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.

CRISPR/Cas9 was described as a bacterial immune system that uses targeted introduction of DNA double-strand breaks (DSBs) to destroy invaders. We hypothesized that we can analogously employ CRISPR/Cas9 nucleases to kill cancer cells by inducing maximal numbers of DSBs in their genome and thus triggering programmed cell death. To do so, we generated CRISPR-to-kill (C2K) lentiviral particles targeting highly repetitive Short Interspersed Nuclear Element-Alu sequences. Our Alu-specific sgRNA has more than 15,000 perfectly matched target sites within the human genome. C2K-Alu-vectors selectively killed human, but not murine cell lines. More importantly, they efficiently inhibited the growth of cancer cells including patient-derived glioblastoma cell lines resistant to high-dose irradiation. Our data provide proof-of-concept for the potential of C2K as a novel treatment strategy overcoming common resistance mechanisms. In combination with tumor-targeting approaches, the C2K system might therefore represent a promising tool for cancer gene therapy.

Ex-vivo gene editing in T lymphocytes paves the way for novel concepts of immunotherapy. One of those strategies is directed at the protection of CD4+-T helper cells from HIV infection in HIV-positive individuals. To this end, we have developed and optimised a CCR5-targeting TALE nuclease, CCR5-Uco-hetTALEN, mediating high-efficiency knockout of C-C motif chemokine receptor 5 (CCR5), the HIV co-receptor essential during initial infection. Clinical translation of the knockout approach requires up-scaling of the manufacturing process to clinically relevant cell numbers in accordance with good manufacturing practice (GMP). Here we present a GMP-compatible mRNA electroporation protocol for the automated production of CCR5-edited CD4+-T cells in the closed CliniMACS Prodigy system. The automated process reliably produced high amounts of CCR5-edited CD4+-T cells (>1.5 × 109 cells with >60% CCR5 editing) within 12 days. Of note, about 40% of total large-scale produced cells showed a biallelic CCR5 editing, and between 25 and 42% of produced cells had a central memory T-cell phenotype. In conclusion, transfection of primary T cells with CCR5-Uco-hetTALEN mRNA is readily scalable for GMP-compatible production and hence suitable for application in HIV gene therapy.

The Gibbon Ape Leukemia Virus envelope protein (GALV-Env) mediates efficient transduction of human cells, particularly primary B and T lymphocytes, and is therefore of great interest in gene therapy. Using internal domains from murine leukemia viruses (MLV), chimeric GALV-Env proteins such as GALV-C4070A were derived, which allow pseudotyping of lentiviral vectors. In order to improve expression efficiency and vector titers, we developed a codon-optimized (co) variant of GALV-C4070A (coGALV-Env). We found that coGALV-Env mediated efficient pseudotyping not only of γ-retroviral and lentiviral vectors, but also α-retroviral vectors. The obtained titers on HEK293T cells were equal to those with the classical GALV-Env, whereas the required plasmid amounts for transient vector production were significantly lower, namely, 20 ng coGALV-Env plasmid per 106 293T producer cells. Importantly, coGALV-Env-pseudotyped γ- and α-retroviral, as well as lentiviral vectors, mediated efficient transduction of primary human T cells. We propose that the novel chimeric coGALV-Env gene will be very useful for the efficient production of high-titer vector preparations, e.g., to equip human T cells with novel specificities using transgenic TCRs or CARs. The considerably lower amount of plasmid needed might also result in a significant cost advantage for good manufacturing practice (GMP) vector production based on transient transfection.

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