ORCID Profile
0000-0002-8632-641X
Current Organisations
Murdoch University
,
PYC Therapeutics
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Publisher: Wiley
Date: 27-08-2003
DOI: 10.1016/S0014-5793(03)00904-9
Abstract: The use of antisense oligonucleotides (AOs) to induce exon skipping leading to generation of an in-frame dystrophin protein product could be of benefit in around 70% of Duchenne muscular dystrophy patients. We describe the use of hyaluronidase enhanced electrotransfer to deliver uncomplexed 2'-O-methyl modified phosphorothioate AO to adult dystrophic mouse muscle, resulting in dystrophin expression in 20-30% of fibres in tibialis anterior muscle after a single injection. Although expression was transient, many of the corrected fibres initially showed levels of dystrophin expression well above the 20% of endogenous previously shown to be necessary for phenotypic correction of the dystrophic phenotype.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 10-2000
DOI: 10.1097/00019052-200010000-00008
Abstract: Gene therapy for inherited muscle disease is an active area of research and development. Initial emphasis has been on gene replacement but alternative approaches are increasingly being considered in order to overcome difficulties, such as the immune rejection of transduced cells, the need for appropriate and tissue-specific control of expression, and the requirement for systemic spread in some conditions. However, the most significant obstacles to the clinical success of gene therapy are still the lack of efficiency and accuracy of gene medicine delivery.
Publisher: Elsevier BV
Date: 10-2011
Publisher: Mary Ann Liebert Inc
Date: 06-2023
Publisher: MDPI AG
Date: 25-06-2020
DOI: 10.3390/IJMS21124511
Abstract: Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease typically caused by protein-truncating mutations that preclude synthesis of a functional dystrophin. Exonic deletions are the most common type of DMD lesion, however, whole exon duplications account for between 10–15% of all reported mutations. Here, we describe in vitro evaluation of antisense oligonucleotide-induced splice switching strategies to re-frame the transcript disrupted by a multi-exon duplication within the DMD gene. Phosphorodiamidate morpholino oligomers and phosphorodiamidate morpholino oligomers coupled to a cell penetrating peptide were evaluated in a Duchenne muscular dystrophy patient cell strain carrying an exon 14–17 duplication. Two strategies were employed the conventional approach was to remove both copies of exon 17 in addition to exon 18, and the second strategy was to remove only the first copy of exon 17. Both approaches result in a larger than normal but in-frame DMD transcript, but surprisingly, the removal of only the first exon 17 appeared to be more efficient in restoring dystrophin, as determined using western blotting. The emergence of a normal sized DMD mRNA transcript that was not apparent in untreated s les may have arisen from back splicing and could also account for some of the dystrophin protein being produced.
Publisher: Elsevier BV
Date: 08-2009
DOI: 10.1038/MT.2009.49
Publisher: Elsevier BV
Date: 07-2007
Abstract: Protein-truncating mutations in the dystrophin gene lead to the most common childhood form of muscle wasting, Duchenne muscular dystrophy. Becker muscular dystrophy, a condition that typically arises from dystrophin gene lesions that do not disrupt the reading frame, clearly indicates that substantial domains of the dystrophin protein are not essential. Potential therapeutic intervention exists during pre-mRNA splicing, whereby selected exons are excised to either remove nonsense mutations or restore the reading frame around frame-shifting mutations from the mature mRNA. Appropriately designed antisense oligonucleotides (AOs), directed at amenable splicing motifs across the dystrophin gene transcript, block exon recognition and/or spliceosome assembly so that targeted exons are removed from the mature mRNA. We describe a panel of AOs designed to induce skipping of every exon within the human dystrophin gene transcript, with the exception of the first and last exons. Every exon targeted in vitro could be removed from the dystrophin mRNA, although some exons are more efficiently excluded than others. No single motif has emerged as a universal AO annealing site for redirection of dystrophin pre-mRNA processing, although the general trend is that the most efficient compounds are directed at motifs in the first half of the target exon.
Publisher: Public Library of Science (PLoS)
Date: 03-06-2014
Publisher: Elsevier BV
Date: 07-1997
DOI: 10.1016/S0960-8966(97)00057-6
Abstract: Golden retriever muscular dystrophy (GRMD) is an excellent model for the study of the efficacy of gene therapy in dystrophin deficient myopathies for there are many similarities between affected dogs and Duchenne muscular dystrophy (DMD) in boys. GRMD is not caused by deletion mutation but results from a point mutation in the consensus splice acceptor in intron 6 of the canine dystrophin gene. As a result exon 7 is skipped during processing of the GRMD dystrophin messenger RNA. We have developed a rapid test which makes direct use of exon 7 specific genomic PCR products. We have undertaken preliminary experiments on gene therapy using the mini-gene and the full length gene alone and in combination with lipofectin and/or the bacterial beta-galactosidase reporter gene Lac Z. Following direct injection of the Lac Z plasmid, either alone or with lipofectin, about 50% of the sites showed expression when biopsied some 14 days later. The beta-galactosidase activity was present in muscle and granulation tissue but was never abundant. Pups injected intraperitoneally with Lac Z were found to have positive material in their mesenteric lymph nodes, liver and spleen. Those injected with Lac Z and lipofectin also had positive material in the diaphragm, intercostal muscles and abdominal muscles, but again only a small amount of positive material was present at any of the sites. In animals directly injected into the muscle with the dystrophin mini-gene, half had positive staining for dystrophin in biopsies taken 14 days later. Of the 6 sites in the muscles of animals given the mini-gene and lipofectin only one had fibres positive for dystrophin when examined 14 days later. Six pups were injected directly with full-length gene construct and when biopsies were taken 10 days later two of the animals had strongly stained peripheries to a small number of fibres.
Publisher: Humana Press
Date: 2012
DOI: 10.1007/978-1-61779-767-5_11
Abstract: We have taken an empirical approach in designing splice-switching oligomers to induce targeted dystrophin exon skipping. The nucleotide sequence of the exon under examination is first analyzed for potential exon recognition motifs and then a set of oligomers complementary to the acceptor and donor splice sites, as well as intra-exonic regions predicted to contain exon splice enhancers, are designed and synthesized as 2'-O-methyl-modified bases on a phosphorothioate backbone (2OMeAOs). The 2OMeAOs can be readily transfected into cultured normal myogenic cells as cationic lipoplexes, and are incubated for 24 h before total RNA extraction and subsequent analysis by semi-quantitative RT-PCR. The lification conditions used for each dystrophin transcript region under investigation minimize preferential production of shorter licons and do not exaggerate the level of induced RT-PCR products, compared to the endogenous dystrophin transcript product. It is imperative that the test oligomers are transfected over a range of concentrations and that the target exon is excised in a reproducible and dose-dependent manner.Once it has been demonstrated that an oligomer can induce some degree of exon skipping, that target region of the pre-mRNA is assumed to be involved in splicing of the exon. A series of overlapping oligomers are prepared and evaluated by transfection into normal myogenic cells at lower concentrations to identify the more effective compounds. Clinical application requires antisense compounds that efficiently modulate splicing at low dosages, delivering the greatest benefits in terms of efficacy, safety, and cost.
Publisher: Elsevier BV
Date: 08-1992
DOI: 10.1016/1044-7431(92)90029-2
Abstract: The regeneration of skeletal muscle is dependent upon proliferation and fusion of activated mononuclear muscle precursor cells. Early and specific markers of this population of activated cells are the transcription factors MyoD and myogenin. Northern analysis was used to determine levels of MyoD and myogenin mRNA in (i) muscles regenerating after experimental crush injury and (ii) in limb muscles of dystrophic mdx mice at various ages in comparison to controls. In crush-injured muscle, MyoD and myogenin mRNA increased at 24 h, peaked between 2 to 6 days, and returned to uninjured control levels by 15 days after injury. In both mdx and control mice, MyoD and myogenin mRNA levels were high in fetal muscles and decreased rapidly during the 2 weeks after birth. In mdx muscles, the mRNA levels increased significantly from about 21 days, remained high until around 40 days, and then decreased to a relatively constant yet elevated level when compared to control muscles. The elevated levels persisted to 420 days of age. The results show that this technique can be used to provide sensitive quantitative information on the size of the population of activated precursor cells in skeletal muscle. As such, it represents a novel and convenient means of measuring regenerative activity in vivo in whole muscles.
Publisher: Bentham Science Publishers Ltd.
