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RNA-based therapeutics have entered the mainstream with seemingly limitless possibilities to treat all categories of neurological disease. Here, common RNA-based drug modalities such as antisense oligonucleotides, small interfering RNAs, RNA aptamers, RNA-based vaccines and mRNA drugs are reviewed highlighting their current and potential applications. Rapid progress has been made across rare genetic diseases and neurodegenerative disorders, but safe and effective delivery to the brain remains a significant challenge for many applications. The advent of individualized RNA-based therapies for ultra-rare diseases is discussed against the backdrop of the emergence of this field into more common conditions such as Alzheimer’s disease and ischaemic stroke. There remains significant untapped potential in the use of RNA-based therapeutics for behavioural disorders and tumours of the central nervous system; coupled with the accelerated development expected over the next decade, the true potential of RNA-based therapeutics to transform the therapeutic landscape in neurology remains to be uncovered.

Item does not contain fulltext Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin, but many patients have rare revertant fibers that express dystrophin. The skeletal muscle pathology of DMD patients includes immune cell infiltration and inflammatory cascades. There are several strategies to restore dystrophin in skeletal muscles of patients, including exon skipping and gene therapy. There is some evidence that dystrophin restoration leads to a reduction in immune cells, but dystrophin epitopes expressed in revertant fibers or following genome editing, cell therapy, or microdystrophin delivery after adeno-associated viral gene therapy may elicit T cell production in patients. This may affect the efficacy of the therapeutic intervention, and potentially lead to serious adverse events. To confirm and extend previous studies, we performed annual enzyme- linked immunospot interferon-gamma assays on peripheral blood mononuclear cells from 77 pediatric boys with DMD recruited into a natural history study, 69 of whom (89.6%) were treated with corticosteroids. T cell responses to dystrophin were quantified using a total of 368 peptides spanning the entire dystrophin protein, organized into nine peptide pools. Peptide mapping pools were used to further localize the immune response in one positive patient. Six (7.8%) patients had a T cell-mediated immune response to dystrophin at at least one time point. All patients who had a positive result had been treated with corticosteroids, either prednisolone or prednisone. Our results show that ∼8% of DMD individuals in our cohort have a pre-existing T cell-mediated immune response to dystrophin, despite steroid treatment. Although these responses are relatively low level, this information should be considered a useful immunological baseline before undertaking clinical trials and future DMD studies. We further highlight the importance for a robust, reproducible standard operating procedure for collecting, storing, and shipping samples from multiple centers to minimize the number of inconclusive data.

Duchenne muscular dystrophy (DMD) is caused by frameshift mutations in theDMDgene that prevent the body-wide translation of its protein product, dystrophin. Besides a severe muscle phenotype, cognitive impairment and neuropsychiatric symptoms are prevalent. Dystrophin protein 71 (Dp71) is the majorDMDgene product expressed in the brain and mutations affecting its expression are associated with the DMD neuropsychiatric syndrome. As with dystrophin in muscle, Dp71 localises to dystrophin-associated protein complexes in the brain. However, unlike in skeletal muscle; in the brain, Dp71 is alternatively spliced to produce many isoforms with differential subcellular localisations and diverse cellular functions. These include neuronal differentiation, adhesion, cell division and excitatory synapse organisation as well as nuclear functions such as nuclear scaffolding and DNA repair. In this review, we first describe brain involvement in DMD and the abnormalities observed in the DMD brain. We then review the gene expression, RNA processing and functions of Dp71. We review genotype-phenotype correlations and discuss emerging cellular/tissue evidence for the involvement of Dp71 in the neuropathophysiology of DMD. The literature suggests changes observed in the DMD brain are neurodevelopmental in origin and that their risk and severity is associated with a cumulative loss of distalDMDgene products such as Dp71. The high risk of neuropsychiatric syndromes in Duchenne patients warrants early intervention to achieve the best possible quality of life. Unravelling the function and pathophysiological significance of dystrophin in the brain has become a high research priority to inform the development of brain-targeting treatments for Duchenne.

BackgroundMutation of the Duchenne muscular dystrophy (DMD)gene causes Duchenne and Becker muscular dystrophy, degenerative neuromuscular disorders that primarily affect voluntary muscles. However, increasing evidence implicatesDMDin the development of all major cancer types.DMDis a large gene with 79 exons that codes for the essential muscle protein dystrophin. Alternative promotor usage drives the production of several additional dystrophin protein products with roles that extend beyond skeletal muscle. The importance and function(s) of these gene products outside of muscle are not well understood.ConclusionsWe highlight a clear role forDMDin the pathogenesis of several cancers, including sarcomas, leukaemia’s, lymphomas, nervous system tumours, melanomas and various carcinomas. We note that the normal balance ofDMDgene products is often disrupted in cancer. The short dystrophin protein Dp71 is, for example, typically maintained in cancer whilst the full-length Dp427 gene product, a likely tumour suppressor, is frequently inactivated in cancer due to a recurrent loss of 5’ exons. Therefore, the ratio of short and long gene products may be important in tumorigenesis. In this review, we summarise the tumours in whichDMDis implicated and provide a hypothesis for possible mechanisms of tumorigenesis, although the question of cause or effect may remain. We hope to stimulate further study into the potential role ofDMDgene products in cancer and the development of novel therapeutics that targetDMD.

