Restoration of correct ßIVS2-654-globin mRNA splicing and HbA production by engineered U7 snRNA in ß-thalassaemia/HbE erythroid cells.

A cytosine to thymine mutation at nucleotide 654 of human ß-globin intron 2 (ßIVS2-654) is one of the most common mutations causing ß-thalassaemia in Chinese and Southeast Asians. This mutation results in aberrant ß-globin pre-mRNA splicing and prevents synthesis of ß-globin protein. Splicing correction using synthetic splice-switching oligonucleotides (SSOs) has been shown to restore expression of the ß-globin protein, but to maintain therapeutically relevant levels of ß-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 ßIVS2-654-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 ßIVS2-654-thalassaemia/HbE patients, which showed restoration of correctly spliced ß-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 ß-thalassaemia.