Preclinical Evaluation of a Novel TALEN Targeting CCR5 Confirms Efficacy and Safety in Conferring Resistance to HIV‐1 Infection,Biotechnology Journal

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Preclinical Evaluation of a Novel TALEN Targeting CCR5 Confirms Efficacy and Safety in Conferring Resistance to HIV‐1 Infection
Biotechnology Journal ( IF 3.2 ) Pub Date : 2020-10-25 , DOI: 10.1002/biot.202000023
Marianna Romito _{1,

2} , Alexandre Juillerat ₃ , Yik Lim Kok _{4,

5} , Markus Hildenbeutel _{1,

2} , Manuel Rhiel _{1,

2} , Geoffroy Andrieux _{2,

6,

7} , Johannes Geiger ₈ , Carsten Rudolph ₈ , Claudio Mussolino _{1,

2} , Aymeric Duclert ₉ , Karin J. Metzner _{4,

5} , Philippe Duchateau ₉ , Toni Cathomen _{1,

2} , Tatjana I. Cornu _{1,

2}

Affiliation

Therapies to treat patients infected with human immunodeficiency virus (HIV) aim at preventing viral replication but fail to eliminate the virus. Although transplantation of allogeneic CCR5Δ32 homozygous stem cell grafts provided a cure for a few patients, this approach is not considered a general therapeutic strategy because of potential side effects. Conversely, gene editing to disrupt the C‐C chemokine receptor type 5 (CCR5) locus, which encodes the major HIV coreceptor, has shown to confer resistance to CCR5‐tropic HIV strains. Here, an engineered transcription activator‐like effector nuclease (TALEN) that enables efficient CCR5 editing in hematopoietic cells is presented. After transferring TALEN‐encoding mRNA into primary CD4+ T cells, up to 89% of CCR5 alleles are disrupted. Genotyping confirms the genetic stability of the CCR5‐edited cells, and genome‐wide off‐target analyses established the absence of relevant mutagenic events. When challenging the edited T cells with CCR5‐tropic HIV, protection in a dose‐dependent manner is observed. Functional assessments reveal no significant differences between edited and control cells in terms of proliferation and their ability to secrete cytokines upon exogenous stimuli. In conclusion, a highly active and specific TALEN to disrupt CCR5 is successfully engineered, paving the way for its clinical application in hematopoietic stem cell grafts.

中文翻译：

对新型TALEN靶向CCR5的临床前评估证实了抗HIV-1感染的有效性和安全性

治疗感染人类免疫缺陷病毒（HIV）的患者的疗法旨在防止病毒复制，但无法消除该病毒。尽管同种异体CCR5Δ32纯合干细胞移植物的移植为少数患者提供了治愈方法，但由于潜在的副作用，该方法未被认为是一般的治疗策略。相反，基因编辑破坏了5型CC趋化因子受体（CCR5）的基因位点，该位点编码主要的HIV共同受体，已显示出对CCR5嗜性HIV菌株的抗性。本文介绍了一种工程化的转录激活因子样效应物核酸酶（TALEN），该酶可在造血细胞中进行有效的CCR5编辑。将编码TALEN的mRNA转移至原代CD4 + T细胞后，多达89％CCR5等位基因被破坏。基因分型证实了CCR5编辑的细胞的遗传稳定性，全基因组脱靶分析确定了不存在相关的诱变事件。当用CCR5嗜性HIV攻击编辑过的T细胞时，观察到剂量依赖性的保护作用。功能评估表明，经编辑的细胞与对照细胞在增殖及其在外源性刺激下分泌细胞因子的能力方面无显着差异。总之，成功设计出了一种高活性和特异性的TALEN来破坏CCR5，为其在造血干细胞移植中的临床应用铺平了道路。

更新日期：2020-10-25

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