Restoration of Human β-Globin Gene Expression in Murine and Human IVS2–654 Thalassemic Erythroid Cells by Free Uptake of Antisense Oligonucleotides

Suwanmanee, Thipparat; Sierakowska, Halina; Lacerra, Giuseppina; Svasti, Saovaros; Kirby, Suzanne L.; Walsh, Christopher E.; Fucharoen, Suthat; Kole, Ryszard

doi:10.1124/mol.62.3.545

Cited by 59 publications

(46 citation statements)

References 26 publications

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“…We have found that repair of splicing-defective thalassemic pre-mRNA and concomitant production of adult hemoglobin (HbA) can be accomplished ex vivo in erythroid progenitor cells from thalassemic patients treated with SSOs with modified backbones (10,11), including peptide-conjugate morpholino, SSO 654-P005 (data not shown). Thus, this treatment is compatible with and effective in the human cells that will constitute its target in the clinic.…”

Section: Resultsmentioning

confidence: 99%

“…The profiles of morpholino and PNA-4K oligonucleotides, including their sequences, were reported elsewhere (11,14,15). The 18-mer morpholino (5Ј-GCTATTACCTTAACCCAG-3Ј)-peptide-[(D)R(D)RRQRRKKRFFC] conjugate (P005) antisense to the aberrant 5Ј splice site in the IVS2-654 ␤-globin pre-mRNA was synthesized at AVI BioPharma.…”

Section: Methodsmentioning

confidence: 99%

“…The oligonucleotides were tested ex vivo in IVS2-654 mouse erythroid progenitor cells, which were plated 18 h before SSO treatment using a free uptake technique as described (11). SSO 654-P005 concentrations were as shown in Fig.…”

Section: Oligonucleotide Treatmentmentioning

confidence: 99%

“…Work in this laboratory showed that splice-switching oligonucleotides (SSOs), which block aberrant splice sites in IVS2-654 and other pre-mRNAs (IVS1-5, IVS1-6, IVS1-110, IVS2-705, and IVS2-745) as well as in the coding sequence (HbE) of the ␤-globin gene, force the splicing machinery to reselect the existing correct splice sites, repairing the splicing pattern of ␤-globin pre-mRNA. This repair, which restores production of correctly spliced ␤-globin mRNA and protein, was accomplished in several in vitro systems and ex vivo in erythroid progenitor cells from thalassemic patients (6)(7)(8)(9)(10)(11)(12). In this study, we investigated the effectiveness in thalassemic splicing correction of a modified morpholino oligomer, SSO 654-P005, in a mouse model of IVS2-654 ␤-thalassemia.…”

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RNA repair restores hemoglobin expression in IVS2–654 thalassemic mice

Svasti

Suwanmanee

Fucharoen

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Repair of ␤-globin pre-mRNA rendered defective by a thalassemiacausing splicing mutation, IVS2-654, in intron 2 of the human ␤-globin gene was accomplished in vivo in a mouse model of IVS2-654 thalassemia. This was effected by a systemically delivered splice-switching oligonucleotide (SSO), a morpholino oligomer conjugated to an arginine-rich peptide. The SSO blocked the aberrant splice site in the targeted pre-mRNA and forced the splicing machinery to reselect existing correct splice sites. Repaired ␤-globin mRNA restored significant amounts of hemoglobin in the peripheral blood of the IVS2-654 mouse, improving the number and quality of erythroid cells.oligonucleotides ͉ RNA splicing ͉ thalassemia ͉ therapy ͉ morpholino oligomers O ne of the most common inherited diseases, ␤-thalassemia, is caused by defects in the ␤-globin gene that affect production of ␤-globin, a subunit of hemoglobin (1). Current therapy consists of frequent blood transfusions combined with iron chelation (2). The only cure, bone marrow transplantation, is limited by the scarcity of suitable histocompatible donors (3). Although more than 200 mutations cause the disease, some of the most common ones induce aberrant splicing of ␤-globin pre-mRNA, interfering with proper translation of ␤-globin protein. The IVS2-654 (C Ͼ T) mutation creates an aberrant 5Ј splice site and activates a cryptic 3Ј splice site within intron 2 of the ␤-globin pre-mRNA, leading to retention of the intron fragment in the spliced mRNA even though the correct splice sites remain undamaged and potentially functional ( Fig. 1A) (4). The retained fragment prevents proper translation of ␤-globin, leading to hemoglobin deficiency and ␤-thalassemia. The IVS2-654 mutation is a common cause of thalassemia in Thailand and other countries of Southeast Asia (5). Work in this laboratory showed that splice-switching oligonucleotides (SSOs), which block aberrant splice sites in IVS2-654 and other pre-mRNAs (IVS1-5, IVS1-6, IVS1-110, IVS2-705, and IVS2-745) as well as in the coding sequence (HbE) of the ␤-globin gene, force the splicing machinery to reselect the existing correct splice sites, repairing the splicing pattern of ␤-globin pre-mRNA. This repair, which restores production of correctly spliced ␤-globin mRNA and protein, was accomplished in several in vitro systems and ex vivo in erythroid progenitor cells from thalassemic patients (6)(7)(8)(9)(10)(11)(12). In this study, we investigated the effectiveness in thalassemic splicing correction of a modified morpholino oligomer, SSO 654-P005, in a mouse model of IVS2-654 ␤-thalassemia. Results and DiscussionTo be effective in splicing repair, SSOs must hybridize tightly to the targeted splicing elements, such as aberrant splice sites, and prevent their recognition by the splicing factors. Furthermore, the resulting double-stranded structures must not be recognized by RNase H, which degrades RNA in RNA-DNA duplexes (13). These conditions are satisfied in cell culture and in vivo by modified oligomers with various backbones, includi...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Oligonucleotide Treatmentmentioning

confidence: 99%

mentioning

confidence: 99%

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RNA repair restores hemoglobin expression in IVS2–654 thalassemic mice

Svasti

Suwanmanee

Fucharoen

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…Les auteurs avaient obtenu la restauration d'un épissage correct en ciblant en système acellulaire, soit le site 5' nouvellement créé, soit le site 3' cryptique activé. Dans les années suivantes, le bien-fondé de cette approche a été vérifié expérimentalement dans des systèmes cellulaires : cellules HeLa, puis précurseurs érythroïdes de sujets thalassémiques [16][17][18][19]. Le passage à l'expérimentation animale in vivo a été franchi grâce à des souris transgéniques dont un des deux locus bêta globine avait été remplacé par le locus bêta humain portant la mutation IVS2-654 [20] [20] ont reçu pendant 3 semaines (4 jours consécutifs, 3 jours d'arrêt) par injection intraveineuse (25 mg/kg) un oligonucléotide antisens long de 18 nt dirigé contre la mutation IVS2-654 (Figure 1).…”

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