The prevalence of childhood obesity remains at approximately 18% among adolescents despite stabilization in its rates between 2007 and 2010.1 Adolescent obesity is associated with an increased risk of severe obesity in adulthood and higher prevalence of obesity related comorbidities, particularly cardiovascular disease (CVD), and Type 2 Diabetes Mellitus (Type 2 DM).2–4 Lifestyle factors play a major role in the development of obesity and its progression to comorbidities such as CVD and Type 2 DM.5–10 Adolescents with obesity frequently display unhealthy eating habits including irregular eating patterns such as skipping breakfast, late-night eating,6 frequent intake of fast food,7 eating in response to emotional issues or boredom, excessive intake of high-sugar beverages,8 inappropriate portion sizes,9 and lacking adequate intake of fruits, vegetables, and nutrients.11 Alterations in various risk factors and/or biomarkers of CVD are evident at an early age in the clinical course of obesity,12–14 but lifestyle interventions based on diet and physical activity have been shown to have favorable effects, both with and without weight reduction.15 The development of cooking skills at an early age may play an important role in promoting a healthy lifestyle, but this has been given limited attention in studies examining the impact of lifestyle approaches in the management and/or prevention of adolescent obesity.16,17 In one study, lack of cooking skills was inversely related to consumption of fruits and vegetables in college students.18 A few studies have also suggested that interventions targeting cooking skills could be an effective strategy to promote healthy eating.19 Cookfresh was designed to study the effect of - as well as feasibility and acceptance of - a community- based cooking skills program, including meal planning and an actual shopping experience, on healthy behavior, quality of life, and health status including Body Mass Index (BMI) and specific biomarkers of cardiometabolic disease.

[Image: see text] Human Macrophage Migration Inhibitory Factor (MIF) is a trimeric cytokine implicated in a number of inflammatory and autoimmune diseases and cancer. We previously reported that the dye p425 (Chicago Sky Blue), which bound MIF at the interface of two MIF trimers covering the tautomerase and allosteric pockets, revealed a unique strategy to block MIF’s pro-inflammatory activities. Structural liabilities, including the large size, precluded p425 as a medicinal chemistry lead for drug development. We report here a rational design strategy linking only the fragment of p425 that binds over the tautomerase pocket to the core of ibudilast, a known MIF allosteric site-specific inhibitor. The chimeric compound, termed L2-4048, was shown by X-ray crystallography to bind at the allosteric and tautomerase sites as anticipated. L2-4048 retained target binding and blocked MIF’s tautomerase CD74 receptor binding, and pro-inflammatory activities. Our studies lay the foundation for the design and synthesis of smaller and more drug-like compounds that retain the MIF inhibitory properties of this chimera.

Background: Duchenne muscular dystrophy is a lethal disease caused by lack of dystrophin. Skipping of exons adjacent to out-of-frame deletions has proven to restore dystrophin expression in Duchenne patients. Exon 51 has been the most studied target in both preclinical and clinical settings and the availability of standardized procedures to quantify exon skipping would be advantageous for the evaluation of preclinical and clinical data. Objective: To compare methods currently used to quantify antisense oligonucleotide–induced exon 51 skipping in the DMD transcript and to provide guidance about the method to use. Methods: Six laboratories shared blinded RNA samples from Duchenne patient-derived muscle cells treated with different amounts of exon 51 targeting antisense oligonucleotide. Exon 51 skipping levels were quantified using five different techniques: digital droplet PCR, single PCR assessed with Agilent bioanalyzer, nested PCR with agarose gel image analysis by either ImageJ or GeneTools software and quantitative real-time PCR. Results: Differences in mean exon skipping levels and dispersion around the mean were observed across the different techniques. Results obtained by digital droplet PCR were reproducible and showed the smallest dispersion. Exon skipping quantification with the other methods showed overestimation of exon skipping or high data variation. Conclusions: Our results suggest that digital droplet PCR was the most precise and quantitative method. The quantification of exon 51 skipping by Agilent bioanalyzer after a single round of PCR was the second-best choice with a 2.3-fold overestimation of exon 51 skipping levels compared to digital droplet PCR.

Photo cross-linking of proteins with short RNA oligomers is a classical method to study RNA–protein interactions that are implicated in many aspects of RNA metabolism and function. Most commonly, this involves the use of [γ-32P]-labeled RNA probes. Although very sensitive, these procedures are complicated by the safety issues associated with the use of radioisotopes. Here, we describe a modified UV cross-linking method using oligonucleotide probes end labelled with the infrared dye IRDye®800. After UV cross-linking, proteins are separated by SDS-PAGE and cross-linked products are visualized with the Odyssey® Infrared Imaging system. This end labelling approach provides a streamlined alternative to random labelling which reduces the efficiency of in-vitro transcription. End labelling is also independent of the length of the probe, thus facilitating quantitative comparisons. To validate the method, we have confirmed the binding of HuD to the 3′-UTR of the mRNA for the microtubule-associated protein tau, implicated in the pathogenesis of Alzheimer’s disease. UV cross-linking of HuD with a labeled 21-mer probe was successfully performed using a recombinant purified glutathione-S-transferase–HuD fusion protein as well as with lysates from CHO cells transfected with HuD cDNA. UV cross-linking combined with infrared imaging offers a convenient and robust strategy to analyse RNA–protein interactions and their emerging importance in disease.

