Modification of Alternative Splicing by Antisense Oligonucleotides as a Potential Chemotherapy for Cancer and Other Diseases

Mercatante, Danielle R.; Sazani, Peter; Kole, Ryszard

doi:10.2174/1568009013334124

Cited by 45 publications

(18 citation statements)

References 128 publications

(148 reference statements)

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“…Finally, the systemic administration of these oligonucleotides might not be successful because of their inability to cross the blood-brain barrier. However, progress is being made in this field with synthetic chemistries that are able to cross the blood-brain barrier (35,36), a factor that will be of great importance in terms of therapeutic possibilities for patients with SMA.…”

Section: Discussionmentioning

confidence: 99%

Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulatesSMN2gene expression in patient fibroblasts

Skordis

Dunckley

Yue

et al. 2003

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

248

198

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The multiplicity of proteins compared with genes in mammals owes much to alternative splicing. Splicing signals are so subtle and complex that small perturbations may allow the production of new mRNA variants. However, the flexibility of splicing can also be a liability, and several genetic diseases result from single-base changes that cause exons to be skipped during splicing. Conventional oligonucleotide strategies can block reactions but cannot restore splicing. We describe here a method by which the use of a defective exon was restored. Spinal muscular atrophy (SMA) results from mutations of the Survival Motor Neuron (SMN) gene. Mutations of SMN1 cause SMA, whereas SMN2 acts as a modifying gene. The two genes undergo alternative splicing with SMN1, producing an abundance of full-length mRNA transcripts, whereas SMN2 predominantly produces exon 7-deleted transcripts. This discrepancy is because of a single nucleotide difference in SMN2 exon 7, which disrupts an exonic splicing enhancer containing an SF2͞ASF binding site. We have designed oligoribonucleotides that are complementary to exon 7 and contain exonic splicing enhancer motifs to provide trans-acting enhancers. These tailed oligoribonucleotides increased SMN2 exon 7 splicing in vitro and rescued the incorporation of SMN2 exon 7 in SMA patient fibroblasts. This treatment also resulted in the partial restoration of gems, intranuclear structures containing SMN protein that are severely reduced in patients with SMA. The use of tailed antisense oligonucleotides to recruit positively acting factors to stimulate a splicing reaction may have therapeutic applications for genetic disorders, such as SMA, in which splicing patterns are altered. P roximal spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by the degeneration of the motor neurons in the anterior horn of the spinal cord, resulting in muscular atrophy and weakness. The overall incidence of SMA is 1 in 10,000 live births, with a carrier frequency of 1 in 50. Onset is primarily in childhood, and three different forms are recognized, type I SMA being the most severe form and type III SMA at the milder end of the scale. Children affected by SMA I never sit and usually die within the first year of life, whereas those with type III acquire the ability to walk and have a normal life expectancy. An intermediate category (SMA II) is also recognized, where affected individuals can sit unsupported but cannot walk (1). The gene implicated in SMA is the Survival of Motor Neuron (SMN) gene located on chromosome 5q13 (2). It consists of eight exons, the first seven of which encode a 294-aa protein (3). The human SMN gene exists as a mirror-image duplication with a telomeric (SMN1) and a centromeric (SMN2) copy. Mutations in SMN1 cause SMA (4), whereas the copy number of the residual SMN2 genes is believed to modify the severity of the phenotype, as suggested by the increased copy number in patients with a milder disease course (5). Deletions of both SMN1 and SMN2 have never been observed ...

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Section: Discussionmentioning

confidence: 99%

Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulatesSMN2gene expression in patient fibroblasts

Skordis

Dunckley

Yue

et al. 2003

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

248

198

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“…A striking example is the Bcl-x gene, which has two isoforms, Bcl-xS and Bcl-xL, that arise from two different 59 splice sites in exon 2 (Mercatante et al 2001(Mercatante et al , 2002. The Bcl-xS isoform is pro-apoptotic and sensitizes cells to chemotherapy.…”

Section: Isoform Switchingmentioning

confidence: 99%

Antisense-mediated exon skipping: A versatile tool with therapeutic and research applications

Aartsma‐Rus¹,

Ommen²

2007

RNA

236

177

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Antisense-mediated modulation of splicing is one of the few fields where antisense oligonucleotides (AONs) have been able to live up to their expectations. In this approach, AONs are implemented to restore cryptic splicing, to change levels of alternatively spliced genes, or, in case of Duchenne muscular dystrophy (DMD), to skip an exon in order to restore a disrupted reading frame. The latter allows the generation of internally deleted, but largely functional, dystrophin proteins and would convert a severe DMD into a milder Becker muscular dystrophy phenotype. In fact, exon skipping is currently one of the most promising therapeutic tools for DMD, and a successful first-in-man trial has recently been completed. In this review the applicability of exon skipping for DMD and other diseases is described. For DMD AONs have been designed for numerous exons, which has given us insight into their mode of action, splicing in general, and splicing of the DMD gene in particular. In addition, retrospective analysis resulted in guidelines for AON design for DMD and most likely other genes as well. This knowledge allows us to optimize therapeutic exon skipping, but also opens up a range of other applications for the exon skipping approach.

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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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Modification of Alternative Splicing by Antisense Oligonucleotides as a Potential Chemotherapy for Cancer and Other Diseases

Cited by 45 publications

References 128 publications

Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulatesSMN2gene expression in patient fibroblasts

Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulatesSMN2gene expression in patient fibroblasts

Antisense-mediated exon skipping: A versatile tool with therapeutic and research applications

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

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