Restoration of correct βIVS2-654-globin mRNA splicing and HbA production by engineered U7 snRNA in β-thalassaemia/HbE erythroid cells

Abstract

A cytosine to thymine mutation at nucleotide 654 of human β-globin intron 2 (β IVS2-654 ) 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 β IVS2-654 -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 β IVS2-654 -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.

Designed U7 snRNAs inhibit DUX4 expression and improve FSHD-associated outcomes in DUX4 overexpressing cells and FSHD patient myotubes

“…Recombinant U7-snRNAs were developed previously to induce skipping of mutated exons as potential treatment for Duchenne muscular dystrophy 36 and β-thalassaemia. 37 In these studies, U7-snRNAs were used to restore the expression of frameshifted genes by skipping entire exons. In contrast, our goal here was to develop a novel gene silencing strategy by using U7-snRNAs to interfere with DUX4 pre-mRNA maturation or inhibit translational initiation.…”

U7 snRNA: A tool for gene therapy

“…Therapy was performed using thalassemia patient erythroid progenitor cells, as well as HeLa cells carrying the β IVS2–654 thalassemic mutation. Altogether, this approach showed a positive response by restoring correctly spliced β‐globin mRNA and correcting thalassemic erythroid cell pathology 85 …”

“…Study 85 – used HeLa IVS2–654 cells and erythroid progenitor cells from patients carrying β IVS2–654 thalassemia mutation. U7.…”

“…Aberrant splicing is also associated with mutations in intron 2 of the β‐globin gene at nucleotide positions 654, 705 or 745. These mutations are not the same (in mutation IVS2–654, C is substituted to T; in mutation IVS2–705, T is substituted to G; in mutation IVS2–745, C is substituted to G) and generate aberrant 5′ splice sites at different positions while activating the same cryptic 3′ splice site at the 579 position, leading to the inclusion of intronic sequences in β‐globin pre‐mRNA 84–86 . Mutations in intron 2 result in the generation of a premature stop codon and inhibit translation into full‐length β‐globin, leading to β‐thalassemia 84 .…”

“…Another study reported the restoration of correct β IVS2–654 ‐globin mRNA splicing and hemoglobin production using U7 Sm OPT (U7.BP+623) 85 . U7 BP+623 targeted the cryptic branch point and an exonic splicing enhancer.…”