Structures of C. difficile alanine racemase in complex with the PLP cofactor and cycloserine are presented., Clostridium difficile, a Gram-positive, spore-forming anaerobic bacterium, is the leading cause of infectious diarrhea among hospitalized patients. C. difficile is frequently associated with antibiotic treatment, and causes diseases ranging from antibiotic-associated diarrhea to life-threatening pseudo­membranous colitis. The severity of C. difficile infections is exacerbated by the emergence of hypervirulent and multidrug-resistant strains, which are difficult to treat and are often associated with increased mortality rates. Alanine racemase (Alr) is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that catalyzes the reversible racemization of l- and d-alanine. Since d-alanine is an essential component of the bacterial cell-wall peptidoglycan, and there are no known Alr homologs in humans, this enzyme is being tested as an antibiotic target. Cycloserine is an antibiotic that inhibits Alr. In this study, the catalytic properties and crystal structures of recombinant Alr from the virulent and multidrug-resistant C. difficile strain 630 are presented. Three crystal structures of C. difficile Alr (CdAlr), corresponding to the complex with PLP, the complex with cycloserine and a K271T mutant form of the enzyme with bound PLP, are presented. The structures are prototypical Alr homodimers with two active sites in which the cofactor PLP and cycloserine are localized. Kinetic analyses reveal that the K271T mutant CdAlr has the highest catalytic constants reported to date for any Alr. Additional studies are needed to identify the basis for the high catalytic activity. The structural and activity data presented are first steps towards using CdAlr for the development of structure-based therapeutics for C. difficile infections.

Background: Individuals with Duchenne muscular dystrophy (DMD) often have lower intelligence than that of the general population. Furthermore, the prevalence of cognitive impairment, epilepsy, autism and attention deficit hyperactivity disorder (ADHD) are higher in DMD patients. Evidence suggests a role for the most predominant dystrophin isoform, Dp71, which is expressed in neuronal cells and glia. Aims: Due to limited knowledge of the brain of Duchenne patients and an unclear understanding of the function of Dp71, we sought to investigate how distal mutations in the DMD gene, which lead to an absence of Dp71 expression, are associated with the range of cognitive and behavioural phenotypes in these patients. We have modelled the loss of Dp71 by using DMD patient-derived fibroblasts (which naturally express the Dp71 isoform). We have also employed siRNA-mediated knockdown of Dp71 in a panel of neuronal and glial cell lines. We examined whether the loss of Dp71 was associated with defects in cell migration and proliferation given that alterations in these processes are associated with the cognitive and behavioural co-morbidities observed in DMD patients. Methods: Four patient-derived fibroblast cell lines, containing unique mutations of the DMD gene (resulting in the loss of Dp71) were examined using wound healing and proliferation assays as well as by RT-PCR and immunoblotting. The associated clinical data confirmed that patients exhibit cognitive and/or behavioural symptoms, which were severe for two individuals. Results: We demonstrate that DMD patient fibroblasts lacking Dp71 had reduced metabolic activity than control cells and migrated more rapidly. The increased level of cell migration is accompanied by an increase in focal adhesion kinase (FAK, a regulator of cell migration) expression. Conclusion: Our data highlights a role for Dp71 in cell migration and proliferation; alterations in these cellular processes are known to be associated with behavioural disorders prevalent in DMD patients such as autism. These findings have potential to inform the development of effective brain-targeting treatments.

The genus Mycobacterium includes non-pathogenic species such as M. smegmatis, and pathogenic species such as M. tuberculosis, the causative agent of tuberculosis (TB). Treatment of TB requires a lengthy regimen of several antibiotics, whose effectiveness has been compromised by the emergence of resistant strains. New antibiotics that can shorten the treatment course and those that have not been compromised by bacterial resistance are needed. In this study, we report that thiadiazolidinones, a relatively little-studied heterocyclic class, inhibit the activity of mycobacterial alanine racemase, an essential enzyme that converts L-alanine to D-alanine for peptidoglycan synthesis. Twelve members of the thiadiazolidinone family were evaluated for inhibition of M. tuberculosis and M. smegmatis alanine racemase activity and bacterial growth. Thiadiazolidinones inhibited M. tuberculosis and M. smegmatis alanine racemases to different extents with 50% inhibitory concentrations (IC50) ranging from 150 µM, respectively. The compounds also inhibited the growth of these bacteria, including multidrug resistant strains of M. tuberculosis. The minimal inhibitory concentrations (MIC) for drug-susceptible M. tuberculosis and M. smegmatis ranged from 6.25 µg/ml to 100 µg/ml, and from 1.56 to 6.25 µg/ml for drug-resistant M. tuberculosis. The in vitro activities of thiadiazolidinones suggest that this family of compounds might represent starting points for medicinal chemistry efforts aimed at developing novel antimycobacterial agents.

In the search for the most efficacious antisense oligonucleotides (AOs) aimed at inducing SMN2 exon 7 inclusion, we systematically assessed three AOs, PMO25 (-10, -34), PMO18 (-10, -27), and PMO20 (-10, -29), complementary to the SMN2 intron 7 splicing silencer (ISS-N1). PMO25 was the most efficacious in augmenting exon 7 inclusion in vitro in spinal muscular atrophy (SMA) patient fibroblasts and in vitro splicing assays. PMO25 and PMO18 were compared further in a mouse model of severe SMA. After a single intracerebroventricular (ICV) injection in neonatal mice, PMO25 increased the life span of severe SMA mice up to 30-fold, with average survival greater by 3-fold compared with PMO18 at a dose of 20 μg/g and 2-fold at 40 μg/g. Exon 7 inclusion was increased in the CNS but not in peripheral tissues. Systemic delivery of PMO25 at birth achieved a similar outcome and produced increased exon 7 inclusion both in the CNS and peripherally. Systemic administration of a 10-μg/g concentration of PMO25 conjugated to an octaguanidine dendrimer (VMO25) increased the life span only 2-fold in neonatal type I SMA mice, although it prevented tail necrosis in mild SMA mice. Higher doses and ICV injection of VMO25 were associated with toxicity. We conclude that (1) the 25-mer AO is more efficient than the 18-mer and 20-mer in modifying SMN2 splicing in vitro; (2) it is more efficient in prolonging survival in SMA mice; and (3) naked Morpholino oligomers are more efficient and safer than the Vivo-Morpholino and have potential for future SMA clinical applications.

Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases.

Duchenne muscular dystrophy (DMD) is the most common childhood neuromuscular disorder. It is caused by mutations in the DMD gene that disrupt the open reading frame (ORF) preventing the production of functional dystrophin protein. The loss of dystrophin ultimately leads to the degeneration of muscle fibres, progressive weakness and premature death. Antisense oligonucleotides (AOs) targeted to splicing elements within DMD pre-mRNA can induce the skipping of targeted exons, restoring the ORF and the consequent production of a shorter but functional dystrophin protein. This approach may lead to an effective disease modifying treatment for DMD and progress towards clinical application has been rapid. Less than a decade has passed between the first studies published in 1998 describing the use of AOs to modify the DMD gene in mice and the results of the first intramuscular proof of concept clinical trials. Whilst phase II and III trials are now underway, the heterogeneity of DMD mutations, efficient systemic delivery and targeting of AOs to cardiac muscle remain significant challenges. Here we review the current status of AO-mediated therapy for DMD, discussing the preclinical, clinical and regulatory hurdles and their possible solutions to expedite the translation of AO-mediated exon skipping therapy to clinic.

We previously conducted a proof of principle; dose escalation study in Duchenne muscular dystrophy (DMD) patients using the morpholino splice-switching oligonucleotide AVI-4658 (eteplirsen) that induces skipping of dystrophin exon 51 in patients with relevant deletions, restores the open reading frame and induces dystrophin protein expression after intramuscular (i.m.) injection. We now show that this dystrophin expression was accompanied by an elevated expression of α-sarcoglycan, β-dystroglycan (BDG) and--in relevant cases--neuronal nitric oxide synthase (nNOS) at the sarcolemma, each of which is a component of a different subcomplex of the dystrophin-associated glycoprotein complex (DAPC). As expected, nNOS expression was relocalized to the sarcolemma in Duchenne patients in whom the dystrophin deletion left the nNOS-binding domain (exons 42-45) intact, whereas this did not occur in patients with deletions that involved this domain. Our results indicate that the novel internally deleted and shorter dystrophin induced by skipping exon 51 in patients with amenable deletions, can also restore the dystrophin-associated complex, further suggesting preserved functionality of the newly translated dystrophin.

Methicillin-resistant Staphylococcus aureus (MRSA) is a human pathogen and a major cause of hospital-acquired infections. New antibacterial agents that have not been compromised by bacterial resistance are needed to treat MRSA-related infections. We chose the S. aureus cell wall synthesis enzyme, alanine racemase (Alr) as the target for a high-throughput screening effort to obtain novel enzyme inhibitors, which inhibit bacterial growth. Among the ‘hits’ identified was a thiadiazolidinone with chemical properties attractive for lead development. This study evaluated the mode of action, antimicrobial activities, and mammalian cell cytotoxicity of the thiadiazolidinone family in order to assess its potential for development as a therapeutic agent against MRSA. The thiadiazolidones inhibited Alr activity with 50% inhibitory concentrations (IC50) ranging from 0.36 to 6.4 μM, and they appear to inhibit the enzyme irreversibly. The series inhibited the growth of S. aureus, including MRSA strains, with minimal inhibitory concentrations (MICs) ranging from 6.25 to 100 μg/ml. The antimicrobial activity showed selectivity against Gram-positive bacteria and fungi, but not Gram-negative bacteria. The series inhibited human HeLa cell proliferation. Lead development centering on the thiadiazolidinone series would require additional medicinal chemistry efforts to enhance the antibacterial activity and minimize mammalian cell toxicity.

Intelligence of individuals with Duchenne muscular dystrophy (DMD) is lower than the general population. Cognitive impairment, epilepsy, autism and attention deficit hyperactivity disorder are prevalent. Mounting evidence links these symptoms to the loss of dystrophin in the brain; yet central nervous system involvement in DMD has been largely ignored. The most predominant dystrophin isoform in the brain is the smaller Dp71 isoform expressed in neurons and glia. Distal DMD mutations affecting Dp71 expression are linked to cognitive impairment. The function(s) of Dp71 and how its absence can lead to cognitive and behavioural symptoms are not well understood. We are modelling the loss of Dp71 both endogenously in DMD patient-derived fibroblasts (which, in controls, naturally express Dp71) and with siRNA in neuronal and glial cell lines. Four patient fibroblast cell lines, each harbouring a different DMD gene mutation that naturally results in the loss of Dp71 are being studied. Accompanying clinical data confirms these patients display cognitive and/or behavioural symptoms, which is severe for two individuals. Our preliminary findings highlight a role for Dp71 in cell migration and proliferation; alterations in these processes are associated with psychiatric disorders prevalent in DMD such as autism. We will present would healing and proliferation assay data exploring these role(s) and discuss potential molecular mechanisms behind the cognitive and behavioural symptoms observed in DMD. The high risk of neuropsychiatric syndromes in Duchene warrants early intervention to achieve the best possible quality of life; our findings have significant potential to inform ongoing drug development in this area.

