A nonsense mutation-created intraexonic splice site is active in the lymphocytes,but not in the skeletal muscle of a DMD patient

Production of semi-functional dystrophin mRNA from the dystrophin gene encoding a premature stop codon has been shown to modify the severe phenotype of Duchenne muscular dystrophy (DMD). In this study, we report the tissue-specific production of semi-functional dystrophin mRNA via activation of a nonsense mutation-created intraexonic splice acceptor site. In a DMD patient a novel nonsense mutation was identified in exon 42. In his lymphocytes semi-functional dystrophin mRNA with a 63-nucleotide deletion in exon 42 (dys-63) was found to be produced. In vitro splicing assay using hybrid minigenes disclosed that the mutation-created intraexonic splice acceptor site was activated. In his skeletal muscle cells, however, only the authentically spliced dystrophin mRNA was found. This finding identifies the modulation of the splicing of muscle dystrophin mRNA in cases of DMD as a potential target for therapeutic strategies to generate a milder phenotype for this disease.     

Extensive and prolonged restoration of dystrophin expression with vivo-morpholino-mediated multiple exon skipping in dystrophic dogs

Duchenne muscular dystrophy (DMD) is a severe and the most prevalent form of muscular dystrophy, characterized by rapid progression of muscle degeneration. Antisense-mediated exon skipping is currently one of the most promising therapeutic options for DMD. However, unmodified antisense oligos such as morpholinos require frequent (weekly or bi-weekly) injections. Recently, new generation morpholinos such as vivo-morpholinos are reported to lead to extensive and prolonged dystrophin expression in the dystrophic mdx mouse, an animal model of DMD. The vivo-morpholino contains a cell-penetrating moiety, octa-guanidine dendrimer. Here, we sought to test the efficacy of multiple exon skipping of exons 6-8 with vivo-morpholinos in the canine X-linked muscular dystrophy, which harbors a splice site mutation at the boundary of intron 6 and exon 7. We designed and optimized novel antisense cocktail sequences and combinations for exon 8 skipping and demonstrated effective exon skipping in dystrophic dogs in vivo. Intramuscular injections with newly designed cocktail oligos led to high levels of dystrophin expression, with some samples similar to wild-type levels. This is the first report of successful rescue of dystrophin expression with morpholino conjugates in dystrophic dogs. Our results show the potential of phosphorodiamidate morpholino oligomer conjugates as therapeutic agents for DMD.     

Frameshift deletions of exons 3-7 and revertant fibers in Duchenne muscular dystrophy: mechanisms of dystrophin production.

Duchenne muscular dystrophy (DMD) patients with mutations that disrupt the translational reading frame produce little or no dystrophin. Two exceptions are the deletion of exons 3-7 and the occurrence of rare dystrophin-positive fibers (revertant fibers) in muscle of DMD patients. Antibodies directed against the amino-terminus and the 5' end of exon 8 did not detect dystrophin in muscle from patients who have a deletion of exons 3-7. However, in all cases, dystrophin was detected with an antibody directed against the 3' end of exon 8. The most likely method of dystrophin production in these cases is initiation at a new start codon in exon 8. We also studied two patients who have revertant fibers: one had an inherited duplication of exons 5-7, which, on immunostaining, showed two types of revertant fibers; and the second patient had a 2-bp nonsense mutation in exon 51, which creates a cryptic splice site. An in-frame mRNA that uses this splice site in exon 51 was detected. Immunostaining demonstrated the presence of the 3' end of exon 51, which is in agreement with the use of this mRNA in revertant fibers. The most likely method of dystrophin production in these fibers is a second mutation that restores the reading frame.     

A novel point mutation (G-1 to T) in a 5' splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker muscular dystrophy.

The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. We now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5' splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5' splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G-1-to-T mutation at the 5' splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection.     

Partial deletion of a dystrophin gene leads to exon skipping and to loss of an intra-exon hairpin structure from the predicted mRNA precursor.

In dystrophin Kobe exon 19 of the dystrophin gene is skipped during the process of mRNA precursor splicing even though the splice sites are unchanged (Matsuo et al. J. Clin. Invest. 87:2127-2131,1991). In the predicted secondary structure of the mRNA precursor, exon 19 of dystrophin Kobe is paired with intron sequences, whereas a large part of exon sequence from wild type is paired with itself and folded into a large hairpin structure. As all of 22 additional dystrophin exons analyzed also form intra-exon hairpin structures, these structures may be considered essential components of exons. We suggest that the abolishment of a hairpin structure in the truncated exon of dystrophin Kobe might prevent the splicing machinery from recognizing the splice sites and induce exon skipping.     

