Preclinical Evaluation of a Novel TALEN Targeting <i>CCR5</i> Confirms Efficacy and Safety in Conferring Resistance to HIV‐1 Infection

Romito, Marianna; Juillerat, Alexandre; Kok, Yik Lim; Hildenbeutel, Markus; Rhiel, Manuel; Andrieux, Geoffroy; Geiger, Johannes; Rudolph, Carsten; Mussolino, Claudio; Duclert, Aymeric; Metzner, Karin J.; Duchâteau, Philippe; Cathomen, Toni; Cornu, Tatjana I.

doi:10.1002/biot.202000023

Cited by 20 publications

(19 citation statements)

References 51 publications

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“…Likewise, monitoring of T-cell proliferation after electroporation with or without TALEN revealed no differences. Together, this data supports that CCR5 editing has no influence on T-cell fitness, as described by other groups, as well [ 41 , 64 ].…”

Section: Discussionsupporting

confidence: 91%

“…Higher editing efficiencies were possible but associated with substantially increased off-target activity at the highly similar CCR2 locus. Our editing rates are well in line with previous [ 18 ] and novel data [ 41 ] for selected, high-efficiency TALENs. In comparison to zinc finger nucleases, which were already tested in a clinical study, our CCR5-Uco-hetTALEN showed higher CCR5 -editing rates with better safety profile [ 40 , 42 ].…”

Section: Discussionsupporting

confidence: 90%

“…CCR5 has emerged as a preferred target, particularly for gene-therapeutic strategies, not the least in view of successful HIV elimination in two patients after transplantation of CCR5-negative HSCs [ 14 , 15 ]. A number of groups, including our own [ 18 , 19 ], have developed designer nucleases that target CCR5 to endow gene-modified cells with resistance to CCR5-tropic HIV [ 34 – 41 ].…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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“…This study put forward a broad prospect of using TALENS to generate site-specific, size-controlled, and homozygous DNA deletions within mammalian genomes. Additionally, after transferring TALEN-encoding mRNA into CD4+ T cells, the CCR5 alleles were disrupted by 90%, whereas the off-target activity was absent and the edited cells proliferated normally (30). As a result, TALEN-mediated CCR5 disruption of CD4+ T cells is paving the way for clinical applications in CD34+ HSCs.…”

Section: Transcription-activator-like Effector Nucleasesmentioning

confidence: 99%

Knowledge From London and Berlin: Finding Threads to a Functional HIV Cure

2021

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Despite the ability of combination antiretroviral therapy (cART) to increase the life expectancy of patients infected with human immunodeficiency virus (HIV), viral reservoirs persist during life-long treatment. Notably, two cases of functional cure for HIV have been reported and are known as the “Berlin Patient” and the “London Patient”. Both patients received allogeneic hematopoietic stem cell transplantation from donors with homozygous CCR5 delta32 mutation for an associated hematological malignancy. Therefore, there is growing interest in creating an HIV-resistant immune system through the use of gene-modified autologous hematopoietic stem cells with non-functional CCR5. Moreover, studies in CXCR4-targeted gene therapy for HIV have also shown great promise. Developing a cure for HIV infection remains a high priority. In this review, we discuss the increasing progress of coreceptor-based hematopoietic stem cell gene therapy, cART, milder conditioning regimens, and shock and kill strategies that have important implications for designing potential strategies aiming to achieve a functional cure for the majority of people with HIV.

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“…Finally, the paper by Romito et al. [ 14 ] presents a viral vector‐free ex vivo gene therapy approach based on the use of electroporation in view of gene editing of primary CD4+ T cells for rendering them resistant to HIV‐1 infection (disruption of CCR5 alleles). In addition to the stable modification, the absence of mutagenic events was shown.…”

mentioning

confidence: 99%