Summary Background We report clinical safety and biochemical efficacy from a dose-ranging study of intravenously administered AVI-4658 phosphorodiamidate morpholino oligomer (PMO) in patients with Duchenne muscular dystrophy. Method We undertook an open-label, phase 2, dose-escalation study (0·5, 1·0, 2·0, 4·0, 10·0, and 20·0 mg/kg bodyweight) in ambulant patients with Duchenne muscular dystrophy aged 5–15 years with amenable deletions in DMD. Participants had a muscle biopsy before starting treatment and after 12 weekly intravenous infusions of AVI-4658. The primary study objective was to assess safety and tolerability of AVI-4658. The secondary objectives were pharmacokinetic properties and the ability of AVI-4658 to induce exon 51 skipping and dystrophin restoration by RT-PCR, immunohistochemistry, and immunoblotting. The study is registered, number NCT00844597. Findings 19 patients took part in the study. AVI-4658 was well tolerated with no drug-related serious adverse events. AVI-4658 induced exon 51 skipping in all cohorts and new dystrophin protein expression in a significant dose-dependent (p=0·0203), but variable, manner in boys from cohort 3 (dose 2 mg/kg) onwards. Seven patients responded to treatment, in whom mean dystrophin fluorescence intensity increased from 8·9% (95% CI 7·1–10·6) to 16·4% (10·8–22·0) of normal control after treatment (p=0·0287). The three patients with the greatest responses to treatment had 21%, 15%, and 55% dystrophin-positive fibres after treatment and these findings were confirmed with western blot, which showed an increase after treatment of protein levels from 2% to 18%, from 0·9% to 17%, and from 0% to 7·7% of normal muscle, respectively. The dystrophin-associated proteins α-sarcoglycan and neuronal nitric oxide synthase were also restored at the sarcolemma. Analysis of the inflammatory infiltrate indicated a reduction of cytotoxic T cells in the post-treatment muscle biopsies in the two high-dose cohorts. Interpretation The safety and biochemical efficacy that we present show the potential of AVI-4658 to become a disease-modifying drug for Duchenne muscular dystrophy. Funding UK Medical Research Council; AVI BioPharma.

The importance of aberrant RNA processing in neurodegeneration is becoming increasingly clear; a recent example being the identification of the splicing factor TDP-43 as the major component of inclusions characteristic of a number of neurodegenerative conditions including amyotrophic lateral sclerosis (ALS). Due to the enormous diversity generated by alternative splicing and its importance in the nervous system, it is no surprise that defective alternative splicing in disease has been particularly well documented. However, in addition to splicing, other RNA processing events such as RNA editing, polyadenylation and mRNA stability are also disrupted in some neurological disorders. For instance: the editing efficiency of specific ionotropic receptors is reduced in ALS affecting ion permeability and the function of RNA-processing proteins is affected by their sequestration to trinucleotide repeat expanded mRNAs in several disorders. Due to the extensive coupling between RNA processing events and the multifunctionality of the RNA processing factors that regulate them, it is important to consider RNA processing as a whole. Here we review RNA processing events and their extensive coupling to one another and detail the associations of RNA processing including, but not exclusively, alternative splicing with neurodegeneration.

The authors report a case of a 13-year-old female with recurrent Burkitt lymphoma who was heavily pretreated with chemotherapy. During chemotherapy for relapse, she developed serious aspergillus infection of the palate and sinuses. Despite 10 microg/kg of filgrastim for 5 days, peripheral blood CD34(+) cells remainedor=1/microL. Hematopoietic progenitor cells were subsequently successfully mobilized with a combination of AMD3100 and filgrastim without tumor or fungus contamination. After transplantation, the patient achieved complete engraftment without exacerbation of fungal infection. This is the first case report of a pediatric patient with relapsed Burkitt lymphoma with mobilization of hematopoietic progenitor cells by AMD3100 without any adverse reactions.

West Nile virus is an emerging pathogen that can cause fatal neurological disease. A recombinant human mAb, mAb11, has been described as a candidate for the prevention and treatment of West Nile disease. Using a yeast surface display epitope mapping assay and neutralization escape mutant, we show that mAb11 recognizes the fusion loop, at the distal end of domain II of the West Nile virus envelope protein. Ab mAb11 cross-reacts with all four dengue viruses and provides protection against dengue (serotypes 2 and 4) viruses. In contrast to the parental West Nile virus, a neutralization escape variant failed to cause lethal encephalitis (at higher infectious doses) or induce the inflammatory responses associated with blood-brain barrier permeability in mice, suggesting an important role for the fusion loop in viral pathogenesis. Our data demonstrate that an intact West Nile virus fusion loop is critical for virulence, and that human mAb11 targeting this region is efficacious against West Nile virus infection. These experiments define the molecular determinant on the envelope protein recognized by mAb11 and demonstrate the importance of this region in causing West Nile encephalitis.

Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by mutations in the MAPT gene, encoding the tau protein that accumulates in intraneuronal lesions in a number of neurodegenerative diseases. Several FTDP-17 mutations affect alternative splicing and result in excess exon 10 (E10) inclusion in tau mRNA. RNA reprogramming using spliceosome-mediated RNA trans-splicing (SMaRT) could be a method of choice to correct aberrant E10 splicing resulting from FTDP-17 mutations. SMaRT creates a hybrid mRNA through a trans-splicing reaction between an endogenous target pre-mRNA and a pre-trans-splicing RNA molecule (PTM). However, FTDP-17 mutations affect the strength of cis-splicing elements and could favor cis-splicing over trans-splicing. Excess E10 inclusion in FTDP-17 can be caused by intronic mutations destabilizing a stem-loop protecting the 5' splice site at the E10/intron 10 junction. COS cells transfected with a minigene containing the intronic +14 mutation produce exclusively E10(+) RNA. Generation of E10(-) RNA was restored after co-transfection with a PTM designed to exclude E10. Similar results were obtained with a target containing the exonic N279K mutation which strengthens a splicing enhancer within E10. Conversely, increase or decrease in E10 content was achieved by trans-splicing from a target carrying the Delta280K mutation, which weakens the same splicing enhancer. Thus E10 inclusion can be modulated by trans-splicing irrespective of the strength of the cis-splicing elements affected by FTDP-17 mutations. In conclusion, RNA trans-splicing could provide the basis of therapeutic strategies for impaired alternative splicing caused by pathogenic mutations in cis-acting splicing elements.

