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

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

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

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

MINIMIZATION OF NO EMISSIONS FROM MULTI-BURNER COAL-FIRED BOILERS

The focus of this program is to provide insight into the formation and minimization of NO{sub x} in multi-burner arrays, such as those that would be found in a typical utility boiler. Most detailed studies are performed in single-burner test facilities, and may not capture significant burner-to-burner interactions that could influence NO{sub x} emissions. Thus, investigations of such interactions were made by performing a combination of single and multiple burner experiments in a pilot-scale coal-fired test facility at the University of Utah, and by the use of computational combustion simulations to evaluate full-scale utility boilers. In addition, fundamental studies on nitrogen release from coal were performed to develop greater understanding of the physical processes that control NO formation in pulverized coal flames--particularly under low NO{sub x} conditions. A CO/H{sub 2}/O{sub 2}/N{sub 2} flame was operated under fuel-rich conditions in a flat flame reactor to provide a high temperature, oxygen-free post-flame environment to study secondary reactions of coal volatiles. Effects of temperature, residence time and coal rank on nitrogen evolution and soot formation were examined. Elemental compositions of the char, tar and soot were determined by elemental analysis, gas species distributions were determined using FTIR, and the chemical structure of the tar and soot was analyzed by solid-state {sup 13}C NMR spectroscopy. A laminar flow drop tube furnace was used to study char nitrogen conversion to NO. The experimental evidence and simulation results indicated that some of the nitrogen present in the char is converted to nitric oxide after direct attack of oxygen on the particle, while another portion of the nitrogen, present in more labile functionalities, is released as HCN and further reacts in the bulk gas. The reaction of HCN with NO in the bulk gas has a strong influence on the overall conversion of char-nitrogen to nitric oxide; therefore, any model that aims to predict the conversion of char-nitrogen to nitric oxide should allow for the conversion of char-nitrogen to HCN. The extent of the HCN conversion to NO or N{sub 2} will depend on the composition of the atmosphere surrounding the particle. A pilot-scale testing campaign was carried out to evaluate the impact of multiburner firing on NO{sub x} emissions using a three-burner vertical array. In general, the results indicated that multiburner firing yielded higher NO{sub x} emissions than single burner firing at the same fuel rate and excess air. Mismatched burner operation, due to increases in the firing rate of the middle burner, generally demonstrated an increase in NO{sub x} over uniform firing. Biased firing, operating the middle burner fuel rich with the upper and lower burners fuel lean, demonstrated an overall reduction in NO{sub x} emissions; particularly when the middle burner was operated highly fuel rich. Computational modeling indicated that operating the three burner array with the center burner swirl in a direction opposite to the other two resulted in a slight reduction in NO{sub x}

Cost and Outcome of Behavioural Activation versus Cognitive Behavioural Therapy for Depression (COBRA): a randomised, controlled, non-inferiority trial

Background Depression is a common, debilitating, and costly disorder. Many patients request psychological therapy, but the best-evidenced therapy—cognitive behavioural therapy (CBT)—is complex and costly. A simpler therapy—behavioural activation (BA)—might be as effective and cheaper than is CBT. We aimed to establish the clinical efficacy and cost-effectiveness of BA compared with CBT for adults with depression. Methods In this randomised, controlled, non-inferiority trial, we recruited adults aged 18 years or older meeting Diagnostic and Statistical Manual of Mental Disorders IV criteria for major depressive disorder from primary care and psychological therapy services in Devon, Durham, and Leeds (UK). We excluded people who were receiving psychological therapy, were alcohol or drug dependent, were acutely suicidal or had attempted suicide in the previous 2 months, or were cognitively impaired, or who had bipolar disorder or psychosis or psychotic symptoms. We randomly assigned participants (1:1) remotely using computer-generated allocation (minimisation used; stratified by depression severity [Patient Health Questionnaire 9 (PHQ-9) score of <19 vs ≥19], antidepressant use, and recruitment site) to BA from junior mental health workers or CBT from psychological therapists. Randomisation done at the Peninsula Clinical Trials Unit was concealed from investigators. Treatment was given open label, but outcome assessors were masked. The primary outcome was depression symptoms according to the PHQ-9 at 12 months. We analysed all those who were randomly allocated and had complete data (modified intention to treat [mITT]) and also all those who were randomly allocated, had complete data, and received at least eight treatment sessions (per protocol [PP]). We analysed safety in the mITT population. The non-inferiority margin was 1·9 PHQ-9 points. This trial is registered with the ISCRTN registry, number ISRCTN27473954. Findings Between Sept 26, 2012, and April 3, 2014, we randomly allocated 221 (50%) participants to BA and 219 (50%) to CBT. 175 (79%) participants were assessable for the primary outcome in the mITT population in the BA group compared with 189 (86%) in the CBT group, whereas 135 (61%) were assessable in the PP population in the BA group compared with 151 (69%) in the CBT group. BA was non-inferior to CBT (mITT: CBT 8·4 PHQ-9 points [SD 7·5], BA 8·4 PHQ-9 points [7·0], mean difference 0·1 PHQ-9 points [95% CI −1·3 to 1·5], p=0·89; PP: CBT 7·9 PHQ-9 points [7·3]; BA 7·8 [6·5], mean difference 0·0 PHQ-9 points [–1·5 to 1·6], p=0·99). Two (1%) non-trial-related deaths (one [1%] multidrug toxicity in the BA group and one [1%] cancer in the CBT group) and 15 depression-related, but not treatment-related, serious adverse events (three in the BA group and 12 in the CBT group) occurred in three [2%] participants in the BA group (two [1%] patients who overdosed and one [1%] who self-harmed) and eight (4%) participants in the CBT group (seven [4%] who overdosed and one [1%] who self-harmed). Interpretation We found that BA, a simpler psychological treatment than CBT, can be delivered by junior mental health workers with less intensive and costly training, with no lesser effect than CBT. Effective psychological therapy for depression can be delivered without the need for costly and highly trained professionals. Funding National Institute for Health Research

CONSORT extension for the reporting of randomised controlled trials conducted using cohorts and routinely collected data (CONSORT-ROUTINE): checklist with explanation and elaboration

Randomised controlled trials are increasingly conducted as embedded, nested, or using cohorts or routinely collected data, including registries, electronic health records, and administrative databases, to assess if participants are eligible for the trial and to facilitate recruitment, to deliver an embedded intervention, to collect trial outcome data, or a combination of these purposes. This report presents the Consolidated Standards of Reporting Trials (CONSORT) extension for randomised controlled trials conducted using cohorts and routinely collected data (CONSORT-ROUTINE). The extension was developed to look at the unique characteristics of trials conducted with these types of data with the goal of improving reporting quality in the long term by setting standards early in the process of uptake of these trial designs. The extension was developed with a sequential approach, including a Delphi survey, a consensus meeting, and piloting of the checklist. The checklist was informed by the CONSORT 2010 statement and two reporting guidelines for observational studies, the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement and the REporting of studies Conducted using Observational Routinely collected Data (RECORD) statement. The extension includes eight items modified from the CONSORT 2010 statement and five new items. Reporting items with explanations and examples are provided, including key aspects of trials conducted using cohorts or routinely collected data that require specific reporting considerations