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.