Positron emission tomography (PET) using the tracer [18F]-fluorodeoxyglucose (FDG) is commonly used for measuring metabolic rate of glucose ( MRglc) in the human brain. Conventional PET methods (e.g., the Patlak method) for quantifying MRglc assume the tissue transport and phosphorylation mechanisms to be in steady state during FDG uptake. As FDG and glucose use the same transporters and phosphorylation enzymes, changing blood glucose levels can change the rates of FDG transport and phosphorylation. Compartmental models were used to simulate the effect of rising arterial glucose, from normal to hyperglycemic levels on FDG uptake for a typical PET protocol. The subsequent errors on the values of MRglc calculated using the Patlak method were investigated, and a correction scheme based on measured arterial glucose concentration ( Gp) was evaluated. Typically, with a 40% rise in Gp over the duration of the PET study, the true MRglc varied by only 1%; however, the Patlak method overestimated MRglc by 15%. The application of the correction reduced this error to −2%. In general, the application of the correction resulted in values of MRglc consistently significantly closer to the true steady state calculation of MRglc independently of changes to the parameters defining the model.

The immune response against viral infection relies on the early production of cytokines that induce an antiviral state and trigger the activation of immune cells. This response is initiated by the recognition of virus-associated molecular patterns such as dsRNA, a viral replication intermediate recognized by TLR3 and certain RNA helicases. Infection with West Nile virus (WNV) can lead to lethal encephalitis in susceptible individuals and constitutes an emerging health threat. In this study, we report that WNV envelope protein (WNV-E) specifically blocks the production of antiviral and proinflammatory cytokines induced by dsRNA in murine macrophages. This immunosuppressive effect was not dependent on TLR3 or its adaptor molecule Trif. Instead, our experiments show that WNV-E acts at the level of receptor-interacting protein 1. Our results also indicate that WNV-E requires a certain glycosylation pattern, specifically that of dipteran cells, to inhibit dsRNA-induced cytokine production. In conclusion, these data show that the major structural protein of WNV impairs the innate immune response and suggest that WNV exploits differential vector/host E glycosylation profiles to evade antiviral mechanisms.

Elucidating the mechanisms of alternative splicing in the brain is a prerequisite to the understanding of the pathogenesis of major neurological diseases linked to impairment of pre-mRNA alternative splicing. The gene trinucleotide repeat-containing 4 (TNRC4) is predicted to encode a member of the CELF (CUG-BP- and ETR-3-like factors) family of RNA-binding proteins containing a 15-18-residue polyglutamine sequence. The TNRC4 transcript is selectively expressed in the brain. Using an anti-peptide antibody against the predicted sequence, we establish the presence of TNRC4 as a approximately 50 kDa protein in the brain. Full-length TNRC4 displays nuclear and cytoplasmic localizations in transfected cells, whereas a C-terminally truncated mutant is essentially confined to the cytoplasm. TNRC4 is not recruited into inclusions formed by polyglutamine-expanded ataxin-1 or huntingtin. TNRC4 activates tau exon 10 (E10) inclusion at high efficiency in transfected cells. TNRC4 contains two consecutive N-terminal RNA recognition motifs (RRMs) separated from the C-terminal RRM. Deletion and point mutant analysis show that the activity of TNRC4 on tau E10 splicing is mainly mediated by the RNA-binding activity of the second RRM and involves an intronic element of the tau pre-mRNA. The polyglutamine sequence has no effect on the activity of TNRC4 on tau E10 splicing. This study represents the first characterization of TNRC4 and provides further insight into the mechanisms of brain-specific alternative splicing and their possible pathological implications.

The rising prevalence of obesity and type 2 diabetes is a global challenge. A possible mechanism linking insulin resistance and weight gain would be attenuation of insulin-evoked responses in brain areas relevant to eating in systemic insulin resistance. We measured brain glucose metabolism, using [18F]fluorodeoxyglucose positron emission tomography, in seven insulin-sensitive (homeostasis model assessment of insulin resistance [HOMA-IR] = 1.3) and seven insulin-resistant (HOMA-IR = 6.3) men, during suppression of endogenous insulin by somatostatin, with and without an insulin infusion that elevated insulin to 24.6 ± 5.2 and 23.2 ± 5.8 mU/l (P = 0.76), concentrations similar to fasting levels of the resistant subjects and approximately threefold above those of the insulin-sensitive subjects. Insulin-evoked change in global cerebral metabolic rate for glucose was reduced in insulin resistance (+7 vs. +17.4%, P = 0.033). Insulin was associated with increased metabolism in ventral striatum and prefrontal cortex and with decreased metabolism in right amygdala/hippocampus and cerebellar vermis (P < 0.001), relative to global brain. Insulin’s effect was less in ventral striatum and prefrontal cortex in the insulin-resistant subjects (mean ± SD for right ventral striatum 3.2 ± 3.9 vs. 7.7 ± 1.7, P = 0.017). We conclude that brain insulin resistance exists in peripheral insulin resistance, especially in regions subserving appetite and reward. Diminishing the link be-tween control of food intake and energy balance may contribute to development of obesity in insulin resistance.

West Nile virus , a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specific antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.

