Deliverables Antisense modulation as therapeutic tool Dystrophin deficiency, which leads to severe and progressive muscle degeneration in patients with Duchenne muscular dystrophy (DMD), is caused by frame shifting mutations in the dystrophin gene. A relatively new therapeutic strategy is based on antisense oligonucleotides (AONs) that induce the specific skipping of a single exon, such that the reading frame is restored. This allows the synthesis of a largely functional dystrophin, associated with a milder Becker muscular dystrophy phenotype. We have previously successfully targeted 20 different DMD exons that would, theoretically, be beneficial for >75% of all patients. To further enlarge this proportion, we here studied the feasibility of double and multiexon skipping. Using a combination of AONs, double skipping of exon 43 and 44 was induced, and dystrophin synthesis was restored in myotubes from one patient affected by a nonsense mutation in exon 43. For another patient, with an exon 46-50 deletion, the therapeutic double skipping of exon 45 and 51 was achieved. Remarkably, in control myotubes, the latter combination of AONs caused the skipping of the entire stretch of exons from 45 through 51. This in-frame multiexon skipping would be therapeutic for a series of patients carrying different DMD-causing mutations. In fact, we here demonstrate its feasibility in myotubes from a patient with an exon 48-50 deletion. The application of multiexon skipping may provide a more uniform methodology for a larger group of patients with DMD. Since the initial characterization of the genetic defect for Duchenne muscular dystrophy, much effort has been expended in attempts to develop a therapy for this devastating childhood disease. Gene therapy was the obvious answer but, initially, the dystrophin gene and its product seemed too large and complex for this approach. However, our increasing knowledge of the organization of the gene and the role of dystrophin in muscle function has indicated ways to manipulate them both. Gene therapy for Duchenne muscular dystrophy now seems to be in reach. The dystrophin deficiency leading to the severely progressing muscle degeneration in Duchenne muscular dystrophy (DMD) patients is caused by frame-shifting mutations in the DMD gene. We are developing a reading frame correction therapy aimed at the antisense-induced skipping of targeted exons from the pre-mRNA. Despite introducing a (larger) deletion, an in-frame transcript is generated that allows the synthesis of a slightly shorter, but largely functional dystrophin as found in the mostly milder Becker muscular dystrophy (BMD). We have recently demonstrated both the efficacy and high efficiency of the antisense-induced skipping of numerous exons from the DMD transcript in control muscle cells. In principle, this would restore the reading frame in over 75% of the patients reported in the Leiden DMD mutation database. In this study, we in fact demonstrate the broad therapeutic applicability of this strategy in cultured muscle cells from six DMD patients carrying different deletions and a nonsense mutation. In each case, the specific skipping of the targeted exon was induced, restoring dystrophin synthesis in over 75% of cells. The protein was detectable as soon as 16 h post-transfection, then increased to significant levels at the membrane within 2 days, and was maintained for at least a week. Finally, its proper function was further suggested by the restored membranal expression of four associated proteins from the dystrophin-glycoprotein complex. These results document important progress towards a clinically applicable, small-molecule based therapy. In-vivo analysis of splicing behaviour in XLDC Alternative splicing of pre-mRNA is a versatile regulatory mechanism that significantly expands the range of possible end products from a single gene. We previously identified an intronic rearrangement in dystrophin intron 11 in one family with X-linked dilated cardiomyopathy (XLDC), causing incorporation of an aberrant exon in a tissue-specific manner. In the present study we performed an in vivo splicing assay by using mini-genes containing part of the patient genomic rearrangement in C2C12 (skeletal muscle) myoblasts and myotubes, H9C2 (cardiac muscle) myocytes, and Hela cells. We show that inclusion of the aberrant exon is favored in H9C2 and differentiated C2C12 myotubes. These data suggest that the aberrant exon undergoes a differentiation-specific splicing. Unexpectedly, length of intron has a significant influence upon alternative splicing with longer introns favouring the inclusion of the aberrant exon in the cardiac cells. These data suggest that cardiac cells are more prone to steric hindrance