Repair of a splicing defect in erythroid cells from patients with beta-thalassemia/HbE disorder.
A HeLa cell line stably expressing the human beta-globin gene carrying thalassemic mutations beta(E)/IVS1-6 served as a thalassemia model for repair of aberrant splicing of beta(E)-globin pre-mRNA with antisense oligonucleotides. Treatment of beta(E)/IVS1-6 HeLa cells with a morpholino oligonucleotide targeted immediately upstream of the aberrant 5' splice site activated by the mutations resulted in an increase in the amount of correctly spliced beta(E)-globin mRNA in a dose-dependent and sequence-specific fashion. The repaired beta(E)-globin mRNA was stable and could be translated into full-length beta(E)-globin polypeptide. Application of the same oligonucleotide to erythroid progenitor cells from two beta-thalassemia/HbE patients resulted in an approximately 70% increase in correct beta(E)-globin mRNA and 36% increase in hemoglobin E. The erythroid progenitor cells represent the actual targets for the clinical application of antisense repair of defective pre-mRNAs.
Suwanmanee
Thipparat
T
Thalassemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya Campus, Nakornpathom, Thailand.
Sierakowska
Halina
H
Fucharoen
Suthat
S
Kole
Ryszard
R
D017398:Alternative Splicing / Q000235:genetics*; D001483:Base Sequence; D004912:Erythrocytes / Q000378:metabolism*; D015316:Genetic Therapy / Q000379:methods*; D005914:Globins / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009154:Mutation / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000235:genetics / Q000494:pharmacology; D022821:RNA Splice Sites / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017086:beta-Thalassemia / Q000235:genetics* / Q000628:therapy*
D016376:Oligonucleotides, Antisense; D022821:RNA Splice Sites; D012333:RNA, Messenger; D005914:Globins