Date: 03-2010
DOI: 10.2174/138161210790883480
Abstract: In little more than a decade, induced exon skipping as a therapy to treat Duchenne muscular dystrophy (DMD) has progressed from a concept tested in vitro, to pre-clinical evaluation in mouse and dog models, and recent completion of Phase I clinical trials in man. There is no longer any doubt that antisense oligomers can redirect dystrophin gene processing and by-pass protein truncating mutations after direct injection into muscle. Proof-of-concept has been demonstrated in human dystrophic muscle, with trials in Leiden and London showing that two different oligomer chemistries can restore the reading-frame in selected DMD patients by excising dystrophin exon 51. Systemic delivery of both oligomer types into DMD patients has commenced with promising results but it remains to be established if this therapy will have measurable clinical benefits. Targeted removal of exon 51 will only be directly applicable to about one in ten DMD in iduals, and the immediate challenges include development of appropriate and effective delivery regimens, and extending splice-switching therapies to other dystrophin gene lesions. The success of induced exon skipping has spawned a number of "fusion therapies", including vector-mediated dystrophin exon skipping and ex vivo viral delivery of splice-switching antisense molecules into myogenic stem cells, followed by implantation, which may address long term oligomer delivery issues. This review summarizes the pivotal events leading to the completion of the first proof-of-concept trials and speculates on some of the scientific, ethical, regulatory and commercial challenges facing targeted exon skipping for the treatment of DMD.
Publisher: Mary Ann Liebert Inc
Date: 20-09-2001
DOI: 10.1089/104303401750476249
Abstract: Helper-dependent adenoviruses (HDAd) are Ad vectors lacking all or most viral genes. They hold great promise for gene therapy of diseases such as Duchenne muscular dystrophy (DMD), because they are less immunogenic than E1/E3-deleted Ad (first-generation Ad or FGAd) and can carry the full-length (Fl) dystrophin (dys) cDNA (12 kb). We have compared the transgene expression of a HDAd (HDAdCMVDysFl) and a FGAd (FGAdCMV-dys) in cell culture (HeLa, C2C12 myotubes) and in the muscle of mdx mice (the mouse model for DMD). Both vectors encoded dystrophin regulated by the same cytomegalovirus (CMV) promoter. We demonstrate that the amount of dystrophin expressed was significantly higher after gene transfer with FGAdCMV-dys compared to HDAdCMVDysFl both in vitro and in vivo. However, gene transfer with HDAdCMVDysFl in the presence of a FGAd resulted in a significant increase of dystrophin expression indicating that gene products synthesized by the FGAd increase, in trans, the amount of dystrophin produced. This enhancement occurred in cell culture and after gene transfer in the muscle of mdx mice and dystrophic golden retriever (GRMD) dogs, another animal model for DMD. The E4 region of Ad is required for the enhancement, because no increase of dystrophin expression from HDAdCMVDysFl was observed in the presence of an E1/E4-deleted Ad in vitro and in vivo. The characterization of these enhancing gene products followed by their inclusion into an HDAd may be required to produce sufficient dystrophin to mitigate the pathology of DMD by HDAd-mediated gene transfer.
Publisher: MDPI AG
Date: 04-2022
DOI: 10.3390/IJMS23073937
Abstract: Spinal muscular atrophy (SMA) is a severe, debilitating neuromuscular condition characterised by loss of motor neurons and progressive muscle wasting. SMA is caused by a loss of expression of SMN1 that encodes the survival motor neuron (SMN) protein necessary for the survival of motor neurons. Restoration of SMN expression through increased inclusion of SMN2 exon 7 is known to ameliorate symptoms in SMA patients. As a consequence, regulation of pre-mRNA splicing of SMN2 could provide a potential molecular therapy for SMA. In this study, we explored if splice switching antisense oligonucleotides could redirect the splicing repressor hnRNPA1 to the hnRNPA1b isoform and restore SMN expression in fibroblasts from a type I SMA patient. Antisense oligonucleotides (AOs) were designed to promote exon 7b retention in the mature mRNA and induce the hnRNPA1b isoform. RT-PCR and western blot analysis were used to assess and monitor the efficiency of different AO combinations. A combination of AOs targeting multiple silencing motifs in hnRNPA1 pre-mRNA led to robust hnRNPA1b induction, which, in turn, significantly increased expression of full-length SMN (FL-SMN) protein. A combination of PMOs targeting the same motifs also strongly induced hnRNPA1b isoform, but surprisingly SMN2 exon 5 skipping was detected, and the PMO cocktail did not lead to a significant increase in expression of FL-SMN protein. We further performed RNA sequencing to assess the genome-wide effects of hnRNPA1b induction. Some 3244 genes were differentially expressed between the hnRNPA1b-induced and untreated SMA fibroblasts, which are functionally enriched in cell cycle and chromosome segregation processes. RT-PCR analysis demonstrated that expression of the master regulator of these enrichment pathways, MYBL2 and FOXM1B, were reduced in response to PMO treatment. These findings suggested that induction of hnRNPA1b can promote SMN protein expression, but not at sufficient levels to be clinically relevant.
Publisher: Springer Science and Business Media LLC
Date: 10-09-2020
Publisher: Public Library of Science (PLoS)
Date: 22-04-2013
Publisher: Hindawi Limited
Date: 2009
DOI: 10.1002/HUMU.20806
Abstract: Out of three mutations in the dystrophin gene that cause Duchenne muscular dystrophy (DMD), the most common, serious childhood muscle wasting disease, two are genomic deletions of one or more exons that disrupt the reading frame. Specific removal of an exon flanking a genomic deletion using antisense oligonucleotide intervention during pre-RNA processing can restore the reading frame and could potentially reduce disease severity. We describe a rare dystrophin gene rearrangement inversion of approximately 28 kb, flanked by a 10-bp duplication and an 11-kb deletion, which led to the omission of exons 49 and 50 from the mature mRNA and the variable inclusion of several pseudoexons. In vitro transfection of cultured patient cells with antisense oligonucleotides directed at exon 51 induced efficient removal of that exon, as well as one of the more commonly included pseudoexons, suggesting closely coordinated splicing of these exons. Surprisingly, several antisense oligonucleotides (AOs) directed at this pseudoexon had no detectable effect on the splicing pattern, while all AOs directed at the other predominant pseudoexon efficiently excised that target. Antisense oligomers targeting dystrophin exon 51 for removal are currently undergoing clinical trials. Despite the unique nature of the dystrophin gene rearrangement described here, a personalized multiexon skipping treatment is applicable and includes one compound entering clinical trials for DMD.
Publisher: Medknow
Date: 2008
Abstract: Duchenne muscular dystrophy (DMD), the most common and serious form of childhood muscle wasting is generally caused by protein-truncating mutations in the large DMD gene. Specific removal of an exon from a defective DMD gene transcript has the potential to allow synthesis of a semi-functional dystrophin, thereby reducing the severity and presumably progression of muscle wasting. The efficacy of this treatment will vary greatly between the different mutations that preclude the synthesis of a functional dystrophin. Restoration of the reading frame from a large multi-exon genomic deletion, typically greater than 36 exons, may lead to synthesis of a protein with only partial function and limited clinical benefit, whereas excising a nonsense mutation in a redundant exon should generate a near normal dystrophin. A clinical trial has recently confirmed proof-of-principle that exclusion of Exon 51 from human dystrophin mRNAs, carrying frame-shifting deletions adjacent to this exon, results in dystrophin expression. No major side-effects after local administration of the antisense oligomer were reported. Additional trials are underway, targeting the same exon but using an oligomer of different backbone chemistry. If functional dystrophin synthesis is demonstrated, and safety issues are addressed, subsequent trials will involve systemic delivery. Great challenges are ahead, some technical establishing an effective delivery regimen, some ethical choosing subsequent targets for therapy, and others of an administrative and regulatory nature.
Publisher: Public Library of Science (PLoS)
Date: 08-01-2012
Publisher: Informa UK Limited
Date: 03-04-2015
Publisher: Cold Spring Harbor Laboratory
Date: 18-10-2018
DOI: 10.1101/446773
Abstract: Oligonucleotides and nucleic acid analogues that alter gene expression are showing therapeutic promise for selected human diseases. The modification of synthetic nucleic acids to protect against nuclease degradation and to influence drug function is common practice, however, such modifications may also confer unexpected physicochemical and biological properties. Here we report backbone-specific effects of modified oligonucleotides on subnuclear organelles, altered distribution of nuclear proteins, the appearance of novel structured nuclear inclusions, and modification of RNA processing in cultured cells transfected with antisense oligonucleotides on a phosphorothioate backbone. Phosphodiester and phosphorodiamidate morpholino oligomers elicited no such consequences. Disruption of subnuclear structures and proteins elicit severe phenotypic disturbances, revealed by transcriptomic analysis of fibroblasts exhibiting such disruption. These data suggest that the toxic effects and adverse events reported after clinical evaluation of phosphorothioate nucleic acid drugs may be mediated, at least in part, by non-specific interaction of nuclear components with the phosphorothioate backbone.
Publisher: Wiley
Date: 29-01-2009
Publisher: Bentham Science Publishers Ltd.
