Therapeutic potential of antisense oligonucleotides as modulators of alternative splicing

Sazani, Peter; Kole, Ryszard

doi:10.1172/jci200319547

Cited by 189 publications

(63 citation statements)

References 34 publications

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“…These results, together with those reported here, indicate that the splicing-mediated pre-mRNA repair is not limited to a single disease. In fact, modulation of aberrant and alternative splicing by antisense oligonucleotides is likely to be useful in numerous indications, because 70% of human genes are alternatively spliced, with a number of them associated with cancer (26) and other diseases (27)(28)(29).…”

Section: Resultsmentioning

confidence: 99%

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%

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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“…Antisense oligonucleotide-based therapy might have utility in a number of human diseases associated with aberrant splicing (10,13,25,49). A recent study demonstrated antisense oligonucleotide-mediated correction of aberrant splicing resulting in the restoration of expression of dystrophin in bodywide skeletal muscles of the mouse model of Duchenne muscular dystrophy (33).…”

Section: Discussionmentioning

confidence: 99%

Splicing of a Critical Exon of Human Survival Motor Neuron Is Regulated by a Unique Silencer Element Located in the Last Intron

Singh

Androphy

et al. 2006

Molecular and Cellular Biology

385

429

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Humans have two nearly identical copies of the Survival Motor Neuron (SMN) gene, SMN1 and SMN2. In spinal muscular atrophy (SMA), SMN2 is not able to compensate for the loss of SMN1 due to exclusion of exon 7. Here we describe a novel inhibitory element located immediately downstream of the 5 splice site in intron 7. We call this element intronic splicing silencer N1 (ISS-N1). Deletion of ISS-N1 promoted exon 7 inclusion in mRNAs derived from the SMN2 minigene. Underlining the dominant role of ISS-N1 in exon 7 skipping, abrogation of a number of positive cis elements was tolerated when ISS-N1 was deleted. Confirming the silencer function of ISS-N1, an antisense oligonucleotide against ISS-N1 restored exon 7 inclusion in mRNAs derived from the SMN2 minigene or from endogenous SMN2. Consistently, this oligonucleotide increased the levels of SMN protein in SMA patient-derived cells that carry only the SMN2 gene. Our findings underscore for the first time the profound impact of an evolutionarily nonconserved intronic element on SMN2 exon 7 splicing. Considering that oligonucleotides annealing to intronic sequences do not interfere with exon-junction complex formation or mRNA transport and translation, ISS-N1 provides a very specific and efficient therapeutic target for antisense oligonucleotide-mediated correction of SMN2 splicing in SMA.Alternative splicing increases the coding potential of the human genome by producing multiple proteins from a single gene (2). It is also associated with a growing number of human diseases (13,15,47). Regulation of alternative splicing is dependent upon the relative concentrations of spliceosomal and nonspliceosomal proteins, namely, serine-arginine-rich proteins (SR proteins), SR-like proteins, and heterogeneous nuclear ribonucleoproteins (hnRNPs) (16,35,43). Some of these proteins bind to pre-mRNA sequences called exonic splicing enhancers (ESEs), intronic splicing enhancers, exonic splicing silencers, and intronic splicing silencers (ISSs). Enhancers and silencers promote or suppress splice site (ss) selection, respectively. Over the last several years, methods have been developed to predict exonic cis elements (7,12,58). Analogous methods to predict intronic cis elements do not exist. Local RNA structure presents an additional level of splicing regulation. Several studies have focused on RNA structures that facilitate specific interactions during pre-mRNA splicing (3). However, the role of critical RNA structures in pre-mRNA splicing remains largely unpredictable.Spinal muscular atrophy (SMA), the second most common autosomal recessive disorder, is caused by the absence of the Survival of Motor Neuron 1 (SMN1) gene (27). SMN1 encodes a ubiquitously expressed 38-kDa SMN protein that is necessary for snRNP assembly, an essential process for cell survival (57).A nearly identical copy of the gene, SMN2, fails to compensate for the loss of SMN1 because of exon 7 skipping, producing an unstable truncated protein, SMN⌬7 (30). SMN1 and SMN2 differ by a critical C-to-T substitution at po...

