Abstract
A cytosine to thymine mutation at nucleotide 654 of human β-globin intron 2 (βIVS2-654) is one of the most common mutations causing β-thalassaemia in Chinese and Southeast Asians. This mutation results in aberrant β-globin pre-mRNA splicing and prevents synthesis of β-globin protein. Splicing correction using synthetic splice-switching oligonucleotides (SSOs) has been shown to restore expression of the β-globin protein, but to maintain therapeutically relevant levels of β-globin it would require lifelong administration. Here, we demonstrate long-term splicing correction using U7 snRNA lentiviral vectors engineered to target several pre-mRNA splicing elements on the βIVS2-654-globin pre-mRNA such as cryptic 3′ splice site, aberrant 5′ splice site, cryptic branch point and an exonic splicing enhancer. A double-target engineered U7 snRNAs targeted to the cryptic branch point and an exonic splicing enhancer, U7.BP + 623, was the most effective in a model cell line, HeLa IVS2-654. Moreover, the therapeutic potential of the vector was demonstrated in erythroid progenitor cells derived from βIVS2-654-thalassaemia/HbE patients, which showed restoration of correctly spliced β-globin mRNA and led to haemoglobin A synthesis, and consequently improved thalassaemic erythroid cell pathology. These results demonstrate proof of concept of using the engineered U7 snRNA lentiviral vector for treatment of β-thalassaemia.
Highlights
Β-Thalassaemia is a common genetic disorder of haemoglobin synthesis, caused by defects in β-globin chain production
We show that lentiviral vectors carrying the optimal splice-switching oligonucleotides (SSOs) sequences restored correct βIVS2-654-globin pre-mRNA splicing and HbA production in erythroid progenitor cells from βIVS2-654-thalassaemia/HbE patients which led to phenotypic improvements of β-thalassaemic erythroblast
In previous work, the lentiviral vector carrying an engineered U7 small nuclear RNA (snRNA) targeted to an exonic splicing enhancer (ESE) at nucleotide position 623 of β-globin intron 2 (U7.623) could barely restore correct splicing in HeLa IVS2-654 cells[10]
Summary
Β-Thalassaemia is a common genetic disorder of haemoglobin synthesis, caused by defects in β-globin chain production. The SSO treatment of patients would require life-long administration to maintain therapeutically relevant levels of β globin To address this issue and to generate long-term effects, the SSO sequences were incorporated into the murine U7 small nuclear RNA (snRNA) as a vehicle to maintain stable levels of the SSO and modulate pre-mRNA splicing in target cells. This approach resulted in restoration of β-globin pre-mRNA correct splicing for at least 6 months in HeLa cells expressing βIVS2-654-, βIVS2-705- and βIVS2-745-globin (HeLa IVS2-654, HeLa IVS2705 and HeLa IVS2-745, respectively)[8,9,10]. We show that lentiviral vectors carrying the optimal SSO sequences restored correct βIVS2-654-globin pre-mRNA splicing and HbA production in erythroid progenitor cells from βIVS2-654-thalassaemia/HbE patients which led to phenotypic improvements of β-thalassaemic erythroblast
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