ORCID Profile
0000-0002-6379-2303
Current Organisations
Oak Ridge National Laboratory
,
Murdoch University
,
Perron Institute for Neurological and Translational Science
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Publications
Kinectin Anchors the Translation Elongation Factor-1δ to the Endoplasmic Reticulum
Publisher: Elsevier BV
Date: 08-2003
Phenotype–genotype correlations in a pseudodominant Stargardt disease pedigree due to a novel ABCA4 deletion–insertion variant causing a splicing defect
Publisher: Wiley
Date: 23-04-2020
DOI: 10.1002/MGG3.1259
Stargardt disease and progress in therapeutic strategies
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.
Changes in reactive oxygen species begin early during replicative aging of Saccharomyces cerevisiae cells
Publisher: Elsevier BV
Date: 04-2011
DOI: 10.1016/J.FREERADBIOMED.2011.01.013
Abstract: Increased reactive oxygen species (ROS) are a feature of aging cells, but little is known about when ROS generation begins as cells age. Here we show how ROS change in Saccharomyces cerevisiae cells throughout their early replicative life span using the fluorescent ROS indicator dihydroethidium (DHE), which has some specificity for the superoxide anion. Cells in a particular age range were heterogeneous with respect to their ROS burden. Surprisingly, some cells as young as 5-7 generations acquired a greatly increased level of ROS detected by DHE relative to virgin cells. By 12 generations 50% of cells had a substantial ROS burden despite being only halfway through their life span. In contrast to the wild type, cells of a sir2 mutant had lower levels of ROS reacting with DHE. Daughters from older mothers had low ROS levels, and this asymmetric distribution of ROS was SIR2-independent. Mitochondrial fragmentation also began to occur in cells after 4 generations and increased markedly as cells aged. Daughter cells regenerated normal tubular mitochondria despite the fragmentation of mitochondria in the mother cells, whereas daughters of the sir2 mutant had fragmented mitochondria at all ages.
Pathogenesis and Treatment of Usher Syndrome Type IIA
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 07-2022
DOI: 10.1097/APO.0000000000000546
Abstract: Usher syndrome (USH) is the most common form of deaf-blindness, with an estimated prevalence of 4.4 to 16.6 per 100,000 people worldwide. The most common form of USH is type IIA (USH2A), which is caused by homozygous or compound heterozygous mutations in the USH2A gene and accounts for around half of all USH cases. USH2A patients show moderate to severe hearing loss from birth, with diagnosis of retinitis pigmentosa in the second decade of life and variable vestibular involvement. Although hearing aids or cochlear implants can provide some mitigation of hearing deficits, there are currently no treatments aimed at preventing or restoring vision loss in USH2A patients. In this review, we first provide an overview of the molecular biology of the USH2A gene and its protein isoforms, which include a transmembrane protein (TM usherin) and an extracellular protein (EC usherin). The role of these proteins in the inner ear and retina and their impact on the pathogenesis of USH2A is discussed. We review animal cell-derived and patient cell-derived models currently used in USH2A research and conclude with an overview of potential treatment strategies currently in preclinical development and clinical trials.
Engaging patients and families in communication across transitions of care: an integrative review protocol
Publisher: Wiley
Date: 18-03-2016
DOI: 10.1111/JAN.12953
Abstract: To describe an integrative review protocol to analyse and synthesize peer-reviewed research evidence in relation to engagement of patients and their families in communication during transitions of care to, in and from acute care settings. Communication at transitions of care in acute care settings can be complex and challenging, with important information about patients not always clearly transferred between responsible healthcare providers. Involving patients and their families in communication during transitions of care may improve the transfer of clinical information and patient outcomes and prevent adverse events during hospitalization and following discharge. Recently, optimizing patient and family participation during care transitions has been acknowledged as central to the implementation of patient-centred care. Integrative review with potential for meta-analysis and application of framework synthesis. The review will evaluate and synthesize qualitative and quantitative research evidence identified through a systematic search. Primary studies will be selected according to inclusion criteria. Data collection, quality appraisal and analysis of the evidence will be conducted by at least two authors. Nine electronic databases (including CINAHL and Medline) will be searched. The search will be restricted to 10 years up to December 2013. Data analysis will include content and thematic analysis. The review will seek to identify all types of patient engagement activities employed during transitions of care communication. The review will identify enablers for and barriers to engagement for patients, families and health professionals. Key strategies and tools for improving patient engagement, clinical communication and promoting patient-centred care will be recommended based on findings.