Objective: We formed a multi-institution collaboration in order to compare dystrophin quantification methods, reach a consensus on the most reliable method, and report its biological significance in the context of clinical trials. Methods: Five laboratories with expertise in dystrophin quantification performed a data-driven comparative analysis of a single reference set of normal and dystrophinopathy muscle biopsies using quantitative immunohistochemistry and Western blotting. We developed standardized protocols and assessed inter- and intralaboratory variability over a wide range of dystrophin expression levels. Results: Results from the different laboratories were highly concordant with minimal inter- and intralaboratory variability, particularly with quantitative immunohistochemistry. There was a good level of agreement between data generated by immunohistochemistry and Western blotting, although immunohistochemistry was more sensitive. Furthermore, mean dystrophin levels determined by alternative quantitative immunohistochemistry methods were highly comparable. Conclusions: Considering the biological function of dystrophin at the sarcolemma, our data indicate that the combined use of quantitative immunohistochemistry and Western blotting are reliable biochemical outcome measures for Duchenne muscular dystrophy clinical trials, and that standardized protocols can be comparable between competent laboratories. The methodology validated in our study will facilitate the development of experimental therapies focused on dystrophin production and their regulatory approval.

Background I felt strongly about improving the services for end of life relatives staying for long periods with their loved ones. I wanted to find out what they felt was missing from our care, and how we could improve their time with us. Method I conducted a survey of twenty EOL relatives asking about their experience generally and what could be improved. I asked the questions in a safe place, questions around this are obviously of a sensitive nature and I wanted them to feel free to answer openly and honestly. Results/findings Relatives want more information about what to expect during the dying process, such as physical signs and symptoms, as well as around what services are available. No facilities for relatives to make their own drinks and often feel they cannot approach busy staff. No facilities available for relatives who arrive without any of their own belongings. Conclusion/actions I created A relative’s guide for loved ones in the last days of life. This booklet contains information about signs and symptoms they may see in a dying loved one; services available across the hospital; and a summary about what support they offer. I instated a scheme where EOL relatives are entitled to a meal card activated with credit worth £5 per day so they are able to buy refreshments from the canteen and restaurant. This reactivates at midnight and can be used throughout their stay. I compiled a relative’s overnight pack-a mini washbag, containing toothbrush, toothpaste, wipes and tissues for when they need to stay unexpectedly. Good palliative care is essential ­­- for a patient’s loved ones as well as themselves. I would like to continue to make changes and create some positives in what can be otherwise a wholly negative experience.

The microtubule-associated protein tau is predominantly localized in the axonal compartment over the entire length of the axon in neurons. The mechanisms responsible for the localization of tau in axons at long distance from the cell body are not properly understood. Using fluorescence in situ hybridization, we show that tau mRNA is present in the central and distal parts of the axons of cultured rat cortical neurons. Axonal tau mRNA is associated with granules which are distributed throughout the entire length of the axon, including the growth cone. We also show that tau mRNA-containing axonal particles are associated with elongation factor 1A, a component of the protein translation machinery. The presence of tau mRNA in axons might be at least part of the process by which tau is localized to distal axons.

Importance: In Duchenne muscular dystrophy (DMD), the reading frame of an out-of-frame DMD deletion can be repaired by antisense oligonucleotide (AO)–mediated exon skipping. This creates a shorter dystrophin protein, similar to those expressed in the milder Becker muscular dystrophy (BMD). The skipping of some exons may be more efficacious than others. Patients with exon 44 or 45 skippable deletions (AOs in clinical development) have a less predictable phenotype than those skippable for exon 51, a group in advanced clinical trials. A way to predict the potential of AOs is the study of patients with BMD who have deletions that naturally mimic those that would be achieved by exon skipping. / Objective: To quantify dystrophin messenger RNA (mRNA) and protein expression in patients with DMD deletions treatable by, or mimicking, exon 44 or 45 skipping. / Design, Setting, and Participants: Retrospective study of nondystrophic controls (n = 2), patients with DMD (n = 5), patients with intermediate muscular dystrophy (n = 3), and patients with BMD (n = 13) at 4 university-based academic centers and pediatric hospitals. Biochemical analysis of existing muscle biopsies was correlated with the severity of the skeletal muscle phenotype. / Main Outcomes and Measures: Dystrophin mRNA and protein expression. / Results: Patients with DMD who have out-of-frame deletions skippable for exon 44 or 45 had an elevated number of revertant and trace dystrophin expression (approximately 19% of control, using quantitative immunohistochemistry) with 4 of 9 patients presenting with an intermediate muscular dystrophy phenotype (3 patients) or a BMD-like phenotype (1 patient). Corresponding in-frame deletions presented with predominantly mild BMD phenotypes and lower dystrophin levels (approximately 42% of control) than patients with BMD modeling exon 51 skipping (approximately 80% of control). All 12 patients with in-frame deletions had a stable transcript compared with 2 of 9 patients with out-of-frame deletions (who had intermediate muscular dystrophy and BMD phenotypes). / Conclusions and Relevance: Exon 44 or 45 skipping will likely yield lower levels of dystrophin than exon 51 skipping, although the resulting protein is functional enough to often maintain a mild BMD phenotype. Dystrophin transcript stability is an important indicator of dystrophin expression, and transcript instability in DMD compared with BMD should be explored as a potential biomarker of response to AOs. This study is beneficial for the planning, execution, and analysis of clinical trials for exon 44 and 45 skipping.

This paper argues that the current debate surrounding new methods of charging for water from the year 2000 should incorporate issues of social equity and must consequently involve not just the water industry but also central government and the Department of Social Security in particular. The water industry in England and Wales is currently fragmented into almost 30 separate companies, each with its own costs and prices for water services. Average water bills for domestic customers vary with company area but this variation is not wholly matched by patterns in the extent of use of the DSS direct payment scheme. In some areas people dependent for their income on means‐tested social assistance benefits can face high water bills yet lack access to budgeting schemes to help with payment.

