565. CCR5-Uco-TALEN – A Novel Transcription Activator-Like Effector Nuclease That Mediates High-Efficiency Knockout of HIV Co-Receptor CCR5 in Primary T Cells After mRNA Transfection

Besides its physiological role, the chemokine receptor CCR5 plays an essential part during HIV infection, acting as the co-receptor for so-called R5-tropic strains that usually mediate initial infection. Notably, homozygosity for CCR5D32, a natural deletion variant of CCR5, largely confers resistance towards HIV infection. Consequently, CCR5 has been proposed as a promising target for HIV therapy. The interest in using CCR5 as a therapeutic target has increased even further after the first report on the cure from HIV in an individual (“Berlin patient”) who received an allogeneic stem cell graft from a CCR5D32-homozygous donor to treat his acute leukemia. Since allogeneic transplantation is not a realistic treatment scenario for HIV, genetic disruption of CCR5 by the means of designer nucleases was proposed as an auspicious gene-therapy approach. We developed a novel transcription activator-like effector nuclease (TALEN), CCR5-Uco-TALEN for highly specific CCR5 knockout. We established efficient TALEN delivery into primary T lymphocytes using mRNA electroporation, a gentle and truly transient gene-transfer technique. After mRNA transfection, CCR5-Uco-TALEN routinely mediated high-rate CCR5 knockout (>90% in PM1 and >50 % in primary T cells) combined with low off-target activity, as demonstrated by flow cytometry, next-generation sequencing and a newly devised, very handy digital-PCR technique. Importantly, CCR5-edited cells were not only resistant towards HIV-derived lentiviral vectors, but also protected from infection with the wild-type CCR5-tropic HIV-1BaL strain. Indeed, 12-day exposure to HIV-1BaL resulted in almost complete suppression of viral replication as well as selection of CCR5-gene edited T cells. In summary, we have introduced a novel TALEN for high-efficiency targeted CCR5 knockout. The proposed mRNA-based gene-editing protocol does not rely on gene-modified organisms and is thus highly compatible with large-scale production and GMP requirements. Together, CCR5-Uco-TALEN and the proposed mRNA-based transfection strategy should therefore be excellently suited for clinical translation.