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

Author:

Wu Jiahui12,Chen Yuan12,Shen Wenchen12,Zhang Jingzhi12,Zeng Fanyi1234

Affiliation:

1. Shanghai Institute of Medical Genetics, Shanghai Children’s Hospital Shanghai Jiao Tong University School of Medicine Shanghai China

2. NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering Shanghai China

3. Department of Histo‐Embryology, Genetics and Developmental Biology Shanghai Jiao Tong University School of Medicine Shanghai China

4. School of Pharmacy Macau University of Science and Technology Macau China

Abstract

AbstractBackgroundLentiGlobin 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.MethodsIn 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.ResultsWe 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.ConclusionsThe 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.

Funder

National Key Research and Development Program of China

National Natural Science Foundation of China

Innovative Research Team of High-level Local University in Shanghai

Publisher

Wiley

Subject

Genetics (clinical),Drug Discovery,Genetics,Molecular Biology,Molecular Medicine

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