between trans-acting factors, involved in the inclusion of the Alu exon. Comparative analysis of the intronic regions involved in the genomic rearrangement We perform a comparative study of a region of intron 11 of the dmd gene involved in the determination of the pathological phenotype in one XLDC patient. The region covers about 10kb located in the central part of intron 11 and contains a repetitive element of the LINE 1 family (L1PMA2). To estimate the approximate age of the L1PMA2 element insertion into the dystrophin intron 11 the orthologous genomic region was examined in selected species belonging to all major taxonomic groups of Primates (Gorilla and Orango from APES, Macaco from Old World Monkey, Marmoset from New World Monkey and Lemur from Prosimians). A L1PMA2 copy, located in the same position as in the human genome was found in all the species tested but lemur. From the latter species the partial sequence of the othologous genomic region revealed the lack of the L1PMA2 copy and a high similarity (80%) to the regions of human dystrophin intron 11 flanking the insertion site of the element. A PCR survey aimed at investigating the evolutionary pattern of the L1PMA2 element in the genomes of primates was also carried out. This was done by randomly amplifying part of the L1P_MA2 5 tail (Tail700) sequences in the same primate species reported above by using a primer pair designed on the L1PMA2 human consensus. We then cloned the PCR product and sequenced several clones. These sequences were used as a query to sort their human orthologues out from EMBL database. The complete alignment of all these sequences was used to reconstruct the relationships among human and other primate L1P_MA2 copies. - Cardazzo B, Bargelloni L, Toffolatti L, Rimessi P, Ferlini A, Patarnello T. «Tempo and mode of evolution of a primate specific retrotransposon belonging to the LINE 1 family.» (2003) J. Mol. Evol. 57 Suppl. 1: S268-S276. - B. Cardazzo, L. Bargelloni, L. Toffolatti, T. Patarnello. «Intervening sequences in paralogous genes: a comparative genomic approach to study the evolution of X chromosome introns.» (2003) Mol. Bio. Evol. 20(12): 2034-2041. Demonstration that a patient-derived pseudo-exon in dystrophin responds to hnRNP G and Tra2beta The hnRNP G family comprises three closely related proteins, hnRNP G, RBMY and hnRNP G-T. We showed previously that they interact with splicing activator proteins, particularly hTra2beta, and suggested that they were involved in regulating Tra2-dependent splicing. We show here that hnRNP G and hTra2beta have opposite effects upon the incorporation of several exons, both being able to act as either an activator or a repressor. HnRNP G acts via a specific sequence to repress the skeletal muscle-specific exon (SK) of human slow skeletal alpha-tropomyosin, TPM3, and stimulates inclusion of the alternative non-muscle exon. The binding of hnRNP G to the exon is antagonized by hTra2beta. The two proteins also have opposite effects upon a dystrophin pseudo-exon. This exon is incorporated in a patient to a higher level in heart muscle than skeletal muscle, causing X-linked dilated cardiomyopathy. It is included to a higher level after transfection of a mini-gene into rodent cardiac myoblasts than into skeletal muscle myoblasts. Co-transfection with hnRNP G represses incorporation in cardiac myoblasts, whereas hTra2beta increases it in skeletal myoblasts. Both the cell specificity and the protein responses depend upon exon sequences. Since the ratio of hnRNP G to Tra2beta mRNA in humans is higher in skeletal muscle than in heart muscle, we propose that the hnRNP G/Tra2beta ratio contributes to the cellular splicing preferences and that the higher proportion of hnRNP G in skeletal muscle plays a role in preventing the incorporation of the pseudo-exon and thus in preventing skeletal muscle dystrophy. Further work showed also that several well-known proteins that generally stimulate splicing inhibit the splicing of this pseudo-exon. One of these, SF2/ASF, was studied in more detail. The site of action on the pseudo-exon was mapped, and a number of mutant proteins were studied to identify the protein domains involve in repression. Molecular definition of the mutation breakpoint We characterised the molecular breakpoint of patients with deletion covering the region between exon 44 and exon 50 of dystrophin gene. We analysed introns 44, 45, 47, 48 and 49 which sequence was completely available in public database and that measure 248, 36, 54, 37 and 17 kb respectively. These regions were characterised for repetitive elements using the RepeatMasker program (http://www.hgmp.mrc.ac.uk) and matrix attachment regions (MARS) using the MAR-WIZ program (http://www.futuresoft.org/MAR-Wiz). After masking the repetitive regions we designed a set of PCR primers and performing multiple PCR amplification of non overlapping fragments we determined the sublocalization of the deletion breakpoint in all the DNA samples with at least one end lying in that region. To PCR amplify and analyse in more detail the deletion junctions, we performed a long range PCR using primers as close as possible to the breakpoint extremities or utilised a PCR based genome walking technique (Clontech) starting from the last known region on one side of the breakpoint. The PCR fragments so amplify were then cloned and sequenced. In this way we succeeded in characterising 12 genomic junctions. We detected no substantial homologies between the normal DNA sequences located across the breakpoints. These data suggest that some mechanisms other than homologous recombination operated in the studied cases. In the breakpoint sequences analysed, we identified some elements that could be involved in double-strand break events. Inverted repeats, able to form stem-loop structures and associated with a strong topoisomerase II cleavage site, are found at or near to six breakpoints. The sequence TTTAAA, known to be able to induce a curvature in the DNA molecule (which may predispose it to recombination), is found in the 50 bp flanking the studied deletion breakpoints with a frequency that is higher than the average frequency of this sequence in the whole introns (1/560 versus 1/712), as previously found in dystrophin intron 49. With regard to the breakpoint distribution, the results obtained indicate that they are widely scattered in intron 48 and 49, as also observed in other regions of the dystrophin gene. Although the number of breakpoints analysed is not high, in intron 47 three of 11 breakpoint are clustered in a region of 2,5kb next to the only matrix attachment regions (MARs) identified. The analysis of DNA sequences across the breakpoints revealed in three cases an insertion of a few nucleotides (1-5 bp), in five cases a short homology region (2-4 bp), and more interestingly, in 4 junctions a duplication of variable length (9-24 bp). This molecular configuration at junction ends can be explained with the processes underlying nonhomologous end joining. Repair of double-strand breaks by this mechanism is achieved after limited processing of the DNA ends, followed by joining and re-ligation, which requires little or no sequence homology at the ends. This process, however, may lead to loss of or short insertions of nucleotides at the join, hence the alternative names of illegitimate recombination or errorprone recombination. We have compared the frequencies of repetitive sequences in introns 47 and 48 with that of the whole dystrophin gene. We observed that introns 47 and 48 are not only characterized by the highest recombination rate in the gene of healthy individuals and in BMD/DMD patients, but present a very high percentage of repetitive sequence as well. A similar correlation among recombination rate, deletion frequency, and percentage of repetitive elements was also observed in intron 7. Deletion breakpoints in introns 47 and 48 do not appear associated to any particular type of repetitive sequences, as also observed in other dystrophin introns (intron 7, intron 44). However, repetitive sequences often contain microsatellites, palindromes, and Alu-associated regions that could form hairpin loops predisposing to double-strand DNA breaks. Nonhomologous end joining is sometimes associated with the insertion of novel fragments of DNA into the double-strand break in yeast, plant, and also mammalian chromosomes. The sequences captured at double-strand molecular breaks had various sources, including microsatellites, retrotransposable elements, reverse transcripts of spliced introns, and exogenous DNA sequences. On the basis of these observations we hypothesize that the ancestral nucleotide sequence of large introns might have contained nucleotide sequence that predisposed to doublestrand molecular breaks, recombination, and insertion of DNA fragments. The inserted sequences might then have favoured further insertions and recombination increasing intron size and deletion frequencies that are characteristic of these intronic regions. Toffolatti L, et al. Investigating the mechanism of chromosomal deletion: characterization of 39 deletion breakpoints in introns 47 and 48 of the human dystrophin gene. Genomics. 