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

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.

The Ins and Outs of Messenger RNA Electroporation for Physical Gene Delivery in Immune Cell-Based Therapy

Messenger RNA (mRNA) electroporation is a powerful tool for transient genetic modification of cells. This non-viral method of genetic engineering has been widely used in immunotherapy. Electroporation allows fine-tuning of transfection protocols for each cell type as well as introduction of multiple protein-coding mRNAs at once. As a pioneering group in mRNA electroporation, in this review, we provide an expert overview of the ins and outs of mRNA electroporation, discussing the different parameters involved in mRNA electroporation as well as the production of research-grade and production and application of clinical-grade mRNA for gene transfer in the context of cell-based immunotherapies.

Electroporation of Dicer-Substrate siRNA Duplexes Targeting Endogenous TCR Enhance Tumor Killing Activity of Wilms' Tumor 1 (WT1)-Specific TCR-Redirected Cytotoxic T Cells

In adoptive cellular immunotherapy, T cells can be genetically engineered to express a novel T-cell receptor (TCR) that recognizes a tumor-associated antigen. However, mispairing between transgene and endogenous TCR chains may result in a reduction of transgene TCR expression and potentially harmful off-target reactivities. Here, we sought to develop a novel clinically safe strategy to promote transgene expression of a Wilms' tumor 1 (WT1)-specific TCR by Dicer-substrate small interfering RNA (DsiRNA)-mediated silencing of the endogenous TCR, using a double electroporation protocol. First, we isolated and cloned an HLA-A*0201-restricted WT1 peptide-specific TCR derived from a leukemia patient who demonstrated clinical benefit after receiving a WT1-targeted DC vaccine. Next, we produced a codon-optimized TCR sequence from the wild-type TCR construct and both TCR mRNAs were generated by in vitro transcription. TCR expression levels were validated by electroporation of TCR-deficient Jurkat J76.7 cells stably transduced with CD8 and an NFAT-driven GFP reporter gene. TCR functionality was confirmed by high expression levels of GFP (70% GFP+cells) upon TCR signaling after co-culture with WT1 peptide-pulsed T2 cells. In order to suppress the translation of endogenous TCR mRNA in CD8+ T cells, DsiRNA duplexes were designed to specifically target the constant regions of wild-type TCR α- and β-chains, but not the codon-optimized TCR. We further developed a double electroporation protocol combining DsiRNA and TCR mRNA transfection in which DsiRNA electroporation was performed 24 hours prior to TCR mRNA electroporation. Our results show more than 2-fold increase in WT1-specific TCR expression by HLA-A2/WT1 tetramer staining after DsiRNA treatment as compared to TCR mRNA electroporation only. This specific TCR expression was maintained at least 5 days after TCR mRNA electroporation in resting peripheral blood CD8+ lymphocytes from healthy donors. The enhanced TCR expression in DsiRNA-transfected CD8+T cells was also correlated with an increase of epitope recognition as shown by interferon (IFN)-γ ELISpot. To determine the killing capacity of DsiRNA/TCR mRNA-transfected CD8+ T cells against epitope-bearing target cells, we performed a flow cytometry-based cytotoxicity assay using WT1 peptide-pulsed T2 cells. Specific cytotoxicity, which was already present in WT1 TCR-transfected cells, was significantly enhanced in TCR mRNA-electroporated T cells following suppression of the endogenous TCR expression by DsiRNA treatment. Accordingly, DsiRNA-treated TCR mRNA transfected CD8+T cells presented higher levels of CD137 and CD69 activation markers and secretion of cytokines (IFN-γ and tumor necrosis factor-α), granzyme B, and perforin upon TCR triggering as compared to the non-DsiRNA treated T cells. In summary, we show a marked enhancement of transgene WT1-specific TCR expression upon silencing of the endogenous TCR using DsiRNA electroporation prior to TCR mRNA electroporation. Importantly, this enhancement in TCR expression was correlated with a significant increase in WT1-specific CD8+ T-cell killing activity, expression of CD69 and CD137 activation markers and cytokine secretion after recognition of WT1 peptide-bearing target cells. These results pave the way for developing a clinically safer strategy for T cell-based adoptive immunotherapy of patients with WT1-expressing malignancies. Disclosures No relevant conflicts of interest to declare.