Proteasome activity restricts lentiviral gene transfer into hematopoietic stem cells and is down-regulated by cytokines that enhance transduction
Proteasome activity restricts lentiviral gene transfer into hematopoietic stem cells and is down-regulated by cytokines that enhance transduction
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DOI：
10.1182/blood-2005-10-4047
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发表时间：
2006-06-01
期刊：
影响因子：
20.3
通讯作者：
Naldini, Luigi
中科院分区：
文献类型：
--
作者：
de Sio, Francesca Romana Santoni;Cascio, Paolo;Naldini, Luigi
造血干细胞（HSC）基因治疗的治疗潜力可以充分利用，只有达到有效的基因转移到HSC，而不损害其生物学特性。尽管HSC可以在短的离体培养中被HIV衍生的慢病毒载体（LV）转导，但它们对载体表现出低的依赖性，需要细胞因子刺激以达到高频率转导。使用严格的竞争性异种移植再增殖试验，我们表明，早期作用的细胞因子协同增强人类HSC基因转移的LV不损害植入和再增殖能力。使用S期自杀试验，我们发现细胞因子的转导增强不依赖于细胞周期的进展，LV可以使静止的HSC恢复。在转导过程中蛋白酶体的药理学抑制显著增强了HSC基因转移，允许在其体内子代中达到非常高水平的载体整合。因此，LV在试验后步骤中被该蛋白水解复合物的活性有效地限制。出乎意料的是，细胞因子刺激迅速和大幅下调造血祖细胞中的蛋白酶体活性，突出了细胞因子可能增强LV基因转移的容许性的一种机制。这些发现表明，最终由蛋白酶体介导的抗病毒反应强烈限制了LV转导HSC的效率，并为基于HSC的基因治疗建立了改善的条件。
The therapeutic potential of hematopoietic stem cell (HSC) gene therapy can be fully exploited only by reaching efficient gene transfer into HSCs without compromising their biologic properties. Although HSCs can be transduced by HIV-derived lentiviral vectors (LVs) in short ex vivo culture, they display low permissivity to the vector, requiring cytokine stimulation to reach high-frequency transduction. Using stringent assays of competitive xenograft repopulation, we show that early-acting cytokines synergistically enhanced human HSC gene transfer by LVs without impairing engraftment and repopulation capacity. Using S-phase suicide assays, we show that transduction enhancement by cytolkines was not dependent on cell cycle progression and that LVs can transduce quiescent HSCs. Pharmacologic inhibition of the proteasome during transduction dramatically enhanced HSC gene transfer, allowing the reach of very high levels of vector integration in their progeny in vivo. Thus, LVs are effectively restricted at a postentry step by the activity of this proteolytic complex. Unexpectedly, cytokine stimulation rapidly and substantially down-regulated proteasome activity in hematopoietic progenitors, highlighting one mechanism by which cytolkines may enhance permissiveness to LV gene transfer. These findings demonstrate that antiviral responses ultimately mediated by proteasomes strongly limit the efficiency of HSC transduction by LVs and establish improved conditions for HSC-based gene therapy.