Date: 2009
DOI: 10.2174/1874467210902010110
Abstract: Duchenne muscular dystrophy (DMD) arises from protein-truncating mutations in the large dystrophin gene that preclude synthesis of a functional protein that primarily stabilizes muscle fibre membranes. The absence of dystrophin leads to this most common and serious form of childhood muscle-wasting. Since the identification of the dystrophin gene in 1987, cell and gene repair or replacement therapies have been evaluated for DMD treatment and one genetic intervention, exon skipping, is now in clinical trials. Antisense oligomers have been designed to redirect dystrophin splicing patterns so that targeted exons may be removed from a defective dystrophin pre-mRNA to either restore the reading frame of a deletion, or excise an in-frame exon corrupted by a nonsense mutation or micro-insertion/deletion. This review discusses the evolution of oligomer induced exon skipping, including in vitro applications, evaluation of different oligomer chemistries, the treatment of animal models and alternative exon skipping strategies involving viral expression cassettes and ex vivo manipulation of stem cells. The discussion culminates with the current clinical trials and the great challenges that lie ahead. The major obstacle to the implementation of personalised genetic treatments to address the many different mutations that can lead to DMD, are considered to be establishing effective treatments for the different patients and their mutations. Furthermore, the view of regulatory authorities in assessing preclinical data on potentially scores of different but class-specific compounds will be of paramount importance in expediting the clinical application of exon skipping therapy for this serious and relentlessly progressive muscle wasting disease.
Publisher: Elsevier BV
Date: 10-2006
DOI: 10.1016/J.NMD.2006.05.017
Abstract: Antisense oligonucleotide (AO) manipulation of pre-mRNA splicing of the dystrophin gene is showing promise in overcoming Duchenne muscular dystrophy (DMD)-causing mutations. To date, this approach has been limited to studies using animal models or cultured human muscle cells, and evidence that AOs can induce exon skipping in human muscle has yet to be shown. In this study, we used different AO analogues to induce exon skipping in muscle explants derived from normal and DMD human tissue. We propose that inducing exon skipping in human muscle explants is closer to in vivo conditions than cells in monolayer cultures, and may minimize the numbers of participants in Phase I clinical studies to demonstrate proof of principle of exon skipping in human muscle.
Publisher: MDPI AG
Date: 18-10-2020
DOI: 10.3390/IJMS21207705
Abstract: The COL7A1 gene encodes homotrimer fibrils essential for anchoring dermal and epidermal layers, and pathogenic mutations in COL7A1 can cause recessive or dominant dystrophic epidermolysis bullosa. As a monogenic disease gene, COL7A1 constitutes a potential target for antisense oligomer-mediated exon skipping, a therapy applicable to a growing number of other genetic disorders. However, certain characteristics of COL7A1: many exons, low average intron size, and repetitive and guanine-cytosine rich coding sequence, present challenges to the design of specific and effective antisense oligomers. While targeting COL7A1 exons 10 and 73 for excision from the mature mRNA, we discovered that antisense oligomers comprised of 2′-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers produced similar, but distinctive, splicing patterns including excision of adjacent nontargeted exons and/or retention of nearby introns in some transcripts. We found that the nonsequential splicing of certain introns may alter pre-mRNA processing during antisense oligomer-mediated exon skipping and, therefore, additional studies are required to determine if the order of intron removal influences multiexon skipping and/or intron retention in processing of the COL7A1 pre-mRNA.
Publisher: Elsevier BV
Date: 10-2005
DOI: 10.1016/J.COPH.2005.06.001
Abstract: The manipulation of pre-mRNA to alter gene transcript splicing patterns offers considerable potential for many genetic disorders. In particular, the targeted removal of one or more exons from a gene transcript can skip over, or compensate for, disease-causing mutations. Duchenne muscular dystrophy (DMD), the most common and severe form of muscular dystrophy, is one such disorder that could benefit from this strategy. Splicing modulation can convert a DMD phenotype into the less severe allelic Becker-like phenotype. Recent studies using antisense oligonucleotide-targeted exon skipping to induce near normal dystrophin in vivo in animal models, and in vitro in DMD cell lines, highlight the promise of this approach. On the basis of these successes, human clinical trials could be realized in the near future.
Publisher: Elsevier BV
Date: 06-2005
Publisher: Cold Spring Harbor Laboratory
Date: 05-2020
DOI: 10.1101/2020.05.01.071696
Abstract: Many long non-coding RNAs (lncRNA) are highly dysregulated in cancer and are emerging as therapeutic targets. One ex le is NEAT1, which consists of two overlapping lncRNA isoforms, NEAT1_1 (3.7kb) and NEAT1_2 (23kb), that are functionally distinct. The longer NEAT1_2 is responsible for scaffolding gene-regulatory nuclear bodies termed paraspeckles, whereas NEAT1_1 is involved in paraspeckle-independent function. The NEAT1 isoform ratio is dependent on the efficient cleavage and polyadenylation of NEAT1_1 at the expense of NEAT1_2. Here we developed a targeted antisense oligonucleotide (ASO) approach to sterically block NEAT1_1 polyadenylation processing, achieving upregulation of NEAT1_2 and abundant paraspeckles. We have applied these ASOs to cells of the heterogeneous infant cancer, neuroblastoma, as we found higher NEAT1_1:NEAT1_2 ratio and lack of paraspeckles in high-risk neuroblastoma cells. These ASOs decrease NEAT1_1 levels, increase NEAT1_2 araspeckles and concomitantly reduce cell viability in high-risk neuroblastoma specifically. In contrast, overexpression of NEAT1_1 has the opposite effect, increasing cell-proliferation. Transcriptomic analyses of high-risk neuroblastoma cells with altered NEAT1 ratios and increased paraspeckle abundance after ASO treatment showed an upregulation of differentiation pathways, as opposed to the usual aggressive neuroblastic phenotype. Thus, we have developed potential anti-cancer ASO drugs that can transiently increase growth-inhibiting NEAT1_2 RNA at the expense of growth-promoting NEAT1_1 RNA. These ASOs, unlike others that degrade lncRNAs, provide insights into the importance of altering lncRNA polyadenylation events to suppress tumorigenesis as a strategy to combat cancer.
Publisher: Wiley
Date: 28-10-2021
DOI: 10.1002/MGG3.1840
Abstract: Cryptic exons are typically characterised as deleterious splicing aberrations caused by deep intronic mutations. However, low‐level splicing of cryptic exons is sometimes observed in the absence of any pathogenic mutation. Five recent reports have described how low‐level splicing of cryptic exons can be modulated by common single‐nucleotide polymorphisms (SNPs), resulting in phenotypic differences amongst different genotypes. We sought to investigate whether additional ‘SNPtic’ exons may exist, and whether these could provide an explanatory mechanism for some of the genotype–phenotype correlations revealed by genome‐wide association studies. We thoroughly searched the literature for reported cryptic exons, cross‐referenced their genomic coordinates against the dbSNP database of common SNPs, then screened out SNPs with no reported phenotype associations. This method discovered five probable SNPtic exons in the genes APC , FGB , GHRL , MYPBC3 and OTC . For four of these five exons, we observed that the phenotype associated with the SNP was compatible with the predicted splicing effect of the nucleotide change, whilst the fifth (in GHRL ) likely had a more complex splice‐switching effect. Application of our search methods could augment the knowledge value of future cryptic exon reports and aid in generating better hypotheses for genome‐wide association studies.
Publisher: Wiley
Date: 14-11-2005
DOI: 10.1002/JGM.838
Abstract: Duchenne and Becker muscular dystrophies are allelic disorders arising from mutations in the dystrophin gene. Duchenne muscular dystrophy is characterised by an absence of functional protein, while Becker muscular dystrophy is usually caused by in-frame deletions allowing synthesis of some functional protein. Treatment options are limited, and we are investigating the potential of transcript manipulation to overcome disease-causing mutations. Antisense oligonucleotides have been used to induce specific exon removal during processing of the dystrophin primary transcript and thereby by-pass protein-truncating mutations. The antisense oligonucleotide chemistry most widely used to alter pre-mRNA processing is 2'-O-methyl-modified bases on a phosphorothioate backbone. The present studies evaluate 2'-O-methylphosphorothioate, peptide nucleic acid and morpholino antisense oligonucleotides in the mdx mouse model of muscular dystrophy, which has a nonsense mutation in exon 23 of the dystrophin gene. We demonstrate dystrophin expression in mdx mouse tissues after localised and systemic delivery of a morpholino antisense oligonucleotide designed to target the dystrophin exon 23 donor splice site. The stability of the morpholino structural type, and the fact that it can be delivered to muscle in the absence of a delivery reagent, render this compound eminently suitable for consideration for therapeutic exon skipping to address dystrophin mutations.
Publisher: Bentham Science Publishers Ltd.