2002; 80(5):523-30. Patient recruitment, breakpoint definition, RNA profiling, antisense modulation in patients with dystrophin mutations We performed a prenatal diagnosis in a family with X-linked dilated cardiomyopathy (XLDC) in which the causative mutation was a pure intronic deletion which causes the splicing of a novel, aberrant, out-of-frame exon into the dystrophin transcript. Our genetic test was performed by defining both the DNA (villous) and the RNA (amniocytes) configuration. The prenatal diagnosis resulted in a female foetus, proven to be a carrier of the genomic deletion. RNA analysis on cultured amniocytes revealed the presence of a well detectable dystrophin transcript as well as the coexistence of both the wild type and the abnormal splicing profile. This finding suggests that dystrophin splicing pattern in amniocytes and skeletal muscle is similar and that therefore this approach could be used in other prenatal dystrophin mutation detection, where abnormal RNA splicing is thought to play a role or for specific cases in which no mutations have been identified in the coding regions. We have precisely characterized genomic breakpoints within introns 2, 6 and 7 and identified the splicing profiles in a cohort of DMD/BMD patients with deletion of exons 3-7, 3-6 and duplication of exons 2-4. The findings of our study support the possibility that the re-initiation of translation mechanism play a role in exons 3-7 deleted patients phenotypic variability. Furthermore, we observed that the out-of-frame exon 2a is constantly spliced into a proportion of the dystrophin transcripts in all patients analysed. We identified and functionally characterised a purine-rich sequence located within dystrophin intron 11 and involved in splicing regulation. A functional role of this motif was suggested by an intronic dystrophin mutation causing X-linked dilated cardiomyopathy and determining the tissue-specific incorporation of an aberrant exon into the dystrophin transcript. This motif, as well as the 5 cryptic splice site used by the aberrant exon in the XLDC family, is contained within an atypical LINE1 5 region. The splicing sequence is contained within the aberrant XLDC exon and behaves in vitro as a splicing enhancer because its deletion strongly interferes with the dystrophin exon inclusion. Furthermore, by using RNA electro-mobility shift assay (REMSA) and sequence-specific UV cross-linking, we demonstrated that the splicing motif displays a nuclear protein binding affinity, further supporting a functional role in splicing modulation. We characterised a dystrophin gene rearrangement in a previously described family with X-linked dilated cardiomyopathy and we demonstrated that it represents an 11 kb deletion occurring within intron 11. This unique deletion joined two physiologically distant intronic regions and brought adjacent two crytpic splice sites, generating a 159 bp sequence recognised as a novel alternative exon and spliced into the dystrophin transcript. Comparative analysis of the intronic region involved in the breakpoint revealed the presence of a LINE1 element (L1P_MA2), containing a 5unconventional region (L1M1_5). This region provides the 5cryptic splice site utilised by the novel exon, includes part of the region spliced into the dystrophin transcript and contains a GA rich region compatible with a splicing motif. We performed an in vitro splicing assay by using a minigene containing the patient minimal genomic rearrangement and we reproduced the inclusion of the novel alternative exon seen in the patient tissues. Antisense splicing modulation targeting the 3crytpic splice site succeeded in restoring the canonical splicing. This represents a novel intronic mutational mechanism affecting the dystrophin gene and generating a splicing pathology. The definition of this mechanism might open perspectives in unravelling splicing regulatory motifs and their involvement in human genetic diseases. We have studied two Duchenne patients carrying the in-frame isolated deletion of dystrophin exon 5 and we have defined the genomic deletion breakpoints. Transcription analysis in skeletal muscle from one patient revealed a complex RNA configuration, combining an unfavorable exon skipping event (involving exon 6 and leading to an out-of-frame transcript) with the production of scrambled, circular RNA molecules. RNA circularization specifically involved the in-frame transcripts (retaining exon 6) with the consequence of depletion of the functional messenger. In addition, we also documented abundant in-frame exon 9 skipping. This peculiar splicing behaviour leading to a defect in the in-frame messenger RNA and in critical protein domains, might represent the pathogenic background underlying the severe clinical impact of the rare exon 5 deletion. The circular molecule formation focuses attention on the role that RNA scrambling might have in contributing to the clinical severity in dystrophin deletions. Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
