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

Abstract

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 ...

Identification of a Novel Cyclic AMP-response Element (CRE-II) and the Role of CREB-1 in the cAMP-induced Expression of the Survival Motor Neuron (SMN) Gene

“…The presence of SMN2 in all SMA patients, the ability of more copies of SMN2 to modify the SMA phenotype, and the rescue of SMA mice by multiple copies of SMN2 make it an attractive therapeutic candidate. Molecules capable of inducing SMN2 expression (30,31) or altering the splicing of SMN2 such that more full-length SMN transcript is produced have been identified (32)(33)(34)(35)(36)(37). At the present time, there is limited information on the mode of action of these compounds as well as the protein complexes that interact with the SMN promoter.…”

Reprogramming of tau alternative splicing by spliceosome-mediated RNA trans-splicing: Implications for tauopathies

“…Other, very elegant strategies to interfere with splicing events are based on the design of bifunctional oligoribonucleotides with a first domain complementary to an exonic enhancer or silencer element in the target pre-mRNA, and a second domain recruiting specific splicing factors near the splice site (35)(36)(37). Although effective, these technologies require the precise knowledge of cis-acting elements regulating splicing of the targeted exon.…”

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

“…These compounds have been used to modify alternative splicing in the SMN2 (spinal muscular atrophy), Bcl-x, and C-myc genes (29)(30)(31)(32)(33)(34)(35)(36). Besides blocking cryptic splice sites, antisense oligonucleotides can block splice regulation sequences in the FGFR1 gene (37).…”