Date: 08-2011
DOI: 10.2174/156652311796150381
Abstract: Antisense oligomers initially showed promise as compounds to modify gene expression, primarily through RNaseH induced degradation of the target transcript. Expansion of the field has led to new chemistries capable of invoking different mechanisms, including suppression of protein synthesis by translational blockade and gene silencing using short interfering RNAs. It is now apparent that the majority of the eukaryotic genome is transcribed and non-protein coding RNAs have been implicated in the regulation of gene expression at many levels. This review considers potential therapeutic applications of antisense oligomers to modify gene expression, primarily by interfering with the process of exon recognition and intron removal during gene transcript splicing. While suppression of gene expression will be necessary to address some conditions, it is likely that antisense oligomer splice modification will have extensive clinical application. Pre-mRNA splicing is a tightly co-ordinated, multifactorial process that can be disrupted by antisense oligomers in a highly specific manner to suppress aberrant splicing, remove exons to by-pass nonsense or frame-shifting mutations or influence exon selection to alter spliceoform ratios. Manipulation of splicing patterns has been applied to a erse range of conditions, including b-thalassemia, Duchenne muscular dystrophy, spinal muscular atrophy and certain cancers. Alternative exon usage has been identified as a major mechanism for generating ersity from a limited repertoire of genes in higher eukaryotes. Considering that the majority of all human primary gene transcripts are reportedly alternatively spliced, intervention at the level of pre-mRNA processing is likely to become increasingly significant in the fight against genetic and acquired disorders.
Publisher: Elsevier BV
Date: 12-2014
Publisher: Elsevier BV
Date: 04-2001
DOI: 10.1016/S0960-8966(00)00187-5
Abstract: Golden retriever muscular dystrophy arises from a mutation in the acceptor splice site of intron 6 of the dystrophin gene. Skipping of exon 7 disrupts the mRNA reading frame and results in premature termination of translation. We are using this animal model to evaluate treatments for Duchenne muscular dystrophy, including gene repair induced by chimeric oligonucleotides. After injection of golden retriever muscular dystrophy (GRMD) muscle with a chimeric oligonucleotide to repair the lesion, immunostaining revealed a modest increase in the number of dystrophin-positive fibres at the injection sites. Dystrophin gene transcripts containing exon 7 were detected by reverse transcription-polymerase chain reaction, suggesting that low levels of splice site correction may have occurred. However, DNA sequencing of these apparently normal dystrophin gene transcripts revealed that the first five bases of exon 7 were missing. It will be important to be aware of this phenomenon with respect to further gene correction studies in the canine model.
Publisher: Public Library of Science (PLoS)
Date: 28-08-2013
Publisher: Elsevier BV
Date: 10-2005
DOI: 10.1016/J.NMD.2005.06.009
Abstract: Induction of specific exon skipping during the processing of the dystrophin gene transcript is being pursued as a potential therapy for Duchenne muscular dystrophy. Antisense oligonucleotides directed at motifs involved in pre-mRNA processing can manipulate dystrophin exon incorporation in the mature gene transcript. We have compared the exon skipping ability of oligodeoxyribonucleotides with compounds of the identical sequence incorporating 2'-O-methyl modified bases. Antisense oligonucleotides composed entirely of 2'-O-methyl modified bases on a phosphorothioate backbone were consistently more efficient at inducing exon skipping than comparable oligodeoxyribonucleotides. Chimeric antisense oligonucleotides, mixtures of unmodified and 2'-O-methyl modified bases, induced intermediate levels of exon skipping. In addition, we describe terminal modifications that may be incorporated into the 2'-O-methyl antisense oligonucleotides to further enhance efficiency of exon skipping. Our findings suggest that 2'-O-methyl antisense oligonucleotides should be considered for human clinical trials involving targeted exon skipping in dystrophin gene expression in preference to oligodeoxyribonucleotides.
Publisher: Springer Science and Business Media LLC
Date: 25-05-2006
Abstract: Manipulation of pre-mRNA splicing by antisense oligonucleotides (AOs) offers considerable potential for a number of genetic disorders. One of these is Duchenne muscular dystrophy (DMD), where mutations in the dystrophin gene typically result in premature termination of translation that causes a loss of functional protein. AOs can induce exon skipping such that the mutation is by-passed and the reading frame restored, producing an internally deleted protein similar to that found in the milder Becker muscular dystrophy. To date, this approach has been applied to the mdx mouse model in vitro and in vivo and in human myoblast cultures. Here, we report the application of AO-directed exon skipping to induce dystrophin expression in vitro in a canine model of DMD, golden retriever muscular dystrophy (GRMD). The efficacy of 2'-O-methyl phosphorothioate (2OMe), phosphorodiamidate morpholino oligomers (PMOs) and peptide-linked PMOs (PMO-Pep) to induce dystrophin expression was assessed. The 2OMe chemistry was only effective for short-term induction of corrected transcript and could not induce detectable dystrophin protein. The PMO chemistry generally induced limited exon skipping at only high concentrations however, a low level of dystrophin protein was produced in treated cells. Use of the PMO-Pep, applied here for the first time to a DMD model, was able to induce high and sustained levels of exon skipping and induced the highest level of dystrophin expression with no apparent adverse effects upon the cells. The induction of dystrophin in the GRMD model offers the potential for further testing of AO delivery regimens in a larger animal model of DMD, in preparation for application in human clinical trials.
Publisher: Proceedings of the National Academy of Sciences
Date: 26-06-2014
Abstract: Duchenne muscular dystrophy (DMD) is a fatal X-linked disease that results in cardiomyopathy and heart failure. The cardiomyopathy is characterized by cytoskeletal protein disarray, contractile dysfunction, and reduced energy production. The mechanisms for altered energy metabolism are not yet fully clarified. The L-type Ca 2+ channel regulates excitation and contraction in the heart, and can regulate mitochondrial function via the movement of cytoskeletal proteins. Here, we find that myocytes from the murine model of DMD ( mdx ) exhibit impaired communication between the L-type Ca 2+ channel and the mitochondria that results in poor energy production. Morpholino oligomer therapy targeting dystrophin or block of the mitochondrial voltage-dependent anion channel (VDAC) “rescues” metabolic function, indicating that impaired communication between the L-type Ca 2+ channel and VDAC contributes to the cardiomyopathy.
Publisher: Elsevier BV
Date: 03-1998
DOI: 10.1016/S1357-2725(97)00108-8
Abstract: Pax7 is a paired-type homeobox gene which has previously been shown to play an important role in skeletal muscle formation. It is expressed in skeletal muscle of the limbs during embryogenesis and in adulthood. The aims of this study were firstly to determine the degree of polymorphism of Pax7 amongst inbred laboratory mice using Southern blotting and Pax7 regional specific sub-probes. Secondly, functional studies were performed on mice with each of the different structural forms of Pax7 to determine whether they were associated with differences in the ability to regenerate damaged skeletal muscle. Four different allelic forms of Pax7 have now been identified in laboratory mice indicating that the previously reported DNA sequence of Pax7 is not applicable to all laboratory mice. Hybridisation patterns of TaqI digested DNA representing each of the different Pax7 alleles with the Pax7 specific sub-probes suggested that in contrast to previous findings, Pax7 is associated with two highly polymorphic homeoboxes. The presence of two homeoboxes in BALB/c mice has been confirmed by DNA sequencing. Results of functional studies have also shown that the ability to regenerate damaged skeletal muscle in adult mice is strongly associated with the presence of a 0.15-kb TaqI fragment derived from one of the homeoboxes.
Publisher: Elsevier BV
Date: 2014
DOI: 10.1038/MTNA.2014.8
Publisher: Wiley
Date: 2003
DOI: 10.1002/JGM.361
Abstract: Duchenne muscular dystrophy (DMD) is an X-linked recessive muscle wasting disorder characterised by the absence of the protein dystrophin. Antisense oligonucleotides have been used to re-direct dystrophin pre-mRNA processing by blocking sequences crucial to pre-mRNA splicing, thereby inducing skipping of specific exons. We wished to determine which splicing motifs are most amenable as targets for antisense oligonucleotide induction of efficient and specific skipping of selected exons. Antisense oligonucleotides were directed at regions of dystrophin exon 19 involved in pre-mRNA splicing, including the donor and acceptor splice sites and the exon splicing enhancer (ESE). Cultured myotubes were transfected with antisense oligonucleotides at various concentrations and studies undertaken to determine both specificity and efficiency of induced exon 19 skipping. Antisense oligonucleotides as small as 12 nucleotides targeting the ESE induced consistent and specific skipping of only exon 19 in both human and normal and mdx mouse myotubes. Antisense oligonucleotides directed at the donor and acceptor splice sites also induced specific exon 19 skipping while mismatched antisense oligonucleotides could only induce skipping when delivered at higher concentrations. No other dystrophin exons were removed from the mature mRNA as a consequence of these antisense oligonucleotides treatments. Antisense oligonucleotides directed at the ESE tended to be marginally more efficient than those which targeted the donor or acceptor splice sites, based on their ability to induce specific skipping at lower concentrations. The specificity of exon removal does not appear to be a function of target selection, but may reflect the combination of the splicing motifs and position of that exon in the pre-mRNA.
