Restoration of correct β-globin mRNA splicing and HbA production by engineered U7 snRNA in β-thalassaemia/HbE erythroid cells.

A cytosine to thymine mutation at nucleotide 654 of human β-globin intron 2 (β) 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 β-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 β-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.