Antisense oligonucleotides induced splice switching of FN1 transcript
Publisher: Wiley
Date: 09-08-2015
DOI: 10.1002/JGM.2834
Systematic Approach to Developing Splice Modulating Antisense Oligonucleotides
Publisher: MDPI AG
Date: 11-10-2019
DOI: 10.3390/IJMS20205030
Abstract: The process of pre-mRNA splicing is a common and fundamental step in the expression of most human genes. Alternative splicing, whereby different splice motifs and sites are recognised in a developmental and/or tissue-specific manner, contributes to genetic plasticity and ersity of gene expression. Redirecting pre-mRNA processing of various genes has now been validated as a viable clinical therapeutic strategy, providing treatments for Duchenne muscular dystrophy (inducing specific exon skipping) and spinal muscular atrophy (promoting exon retention). We have designed and evaluated over 5000 different antisense oligonucleotides to alter splicing of a variety of pre-mRNAs, from the longest known human pre-mRNA to shorter, exon-dense primary gene transcripts. Here, we present our guidelines for designing, evaluating and optimising splice switching antisense oligomers in vitro. These systematic approaches assess several critical factors such as the selection of target splicing motifs, choice of cells, various delivery reagents and crucial aspects of validating assays for the screening of antisense oligonucleotides composed of 2′-O-methyl modified bases on a phosphorothioate backbone.
Cryptic U2-dependent pre-mRNASplice site usage induced by splice switching antisense oligonucleotides
Publisher: Research Square Platform LLC
Date: 18-01-2021
DOI: 10.21203/RS.3.RS-144809/V1
Abstract: Antisense oligomers (AOs) are increasingly being used for modulating RNA splicing in live cells, both for research and for therapeutic purposes. While the most common intended effect of these AOs is to induce skipping of whole exons, rare ex les are emerging of AOs that induce skipping of only part of an exon, through activation of an internal cryptic splice site. In this report, we examined seven such ex les of AO-induced cryptic splice site activation – five new ex les from our own experiments and three from reports published by others. We modelled the predicted effects that AO binding would have on the secondary structure of each of the RNA targets, and how these alterations would in turn affect the accessibility of the RNA to splice factors. We observed that a common predicted effect of AO binding was a disruption to the exon definition signal within the exon’s excluded segment.
Single Stranded Fully Modified-Phosphorothioate Oligonucleotides can Induce Structured Nuclear Inclusions, Alter Nuclear Protein Localization and Disturb the Transcriptome In Vitro.
Publisher: Frontiers Media SA
Date: 06-04-2022
DOI: 10.3389/FGENE.2022.791416
Abstract: Oligonucleotides and nucleic acid analogues that alter gene expression are now showing therapeutic promise in human disease. Whilst the modification of synthetic nucleic acids to protect against nuclease degradation and to influence drug function is common practice, such modifications may also confer unexpected physicochemical and biological properties. Gapmer mixed-modified and DNA oligonucleotides on a phosphorothioate backbone can bind non-specifically to intracellular proteins to form a variety of toxic inclusions, driven by the phosphorothioate linkages, but also influenced by the oligonucleotide sequence. Recently, the non-antisense or other off-target effects of 2′ O - fully modified phosphorothioate linkage oligonucleotides are becoming better understood. Here, we report chemistry-specific effects of oligonucleotides composed of modified or unmodified bases, with phosphorothioate linkages, on subnuclear organelles and show altered distribution of nuclear proteins, the appearance of highly stable and strikingly structured nuclear inclusions, and disturbed RNA processing in primary human fibroblasts and other cultured cells. Phosphodiester, phosphorodiamidate morpholino oligomers, and annealed complimentary phosphorothioate oligomer duplexes elicited no such consequences. Disruption of subnuclear structures and proteins elicit severe phenotypic disturbances, revealed by transcriptomic analysis of transfected fibroblasts exhibiting such disruption. Our data add to the growing body of evidence of off-target effects of some phosphorothioate nucleic acid drugs in primary cells and suggest alternative approaches to mitigate these effects.
