S12 Dataset -

Drug-based antiretroviral therapies (ART) efficiently suppress HIV replication in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. Importantly, ART cannot eliminate HIV from an infected individual, since it does not target the integrated provirus. Therefore, genome editing-based strategies that can inactivate or excise HIV genomes would provide the technology for novel curative therapies. In fact, the HIV-1 LTR-specific designer-recombinase Brec1 has been shown to remove integrated proviruses from infected cells and is highly efficacious on clinical HIV-1 isolates in vitro and in vivo, suggesting that Brec1 has the potential for clinical development of advanced HIV-1 eradication strategies in people living with HIV. In line with the preparation of a first-in-human advanced therapy medicinal product gene therapy trial, we here present an extensive preclinical evaluation of Brec1 and lentiviral vectors expressing the Brec1 transgene. This included detailed functional analysis of potential genomic off-target sites, assessing vector safety by investigating vector copy number (VCN) and the risk for potential vector-related insertional mutagenesis, as well as analyzing the potential of Brec1 to trigger an undesired strong T cell immune response. In conclusion, the antiviral designer-recombinase Brec1 is shown to lack any detectable cytopathic, genotoxic or T cell-related immunogenic effects, thereby meeting an important precondition for clinical application of the therapeutic lentiviral vector LV-Brec1 in novel HIV-1 curative strategies.基于药物的抗逆转录病毒疗法（antiretroviral therapies, ART）可高效抑制人体内的HIV复制，但病毒会以整合型前病毒储库的形式持续存在于少量细胞中。值得注意的是，ART无法从感染者体内清除HIV，因其无法靶向整合型前病毒。因此，能够灭活或切除HIV基因组的基于基因组编辑的策略，将为新型治愈疗法提供技术支撑。事实上，特异性靶向HIV-1长末端重复序列（Long Terminal Repeat, LTR）的定制重组酶Brec1已被证实可从感染细胞中切除整合型前病毒，且在体外与体内对临床分离的HIV-1毒株均具有极高效力，这表明Brec1具备开发先进HIV-1根除策略的临床转化潜力，以造福HIV感染者。为筹备首次用于人体的先进治疗药物基因治疗临床试验，本文对Brec1以及表达Brec1转基因的慢病毒载体开展了全面的临床前评价。该评价涵盖了潜在基因组脱靶位点的详细功能分析，通过检测载体拷贝数（vector copy number, VCN）以及潜在载体相关插入诱变风险来评估载体安全性，同时分析了Brec1引发不期望出现的强烈T细胞免疫应答的可能性。综上，本研究证实抗病毒定制重组酶Brec1未表现出任何可检测到的细胞病变、遗传毒性或T细胞相关免疫原性效应，从而满足了治疗性慢病毒载体LV-Brec1应用于新型HIV-1治愈策略的临床应用所需的重要前提条件。