Publisher: MDPI AG
Date: 10-2020
DOI: 10.3390/IJMS21197282
Abstract: Parkin-type autosomal recessive juvenile-onset Parkinson’s disease is caused by mutations in the PRKN gene and accounts for 50% of all autosomal recessive Parkinsonism cases. Parkin is a neuroprotective protein that has dual functions as an E3 ligase in the ubiquitin–proteasome system and as a transcriptional repressor of p53. While genomic deletions of PRKN exon 3 disrupt the mRNA reading frame and result in the loss of functional parkin protein, deletions of both exon 3 and 4 maintain the reading frame and are associated with a later onset, milder disease progression, indicating this particular isoform retains some function. Here, we describe in vitro evaluation of antisense oligomers that restore functional parkin expression in cells derived from a Parkinson’s patient carrying a heterozygous PRKN exon 3 deletion, by inducing exon 4 skipping to correct the reading frame. We show that the induced PRKN transcript is translated into a shorter but semi-functional parkin isoform able to be recruited to depolarised mitochondria, and also transcriptionally represses p53 expression. These results support the potential use of antisense oligomers as a disease-modifying treatment for selected pathogenic PRKN mutations.
Publisher: Wiley
Date: 06-08-2020
DOI: 10.1002/MED.21718
Publisher: Elsevier BV
Date: 07-1997
DOI: 10.1016/S1357-2725(97)00023-X
Abstract: Pax7 is associated with formation of skeletal muscle and the neural tube in developing embryos. Interestingly, in adult mice, rearrangements of Pax7 are associated with differences in the efficiency of skeletal muscle regrowth between mouse strains. The aim of this study was to investigate the possibility that Pax7 is expressed in skeletal muscle or other tissues from adult mice. Total RNA was isolated from adult mouse tissues and the polymerase chain reaction was performed on reverse transcribed mRNA using primers specific for regions that encode the paired and homeodomain of Pax7. At least four different Pax7 transcripts were found. A full-length transcript similar in sequence to that published previously was identified in skeletal muscle, brain and spleen cells of adult mice. Further putative full-length Pax7 transcripts, including one that contains a hexanucleotide insertion in the paired box and one in which approximately 10 bp have been deleted in the homeobox, were found to be expressed in skeletal muscle and brain of adult mice, respectively. A truncated Pax7 splice product comprising the paired box only was found to be expressed in most adult tissues except liver. Results of these studies demonstrate that there are alternate transcripts of Pax7, some of which are expressed exclusively in adult skeletal muscle and brain. It is possible that one of these transcripts may specify an alternate myogenic pathway involved in regeneration of damaged skeletal muscle in adult mice.
Publisher: Elsevier BV
Date: 12-2010
Publisher: F1000 Research Ltd
Date: 22-05-2019
DOI: 10.12688/F1000RESEARCH.18466.1
Abstract: Recent approvals of oligonucleotide analogue drugs to alter gene expression have been welcomed by patient communities but not universally supported. These compounds represent a class of drugs that are designed to target a specific gene transcript, and they include a number of chemical entities to evoke different antisense mechanisms, depending upon the disease aetiology. To date, oligonucleotide therapeutics that are in the clinic or at advanced stages of translation target rare diseases, posing challenges to clinical trial design, recruitment and evaluation and requiring new evaluation paradigms. This review discusses the currently available and emerging therapeutics that alter exon selection through an effect on pre-mRNA splicing and explores emerging concerns over safety and efficacy. Although modification of synthetic nucleic acids destined for therapeutic application is common practice to protect against nuclease degradation and to influence drug function, such modifications may also confer unexpected physicochemical and biological properties. Negatively charged oligonucleotides have a strong propensity to bind extra- and intra-cellular proteins, whereas those analogues with a neutral backbone show inefficient cellular uptake but excellent safety profiles. In addition, the potential for incorporation of chemically modified nucleic acid monomers, yielded by nuclease degradation of exogenous oligonucleotides, into biomolecules has been raised and the possibility not entirely discounted. We conclude with a commentary on the ongoing efforts to develop novel antisense compounds and enhance oligonucleotide delivery in order to further improve efficacy and accelerate implementation of antisense therapeutics for human disease.
Publisher: Wiley
Date: 23-04-2020
DOI: 10.1002/MGG3.1259
Publisher: Informa UK Limited
Date: 29-08-2022
DOI: 10.1080/13816810.2021.1966053
Abstract: Stargardt disease (STGD1) is an autosomal recessive retinal dystrophy due to mutations in ABCA4, characterized by subretinal deposition of lipofuscin-like substances and bilateral centrifugal vision loss. Despite the tremendous progress made in the understanding of STGD1, there are no approved treatments to date. This review examines the challenges in the development of an effective STGD1 therapy. A literature review was performed through to June 2021 summarizing the spectrum of retinal phenotypes in STGD1, the molecular biology of ABCA4 protein, the in vivo and in vitro models used to investigate the mechanisms of ABCA4 mutations and current clinical trials. STGD1 phenotypic variability remains an challenge for clinical trial design and patient selection. Pre-clinical development of therapeutic options has been limited by the lack of animal models reflecting the erse phenotypic spectrum of STDG1. Patient-derived cell lines have facilitated the characterization of splice mutations but the clinical presentation is not always predicted by the effect of specific mutations on retinoid metabolism in cellular models. Current therapies primarily aim to delay vision loss whilst strategies to restore vision are less well developed. STGD1 therapy development can be accelerated by a deeper understanding of genotype-phenotype correlations.
Publisher: Oxford University Press (OUP)
Date: 21-07-2003
DOI: 10.1093/HMG/DDG196
Abstract: The mdx mouse model of muscular dystrophy arose due to a nonsense mutation in exon 23 of the dystrophin gene. We have previously demonstrated that 2'-O-methyl phosphorothioate antisense oligonucleotides (AOs) can induce removal of exon 23 during processing of the primary transcript. This results in an in-frame mRNA transcript and subsequent expression of a slightly shorter dystrophin protein in mdx muscle. Refinement of AO design has allowed efficient exon skipping to be induced in mdx mouse muscle cultures at nanomolar concentrations. In contrast, splicing intervention by morpholino AOs has been applied to the beta-globin gene pre-mRNA in cultured cells to correct aberrant splicing when delivered in the micromolar range. The morpholino chemistry produces a neutral molecule that has exceptional biological stability but poor cellular delivery. We present data showing that exon skipping in mdx cells may be induced by morpholino AOs at nanomolar concentrations when annealed to a sense oligonucleotide or "leash", and delivered as a cationic lipoplex. We have investigated a number of leash designs and chemistries, including mixed backbone oligonucleotides, and their ability to influence delivery and efficacy of the morpholino AO. Significantly, we detected dystrophin protein synthesis and correct sarcolemmal localisation after intramuscular injection of morpholino AO : leash lipoplexes in mdx muscle in vivo. We show enhanced delivery of a morpholino AO, enabling the advantageous properties to be exploited for potentially therapeutic outcomes.
Publisher: Elsevier BV
Date: 06-2014
Publisher: Bentham Science Publishers Ltd.
Date: 10-2005
DOI: 10.2174/156652305774329249
Abstract: Antisense oligonucleotides initially offered great hope as specific compounds to modify gene expression, primarily through RNaseH induced degradation of the target transcript. Expansion of the field led to new chemistries capable of invoking different mechanisms, including suppression of protein synthesis by translational blockade, and there is now a major interest in downregulation of gene expression using short interfering RNAs to induce RNA silencing. Naturally occurring microRNAs have been implicated in the regulation of gene expression. This review considers ex les of antisense oligonucleotides redirecting the process of exon recognition and intron removal during gene transcript splicing. While suppression of gene expression is necessary to address some conditions, it appears likely that there may be many more clinical applications for antisense oligonucleotides in re-directing splicing patterns. Pre-mRNA splicing is a tightly co-ordinated, multifactorial process, which can be disrupted by antisense oligonucleotides in a highly specific manner, allowing either suppression of aberrant splicing, by-pass of nonsense or frame-shifting mutations or alteration of spliceoform ratios. Manipulation of splicing patterns has been applied to a erse range of conditions, including beta-thalassemia, Duchenne muscular dystrophy, spinal muscular atrophy and certain cancers. Alternative exon usage has been identified as a major mechanism for generating ersity from a limited repertoire of genes in higher eukaryotes. Considering that up to 75% of all human primary gene transcripts are reported to be alternatively spliced, intervention at the level of pre-mRNA processing is likely to become increasingly significant in the fight against genetic and acquired disorders.
