METHODS: In the present study, a read-through reducing agent (C-U+ or WPRE) was designed to be placed at the 3\' UTR of the β-globin gene. The Enhancer Activities and/or Transcriptional Read-Through (EATRT) rate at the mRNA level and the protein expression level regarding lentiviral preparation titer were examined.
RESULTS: We found that the insertion of the element (C-U+ or WPRE) reduced the EATRT effectively by 53% or 41%, respectively. C-U+ has less impact on virus package efficiency. Furthermore, there was no significant difference in the protein expression level after the C-U+ or WPRE insertion.
CONCLUSIONS: The results of the present study show that inserting C-U+ or WPRE before the polyA sequence of the BB305 would reduce the EATRT rate at no cost of its expressing efficacy and viral preparation titers. Thus, we present an alternative improvement for a safer lentiviral vector for β-thalassemia clinical trials.
方法:在本研究中,设计了一种通读还原剂(C-U或WPRE),将其置于β-珠蛋白基因的3UTR处。检查mRNA水平的增强子活性和/或转录通读(EATRT)率和关于慢病毒制剂滴度的蛋白质表达水平。
结果:我们发现元素(C-U或WPRE)的插入有效地将EATRT降低了53%或41%,分别。C-U+对病毒封装效率影响较小。此外,C-U+或WPRE插入后蛋白表达水平无显著差异。
结论:本研究的结果表明,在BB305的polyA序列之前插入C-U+或WPRE将降低EATRT率,而不损害其表达功效和病毒制备滴度。因此,我们提出了一种用于β-地中海贫血临床试验的更安全的慢病毒载体的替代改进。