Reduction of integrin alpha 4 activity through splice modulating antisense oligonucleotides.
Publisher: Springer Science and Business Media LLC
Date: 10-09-2019
DOI: 10.1038/S41598-019-49385-6
Abstract: With recent approvals of antisense oligonucleotides as therapeutics, there is an increasing interest in expanding the application of these compounds to many other diseases. Our laboratory focuses on developing therapeutic splice modulating antisense oligonucleotides to treat diseases potentially amendable to intervention during pre-mRNA processing, and here we report the use of oligomers to down-regulate integrin alpha 4 protein levels. Over one hundred antisense oligonucleotides were designed to induce skipping of in idual exons of the ITGA4 transcript and thereby reducing protein expression. Integrin alpha 4-mediated activities were evaluated in human dermal fibroblasts and Jurkat cells, an immortalised human T lymphocyte cell line. Peptide conjugated phosphorodiamidate morpholino antisense oligomers targeting ITGA4 were also assessed for their effect in delaying disease progression in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. With the promising results in ameliorating disease progression, we are optimistic that the candidate oligomer may also be applicable to many other diseases associated with integrin alpha 4 mediated inflammation. This highly specific strategy to down-regulate protein expression through interfering with normal exon selection during pre-mRNA processing should be applicable to many other gene targets that undergo splicing during expression.
Consequences of Making the Inactive Active Through Changes in Antisense Oligonucleotide Chemistries
Publisher: Frontiers Media SA
Date: 20-12-2019
Deletion of dystrophin in-frame exon 5 leads to a severe phenotype: Guidance for exon skipping strategies
Publisher: Public Library of Science (PLoS)
Date: 08-01-2012
Expression phenotypes suggest that Der participates in a specific, high affinity interaction with membranes
Publisher: Elsevier BV
Date: 07-2011
DOI: 10.1016/J.PEP.2011.02.009
Abstract: The GTPase Der is universally conserved in bacteria and is structurally unique as it consists of two GTP-binding domains in tandem (G-domain 1 and G-domain 2) whereas all the other GTPases posses a single GTPase domain. In order to assess the function of Der we have fractionated whole cell lysates containing over expressed Der. This analysis indicated that Der was present in sucrose gradient fractions containing membrane proteins. The interaction with the membrane fraction was specific for Der, since the related GTPase, Era, did not form the membrane complex. In addition, three independent criteria suggested a high affinity interaction (1) the interaction can be detected under partially denaturing conditions using a gel electrophoresis co-migration assay, (2) the interaction survived 16 h sucrose gradient centrifugation, and (3) the complex could be efficiently reconstituted from purified components. Microscopic examination of cells containing over expressed Der showed that the cell wall structure was disrupted at both cell poles. This phenotype required Der domain three since domain deletion mutations showed no affect on cell wall structure. Surprisingly point mutations that ablate nucleotide binding of either GTP binding domain result in a defect in cell wall structure at only a single cell pole. The data reported here were considered together with results presented previously to suggest that Der may engage in a functional cyclic interaction between ribosomes and the membrane in Escherichia coli.