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“…Here, we preferred to focus first on individual exons because their identification is sufficient for further analysis and manipulation of experimental systems. In addition to the use of realtime-PCR, individual exons can be targeted by specific antisense oligonucleotides [33][34][35] or antibodies against exon-encoded protein domains 36,37 by approaches already established for drug development and pharmacogenomics 38 . Thus, in combination with ASLs, exon-specific antisense oligonucleotides could be used to analyze the contribution of individual splicing events to gene function.…”

Section: Discussionmentioning

confidence: 99%

Libraries enriched for alternatively spliced exons reveal splicing patterns in melanocytes and melanomas

et al. 2004

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It is becoming increasingly clear that alternative splicing enables the complex development and homeostasis of higher organisms. To gain a better understanding of how splicing contributes to regulatory pathways, we have developed an alternative splicing library approach for the identification of alternatively spliced exons and their flanking regions by alternative splicing sequence enriched tags sequencing. Here, we have applied our approach to mouse melan-c melanocyte and B16-F10Y melanoma cell lines, in which 5,401 genes were found to be alternatively spliced. These genes include those encoding important regulatory factors such as cyclin D2, Ilk, MAPK12, MAPK14, RAB4, melastatin 1 and previously unidentified splicing events for 436 genes. Real-time PCR further identified cell line-specific exons for Tmc6, Abi1, Sorbs1, Ndel1 and Snx16. Thus, the ASL approach proved effective in identifying splicing events, which suggest that alternative splicing is important in melanoma development.The results of the whole-genome sequencing projects have thus far identified far fewer genes than previously expected, and it is particularly puzzling that highly diverse organisms such as fruit fly, mouse and human seem to have rather similar numbers of genes. Therefore, the number of genes alone does not account for the increased complexity of higher organisms, and other regulatory principles must contribute to the use of genetic information during development and homeostasis 1,2 . This phenomenon is attributed in part to the mechanisms of alternative splicing, which allow for combinatorial assembly of different exons from the same primary transcript. It was estimated that B38-59% of the genes in mouse and human are subject to alternative splicing 2-6 and that, furthermore, B15% of the genetic disorders in humans are caused by mutations related to defects in pre-mRNA splicing [7][8][9] .Although the identification of all splice variants is important for understanding the transcriptome, only a few large-scale analyses have been made by computational means for exon prediction within genomes or using expressed-sequence tags (ESTs) and cDNA sequences. Predicted exons were used to design exon-and intron 10 -specific oligonucleotide arrays to monitor pre-mRNA splicing on a genome-wide scale 11 , including experiments using exon-junction arrays 12 or predicted exons on chromosome 22 (ref. 1). But these approaches are limited to predicted or known exons and do not allow new exon discovery.To identify genes regulated at the level of alternative splicing by experimental means, we developed an approach to selectively clone differentially spliced exons from distinct biological samples into alternative splicing libraries (ASLs) and to sequence alternative splicing sequence enriched tags (ASSETs). As ASSETs encompass exons along with their related splice junctions, they not only allow exon and gene identification but also enable the analysis of different splicing types, which are beyond the reach of present computational predictions.As cancer-r...

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Therapeutic potential of antisense oligonucleotides as modulators of alternative splicing

Cited by 189 publications

References 34 publications

RNA repair restores hemoglobin expression in IVS2–654 thalassemic mice

RNA repair restores hemoglobin expression in IVS2–654 thalassemic mice

Splicing of a Critical Exon of Human Survival Motor Neuron Is Regulated by a Unique Silencer Element Located in the Last Intron

Libraries enriched for alternatively spliced exons reveal splicing patterns in melanocytes and melanomas

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