Correction of Aberrant pre mRNA splicing using antisense oligonucleotides in B thalassemia patients with known IVS-I,nt 110 mutation

ABSTRACT

1VS In l 110 mutation is the most common mutation in Egyptian thalassemics. The mutation causes aberrant splicing ofpre-mRNA resulting in labile RNA causing deficient B-globin chain synthesis. Antisense oligonucleotide strategy is one of the more simple technique in gene therapy. Using oligonucleotides covering the aberrant splice site gives chance for normal splicing and correction of the anomaly at the RNA level. Blocking of aberrant splicing at IVS 1nt 110 site of B-globin pre-mRNA using antisense oligonucleotides results in subsequent restoration of normal mRNA production in B-thalasseamia patients with IVS 1nt 110 mutation This study involved 10 patients with known IVS IntllO mutation; 6 homozygous and 4 heterozygous patients peripheral blood mononuclear cells were separated and duplicate liquid culture systems were set using erythropoietin and stem cell factor with and without antisense oligonucleotides [20 micro. mol/ml]. Correction of aberrant splicing was evaluated by estimation of total hemoglobin, hemoglobin F, and a reverse transcriptase polymerase chain reaction followed by agar gel electrophoresis was used to amplify and detect both aberrant mRNA and normal mRNA in duplicate samples. Five cases [50%] showed correction after antisense oligonucleotide treatment, two cases showed the appearance of normal mRNA band with absence of aberrant band and in 3 cases an increased ratio of normal mRNA band to aberrant band was found [from 21 to 31 in two cases and from 21 to 41 in the third case]. The 5 corrected cases showed significant increase in total Hb which varied between 4 to 6 folds increase. Antisense oligonucleotide treatment corrects splicing ofpre-mRNA leading to appropriate expression of B-globin mRNA which may pave the way for treatment of thalassaemia.