Removal of the polyglutamine repeat of ataxin-3 by redirecting pre-mRNA processing
Publisher: MDPI AG
Date: 31-10-2019
DOI: 10.3390/IJMS20215434
Abstract: Spinocerebellar ataxia type 3 (SCA3) is a devastating neurodegenerative disease for which there is currently no cure, nor effective treatment strategy. One of nine polyglutamine disorders known to date, SCA3 is clinically heterogeneous and the main feature is progressive ataxia, which in turn affects speech, balance and gait of the affected in idual. SCA3 is caused by an expanded polyglutamine tract in the ataxin-3 protein, resulting in conformational changes that lead to toxic gain of function. The expanded glutamine tract is located at the 5′ end of the penultimate exon (exon 10) of ATXN3 gene transcript. Other studies reported removal of the expanded glutamine tract using splice switching antisense oligonucleotides. Here, we describe improved efficiency in the removal of the toxic polyglutamine tract of ataxin-3 in vitro using phosphorodiamidate morpholino oligomers, when compared to antisense oligonucleotides composed of 2′-O-methyl modified bases on a phosphorothioate backbone. Significant downregulation of both the expanded and non-expanded protein was induced by the morpholino antisense oligomer, with a greater proportion of ataxin-3 protein missing the polyglutamine tract. With growing concerns over toxicity associated with long-term administration of phosphorothioate oligonucleotides, the use of a phosphorodiamidate morpholino oligomer may be preferable for clinical application. These results suggest that morpholino oligomers may provide greater therapeutic benefit for the treatment of spinocerebellar ataxia type 3, without toxic effects.
Splice correction therapies for familial hypercholesterolemic patients with low-density lipoprotein receptor mutations.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 13-10-2021
Neurodegenerative diseases: A hotbed for splicing defects and the potential therapies
Publisher: Springer Science and Business Media LLC
Date: 20-05-2021
DOI: 10.1186/S40035-021-00240-7
Abstract: Precursor messenger RNA (pre-mRNA) splicing is a fundamental step in eukaryotic gene expression that systematically removes non-coding regions (introns) and ligates coding regions (exons) into a continuous message (mature mRNA). This process is highly regulated and can be highly flexible through a process known as alternative splicing, which allows for several transcripts to arise from a single gene, thereby greatly increasing genetic plasticity and the ersity of proteome. Alternative splicing is particularly prevalent in neuronal cells, where the splicing patterns are continuously changing to maintain cellular homeostasis and promote neurogenesis, migration and synaptic function. The continuous changes in splicing patterns and a high demand on many cis- and trans- splicing factors contribute to the susceptibility of neuronal tissues to splicing defects. The resultant neurodegenerative diseases are a large group of disorders defined by a gradual loss of neurons and a progressive impairment in neuronal function. Several of the most common neurodegenerative diseases involve some form of splicing defect(s), such as Alzheimer’s disease, Parkinson’s disease and spinal muscular atrophy. Our growing understanding of RNA splicing has led to the explosion of research in the field of splice-switching antisense oligonucleotide therapeutics. Here we review our current understanding of the effects alternative splicing has on neuronal differentiation, neuronal migration, synaptic maturation and regulation, as well as the impact on neurodegenerative diseases. We will also review the current landscape of splice-switching antisense oligonucleotides as a therapeutic strategy for a number of common neurodegenerative disorders.
Constraints and potentials of future irrigation water availability on agricultural production under climate change
Publisher: Proceedings of the National Academy of Sciences
Date: 16-12-2013
Abstract: Freshwater availability is relevant to almost all socioeconomic and environmental impacts of climate and demographic change and their implications for sustainability. We compare ensembles of water supply and demand projections driven by ensemble output from five global climate models. Our results suggest reasons for concern. Direct climate impacts to maize, soybean, wheat, and rice involve losses of 400–2,600 Pcal (8–43% of present-day total). Freshwater limitations in some heavily irrigated regions could necessitate reversion of 20–60 Mha of cropland from irrigated to rainfed management, and a further loss of 600–2,900 Pcal. Freshwater abundance in other regions could help ameliorate these losses, but substantial investment in infrastructure would be required.
Nonsequential Splicing Events Alter Antisense-Mediated Exon Skipping Outcome in COL7A1
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.
