Abstract：
BACKGROUND: LentiGlobin BB305 is a self-inactivating lentiviral vector carrying a human β-globin expressing cassette for treating β-thalassemia. Initially, a 2 × 250 bp chicken Locus Control Region fragment of cHS4, functioning as an insulator, was placed at its ΔU3, which was removed after the first clinical trial led by a French team to avoid abnormal splicing, etc. This action could potentially lead to an increasing risk of the transcriptional read-through rate driven by the β-globin promoter to a significant level, posing a biosafety risk in clinical trials.
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.
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.
摘要：
背景:慢球蛋白BB305是携带用于治疗β-地中海贫血的人β-球蛋白表达盒的自我失活慢病毒载体。最初，cHS4的2×250bp鸡基因座控制区片段，起到绝缘子的作用，被置于其ΔU3，在由法国团队领导的避免异常剪接的第一次临床试验后被移除，等。这种作用可能导致由β-珠蛋白启动子驱动的转录通读速率增加到显著水平的风险。在临床试验中构成生物安全风险。
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。
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。
