A splice intervention therapy for autosomal recessive juvenile Parkinson’s disease arising from Parkin mutations

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

Novel mutations found in individuals with adult-onset Pompe disease

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

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-NucleofectorTM 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 3000TM for delivering phosphorodiamidate morpholino oligomers into fibroblasts and Lipofectamine 3000TM or Lipofectamine 2000. for myoblasts/myotubes. We also provide optimal programs for nucleofection into various cell lines using the P3 Primary Cell 4D-NucleofectorTM 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

Consequences of making the inactive active through changes in antisense oligonucleotide chemistries

Antisense oligonucleotides are short, single-stranded nucleic acid analogues that can interfere with pre-messenger RNA (pre-mRNA) processing and induce excision of a targeted exon from the mature transcript. When developing a panel of antisense oligonucleotides to skip every dystrophin exon, we found great variation in splice switching efficiencies, with some antisense oligonucleotides ineffective, even when directed to canonical splice sites and transfected into cells at high concentrations. In this study, we re-evaluated some of these ineffective antisense oligonucleotide sequences after incorporation of locked nucleic acid residues to increase annealing potential. Antisense oligonucleotides targeting exons 16, 23, and 51 of human DMD transcripts were synthesized as two different chemistries, 2 '-O-methyl modified bases on a phosphorothioate backbone or mixmers containing several locked nucleic acid residues, which were then transfected into primary human myotubes, and DMD transcripts were analyzed for exon skipping. The ineffective 2 '-O-methyl modified antisense oligonucleotides induced no detectable exon skipping, while all corresponding mixmers did induce excision of the targeted exons. Interestingly, the mixmer targeting exon 51 induced two unexpected transcripts arising from partial skipping of exon 51 with retention of 95 or 188 bases from the 5 ' region of exon 51. These results indicated that locked nucleic acid/2 '-O-methyl mixmers are more effective at inducing exon skipping, however, this improvement may come at the cost of activating alternative cryptic splice sites and off-target effects on gene expression

Pathogenesis and Treatment of Usher Syndrome Type IIA

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

Near infrared spectrometry for rapid non-invasive modelling of Aspergillus-contaminated maturing kernels of maize (Zea mays L.)

Open Access JournalAflatoxin-producing Aspergillus spp. produce carcinogenic metabolites that contaminate maize. Maize kernel absorbance patterns of near infrared (NIR) wavelengths (800–2600 nm) were used to non-invasively identify kernels of milk-, dough- and dent-stage maturities with four doses of Aspergillus sp. contamination. Near infrared spectrometry (NIRS) spectral data was pre-processed using first derivative Savitzky-Golay (1d-SG) transformation and multiplicative scatter correction on spectral data. Contaminated kernels had higher absorbance between 800–1134 nm, while uninoculated samples had higher absorbance above 1400 nm. Dose and maturity clusters seen in Principal Component Analysis (PCA) score plots were due to bond stretches of combination bands, CH and C=O functional groups within grain macromolecules. The regression model at 2198 nm separated uninoculated and inoculated kernels (p < 0.0001, R2 = 0.88, root mean square error = 0.15). Non-invasive identification of Aspergillus-contaminated maize kernels using NIR spectrometry was demonstrated in kernels of different maturities

Observations of the post shock break-out emission of SN 2011dh with XMM-Newton

After the occurrence of the type cIIb SN 2011dh in the nearby spiral galaxy M 51 numerous observations were performed with different telescopes in various bands ranging from radio to gamma-rays. We analysed the XMM-Newton and Swift observations taken 3 to 30 days after the SN explosion to study the X-ray spectrum of SN 2011dh. We extracted spectra from the XMM-Newton observations, which took place ~7 and 11 days after the SN. In addition, we created integrated Swift/XRT spectra of 3 to 10 days and 11 to 30 days. The spectra are well fitted with a power-law spectrum absorbed with Galactic foreground absorption. In addition, we find a harder spectral component in the first XMM-Newton spectrum taken at t ~ 7 d. This component is also detected in the first Swift spectrum of t = 3 - 10 d. While the persistent power-law component can be explained as inverse Compton emission from radio synchrotron emitting electrons, the harder component is most likely bremsstrahlung emission from the shocked stellar wind. Therefore, the harder X-ray emission that fades away after t ~ 10 d can be interpreted as emission from the shocked circumstellar wind of SN 2011dh.Comment: Accepted for publication as a Research Note in Astronomy and Astrophysic

Systematic approach to developing splice modulating antisense oligonucleotides

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 diversity 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

Induction of cryptic pre-mRNA splice-switching by antisense oligonucleotides

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 examples 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

A pot of gold at the end of the cosmic "raynbow"?

We critically review the common belief that ultrahigh energy cosmic rays are protons or atomic nuclei with masses not exceeding that of iron. We find that heavier nuclei are indeed possible, and discuss possible sources and acceleration mechanisms for such primaries. We also show detailed simulations of extensive air showers produced by ``superheavy'' nuclei, and discuss prospects for their detection in future experiments.Comment: Talk to be presented at the International Symposium on Very High Energy Cosmic Ray Interactions X