Polyglutamine Ataxias: Our Current Molecular Understanding and What the Future Holds for Antisense Therapies
Publisher: MDPI AG
Date: 20-10-2021
DOI: 10.3390/BIOMEDICINES9111499
Abstract: Polyglutamine (polyQ) ataxias are a heterogenous group of neurological disorders all caused by an expanded CAG trinucleotide repeat located in the coding region of each unique causative gene. To date, polyQ ataxias encompass six disorders: spinocerebellar ataxia types 1, 2, 3, 6, 7, and 17 and account for a larger group of disorders simply known as polyglutamine disorders, which also includes Huntington’s disease. These diseases are typically characterised by progressive ataxia, speech and swallowing difficulties, lack of coordination and gait, and are unfortunately fatal in nature, with the exception of SCA6. All the polyQ spinocerebellar ataxias have a hallmark feature of neuronal aggregations and share many common pathogenic mechanisms, such as mitochondrial dysfunction, impaired proteasomal function, and autophagy impairment. Currently, therapeutic options are limited, with no available treatments that slow or halt disease progression. Here, we discuss the common molecular and clinical presentations of polyQ spinocerebellar ataxias. We will also discuss the promising antisense oligonucleotide therapeutics being developed as treatments for these devastating diseases. With recent advancements and therapeutic approvals of various antisense therapies, it is envisioned that some of the studies reviewed may progress into clinical trials and beyond.
Generation of two induced pluripotent stem cell lines from a patient with Stargardt disease caused by compound heterozygous mutations in the ABCA4 gene
Publisher: Elsevier BV
Date: 07-2021
Correction to: Neurodegenerative diseases: A hotbed for splicing defects and the potential therapies
Publisher: Springer Science and Business Media LLC
Date: 25-10-2021
Induction of cryptic pre-mRNA splice-switching by antisense oligonucleotides
Publisher: Springer Science and Business Media LLC
Date: 23-07-2021
DOI: 10.1038/S41598-021-94639-X
Abstract: Antisense oligomers (AOs) are increasingly being used to modulate RNA splicing in live cells, both for research and for the development of therapeutics. While the most common intended effect of these AOs is to induce skipping of whole exons, rare ex les are emerging of AOs that induce skipping of only part of an exon, through activation of an internal cryptic splice site. In this report, we examined seven AO-induced cryptic splice sites in six genes. Five of these cryptic splice sites were discovered through our own experiments, and two originated from other published reports. We modelled the predicted effects of AO binding on the secondary structure of each of the RNA targets, and how these alterations would in turn affect the accessibility of the RNA to splice factors. We observed that a common predicted effect of AO binding was disruption of the exon definition signal within the exon’s excluded segment.
Maintenance of Mitochondrial Morphology by Autophagy and Its Role in High Glucose Effects on Chronological Lifespan ofSaccharomyces cerevisiae
Publisher: Hindawi Limited
Date: 2013
DOI: 10.1155/2013/636287
Abstract: In Saccharomyces cerevisiae , mitochondrial morphology changes when cells are shifted between nonfermentative and fermentative carbon sources. Here, we show that cells of S. cerevisiae grown in different glucose concentrations display different mitochondrial morphologies. The morphology of mitochondria in the cells growing in 0.5% glucose was similar to that of mitochondria in respiring cells. However, the mitochondria of cells growing in higher glucose concentrations (2% and 4%) became fragmented after growth in these media, due to the production of acetic acid however, the fragmentation was not due to intracellular acidification. From a screen of mutants involved in sensing and utilizing nutrients, cells lacking TOR1 had reduced mitochondrial fragmentation, and autophagy was found to be essential for this reduction. Mitochondrial fragmentation in cells grown in high glucose was reversible by transferring them into conditioned medium from a culture grown on 0.5% glucose. Similarly, the chronological lifespan of cells grown in high glucose medium was reduced, and this phenotype could be reversed when cells were transferred to low glucose conditioned medium. These data indicate that chronological lifespan seems correlated with mitochondrial morphology of yeast cells and that both phenotypes can be influenced by factors from conditioned medium of cultures grown in low glucose medium.
