人T细胞白血病病毒1型（HTLV-1）是一种致病性逆转录病毒，作为原生质体存在于感染细胞的基因组中，并可能导致成人T细胞白血病（ATL）。全球有超过1000万人感染，其中约有5％的人将发展成高度侵袭性且目前无法治愈的癌症ATL。近年来，基因组编辑工具已成为有前途的抗病毒药物。在这个概念验证研究中，我们使用基质链路定向进化（SLiDE）来设计Cre衍生的位点特异性重组酶，以从感染的细胞中切除HTLV-1原生质体基因组。我们确定一种类似loxP的保守序列（loxHTLV），存在于大部分病毒分离物的长末端重复里。经过181个SLiDE循环，我们分离出一个设计重组酶（命名为RecHTLV），它可以在细菌和人类细胞中高特异性地重组loxHTLV序列。在人类Jurkat T细胞中表达RecHTLV，挑战HTLV-1感染时表现出抗病毒活性。此外，在慢性感染的SP细胞中表达RecHTLV导致HTLV-1原生质体DNA的切除。我们的数据表明，重组酶介导的HTLV-1原生质体的切除是降低HTLV-1感染个体原生质量的有前途的方法，可以预防HTLV-1相关疾病的发展。版权所有©2023作者。由Elsevier Inc.出版。保留所有权利。

The Human T-cell leukaemia virus type 1 (HTLV-1) is a pathogenic retrovirus that persists as a provirus in the genome of infected cells and can lead to adult T-cell leukaemia (ATL). Worldwide, more than 10 million people are infected and approximately 5% of these individuals will develop ATL, a highly aggressive cancer that is currently incurable. In the last years, genome editing tools have emerged as promising antiviral agents. In this proof-of-concept study, we used substrate linked directed evolution (SLiDE) to engineer Cre-derived site-specific recombinases to excise the HTLV-1 proviral genome from infected cells. We identified a conserved loxP-like sequence (loxHTLV) present in the long terminal repeats of the majority of virus isolates. After 181 cycles of SLiDE, we isolated a designer-recombinase (designated RecHTLV), which efficiently recombines the loxHTLV sequence in bacteria and human cells with high specificity. Expression of RecHTLV in human Jurkat T-cells resulted in antiviral activity when challenged with an HTLV-1 infection. Moreover, expression of RecHTLV in chronically infected SP cells led to the excision of HTLV-1 proviral DNA. Our data suggest that recombinase mediated excision of the HTLV-1 provirus represents a promising approach to reduce proviral load in HTLV-1 infected individuals, potentially preventing the development of HTLV-1 associated diseases.Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.