic animals / Transgenic mice / viral latency