mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5

• Published in: Nucleic acids research Vol. 43; no. 11; pp. 5560 - 5571
• Main Authors: Mock, Ulrike, Machowicz, Rafał, Hauber, Ilona, Horn, Stefan, Abramowski, Pierre, Berdien, Belinda, Hauber, Joachim, Fehse, Boris
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 23.06.2015
• Subjects: Cell Line; Deoxyribonucleases - chemistry; Deoxyribonucleases - genetics; DNA End-Joining Repair; Electroporation; Gene Knockout Techniques - methods; HIV - physiology; HIV Infections - genetics; Human immunodeficiency virus; Humans; Mutation; Nucleic Acid Enzymes; Polymerase Chain Reaction; Receptors, CCR5 - genetics; RNA, Messenger; T-Lymphocytes - virology; Transfection
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkv469

Homozygosity for a natural deletion variant of the HIV-coreceptor molecule CCR5, CCR5Δ32, confers resistance toward HIV infection. Allogeneic stem cell transplantation from a CCR5Δ32-homozygous donor has resulted in the first cure from HIV ('Berlin patient'). Based thereon, genetic disruption of CCR5 using designer nucleases was proposed as a promising HIV gene-therapy approach. Here we introduce a novel TAL-effector nuclease, CCR5-Uco-TALEN that can be efficiently delivered into T cells by mRNA electroporation, a gentle and truly transient gene-transfer technique. CCR5-Uco-TALEN mediated high-rate CCR5 knockout (>90% in PM1 and >50% in primary T cells) combined with low off-target activity, as assessed by flow cytometry, next-generation sequencing and a newly devised, very convenient gene-editing frequency digital-PCR (GEF-dPCR). GEF-dPCR facilitates simultaneous detection of wild-type and gene-edited alleles with remarkable sensitivity and accuracy as shown for the CCR5 on-target and CCR2 off-target loci. CCR5-edited cells were protected from infection with HIV-derived lentiviral vectors, but also with the wild-type CCR5-tropic HIV-1BaL strain. Long-term exposure to HIV-1BaL resulted in almost complete suppression of viral replication and selection of CCR5-gene edited T cells. In conclusion, we have developed a novel TALEN for the targeted, high-efficiency knockout of CCR5 and a useful dPCR-based gene-editing detection method.