Reducing the transcriptional read-through rate of a lentiviral vector for β-thalassemia gene therapy.

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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. 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. 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. 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.