Correction of Aberrant Pre-mRNA Splicing by Antisense Oligonucleotides in β-Thalassemia Egyptian Patients With IVSI-110 Mutation

Abstract

The splicing mutation in intron 1 of beta-globin gene (IVS1-110) is the most common mutation in Egyptian thalassemics that causes aberrant splicing of pre-mRNA and deficient beta-globin chain synthesis. Antisense oligonucleotides (ASONs) are compounds that redirect pre-mRNA splicing and modify gene expression. Our aim was ex vivo correction of the aberrant splicing of beta-globin110 pre-mRNA by ASON against the 3' aberrant splice site. Peripheral blood mononuclear cells of 10 thalassemic patients with IVS1-110 mutation were duplicated and 1 was treated with 20 micromoL/mL morpholino ASON targeted against the 3' aberrant splice site. The level of total hemoglobin (Hb), fetal Hb, and mRNA were estimated in the duplicate samples. Five cases (50%) showed correction with ASON treatment, of which 2 cases showed the appearance of corrected mRNA band with absence of the aberrant band and 3 cases showed an increased ratio of the corrected to the aberrant mRNA band from 2:1 to 3:1, and 4:1. The total Hb showed significant increase in the 5 corrected cases. In conclusion, ASON can restore correct splicing of beta-globin pre-mRNA leading to correct gene product in cultured erythropoietic cells. These results suggest the applicability of ASON for the treatment of thalassemia.

Targeting RNA splicing for disease therapy

“…Blocking cryptic 5′ss with ASOs has been used in several disease models to correct splicing (Table 1, cryptic splice sites ) . ASO‐directed blocking of cryptic splice sites caused by mutations in HBB (β ‐globin), FKTN, LMNA , and CLC1 has been shown to rescue normal splicing in mouse models of β ‐thalassemia,39–43 Fukuyama congenital muscular dystrophy,50 Hutchinson‐Gilford progeria,53 and myotonic dystrophy,60 respectively (Table 1, cryptic splice sites). Also, rescue of disease phenotype in a mouse model has been shown using ASOs targeting a mutation the USH1C gene that creates a cryptic 5′ss and causes Usher syndrome.…”

Generation and Characterization of a Transgenic Mouse Carrying a Functional Humanβ-Globin Gene with the IVSI-6 Thalassemia Mutation

“…In this respect, ex vivo experiments based on the correction of splicing defects causing β -thalassemia have been reported by several research groups using antisense phosphorothioate 2′-O-methyl-oligonucleotides [ 45 , 46 ], morpholino-oligonucleotides [ 18 , 47 ], 2′-O-(2-methoxy) ethyl-oligonucleotides [ 47 ], and peptide nucleic acids [ 48 ]. These antisense molecules have been used either free [ 45 , 46 ] or delivered with peptides and lipid-based strategies [ 49 ].…”

“…These antisense molecules have been used either free [ 45 , 46 ] or delivered with peptides and lipid-based strategies [ 49 ]. For instance, El-Beshlawy et al [ 18 ] reported the ex vivo correction of the aberrant splicing of IVSI-110 β -globin pre-mRNA by antisense oligonucleotides (ASONs) against the 3′ aberrant splicing site. In their study, ErPCs with the IVSI-110 mutation were treated with 20 μ mol/mL morpholino ASONs targeting the 3′ aberrant splicing site.…”

“…Lonkar et al described a PNA-based approach method for targeted correction of a thalassemia-associated β -globin mutation [ 16 ]. In addition, other groups approached a therapy based on the correction of aberrant pre-mRNA splicing [ 17 , 18 ].…”

<p>Nucleic Acid Therapy for β-Thalassemia</p>

“…SSOs modulate RNA splicing by redirecting alternative splicing that can be categorized in four mechanisms: exon skipping, exon retention, restoration of correct splicing and displacement of splicing factors from triplets repeats which have been reviewed extensively. 28 The SSOs have been used to correct aberrant splicing in β-thalassemia alleles including the mutations IVS1-110, 34 , 35 IVS2-654, 36 – 38 IVS2-705, 39 IVS2-745, 40 and βE. 36 SSOs successfully induced correct splicing and increased normal β-globin production in cell-free extracts, stable cell lines with a mutated β-globin gene, erythroid mononuclear cells from peripheral blood of patients, thalassemic mouse erythroid progenitors, human iPSCs and β-thalassemic mouse model.…”