CCR5 knockout is genetically stable in CD4+ T cells
The target locus in exon 3 of the CCR5 gene was chosen to be in
the region coding for the N-terminus of the CCR5 protein (Fig. 1A). The
rationale was two-fold: (1) out-of-frame insertion/deletion (indel)
mutations will cause frameshift mutations leading to early termination
of translation, and (2) it encodes a conserved region of CCR5 that is
bound by HIV-1 gp120. Even in-frame indels will remove critical residues
necessary for HIV-1 to bind to CCR5 [32, 33]. We first established
the optimal conditions to achieve high CCR5 disruption in CD4+ T
cells from different donors with TALEN encoding mRNA via
electroporation. Cells were cultivated for several days and subjected to
T7E1 assay at days 7 and 21 of culture to monitor gene disruption
frequencies. A transient cold shock increased allelic CCR5disruption frequency from 50% to 85% (Fig. 1B). The indel frequencies
ranged between 50–90% at day 7 of culture and stabilized around 85%
after 21 days, demonstrating stable editing in long term cultures. We
also evaluated the effect of the gene disruption on the surface
expression of CCR5. CCR5 expression varies between cell subsets and
donors [34] as well as during activation, therefore staining was
performed at the same culture stage. The percentage of CCR5 positive
cells was significantly decreased (p = 0.0009) after editing with
TALEN while no significant decrease in CCR5 expression was detected in
control samples treated with only the left TALEN arm (Fig. 1C).
Reduction of CCR5 protein expression remained significant (p =
0.0314) and stable for 21 days , which paired well with the observations
made on the genomic level (Fig. 1B),
In parallel we edited cells from a
Δ32 heterozygous donor. Allelic gene
disruption rates reached 69% at day 7 and 86% at day 21 (Fig. 1B), so
well within the ranges of the homozygous CCR5 wt donors. When performing
a phenotypic analysis, a drop of 50% in CCR5 expression was also
observed for this donor (Fig. 1C, red dots), in line with the results
seen with the homozygous donors.
In order to better understand the type of indels generated by the
nucleases, we performed next generation sequencing (NGS) analysis on the
edited CCR5 target locus in 3 different donors, one of which
being Δ32 heterozygous, and analyzed the indels at days 7 and 21,
respectively (Fig. 1D-E). The indel patterns and frequencies remained
comparable between samples and over time. There was no significant
enrichment or depletion of a specific indel type detected by a pairedt -test during the culture period. This indicates that the clonal
distribution of the edited cells between the two time points remained
stable. In all 3 donors, the most frequent indel was a 19-nt deletion,
followed by a 9-nt deletion, that combined represented about 20% of the
total indel mutations. Of note, the four most frequent deletions -19,
-9, -13, and -26 are likely due to microhomologies, as detected by the
Microhomology-Predictor tool [35].