Repair of a Splicing Defect in Erythroid Cells from Patients with β-Thalassemia/HbE Disorder

Suwanmanee, Thipparat; Sierakowska, Halina; Fucharoen, Suthat; Kole, Ryszard

doi:10.1006/mthe.2002.0805

Cited by 70 publications

(50 citation statements)

References 33 publications

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“…1 For instance, AONs have restored normal splicing by blocking cryptic splice sites, 2,3 altered the ratio of alternative splicing from malignant to nonmalignant isoforms, 4 and induced exon inclusion for mutated exons that were otherwise skipped. 5 In these studies, the AON treatments aimed at the re-establishment of wild-type mRNA.…”

Section: Introductionmentioning

confidence: 99%

Comparative analysis of antisense oligonucleotide analogs for targeted DMD exon 46 skipping in muscle cells

et al. 2004

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As small molecule drugs for Duchenne muscular dystrophy (DMD), antisense oligonucleotides (AONs) have been shown to restore the disrupted reading frame of DMD transcripts by inducing specific exon skipping. This allows the synthesis of largely functional Becker muscular dystrophy (BMD)-like dystrophins and potential conversion of severe DMD into milder BMD phenotypes. Thus far we have used 2 0 -O-methyl phosphorothioate (2OMePS) AONs. Here, we assessed the skipping efficiencies of different AON analogs containing morpholino-phosphorodiamidate, locked nucleic acid (LNA) or peptide nucleic acid (PNA) backbones. In contrast to PNAs and morpholinos, LNAs have not yet been tested as splice modulators. Compared to the most effective 2OMePS AON directed at exon 46, the LNA induced higher skipping levels in myotubes from a human control (85 versus 20%) and an exon 45 deletion DMD patient (98 versus 75%). The morpholinoinduced skipping levels were only 5-6%, whereas the PNA appeared to be ineffective. Further comparative analysis of LNA and 2OMePS AONs containing up to three mismatches revealed that LNAs, while inducing higher skipping efficiencies, show much less sequence specificity. This limitation increases the risk of adverse effects elsewhere in the human genome. Awaiting further improvements in oligochemistry, we thus consider 2OMePS AONs currently the most favorable compounds, at least for targeted DMD exon 46 skipping.

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Section: Introductionmentioning

confidence: 99%

Comparative analysis of antisense oligonucleotide analogs for targeted DMD exon 46 skipping in muscle cells

et al. 2004

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“…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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“…Antisense oligonucleotides have been used to restore pre-mRNA splicing in other disease models (1)(2)(3)(4)(5)(6)(7)(8); however, the therapeutic rationale for each varies considerably and, to our knowledge, none has addressed an intranuclear or DNA repair disorder. Furthermore, we believe that A-T offers several advantages for exploring the therapeutic potential of AMOs, such as (i) the availability of many surrogate markers for evaluating ATM function, ex vivo and in vivo, and (ii) a well characterized spectrum of ATM mutations supported by an extensive cell repository of lymphoblastoid cell lines (LCLs) derived from patients with those mutations (9)(10)(11)(12).…”

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Correction of prototypic ATM splicing mutations and aberrant ATM function with antisense morpholino oligonucleotides

Du¹,

Pollard²,

Gatti

2007

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

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We used antisense morpholino oligonucleotides (AMOs) to redirect and restore normal splicing of three prototypic splicing mutations in the ataxia-telangiectasia mutated (ATM) gene. Two of the mutations activated cryptic 5 or 3 splice sites within exonic regions; the third mutation activated a downstream 5 splice site leading to pseudoexon inclusion of a portion of intron 28. AMOs were targeted to aberrant splice sites created by the mutations; this effectively restored normal ATM splicing at the mRNA level and led to the translation of full-length, functional ATM protein for at least 84 h in the three cell lines examined, as demonstrated by immunoblotting, ionizing irradiation-induced autophosphorylation of ATM, and transactivation of ATM substrates. Ionizing irradiation-induced cytotoxicity was markedly abrogated after AMO exposure. The ex vivo data strongly suggest that the disease-causing molecular pathogenesis of such prototypic mutations is not the amino acid change of the protein but the mutated DNA code itself, which alters splicing. Such prototypic splicing mutations may be correctable in vivo by systemic administration of AMOs and may provide an approach to customized, mutation-based treatment for ataxia-telangiectasia and other genetic disorders.ataxia-telangiectasia mutated ͉ mutation-based treatment T his study addresses the restoration of normal splicing in three ataxia-telangiectasia mutated (ATM) splicing mutations by exposure of ataxia-telangiectasia (A-T) cells to antisense morpholino oligonucleotides (AMOs). Antisense oligonucleotides have been used to restore pre-mRNA splicing in other disease models (1-8); however, the therapeutic rationale for each varies considerably and, to our knowledge, none has addressed an intranuclear or DNA repair disorder. Furthermore, we believe that A-T offers several advantages for exploring the therapeutic potential of AMOs, such as (i) the availability of many surrogate markers for evaluating ATM function, ex vivo and in vivo, and (ii) a well characterized spectrum of ATM mutations supported by an extensive cell repository of lymphoblastoid cell lines (LCLs) derived from patients with those mutations (9-12). The LCLs allow pre-mRNA mechanisms to be dissected in great detail. The principles gleaned can then be extended to other tissue targets, such as neuronal cells.A-T is a progressive autosomal recessive neurodegenerative disorder resulting from mutations in ATM (13). This gene includes 66 exons and encodes a 13-kb mature transcript with an ORF of 9,168 nt. The ATM protein is a serine/threonine kinase with Ͼ30 phosphorylation targets (14, 15). It affects control of cell cycle checkpoints, repair of dsDNA breaks, responses to oxidative stress, and apoptosis and is a potent tumor suppressor (16)(17)(18)(19). ATM is constitutively expressed in all tissues, primarily in the nucleus. So far no therapy exists for this disorder.ATM protein is not detectable in most A-T patients. A subset of patients with notable protein levels (5-20%) has milder phenotypes (20). Pati...

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Repair of a Splicing Defect in Erythroid Cells from Patients with β-Thalassemia/HbE Disorder

Cited by 70 publications

References 33 publications

Comparative analysis of antisense oligonucleotide analogs for targeted DMD exon 46 skipping in muscle cells

Comparative analysis of antisense oligonucleotide analogs for targeted DMD exon 46 skipping in muscle cells

RNA repair restores hemoglobin expression in IVS2–654 thalassemic mice

Correction of prototypic ATM splicing mutations and aberrant ATM function with antisense morpholino oligonucleotides

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