Splice Switching Oligonucleotides as Potential Therapeutics

Sazani, Peter; Gra̧ziewicz, Maria A.; Kole, Ryszard

doi:10.1201/9780849387951.ch4

Cited by 14 publications

(16 citation statements)

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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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“…This ability to repair the RNA and resurrect the missing protein, leading to amelioration of disease, is the key distinguishing feature of oligonucleotide-induced modulation of pre-mRNA splicing. It cannot be achieved by siRNA or classical antisense gapmer oligonucleotides, which lead only to degradation of the targeted RNA (25). The discovery of alternative splicing showed that this mechanism of competition between splice sites is not limited to just mutant genes, but that it also controls expression of the vast majority of native, properly functioning genes (108).…”

Section: Figurementioning

confidence: 99%

RNA therapeutics: beyond RNA interference and antisense oligonucleotides

Kole

Krainer

Altman³

2012

Nat Rev Drug Discov

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828

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Here we discuss three RNA therapeutic technologies exploiting various oligonucleotides that bind RNA by base-pairing in a sequence-specific manner yet have different mechanisms of action and effects. RNA interference and antisense oligonucleotides downregulate gene expression by enzyme-dependent degradation of targeted mRNA. Steric blocking oligonucleotides block access of cellular machinery to pre-mRNA and mRNA without degrading the RNA. Through this mechanism, blocking oligonucleotides can redirect alternative splicing, repair defective RNA, restore protein production or also downregulate gene expression. Moreover, they can be extensively chemically modified, resulting in more drug-like properties. The ability of RNA blocking oligonucleotides to restore gene function makes them suited for treatment of genetic disorders. Positive results from clinical trials for the treatment of Duchenne muscular dystrophy show that this technology is close to realizing its clinical potential.

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“…[10]). One mechanism of interest and of particular importance for some diseases is the use of ASOs to alter splicing, for example, spinal muscle atrophy [49]. We first identified the key features of splice sites that influence the ability of ASOs to alter splicing [50] and then optimized the design of ASOs to take advantage of this mechanism.…”

Section: Other Posthybridization Mechanismsmentioning

confidence: 99%

Molecular Mechanisms of Antisense Oligonucleotides

Crooke

2017

Nucleic Acid Therapeutics

280

208

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In 1987, when I became interested in the notion of antisense technology, I returned to my roots in RNA biochemistry and began work to understand how oligonucleotides behave in biological systems. Since 1989, my research has focused primarily on this topic, although I have been involved in most areas of research in antisense technology. I believe that the art of excellent science is to frame large important questions that are perhaps not immediately answerable with existing knowledge and methods, and then conceive a long-term (multiyear) research strategy that begins by answering the most pressing answerable questions on the path to the long-term goals. Then, a step-by-step research pathway that will address the strategic questions posed must be implemented, adjusting the plan as new things are learned. This is the approach we have taken at Ionis. Obviously, to create antisense technology, we have had to address a wide array of strategic questions, for example, the medicinal chemistry of oligonucleotides, manufacturing and analytical methods, pharmacokinetics and toxicology, as well as questions about the molecular pharmacology of antisense oligonucleotides (ASOs). Each of these endeavors has consumed nearly three decades of scientific effort, is still very much a work-in-progress, and has resulted in hundreds of publications. As a recipient of the Lifetime Achievement Award 2016 granted by the Oligonucleotide Therapeutic Society, in this note, my goal is to summarize the contributions of my group to the efforts to understand the molecular mechanisms of ASOs.

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Splice Switching Oligonucleotides as Potential Therapeutics

Cited by 14 publications

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RNA repair restores hemoglobin expression in IVS2–654 thalassemic mice

RNA repair restores hemoglobin expression in IVS2–654 thalassemic mice

RNA therapeutics: beyond RNA interference and antisense oligonucleotides

Molecular Mechanisms of Antisense Oligonucleotides

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