Long-term administration of antisense oligonucleotides into the paraspinal muscles of mdx mice reduces kyphosis

The mdx mouse model of muscular dystrophy has a premature stop codon preventing production of dystrophin. This results in a progressive phenotype causing centronucleation of skeletal muscle fibers, muscle weakness, and fibrosis and kyphosis. Antisense oligonucleotides alter RNA splicing to exclude the nonsense mutation, while still maintaining the open reading frame to produce a shorter, but partially functional dystrophin protein that should ameliorate the extent of pathology. The present study investigated the benefits of chronic treatment of mdx mice by once-monthly deep intramuscular injections of antisense oligonucleotides into paraspinal muscles. After 8 mo of treatment, mdx mice had reduced development of kyphosis relative to untreated mdx mice, a benefit that was retained until completion of the study at 18 mo of age (16 mo of treatment). This was accompanied by reduced centronucleation in the latissimus dorsi and intercostals muscles and reduced fibrosis in the diaphragm and latissimus dorsi. These benefits were accompanied by a significant increase in dystrophin production. In conclusion, chronic antisense oligonucleotide treatment provides clear and ongoing benefits to paralumbar skeletal muscle, with associated marked reduction in kyphosis.     

Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing

Besides linear RNAs, pre-mRNA splicing generates three forms of RNAs: lariat introns, Y-structure introns from trans-splicing, and circular exons through exon skipping. To study the persistence of excised introns in total cellular RNA, we used three Escherichia coli 3′ to 5′ exoribonucleases. Ribonuclease R (RNase R) thoroughly degrades the abundant linear RNAs and the Y-structure RNA, while preserving the loop portion of a lariat RNA. Ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase) also preserve the lariat loop, but are less efficient in degrading linear RNAs. RNase R digestion of the total RNA from human skeletal muscle generates an RNA pool consisting of lariat and circular RNAs. RT–PCR across the branch sites confirmed lariat RNAs and circular RNAs in the pool generated by constitutive and alternative splicing of the dystrophin pre-mRNA. Our results indicate that RNase R treatment can be used to construct an intronic cDNA library, in which majority of the intron lariats are represented. The highly specific activity of RNase R implies its ability to screen for rare intragenic trans-splicing in any target gene with a large background of cis-splicing. Further analysis of the intronic RNA pool from a specific tissue or cell will provide insights into the global profile of alternative splicing.     

Human ribosomal protein S26 inhibits splicing of its own pre-mRNA

In vitro splicing was studied for a human ribosomal protein (rp) S26 pre-mRNA fragment containing the first exon, first intron, and a part of the second exon. Splicing yielded two products, the first was corresponded to a fragment of the mature rpS26 mRNA and another was retained the 19 3'-terminal nucleotides of the first intron between the first and second exons. Recombinant rpS26 inhibites generation of both splicing products in vitro. The inhibition was specific, because another recombinant human rp, S19, had no effect on the splicing of the pre-mRNA fragment. Toe-printing was used to map the spS26-binding sites of the per-mRNA within the regions of the conventional and alternative 3' splicing sites of the first intron. On the strength of the rusults, rpS26 was assumed to regulate the expression of its own gene at the level of pre-mRNA splicing via a feedback mechanism.     

Mutation Types and Aging Differently Affect Revertant Fiber Expansion in Dystrophic Mdx and Mdx52 Mice

Duchenne muscular dystrophy (DMD), one of the most common and lethal genetic disorders, and the mdx mouse myopathies are caused by a lack of dystrophin protein. These dystrophic muscles contain sporadic clusters of dystrophin-expressing revertant fibers (RFs), as detected by immunohistochemistry. RFs are known to arise from muscle precursor cells with spontaneous exon skipping (alternative splicing) and clonally expand in size with increasing age through the process of muscle degeneration/regeneration. The expansion of revertant clusters is thought to represent the cumulative history of muscle regeneration and proliferation of such precursor cells. However, the precise mechanisms by which RFs arise and expand are poorly understood. Here, to test the effects of mutation types and aging on RF expansion and muscle regeneration, we examined the number of RFs in mdx mice (containing a nonsense mutation in exon 23) and mdx52 mice (containing deletion mutation of exon 52) with the same C57BL/6 background at 2, 6, 12, and 18months of age. Mdx mice displayed a significantly higher number of RFs compared to mdx52 mice in all age groups, suggesting that revertant fiber expansion largely depends on the type of mutation and/or location in the gene. A significant increase in the expression and clustering levels of RFs was found beginning at 6months of age in mdx mice compared with mdx52 mice. In contrast to the significant expansion of RFs with increasing age, the number of centrally nucleated fibers and embryonic myosin heavy chain-positive fibers (indicative of cumulative and current muscle regeneration, respectively) decreased with age in both mouse strains. These results suggest that mutation types and aging differently affect revertant fiber expansion in mdx and mdx52 mice.