4 Discussion
Approaches to disrupt CCR5 in CD4+ T cells have been previously
described. These reports demonstrated the feasibility of abrogating HIV
entry, albeit with some drawbacks including low gene disruption
efficiencies, high off-target effects or absence of specificity
analyses, missing potency analyses, and/or lack of GMP compatibility
[37-40].
In this study, we present efficient editing of the CCR5 locus in
primary CD4+ T cells in a GMP-compliant manner. Under these conditions,
we disrupted up to 90% of CCR5 alleles with no notable
off-target activity, suggesting that the employed TALEN are highly
specific. Disrupting the region that encodes the N-terminus of the CCR5
protein in CD4+ T cells gave rise to cells resistant to infection with
R5-tropic HIV-1 but, as expected, not X4-tropic virus. Importantly,CCR5 edited CD4+ T cells did not show any differences to
non-edited control cells in the applied proliferation and potency
assays. This indicates that TALEN-mediated editing of CCR5 is
efficacious and safe, opening a window for therapeutic applications.
Our data demonstrate that low CCR5 editing frequency is not
sufficient to abrogate HIV infection, putting forward that efficient
disruption that mediates biallelic gene knockouts are paramount to see
clinical effects [16]. This is in line with a recently published
case report confirming that a CCR5 knockout frequency of 5% in
the T cell compartment is not sufficient to achieve clinical benefit
[41].
A crucial point for a clinical trial is to recruit patients exclusively
positive for R5-tropic HIV variants, since the absence of CCR5 would
favor the propagation of X4-tropic viruses if present in the patient.
The importance of proper examination was seen in the ‘Essen patient’
[42] who suffered from an X4 rebound after allogenic transplantation
of a Δ32 homozygous graft. In a CD4+ T transplantation scenario,
performing a knockout of both coreceptor encoding loci CCR5 andCXCR4 can be considered. Strategies employing ZFN or CRISPR-Cas9
in CD4+ T cells conferred in fact resistance to R5- and X4-tropic
viruses [43-45]. This would prevent a rebound of X4-tropic viruses,
but concomitant generation of two DNA double strand breaks drastically
increase the probability for translocations and thus the risks of
malignancies.
It was recently discovered, that a lack of CCR5 is associated with worse
clinical outcomes in West Nile Virus infections, although CCR5 is not
playing a direct role in the infection cycle [46]. This implies that
one should carefully assess the risks and benefits of applyingCCR5 edited cells in regions where West Nile Virus infections are
prevalent.
A side effect of gene editing approaches is genotoxicity as a
consequence of OT effects. OT activity can induce mutagenesis but also
provoke chromosomal rearrangements, both potentially inducing
malignancies. Employing highly specific designer nucleases is therefore
paramount to mitigate the risk of such effects. Several tools are
available to identify OT events, one of them being in silicopredictions. The reported studies [37, 39] analyzed off-target
events using this tool with the drawback of having some biases, which
leads to overseeing some important off-targets. Another study [38]
performed off-target analysis using whole genome sequencing. This method
does not reach a sufficient depth to detect rare events. In our study,
we carefully evaluated OT using both in silico prediction as well
as by employing an unbiased in vivo oligonucleotide capturing
assay. We detected two rare OT events occurring in introns of genes,
thus minimizing the risks of adverse effects. For instance, the OT site
in CNOT10 (OT1), which was also detected by the OCA (OCA3), was
cleaved in about 0.12% of cells. OCA identified three additional OT
sites, one of them in the CCR2 gene. But again, the OCA scores of
these OTs was similarly low as OCA3, suggesting OT activities in the
range of 0.1% Moreover, targeting terminally differentiated cells, like
CD4+ T cells, furthermore mitigates the risk of developing malignancies.
Besides, all functional assays we performed in this study demonstrated
that the CCR5 edited cells behaved as unedited cells, suggesting
that TALEN expression did not have any impact on these cells.
All in all, we show that we can manufacture high functioning edited CD4+
T cells with low genotoxicity that can be scaled up to clinical relevant
numbers and be applied to patients.