title: TALEN-Mediated Knockout of CCR5 Confers Protection Against Infection of Human Immunodeficiency Virus.
abstract: Transcription activator-like effector nuclease (TALEN) represents a valuable tool for genomic engineering due to its single-nucleotide precision, high nuclease activity, and low cytotoxicity. We report here systematic design and characterization of 28 novel TALENs targeting multiple regions of CCR5 gene (CCR5-TALEN) which encodes the co-receptor critical for entry of human immunodeficiency virus type I (HIV-1). By systemic characterization of these CCR5-TALENs, we have identified one (CCR5-TALEN-515) with higher nuclease activity, specificity, and lower cytotoxicity compared with zinc-finger nuclease (CCR5-ZFN) currently undergoing clinical trials. Sequence analysis of target cell line GHOST-CCR5-CXCR4 and human primary CD4 T cells showed that the double-strand breaks at the TALEN targeted sites resulted in truncated or nonfunctional CCR5 proteins thereby conferring protection against HIV-1 infection in vitro. None of the CCR5-TALENs had detectable levels of off-target nuclease activity against the homologous region in CCR2 although substantial level was identified for CCR5-ZFN in the primary CD4 T cells. Our results suggest that the CCR5-TALENs identified here are highly functional nucleases that produce protective genetic alterations to human CCR5. Application of these TALENs directly to the primary CD4 T cells and CD34 hematopoietic stem cells (HSCs) of infected individuals could help to create an immune system resistant to HIV-1 infection, recapitulating the success of "Berlin patient" and serving as an essential first step towards a "functional" cure of AIDS.
authors.affiliation: *Comprehensive AIDS Research Center and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University, Beijing, China; †Department of Biomedical Engineering, Peking University, Beijing, China; and ‡ViewSolid Biotech, Beijing, China.
authors.forename: Bingjie
authors.initials: B
authors.lastname: Shi
authors.forename: Juan
authors.initials: J
authors.lastname: Li
authors.forename: Xuanling
authors.initials: X
authors.lastname: Shi
authors.forename: Wenxu
authors.initials: W
authors.lastname: Jia
authors.forename: Yi
authors.initials: Y
authors.lastname: Wen
authors.forename: Xiongbing
authors.initials: X
authors.lastname: Hu
authors.forename: Fengfeng
authors.initials: F
authors.lastname: Zhuang
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Xi
authors.forename: Linqi
authors.initials: L
authors.lastname: Zhang
mesh_terms: D000328:Adult; D015496:CD4-Positive T-Lymphocytes / Q000821:virology; D002478:Cells, Cultured; D055786:Gene Knockout Techniques / Q000379:methods*; D015497:HIV-1 / Q000254:growth & development / Q000502:physiology*; D006801:Humans; D019713:Receptors, CCR5 / Q000172:deficiency*; D015728:Receptors, HIV / Q000172:deficiency*; D017384:Sequence Deletion; D000069896:Transcription Activator-Like Effector Nucleases / Q000378:metabolism*
chemical_list: C584933:CCR5 protein, human; D019713:Receptors, CCR5; D015728:Receptors, HIV; D000069896:Transcription Activator-Like Effector Nucleases