mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5

Abstract

Homozygosity for a natural deletion variant of the HIV-coreceptor molecule CCR5, CCR5Δ32, confers resistance toward HIV infection. Allogeneic stem cell transplantation from a CCR5Δ32-homozygous donor has resulted in the first cure from HIV (‘Berlin patient’). Based thereon, genetic disruption of CCR5 using designer nucleases was proposed as a promising HIV gene-therapy approach. Here we introduce a novel TAL-effector nuclease, CCR5-Uco-TALEN that can be efficiently delivered into T cells by mRNA electroporation, a gentle and truly transient gene-transfer technique. CCR5-Uco-TALEN mediated high-rate CCR5 knockout (>90% in PM1 and >50% in primary T cells) combined with low off-target activity, as assessed by flow cytometry, next-generation sequencing and a newly devised, very convenient gene-editing frequency digital-PCR (GEF-dPCR). GEF-dPCR facilitates simultaneous detection of wild-type and gene-edited alleles with remarkable sensitivity and accuracy as shown for the CCR5 on-target and CCR2 off-target loci. CCR5-edited cells were protected from infection with HIV-derived lentiviral vectors, but also with the wild-type CCR5-tropic HIV-1BaL strain. Long-term exposure to HIV-1BaL resulted in almost complete suppression of viral replication and selection of CCR5-gene edited T cells. In conclusion, we have developed a novel TALEN for the targeted, high-efficiency knockout of CCR5 and a useful dPCR-based gene-editing detection method.

Engineering HIV-Resistant, Anti-HIV Chimeric Antigen Receptor T Cells

“…36,37 Individuals with an allelic variant that is not functional (CCR5D32) are protected from CCR5-tropic HIV infection. 38 Hematopoietic stem cell transplant using a CCR5D32 donor led to the only known cure of HIV-1 infection, 39,40 and T cells treated with engineered nucleases that introduce mutations at the CCR5 locus are resistant to HIV, [41][42][43][44][45] accelerating ongoing efforts to develop gene editing-and cell-based therapeutic agents for HIV. 11,46 Using new gene-editing techniques, it has recently become possible to achieve high rates of homology-directed recombination (HDR) of therapeutic cassettes into targeted loci, including CCR5 in primary T cells.…”

Genome-Editing Technologies: Principles and Applications

“…However, TALENs are substantially larger than ZFNs, and have a highly repetitive structure, making their efficient delivery into cells through the use of lentivirus (Holkers et al 2013) or a single adeno-associated virus (AAV) particle challenging. Methods for overcoming these limitations have emerged as TALENs can be readily delivered into cells as mRNA (Mahiny et al 2015;Mock et al 2015) and even protein Liu et al 2014a), although alternative codon usage and amino acid degeneracy can also be leveraged to express RVD arrays that might be less susceptible to recombination (Kim et al 2013a). In addition, adenoviral vectors have also proven particularly useful for mediating TALEN delivery to hard-to-transfect cell types (Holkers et al 2014;Maggio et al 2016).…”

Clinical implementation of genome editing for correction of human diseases

“…For example, highly effective CCR5 modification was demonstrated for human T lymphocytes using TALENbased approach [18,19]. Preliminary results from a joint research group of the First Palvov State Medical University of St. Petersburg and Hamburg University have shown 40 per cent efficiency for the CCR5 gene modification using CCR5-Uco-TALEN system (on behalf of CIC725, unpublished data).…”