Splice modulating antisense oligonucleotides restore some acid-alpha-glucosidase activity in cells derived from patients with late-onset Pompe disease
Publisher: Springer Science and Business Media LLC
Date: 21-04-2020
DOI: 10.1038/S41598-020-63461-2
Abstract: Pompe disease is caused by mutations in the GAA gene, resulting in deficient lysosomal acid-α-glucosidase activity in patients, and a progressive decline in mobility and respiratory function. Enzyme replacement therapy is one therapeutic option, but since not all patients respond to this treatment, alternative interventions should be considered. One GAA mutation, c.-32-13T G, impacts upon normal exon 2 splicing and is found in two-thirds of late-onset cases. We and others have explored a therapeutic strategy using splice modulating phosphorodiamidate morpholino oligomers to enhance GAA exon 2 inclusion in the mature mRNA of patients with one c.-32-13T G allele. We designed 20 oligomers and treated fibroblasts derived from five patients to identify an oligomer sequence that maximally increased enzyme activity in all fibroblasts. The most effective splice correcting oligomer was chosen to treat forced-myogenic cells, derived from fibroblasts from nine patients carrying the c.-32-13T G mutation. After transfection, we show increased levels of the full-length GAA transcript, acid-α-glucosidase protein, and enzyme activity in all patients’ myogenic cells, regardless of the nature of the mutation in the other allele. This data encourages the initiation of clinical trials to assess the therapeutic efficacy of this oligomer for those patients carrying the c.-32-13T G mutation.
The Role of Mitochondria in the Aging Processes of Yeast
Publisher: Springer Netherlands
Date: 2011
Developmentally programmed cell death in Drosophila
Publisher: Elsevier BV
Date: 12-2013
DOI: 10.1016/J.BBAMCR.2013.06.014
Abstract: During the development of metazoans, programmed cell death (PCD) is essential for tissue patterning, removal of unwanted cells and maintaining homeostasis. In the past 20 years Drosophila melanogaster has been one of the systems of choice for studies involving developmental cell death, providing an ideal genetically tractable model of intermediary complexity between Caenorhabditis elegans and mammals. The lessons learned from studies using Drosophila indicate both the conserved nature of the many cell death pathways as well as novel and unexpected mechanisms. In this article we review the understanding of PCD during Drosophila development, highlighting the key mechanisms that are evolutionarily conserved as well as apparently unusual pathways, which indicate ergence, but provide evidence of complexity acquired during organismic evolution. This article is part of a Special Section entitled: Cell Death Pathways.
Oxidative Stresses and Ageing
Publisher: Springer Netherlands
Date: 2011
DOI: 10.1007/978-94-007-2561-4_2
Abstract: Oxidative damage to cellular constituents has frequently been associated with aging in a wide range of organisms. The power of yeast genetics and biochemistry has provided the opportunity to analyse in some detail how reactive oxygen and nitrogen species arise in cells, how cells respond to the damage that these reactive species cause, and to begin to dissect how these species may be involved in the ageing process. This chapter reviews the major sources of reactive oxygen species that occur in yeast cells, the damage they cause and how cells sense and respond to this damage.
A Splice Intervention Therapy for Autosomal Recessive Juvenile Parkinson's Disease Arising from Parkin Mutations
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.
High-Entropy Perovskite as a High-Performing Chromium-Tolerant Cathode for Solid Oxide Fuel Cells
Publisher: American Chemical Society (ACS)
Date: 16-05-2022
Abstract: To achieve chromium tolerance and high performance, a new series of high-entropy perovskites (HEPs) are investigated as cathode materials for solid oxide fuel cells (SOFCs). Multiple rare-earth, alkaline-earth, and high-order transition metal elements are used for the A-site of this ABO
Characterising splicing defects of ABCA4 variants within exons 13–50 in patient-derived fibroblasts
Publisher: Elsevier BV
Date: 12-2022
DOI: 10.1016/J.EXER.2022.109276
Abstract: The ATP-binding cassette subfamily A member 4 gene (ABCA4)-associated retinopathy, Stargardt disease, is the most common monogenic inherited retinal disease. Given the pathogenicity of numerous ABCA4 variants is yet to be examined and a significant proportion (more than 15%) of ABCA4 variants are categorized as splice variants in silico, we therefore established a fibroblast-based splice assay to analyze ABCA4 variants in an Australian Stargardt disease cohort and characterize the pathogenic mechanisms of ABCA4 variants. A cohort of 67 patients clinically diagnosed with Stargardt disease was recruited. Genomic DNA was analysed using a commercial panel for ABCA4 variant detection and the consequences of ABCA4 variants were predicted in silico. Dermal fibroblasts were propagated from skin biopsies, total RNA was extracted and the ABCA4 transcript was lified by RT-PCR. Our analysis identified a total of 67 unique alleles carrying 74 unique variants. The most prevalent splice-affecting complex allele c.[5461-10T>C 5603A>T] was carried by 10% of patients in a compound heterozygous state. ABCA4 transcripts from exon 13 to exon 50 were readily detected in fibroblasts. In this region, aberrant splicing was evident in 10 out of 57 variant transcripts (18%), carried by 19 patients (28%). Patient-derived fibroblasts provide a feasible platform for identification of ABCA4 splice variants located within exons 13-50. Experimental evidence of aberrant splicing contributes to the pathogenic classification for ABCA4 variants. Moreover, identification of variants that affect splicing processes provides opportunities for intervention, in particular antisense oligonucleotide-mediated splice correction.
