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

Abstract

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 CCR5Δ55–60 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.

Detection of CCR5Δ32 Mutant Alleles in Heterogeneous Cell Mixtures Using Droplet Digital PCR

“…Possible competitors to the use of ddPCR are the NGS sequencing methods ( Malicherova et al, 2018 ; Dong et al, 2018 ), which theoretically, with their great depth of reading, can provide the highest efficiency and accuracy. A number of studies have used NGS sequencing data for off-target and on-target editing at CCR5 locus to evaluate the results of inactivation of the CCR5 gene ( Liu et al, 2017 ; Schwarze et al, 2021 ). But the disadvantage of these methods is still the high cost of analysis and the need to use bioinformatics methods when analyzing the results.…”

Targeting CCR5 as a Component of an HIV-1 Therapeutic Strategy

“…No research using TALENs to disrupt CCR5 in HSPC has yet to be published as of this writing. The main TALEN tool developed, CCR5-Uco-hetTALEN, includes a heterodimeric Fok1-cleavage domain and almost completely reduces off-target effects, with the notable exception of the highly homologous CCR2 ( 145 ). This technology has advanced so much that it is now automated and can reliably generate the CCR5 knockdown in frequencies above 60% within primary T cells, 40% of which can be biallelic CCR5 mutations ( 146 ).…”

Closing the Door with CRISPR: Genome Editing of CCR5 and CXCR4 as a Potential Curative Solution for HIV

“…As discussed, CCR5 and CXCR4 are the best described co-receptors [ 9 , 32 , 35 ]. Combined with the fact that the CCR5Δ32/Δ32 mutation confers resistance to infection [ 14 , 43 ], these molecules could be attractive targets as a potential cure, and as such have been investigated as targets to render cells refractive to HIV entry [ 44 , 45 ]. Therefore, the remainder of this review will focus mostly on these two targets.…”