Publisher: Elsevier BV
Date: 02-2018
Publisher: Wiley
Date: 02-1988
DOI: 10.1111/J.1432-1033.1988.TB13842.X
Abstract: During inflammation and tissue injury, there is an increase in the plasma concentration of several proteins, the acute-phase proteins. The levels of some acute-phase proteins have been reported to increase in pregnant and tumour-bearing animals. Rat alpha 2-macroglobulin is classified as an acute-phase protein. In this study we report the expression of alpha 2-macroglobulin in various tissues during development of the rat embryo by analysis of mRNA. The tissues studied are liver, visceral yolk sac, placental labyrinth, decidua and trophoblast. In addition, the sites of alpha 2-macroglobulin expression are localized by in situ hybridization of cDNA for alpha 2-macroglobulin to mid-sagittal cryosections of rat embryos. The level of mRNA coding for alpha 2-macroglobulin is determined in the liver of rats aged between 12 days gestation and 2 days postnatal. alpha 2-Macroglobulin mRNA is first observed in fetal liver from 12 days of gestation and increases after day 17, reaching a maximum on day 20. At this time the level is greater than that found in the liver of an adult rat suffering from acute inflammation. alpha 2-Macroglobulin mRNA is detectable in the yolk sac, placental labyrinth, trophoblast tissue and decidua. In the decidua the alpha 2-macroglobulin message is first detected at 8 days of gestation, with high levels observed from 10 to 21 days of gestation. These observations are supported by in situ hybridization studies. Experiments using cultured hepatocytes show that cells derived from rats at 15 days and 19 days of gestation are capable of synthesizing and secreting alpha 2-macroglobulin. Both synthesis and secretion can be induced by the addition of dexamethasone to the culture medium.
Publisher: MDPI AG
Date: 28-09-2021
Abstract: Retinitis pigmentosa 11 (RP11) is caused by dominant mutations in PRPF31, however a significant proportion of mutation carriers do not develop retinopathy. Here, we investigated the relationship between CNOT3 polymorphism, MSR1 repeat copy number and disease penetrance in RP11 patients and non-penetrant carriers (NPCs). We further characterized PRPF31 and CNOT3 expression in fibroblasts from eight RP11 patients and one NPC from a family carrying the c.1205C T variant. Retinal organoids (ROs) and retinal pigment epithelium (RPE) were differentiated from induced pluripotent stem cells derived from RP11 patients, an NPC and a control subject. All RP11 patients were homozygous for the 3-copy MSR1 repeat in the PRPF31 promoter, while 3/5 NPCs carried a 4-copy MSR1 repeat. The CNOT3 rs4806718 genotype did not correlate with disease penetrance. PRFP31 expression declined with age in adult cadaveric retina. PRPF31 and CNOT3 expression was reduced in RP11 fibroblasts, RO and RPE compared with controls. Both RP11 and NPC RPE displayed shortened primary cilia compared with controls, however a subpopulation of cells with normal cilia lengths was present in NPC RPE monolayers. Our results indicate that RP11 non-penetrance is associated with the inheritance of a 4-copy MSR1 repeat, but not with CNOT3 polymorphisms.
Publisher: Elsevier BV
Date: 2012
DOI: 10.1038/MTNA.2012.40
Publisher: Wiley
Date: 2002
DOI: 10.1002/JGM.295
Abstract: Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by dystrophin gene mutations that preclude synthesis of a functional protein. One potential treatment of the disorder has utilised antisense oligoribonucleotides (AOs) to induce removal of disease-associated exons during pre-mRNA processing. Induced in-frame mRNA transcripts encode a shorter but functional dystrophin. We have investigated and improved the design of AOs capable of removing exon 23, and thus the disease-causing nonsense mutation, from mRNA in the mdx mouse model of DMD. H-2K(b)-tsA58 mdx cultures were transfected with complexes of Lipofectin and AOs. Exon skipping was detected by RT-PCR and subsequent protein production was demonstrated by Western blotting. AOs were delivered at a range of doses in order to compare relative efficiencies. We describe effective and reproducible exon 23 skipping with several AOs, including one as small as 17 nucleotides. Furthermore, the location of a sensitive exon 23 target site has been refined, whilst minimum effective doses have been estimated in vitro. These doses are significantly lower than previously reported and were associated with the synthesis of dystrophin protein in vitro. These results demonstrate the increasing feasibility of an AO-based therapy for treatment of DMD. By refining AO design we have been able to reduce the size and the effective dose of the AOs and have dramatically improved the efficiency of the technique.
Publisher: Elsevier BV
Date: 07-1999
DOI: 10.1016/S0960-8966(99)00010-3
Abstract: The mdx mouse, which carries a nonsense mutation in exon 23 of the dystrophin gene, has been used as an animal model of Duchenne muscular dystrophy to evaluate cell or gene replacement therapies. Despite the mdx mutation, which should preclude the synthesis of a functional dystrophin protein, rare, naturally occurring dystrophin-positive fibres have been observed in mdx muscle tissue. These dystrophin-positive fibres are thought to have arisen from an exon-skipping mechanism, either somatic mutations or alternative splicing. Increasing the frequency of these fibres may offer another therapeutic approach to reduce the severity of Duchenne muscular dystrophy. Antisense oligonucleotides have been shown to block aberrant splicing in the human beta-globin gene. We wished to use a similar approach to re-direct normal processing of the dystrophin pre-mRNA and induce specific exon skipping. Antisense 2'-O-methyl-oligoribonucleotides, directed to the 3' and 5' splice sites of introns 22 and 23, respectively in the mdx pre-mRNA, were used to transfect myoblast cultures. The 5' antisense oligonucleotide appeared to efficiently displace factors normally involved in the removal of intron 23 so that exon 23 was also removed during the splicing of the dystrophin pre-mRNA. Approximately 50% of the dystrophin gene mRNAs were missing this exon 6 h after transfection of primary mdx myotubes, with all transcripts showing skipping of exon 23 after 24 h. Deletion of exon 23 does not disrupt the reading frame and should allow the synthesis of a shorter but presumably functional Becker-like dystrophin. Molecular intervention at dystrophin pre-mRNA splicing has the potential to reduce the severity of a Duchenne mutation to the milder Becker phenotype.
Publisher: MDPI AG
Date: 27-03-2021
DOI: 10.3390/IJMS22073479
Abstract: Marfan syndrome is one of the most common dominantly inherited connective tissue disorders, affecting 2–3 in 10,000 in iduals, and is caused by one of over 2800 unique FBN1 mutations. Mutations in FBN1 result in reduced fibrillin-1 expression, or the production of two different fibrillin-1 monomers unable to interact to form functional microfibrils. Here, we describe in vitro evaluation of antisense oligonucleotides designed to mediate exclusion of FBN1 exon 52 during pre-mRNA splicing to restore monomer homology. Antisense oligonucleotide sequences were screened in healthy control fibroblasts. The most effective sequence was synthesised as a phosphorodiamidate morpholino oligomer, a chemistry shown to be safe and effective clinically. We show that exon 52 can be excluded in up to 100% of FBN1 transcripts in healthy control fibroblasts transfected with PMO52. Immunofluorescent staining revealed the loss of fibrillin 1 fibres with ~50% skipping and the subsequent re-appearance of fibres with % skipping. However, the effect of exon skipping on the function of the induced fibrillin-1 isoform remains to be explored. Therefore, these findings demonstrate proof-of-concept that exclusion of an exon from FBN1 pre-mRNA can result in internally truncated but identical monomers capable of forming fibres and lay a foundation for further investigation to determine the effect of exon skipping on fibrillin-1 function.
Publisher: Elsevier BV
Date: 07-2015
DOI: 10.1016/J.MOLMED.2015.04.006
Abstract: Targeted dystrophin exon removal is a promising therapy for Duchenne muscular dystrophy (DMD) however, dystrophin expression in some reports is not supported by the associated data. As in the account of 'The Emperor's New Clothes', the validity of such claims must be questioned, with critical re-evaluation of available data. Is it appropriate to report clinical benefit and induction of dystrophin as dose dependent when the baseline is unclear? The inability to induce meaningful levels of dystrophin does not mean that dystrophin expression as an end point is irrelevant, nor that induced exon skipping as a strategy is flawed, but demands that drug safety and efficacy, and study parameters be addressed, rather than questioning the strategy or the validity of dystrophin as a biomarker.
