Analysis of factor VIII mediated suppression of lentiviral vector titres
Analysis of factor VIII mediated suppression of lentiviral vector titres
复制标题
DOI：
10.1038/sj.gt.3303080
复制
发表时间：
2008-02-01
期刊：
影响因子：
5.1
通讯作者：
Mitrophanous, K. A.
中科院分区：
文献类型：
--
作者：
Radcliffe, P. A.;Sion, C. J. M.;Mitrophanous, K. A.
血友病A的有效基因治疗需要不受预先存在的免疫应答影响、具有足够的编码能力、提供长期表达并且优选地可以靶向非分裂细胞的载体系统。基于慢病毒如马传染性贫血病毒（EIAV）的载体系统满足递送因子VIII（FVIII）的这些标准。我们发现，当与EIAV载体系统共表达时，B结构域缺失（BDD）FVIII蛋白抑制功能性病毒颗粒的产生。尽管颗粒数量（通过逆转录酶活性测量）接近正常，但RNA基因组水平降低，并且整合拷贝的测量显示病毒在侵袭靶细胞的能力方面存在严重缺陷。这是由于来自表达FVIII的细胞的病毒颗粒上缺乏足够的水泡性口炎病毒糖蛋白（VSV-G）包膜。通过使用在生产细胞中具有低活性的内部组织特异性启动子来驱动FVIII的表达，我们克服了这种抑制作用，使我们产生的滴度接近用编码报告基因的载体基因组获得的滴度。此外，我们报告说，全长FVIII基因的密码子优化增加载体滴度约10倍，除了大大提高每个整合载体拷贝的表达。
Effective gene therapy for haemophilia A necessitates a vector system that is not subject to a pre-existing immune response, has adequate coding capacity, gives long-term expression and preferably can target non-dividing cells. Vector systems based on lentiviruses such as equine infectious anaemia virus (EIAV) fulfil these criteria for the delivery of factor VIII (FVIII). We have found that B domain-deleted (BDD) FVIII protein inhibits functional viral particle production when co-expressed with the EIAV vector system. Although particle numbers (as measured by reverse transcriptase activity) are near normal, RNA genome levels are reduced and measurement of integrated copies revealed the virus is severely defective in its ability to transduce target cells. This is due to the absence of sufficient vesicular stomatitis virus glycoprotein (VSV-G) envelope on viral particles derived from cells expressing FVIII. By using an internal tissue-specific promoter, that has low activity in the producer cells, to drive expression of FVIII we have overcome this inhibitory effect allowing us to generate titres approaching those obtained with vector genomes encoding reporter genes. Furthermore, we report that codon optimization of the full-length FVIII gene increased vector titres approximately 10-fold in addition to substantially improving expression per integrated vector copy.