Creating Higher Titer Lentivirus with Caffeine
Creating Higher Titer Lentivirus with Caffeine
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DOI：
10.1089/hum.2010.068
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发表时间：
2011-01-01
影响因子：
4.2
通讯作者：
Porteus, Matthew H.
中科院分区：
文献类型：
--
作者：
Ellis, Brian L.;Potts, Patrick Ryan;Porteus, Matthew H.
慢病毒载体的使用从实验室延伸到临床，在实验室中，它们用于病毒学的基础研究和作为基因转移载体基因递送，在临床中，使用这些载体进行基因治疗的临床试验目前正在进行中。慢病毒载体可用于基因转移，因为它们具有大的克隆能力和广泛的向性。虽然慢病毒载体生产的程序已经标准化，但在生产过程中产生更高滴度病毒的简单方法将在研究和临床应用中具有广泛和重要的应用。在这里，我们提出了一种简单而廉价的方法，以增加3至8倍的滴度的整合能力慢病毒和整合缺陷慢病毒。这是在标准的慢病毒生产过程中通过添加咖啡因至2-4 mM的最终浓度来实现的。我们发现，丁酸钠，一种先前显示可增加病毒滴度的组蛋白去乙酰化酶抑制剂，其作用仅与50%以及咖啡因相似。我们还表明，DNA-PKcs（DNA依赖性蛋白激酶催化亚基）抑制剂NU 7026也可以增加病毒滴度，但咖啡因和NU 7026的组合并不比单独使用咖啡因更有效。我们表明，咖啡因治疗的时间过程是很重要的，在实现更高滴度的病毒，是最有效的，当咖啡因存在于转染后17至41小时。最后，虽然咖啡因增加慢病毒载体滴度，但它对腺相关病毒2型载体滴度具有相反的作用。总之，这些结果提供了一种新的，简单的，廉价的方式来显着增加慢病毒载体的滴度。
The use of lentiviral vectors extends from the laboratory, where they are used for basic studies in virology and as gene transfer vectors gene delivery, to the clinic, where clinical trials using these vectors for gene therapy are currently underway. Lentiviral vectors are useful for gene transfer because they have a large cloning capacity and a broad tropism. Although procedures for lentiviral vector production have been standardized, simple methods to create higher titer virus during production would have extensive and important applications for both research and clinical use. Here we present a simple and inexpensive method to increase the titer by 3- to 8-fold for both integration-competent lentivirus and integration-deficient lentivirus. This is achieved during standard lentiviral production by the addition of caffeine to a final concentration of 2-4 mM. We find that sodium butyrate, a histone deacetylase inhibitor shown previously to increase viral titer, works only similar to 50% as well as caffeine. We also show that the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) inhibitor NU7026 can also increase viral titer, but that the combination of caffeine and NU7026 is not more effective than caffeine alone. We show that the time course of caffeine treatment is important in achieving a higher titer virus, and is most effective when caffeine is present from 17 to 41 hr posttransfection. Last, although caffeine increases lentiviral vector titer, it has the opposite effect on the titer of adeno-associated virus type 2 vector. Together, these results provide a novel, simple, and inexpensive way to significantly increase the titer of lentiviral vectors.