Publisher: Springer Science and Business Media LLC
Date: 24-05-2006
Abstract: Duchenne muscular dystrophy is a fatal genetic disorder caused by dystrophin gene mutations that result in premature termination of translation and the absence of functional protein. Despite the primary dystrophin gene lesion, immunostaining studies have shown that at least 50% of DMD patients, mdx mice and a canine model of DMD have rare dystrophin-positive or 'revertant' fibres. Fine epitope mapping has shown that the majority of transcripts responsible for revertant fibres exclude multiple exons, one of which includes the dystrophin mutation. The mdx mouse model of muscular dystrophy has a nonsense mutation in exon 23 of the dystrophin gene. We have shown that antisense oligonucleotides (AOs) can induce the removal of this exon, resulting in an in-frame mRNA transcript encoding a shortened but functional dystrophin protein. To emulate one exonic combination associated with revertant fibres, we target multiple exons for removal by the application of a group of AOs combined as a "cocktail". Exons 19–25 were consistently excluded from the dystrophin gene transcript using a cocktail of AOs. This corresponds to an alternatively processed gene transcript that has been sporadically detected in untreated dystrophic mouse muscle, and is presumed to give rise to a revertant dystrophin isoform. The transcript and the resultant correctly localised smaller protein were confirmed by RT-PCR, immunohistochemistry and western blot analysis. This work demonstrates the feasibility of AO cocktails to by-pass dystrophin mutation hotspots through multi-exon skipping. Multi-exon skipping could be important in expediting an exon skipping therapy to treat DMD, so that the same AO formulations may be applied to several different mutations within particular domains of the dystrophin gene.
Publisher: Springer Science and Business Media LLC
Date: 03-2017
DOI: 10.1038/NBT.3819
Publisher: Wiley
Date: 31-10-2013
DOI: 10.1111/IEP.12048
Publisher: Informa UK Limited
Date: 02-11-2017
DOI: 10.1080/14712598.2017.1250880
Abstract: Antisense nucleic acid analogues can interact with pre-mRNA motifs and influence exon or splice site selection and thereby alter gene expression. Design of antisense molecules to target specific motifs can result in either exon exclusion or exon inclusion during splicing. Novel drugs exploiting the antisense concept are targeting rare, life-limiting diseases however, the potential exists to treat a wide range of conditions by antisense-mediated splice intervention. Areas covered: In this review, the authors discuss the clinical translation of novel molecular therapeutics to address the fatal neuromuscular disorders Duchenne muscular dystrophy and spinal muscular atrophy. The review also highlights difficulties posed by issues pertaining to restricted participant numbers, variable phenotype and disease progression, and the identification and validation of study endpoints. Expert opinion: Translation of novel therapeutics for Duchenne muscular dystrophy and spinal muscular atrophy has been greatly advanced by multidisciplinary research, academic-industry partnerships and in particular, the engagement and support of the patient community. Sponsors, supporters and regulators are cooperating to deliver new drugs and identify and define meaningful outcome measures. Non-conventional and adaptive trial design could be particularly suited to clinical evaluation of novel therapeutics and strategies to treat serious, rare diseases that may be problematic to study using more conventional clinical trial structures.
Publisher: Springer Science and Business Media LLC
Date: 2007
Publisher: Springer Science and Business Media LLC
Date: 27-04-2012
Publisher: Elsevier BV
Date: 09-2007
Abstract: Duchenne and Becker muscular dystrophies are allelic disorders arising from mutations in the dystrophin gene. Duchenne muscular dystrophy is characterized by an absence of functional protein, whereas Becker muscular dystrophy, commonly caused by in-frame deletions, shows synthesis of partially functional protein. Anti-sense oligonucleotides can induce specific exon removal during processing of the dystrophin primary transcript, while maintaining or restoring the reading frame, and thereby overcome protein-truncating mutations. The mdx mouse has a non-sense mutation in exon 23 of the dystrophin gene that precludes functional dystrophin production, and this model has been used in the development of treatment strategies for dystrophinopathies. A phosphorodiamidate morpholino oligomer (PMO) has previously been shown to exclude exon 23 from the dystrophin gene transcript and induce dystrophin expression in the mdxmouse, in vivo and in vitro. In this report, a cell-penetrating peptide (CPP)-conjugated oligomer targeted to the mouse dystrophin exon 23 donor splice site was administered to mdxmice by intraperitoneal injection. We demonstrate dystrophin expression and near-normal muscle architecture in all muscles examined, except for cardiac muscle. The CPP greatly enhanced uptake of the PMO, resulting in widespread dystrophin expression.
Publisher: Springer Science and Business Media LLC
Date: 06-07-2003
DOI: 10.1038/NM897
Publisher: Elsevier BV
Date: 2010
DOI: 10.1038/MT.2009.248
Publisher: MDPI AG
Date: 14-05-2021
DOI: 10.3390/BIOMEDICINES9050552
Abstract: The literature surrounding the use of antisense oligonucleotides continues to grow, with new disease and mechanistic applications constantly evolving. Furthermore, the discovery and advancement of novel chemistries continues to improve antisense delivery, stability and effectiveness. For each new application, a rational sequence design is recommended for each oligomer, as is chemistry and delivery optimization. To confirm oligomer delivery and antisense activity, a positive control AO sequence with well characterized target-specific effects is recommended. Here, we describe splice-switching antisense oligomer sequences targeting the ubiquitously expressed human and mouse SMN and Smn genes for use as control AOs for this purpose. We report two AO sequences that induce targeted skipping of SMN1/SMN2 exon 7 and two sequences targeting the Smn gene, that induce skipping of exon 5 and exon 7. These antisense sequences proved effective in inducing alternative splicing in both in vitro and in vivo models and are therefore broadly applicable as controls. Not surprisingly, we discovered a number of differences in efficiency of exon removal between the two species, further highlighting the differences in splice regulation between species.
Publisher: Elsevier BV
Date: 2007
Abstract: Antisense oligonucleotides (AOs) can be used to redirect dystrophin pre-messenger RNA (mRNA) processing, to remove selected exons from the mature dystrophin mRNA, to overcome nonsense mutations, and/or restore the reading frame. Redundancy within the dystrophin protein allows some domains to be removed without seriously compromising function. One of the challenges for splicing blockade is to design AOs that efficiently remove targeted exons across the dystrophin pre-mRNA. AOs are initially designed to anneal to the more obvious motifs implicated in the splicing process, such as acceptor or donor splice sites and in silico predicted exonic splicing enhancers. The AOs are evaluated for their ability to induce targeted exon skipping after transfection into cultured myoblasts. Although no single motif has been implicated in the consistent induction of exon skipping, the length of the AO has emerged as an important parameter in designing compounds that redirect dystrophin pre-mRNA processing. We present data from in vitro studies in murine and human cells showing that appropriately designed AOs of 25-31 nucleotides are generally more effective at inducing exon skipping than shorter counterparts. However, there appears to be an upper limit in optimal length, which may have to be established on a case-by-case basis.
Publisher: Elsevier BV
Date: 06-2010
DOI: 10.1038/MT.2010.45
Publisher: Springer Science and Business Media LLC
Date: 22-08-2018
DOI: 10.1038/S41598-018-30790-2
Abstract: Cell penetrating peptides (CPPs) offer great potential to deliver therapeutic molecules to previously inaccessible intracellular targets. However, many CPPs are inefficient and often leave their attached cargo stranded in the cell’s endosome. We report a versatile platform for the isolation of peptides delivering a wide range of cargos into the cytoplasm of cells. We used this screening platform to identify multiple “Phylomer” CPPs, derived from bacterial and viral genomes. These peptides are amenable to conventional sequence optimization and engineering approaches for cell targeting and half-life extension. We demonstrate potent, functional delivery of protein, peptide, and nucleic acid analog cargos into cells using Phylomer CPPs. We validate in vivo activity in the cytoplasm, through successful transport of an oligonucleotide therapeutic fused to a Phylomer CPP in a disease model for Duchenne’s muscular dystrophy. This report thus establishes a discovery platform for identifying novel, functional CPPs to expand the delivery landscape of druggable intracellular targets for biological therapeutics.
Publisher: Springer Science and Business Media LLC
Date: 20-10-2011
Abstract: Antisense oligomer induced exon skipping aims to reduce the severity of Duchenne muscular dystrophy by redirecting splicing during pre-RNA processing such that the causative mutation is by-passed and a shorter but partially functional Becker muscular dystrophy-like dystrophin isoform is produced. Normal exons are generally targeted to restore the dystrophin reading frame however, an appreciable subset of dystrophin mutations are intra-exonic and therefore have the potential to compromise oligomer efficiency, necessitating personalised oligomer design for some patients. Although antisense oligomers are easily personalised, it remains unclear whether all patient polymorphisms within antisense oligomer target sequences will require the costly process of producing and validating patient specific compounds. Here we report preclinical testing of a panel of splice switching antisense oligomers, designed to excise exon 25 from the dystrophin transcript, in normal and dystrophic patient cells. These patient cells harbour a single base insertion in exon 25 that lies within the target sequence of an oligomer shown to be effective at removing exon 25. It was anticipated that such a mutation would compromise oligomer binding and efficiency. However, we show that, despite the mismatch an oligomer, designed and optimised to excise exon 25 from the normal dystrophin mRNA, removes the mutated exon 25 more efficiently than the mutation-specific oligomer. This raises the possibility that mismatched AOs could still be therapeutically applicable in some cases, negating the necessity to produce patient-specific compounds.