Multitasking by Multivalent Circular DNA Aptamers
Publisher: Wiley
Date: 03-2006
Abstract: Nucleic acid aptamers are finding increasing applications in biology, especially as therapeutic candidates and diagnostic components. An important characteristic in meeting the needs of these applications is improved stability in physiological fluids, which is most often accomplished with chemical modification or unnatural nucleotides. In an alternative approach we have specified the design of a multivalent circular DNA aptamer topology that encompasses a number of properties relevant to nucleic acid therapeutic candidates, especially the ability to multitask by combining different activities together within a modular structure. Improved stability in blood products, greater conformational stability, antidoting by complementary circular antiaptamers, heterovalency, transcription factor decoy activity and minimal unintended effects upon the cellular innate immune response are desirable properties that are described here. Multitasking by circular DNA aptamers could similarly find applications in diagnostics and biomaterials, where the combination of interchangeable modules might generate new functions, such as anticoagulation coupled with reversible cell capture as, described here. These results provide a platform for further exploration of multivalent circular aptamer properties, especially in novel combinations of nucleic acid therapeutic modes.
UTX coordinates steroid hormone-mediated autophagy and cell death
Publisher: Springer Science and Business Media LLC
Date: 13-12-2013
DOI: 10.1038/NCOMMS3916
Nonsequential Pre-mRNA Splicing: From Basic Understanding to Impacts on Splice-Manipulating Therapies
Publisher: Springer International Publishing
Date: 2022
Novel Mutations Found in Individuals with Adult-Onset Pompe Disease
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.
In Vitro Validation of Phosphorodiamidate Morpholino Oligomers.
Publisher: MDPI AG
Date: 12-08-2019
DOI: 10.3390/MOLECULES24162922
Abstract: One of the crucial aspects of screening antisense oligonucleotides destined for therapeutic application is confidence that the antisense oligomer is delivered efficiently into cultured cells. Efficient delivery is particularly vital for antisense phosphorodiamidate morpholino oligomers, which have a neutral backbone, and are known to show poor gymnotic uptake. Here, we report several methods to deliver these oligomers into cultured cells. Although 4D-Nucleofector™ or Neon™ electroporation systems provide efficient delivery and use lower amounts of phosphorodiamidate morpholino oligomer, both systems are costly. We show that some readily available transfection reagents can be used to deliver phosphorodiamidate morpholino oligomers as efficiently as the electroporation systems. Among the transfection reagents tested, we recommend Lipofectamine 3000™ for delivering phosphorodiamidate morpholino oligomers into fibroblasts and Lipofectamine 3000™ or Lipofectamine 2000™ for myoblasts/myotubes. We also provide optimal programs for nucleofection into various cell lines using the P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza), as well as antisense oligomers that redirect expression of ubiquitously expressed genes that may be used as positive treatments for human and murine cell transfections.
Related Organisations
Perron Institute For Neurological And Translational Science
Location: Australia
Related Funding Activities
No related grants have been discovered for May Thandar Aung-Htut.