Karpinski Janet, Chemnitz Jan, Hauber Ilona, Abi-Ghanem Josephine, Paszkowski-Rogacz Maciej, Surendranath Vineeth, Chakrabort Debojyoti, Hackmann Karl, Schröck Evelin, Pisabarro María Teresa, Hauber Joachim, Buchholz Frank
Medical Systems Biology, Medical Faculty, TU Dresden, Dresden, Germany.
Antiviral Strategies, Heinrich Pette Institute - Leibniz Institute for Experimental Virology, Hamburg, Germany.
J Int AIDS Soc. 2014 Nov 2;17(4 Suppl 3):19706. doi: 10.7448/IAS.17.4.19706. eCollection 2014.
Current drugs against HIV can suppress the progression to AIDS but cannot clear the patient from the virus. Because of potential side effects of these drugs and the possible development of drug resistance, finding a cure for HIV infection remains a high priority of HIV/AIDS research. We recently generated a recombinase (termed Tre) tailored to efficiently eradicate the provirus from the host genome of HIV-1 infected cells by specifically targeting a sequence that is present in the long terminal repeats (LTRs) of the viral DNA [1]. In vivo analyses in HIV-infected humanized mice demonstrated highly significant antiviral effects of Tre recombinase [2]. However, the fact that Tre recognizes a particular HIV-1 subtype A strain may limit its broad therapeutic application. To advance our Tre-based strategy towards a universally efficient cure, we have engineered a new, universal recombinase (uTre) applicable to the majority of HIV-1 infections by the various virus strains and subtypes. We employed the search tool SeLOX [3] in order to find a well-conserved HIV-1 proviral sequence that could serve as target site for a universal Tre from sequences compiled in the Los Alamos HIV Sequence Database. We selected a candidate (termed loxLTRu) with a mean conservation rate of 94% throughout the major HIV-1 subtype groups A, B and C. We applied loxLTRu as substrate in our established substrate-linked protein evolution (SLiPE) process [4] and evolved the uTre recombinase in 142 evolution cycles. Highly specific enzymatic activity on loxLTRu is demonstrated for uTre in both Escherichia coli and human cells. Naturally occurring viral variants with single mutations within the loxLTRu sequence are also shown to be efficiently targeted by uTre, further increasing the range of applicability of the recombinase. Potential off-target sites in the human genome are not recombined by uTre. Furthermore, uTre expression in primary human T cells shows no obvious Tre-related cytopathic or genotoxic effects. Finally, uTre expressing mice show no undesired phenotypes during their normal lifespan. We have developed a broad-range HIV-1 LTR specific recombinase that has the potential to be effective against the vast majority of HIV-1 strains and to cure HIV-1 infected cells from the infection. These results strongly encouraged us in our confidence that a Tre recombinase-mediated HIV eradication strategy may become a valuable component of a future therapy for HIV-infected patients.
目前的抗HIV药物可以抑制病情发展至艾滋病，但无法清除患者体内的病毒。由于这些药物存在潜在副作用以及可能产生耐药性，找到治愈HIV感染的方法仍然是HIV/AIDS研究的首要任务。我们最近研发了一种重组酶（称为Tre），通过特异性靶向病毒DNA长末端重复序列（LTRs）中存在的序列，能够有效地从HIV-1感染细胞的宿主基因组中根除前病毒[1]。在感染HIV的人源化小鼠体内进行的分析表明，Tre重组酶具有高度显著的抗病毒效果[2]。然而，Tre识别特定HIV-1 A亚型毒株这一事实可能会限制其广泛的治疗应用。为了推进基于Tre的策略，实现普遍有效的治愈方法，我们设计了一种新的通用重组酶（uTre），它适用于大多数由各种病毒株和亚型引起的HIV-1感染。我们使用搜索工具SeLOX[3]，以便从洛斯阿拉莫斯HIV序列数据库中汇编的序列里找到一个保守的HIV-1前病毒序列，作为通用Tre的靶位点。我们选择了一个候选序列（称为loxLTRu），在主要的HIV-1 A、B和C亚型组中，其平均保守率为94%。我们将loxLTRu作为底物应用于我们已建立的底物连接蛋白进化（SLiPE）过程[4]，并在142个进化循环中进化出uTre重组酶。在大肠杆菌和人类细胞中，uTre对loxLTRu均表现出高度特异性的酶活性。loxLTRu序列内具有单个突变的天然病毒变体也显示能被uTre有效靶向，进一步扩大了重组酶的适用范围。uTre不会重组人类基因组中的潜在脱靶位点。此外，uTre在原代人T细胞中的表达未显示出明显的与Tre相关的细胞病变或基因毒性作用。最后，表达uTre的小鼠在其正常寿命期间未表现出不良表型。我们研发了一种广泛靶向HIV-1 LTR的特异性重组酶，它有可能有效对抗绝大多数HIV-1毒株，并治愈被HIV-1感染的细胞。这些结果极大地增强了我们的信心，即Tre重组酶介导的HIV根除策略可能成为未来治疗HIV感染患者的一种有价值的方法。