Publisher: Elsevier BV
Date: 06-1993
DOI: 10.1016/1050-3862(93)90038-K
Abstract: A novel technique that enabled the specific cloning of a DNA fragment unique to the dog Y chromosome is described. The method involves competitive hybridization of DNA prepared from male dog lymphocytes with biotin-labeled DNA prepared from female dog lymphocytes. The biotinylated female-female and male-female hybrid DNA fragments were removed by capture with streptavidin-coated paramagnetic particles. Full-length double-stranded DNA was generated from the remaining fragments by using the Klenow fragment of DNA polymerase I, followed by direct cloning using a low-background ligation technique. Analysis of putative recombinant clones derived by this method has led to the identification of a fragment that hybridizes specifically to male dog DNA. The clones were selected initially on the basis of a differential signal obtained when hybridized to dilutions of male and female dog DNA immobilized on neural nylon membrane. To evaluate its suitability as a probe for trans-sexually grafted cells in transplantation studies, the fragment was labeled with digoxigenin and hybridized in situ to male and female dog tissue sections. The clone designated number 6.2 hybridized strongly to male dog nuclei. The cloning strategy employed could be extended to other studies in which competitive reassociation can be used to identify unique DNA sequences.
Publisher: Informa Healthcare
Date: 15-12-2015
Publisher: Elsevier BV
Date: 04-2012
Publisher: Wiley
Date: 15-04-2015
DOI: 10.1002/MGG3.144
Publisher: Wiley
Date: 12-11-2008
DOI: 10.1002/JGM.1265
Abstract: Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder, is caused by protein-truncating mutations in the dystrophin gene. Absence of functional dystrophin renders muscle fibres more vulnerable to damage and necrosis. We report antisense oligomer (AO) induced exon skipping in the B6Ros.Cg-Dmd(mdx-4Cv)/J (4(CV)) mouse, a muscular dystrophy model arising from a nonsense mutation in dystrophin exon 53. Both exons 52 and 53 must be excised to remove the mutation and maintain the reading frame. A series of 2'-O-methyl modified oligomers on a phosphorothioate backbone (2OMeAOs) were designed and evaluated for the removal of each exon, and the most effective compounds were then combined to induce dual exon skipping in both myoblast cultures and in vivo. Exon skipping efficiency of 2OMeAOs and phosphorodiamidate morpholino oligomers (PMOs) was evaluated both in vitro and in vivo at the RNA and protein levels. Compared to the original mdx mouse studies, induction of exon skipping from the 4(CV) dystrophin mRNA was far more challenging. PMO cocktails could restore synthesis of near-full length dystrophin protein in cultured 4(CV) myogenic cells and in vivo, after a single intramuscular injection. By-passing the protein-truncating mutation in the 4(CV) mouse model of muscular dystrophy could not be achieved with single oligomers targeting both exons and was only achieved after the application of AO cocktails to remove exons 52 and 53. As in previous studies, the stability and efficiency of PMOs proved superior to 2OMeAOs for consistent and sustained protein induction in vivo.
Publisher: Informa UK Limited
Date: 03-2011
DOI: 10.2147/TACG.S8762
Publisher: Elsevier BV
Date: 1997
DOI: 10.1016/S0378-1119(96)00572-0
Abstract: Structural alterations within the myogenic and neurogenic developmental gene Pax7 which involve TaqI recognition sequences have previously been reported. These alterations are associated with differences in the efficiency of regrowth of damaged skeletal muscle. To identify other structural features of Pax genes which may influence skeletal muscle regrowth, variation in the structure and methylation status of Pax7 and the closely related gene Pax3 has been sought among different mouse strains and during gene expression using the restriction endonucleases MspI and HpaII. Following MspI digestion, RFLPs within Pax7 have been found which most likely reflect intron size variability within the paired box. Differences in the size of MspI and HpaII fragments hybridising with Pax7 and Pax3 region specific sub-probes indicate that the paired boxes are hypomethylated, whereas the region encoding the homeodomain of each gene is highly methylated in the spleen and other tissues from adult mice. In the skeletal muscle precursor cell line C2C12, which expresses Pax7 but not Pax3, the homeodomain encoding region of Pax7 is hypomethylated. In spleen cells, the Pax7 paired box is transcribed but the homeodomain encoding region is not. By contrast, both the paired box and the homeobox of Pax3 are hypermethylated in C2C12 cells indicating that generation of alternate transcripts from Pax genes may be controlled by DNA methylation. In contrast to Pax3, reference to the size of fragments hybridising with a Pax7 homeobox specific probe provides evidence for CpNpG methylation within and immediately downstream from the region encoding the homeodomain. Interestingly, CpNpG methylation remains when the Pax7 homeobox is expressed. Structural variation recognised by MspI digestion and differences in the methylation profile of Pax7 are not associated with the ability to regrow damaged skeletal muscle.
Publisher: Wiley
Date: 20-03-2001
DOI: 10.1002/MUS.1031
Abstract: Seven 2-day-old golden retriever pups were given focal intramuscular injections of a first generation adenovirus-dystrophin minigene construct and adenovirus-beta-galactosidase construct as a 2:1 mixture into the left anterior tibial muscle. The spread of transgene expression within the anterior tibial muscle was compared with the spread of methylene blue dye after identical injection into the contralateral muscle. Transgene expression 5-7 days after intramuscular injection was shown to extend between 5.8 and 11.6 mm along the biopsied muscle length (range of biopsy lengths 11.1-12.2 mm). The level of transgene expression at 2-2.5-mm intervals from the site of injection was significantly related to the distance from the site of injection (dystrophin, P = 0.009 beta-galactosidase, P = 0.015). The spread of methylene blue dye within the anterior tibial muscle < or =24 h after identical intramuscular injection demonstrated a similar pattern to the transgene expression, with dye staining measured between 5.5 and 8.5 mm along the muscle s le length (range of biopsy lengths 5.6-15.6 mm). The greatest transgene expression and dye staining was measured 2-2.5 mm proximal to the site of injection with a maximum of 23% of muscle fibers expressing the dystrophin transgene, 95.2% expressing the beta-galactosidase transgene, and 98% of the tissue section stained with methylene blue dye. These results suggest transgene expression after focal intramuscular injection is relatively localized around the site of injection. Further research is required to develop techniques that will provide transgene expression throughout the length and breadth of a muscle.
Publisher: Elsevier BV
Date: 06-1996
DOI: 10.1016/1357-2725(96)00007-6
Abstract: In order to study transcriptional regulation of hepatic genes during development, a method for transfer of fusion genes to primary cultures of fetal hepatocytes was required. The aim of this study was to assess currently available transfection methods and optimize the best method for use with cultured fetal hepatocytes. The Rous sarcoma virus 5' long terminal repeat controlling transcription of the beta-galactosidase reporter gene (pRSV lac Z II) was used to assess electroporation, lipofection, DEAE-dextran and calcium phosphate transfection in cultured primary fetal hepatocytes. The success of transfection was determined by histochemical detection and quantitation of beta-galactosidase activity. Results showed that calcium phosphate transfection was optimal for fetal hepatocytes with respect to beta-galactosidase activity and cell survival. For maximum transfection of cells, 10 micrograms/ml DNA, HEPES buffered saline transfection buffer at pH 7.05 and a 24 hr expression period for the reporter gene were employed. Glycerol shock did not increase transfection efficiency significantly. The method was simplified by adding calcium chloride solution to DNA diluted in transfection buffer and the resulting co-precipitate added directly to the medium covering the cells. Transfection 24 hr after initial culture and a precipitate incubation time of 20 hr were optimal. The suitability of this method was confirmed with a liver-specific promoter controlling beta-galactosidase and chlor henicol acetyltransferase expression. In conclusion this study shows that a modified calcium phosphate transfection method is most effective for transferring DNA to primary cultured fetal hepatocytes. It is concluded that this method is appropriate for use with fetal hepatocytes and will facilitate studies of gene regulation during liver development.
Publisher: MDPI AG
Date: 28-01-2020
Abstract: Pompe disease, or glycogen storage disease II is a rare, progressive disease leading to skeletal muscle weakness due to deficiency of the acid α-1,4-glucosidase enzyme (GAA). The severity of disease and observed time of onset is subject to the various combinations of heterozygous GAA alleles. Here we have characterized two novel mutations: c.2074C T and c.1910_1918del, and a previously reported c.1082C G mutation of uncertain clinical significance. These mutations were found in three unrelated patients with adult-onset Pompe disease carrying the common c.-32-13T G mutation. The c.2074 C T nonsense mutation has obvious consequences on GAA expression but the c.1910_1918del (deletion of 3 amino acids) and c.1082C G missense variants are more subtle DNA changes with catastrophic consequences on GAA activity. Molecular and clinical analyses from the three patients corresponded with the anticipated pathogenicity of each mutation.
Publisher: American Physiological Society
Date: 08-2008
DOI: 10.1152/JAPPLPHYSIOL.00068.2008
Abstract: 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.
No related grants have been discovered for Sue Fletcher.