Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy.
J Virol. 2020 Sep 15;94(19). doi: 10.1128/JVI.00871-20.
Adeno-associated virus (AAV) has proven to be a promising candidate for gene therapy due to its nonpathogenic nature, ease of production, and broad tissue tropism. However, its transduction capabilities are not optimal due to the interaction with various host factors within the cell. In a previous study, we identified members of the small ubiquitin-like modifier (SUMO) pathway as significant restriction factors in AAV gene transduction. In the present study, we explored the scope of this restriction by focusing on the AAV capsid and host cell proteins as targets. We show that during vector production, the capsid protein VP2 becomes SUMOylated, as indicated by deletion and point mutations of VP2 or the obstruction of its N terminus via the addition of a tag. We observed that SUMOylated AAV capsids display higher stability than non-SUMOylated capsids. Prevention of capsid SUMOylation by VP2 mutations did not abolish transduction restriction by SUMOylation; however, it reduced activation of gene transduction by shutdown of the cellular SUMOylation pathway. This indicates a link between capsid SUMOylation and SUMOylation of cellular proteins in restricting gene transduction. Infection with AAV triggers general SUMOylation of cellular proteins. In particular, the DAXX protein, a putative host cell restriction factor that can become SUMOylated, is able to restrict AAV gene transduction by reducing the intracellular accumulation of AAV vectors. We also observe that the coexpression of a SUMOylation inhibitor with an AAV2 reporter gene vector increased gene transduction significantly. Host factors within the cell are the major mode of restriction of adeno-associated virus (AAV) and keep it from fulfilling its maximum potential as a gene therapy vector. A better understanding of the intricacies of restriction would enable the engineering of better vectors. Via a genome-wide short interfering RNA screen, we identified that proteins of the small ubiquitin-like modifier (SUMO) pathway play an important role in AAV restriction. In this study, we investigate whether this restriction is targeted to the AAV directly or indirectly through host cell factors. The results indicate that both targets act in concert to restrict AAV.
腺相关病毒 (AAV) 因其非致病性、易于生产和广泛的组织嗜性而被证明是基因治疗的有前途的候选者。然而，由于其与细胞内各种宿主因素的相互作用，其转导能力并不理想。在之前的研究中，我们确定了小泛素样修饰物 (SUMO) 途径的成员是 AAV 基因转导的重要限制因素。在本研究中，我们通过关注 AAV 衣壳和宿主细胞蛋白作为靶点，探讨了这种限制的范围。我们表明，在载体生产过程中，衣壳蛋白 VP2 被 SUMO 化，这可以通过删除和点突变 VP2 或通过添加标签来阻止其 N 端来指示。我们观察到，SUMO 化的 AAV 衣壳比非 SUMO 化的衣壳更稳定。通过 VP2 突变阻止衣壳 SUMO 化并没有通过 SUMO 化消除转导限制；然而，它通过关闭细胞 SUMO 化途径来减少基因转导的激活。这表明衣壳 SUMO 化与限制基因转导的细胞蛋白 SUMO 化之间存在联系。AAV 感染会引发细胞蛋白的普遍 SUMO 化。特别是，DAXX 蛋白是一种假定的宿主细胞限制因子，可以通过减少 AAV 载体在细胞内的积累来限制 AAV 基因转导，它能够成为 SUMO 化的靶标。我们还观察到，与 AAV2 报告基因载体共表达 SUMO 化抑制剂可显著增加基因转导。细胞内的宿主因素是腺相关病毒 (AAV) 限制的主要模式，使其无法充分发挥作为基因治疗载体的潜力。更好地了解限制的复杂性将能够设计更好的载体。通过全基因组短干扰 RNA 筛选，我们确定了小泛素样修饰物 (SUMO) 途径的蛋白质在 AAV 限制中发挥重要作用。在这项研究中，我们研究了这种限制是直接针对 AAV 还是通过宿主细胞因子间接针对 AAV。结果表明，这两个靶标协同作用以限制 AAV。
