Optimisation of a TALE nuclease targeting the HIV co-receptor CCR5 for clinical application

• Published in: Gene therapy Vol. 28; no. 9; pp. 588 - 601
• Main Authors: Schwarze, Lea Isabell, Głów, Dawid, Sonntag, Tanja, Uhde, Almut, Fehse, Boris
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.09.2021; Nature Publishing Group
• Subjects: 38; 38/77; 42; 42/109; 45/23; 631/1647/2300/1851; 692/699/255; Acquired immune deficiency syndrome; AIDS; Binding sites; Biomedical and Life Sciences; Biomedicine; Care and treatment; CC chemokine receptors; CCR2 protein; CCR5 protein; Cell Biology; Chemokine receptors; Cloning; Cytosol; Electroporation; Endonucleases - genetics; Gene deletion; Gene Expression; Gene Therapy; Genetic aspects; Genetic research; Genetic vectors; Genome editing; HIV; HIV infection; HIV Infections - genetics; HIV Infections - therapy; Human Genetics; Human immunodeficiency virus; Humans; Infections; Lymphocytes; Lymphocytes T; Methods; Monocyte chemoattractant protein 1; mRNA; Nanotechnology; Nuclease; Receptors, CCR2; Receptors, CCR5 - genetics; Restriction enzymes, DNA; Stem cells; T-Lymphocytes; Transcription Activator-Like Effector Nucleases; Vectors (Biology); Germany
• ISSN: 0969-7128; 1476-5462; 1476-5462
• DOI: 10.1038/s41434-021-00271-9

Disruption of the C-C-Chemokine-receptor-5 ( CCR5 ) gene induces resistance towards CCR5-tropic HIV. Here we optimised our previously described CCR5-Uco-TALEN and its delivery by mRNA electroporation. The novel variant, CCR5-Uco-hetTALEN features an obligatory heterodimeric Fok1-cleavage domain, which resulted in complete abrogation of off-target activity at previously found homodimeric as well as 7/8 in silico predicted, potential heterodimeric off-target sites, the only exception being highly homologous CCR2 . Prevailing 18- and 10-bp deletions at the on-target site revealed microhomology-mediated end-joining as a major repair pathway. Notably, the CCR5 Δ55–60 protein resulting from the 18-bp deletion was almost completely retained in the cytosol. Simultaneous cutting at CCR5 and CCR2 induced rearrangements, mainly 15-kb deletions between the cut sites, in up to 2% of T cells underlining the necessity to restrict TALEN expression. We optimised in vitro mRNA production and showed that CCR5 -on- and CCR2 off-target activities of CCR5-Uco-hetTALEN were limited to the first 72 and 24–48 h post-mRNA electroporation, respectively. Using single-cell HRMCA, we discovered high rates of TALEN-induced biallelic gene editing of CCR5 , which translated in large numbers of CCR5-negative cells resistant to HIVenv-pseudotyped lentiviral vectors. We conclude that CCR5-Uco-hetTALEN transfected by mRNA electroporation facilitates specific, high-efficiency CCR5 gene-editing (30%–56%) and it is highly suited for clinical translation subject to further characterisation of off-target effects.