Restoration of the open reading frame of the DMD gene and dystrophin protein production in Duchenne muscular dystrophy (DMD) can be achieved by exon skipping using antisense oligomers (AOs) targeted to splicing elements. Several such RNA-based gene therapy approaches are in clinical development in which all studies to date have assessed AO efficacy by semiquantitative nested reverse-transcription polymerase chain reaction (RT-PCR). Precise evaluation of dystrophin protein levels is complex and hindered by the large size and low abundance of dystrophin; thus an accurate and standardized measurement of DMD exon skipping at the RNA level remains important to assess and compare patient responses in DMD exon skipping clinical trials. Here we describe the development of a Taqman quantitative (q)RT-PCR assay to quantify exon skipping and highlight its use to determine the levels of exon skipping in DMD patients treated intramuscularly with a morpholino AO to skip exon 51, eteplirsen (AVI-4658). The muscle biopsies of these patients were previously thoroughly characterized, providing a valuable benchmark for the evaluation of novel methodology. We demonstrate that levels of dystrophin protein restoration, and thus patient response, correlate accurately with the RNA level. Furthermore, this sensitive assay detects revertant exon 51 skipped fibers in untreated biopsies, providing an important baseline to precisely quantify treatment success. This study represents the first quantitative assessment of exon skipping in a clinical trial setting. We present a standardized and reproducible method to assess patient response that will complement protein studies in future preclinical and clinical exon skipping-based gene therapy studies for DMD.

Duchenne muscular dystrophy is caused by mutations in the DMD gene that disrupt the open reading frame and prevent the full translation of its protein product, dystrophin. Restoration of the open reading frame and dystrophin production can be achieved by exon skipping using antisense oligonucleotides targeted to splicing elements. This approach aims to transform the Duchenne muscular dystrophy phenotype to that of the milder disorder, Becker muscular dystrophy, typically caused by in-frame dystrophin deletions that allow the production of an internally deleted but partially functional dystrophin. There is ongoing debate regarding the functional properties of the different internally deleted dystrophins produced by exon skipping for different mutations; more insight would be valuable to improve and better predict the outcome of exon skipping clinical trials. To this end, we have characterized the clinical phenotype of 17 patients with Becker muscular dystrophy harbouring in-frame deletions relevant to on-going or planned exon skipping clinical trials for Duchenne muscular dystrophy and correlated it to the levels of dystrophin, and dystrophin-associated protein expression. The cohort of 17 patients, selected exclusively on the basis of their genotype, included 4 asymptomatic, 12 mild and 1 severe patient. All patients had dystrophin levels of >40% of control and significantly higher dystrophin (P = 0.013), ?-dystroglycan (P = 0.025) and neuronal nitric oxide synthase (P = 0.034) expression was observed in asymptomatic individuals versus symptomatic patients with Becker muscular dystrophy. Furthermore, grouping the patients by deletion, patients with Becker muscular dystrophy with deletions with an end-point of exon 51 (the skipping of which could rescue the largest group of Duchenne muscular dystrophy deletions) showed significantly higher dystrophin levels (P = 0.034) than those with deletions ending with exon 53. This is the first quantitative study on both dystrophin and dystrophin-associated protein expression in patients with Becker muscular dystrophy with deletions relevant for on-going exon skipping trials in Duchenne muscular dystrophy. Taken together, our results indicate that all varieties of internally deleted dystrophin assessed in this study have the functional capability to provide a substantial clinical benefit to patients with Duchenne muscular dystrophy.

West Nile virus (WNV) is an emerging human pathogen for which specific antiviral therapy has not been developed. The therapeutic potential of RNA interference (RNAi) as a sequence-specific inhibitor of WNV has been well demonstrated. Although multiple siRNA targets have been identified within the genomic coding region, targets within the untranslated regions (UTR), which encode cis-acting regulatory elements, remain relatively unknown. In WNV and other flaviviruses, UTRs are located at the genomic termini. These regions form complex secondary structures, which pose difficulty when designing effective siRNA targets. In this study, we report the identification of siRNA targets in the WNV 3' UTR. These targets were selected by siRNA predictor algorithms, and synthesized as short hairpin RNA sequences from a plasmid-based expression system. Vero cells stably expressing these sequences had greatly diminished ability to support WNV replication but not the related dengue virus, demonstrating that the siRNAs were effective and suppressed WNV viral replication in a sequence-specific manner. The siRNAs developed in this study could function as potential antiviral therapeutics and as genetic tools to investigate the role of 3' UTR in WNV pathogenesis.

In this study, a recombinant truncated West Nile virus envelope protein antigen (rWNV-E) was produced in serum-free cultures of the expresSF+ insect cell line via baculovirus infection. This production system was selected based on its use in the production of candidate human and animal vaccine antigens. A defined fermentation and purification process for the rWNV-E antigen was established to control for purity and immunogenicity of each protein batch. The material formulated with aluminum hydroxide was stable for greater than 8months at 4 degrees C. The recombinant vaccine candidate was evaluated for immunogenicity and protective efficacy in several animal models. In mouse and hamster WNV challenge models, the vaccine candidate induced viral protection that correlated with anti-rWNV-E immunogenicity and WNV neutralizing antibody titers. The rWNV-E vaccine candidate was used to boost horses previously immunized with the Fort Dodge inactivated WNV vaccine and also to induce WNV neutralizing titers in naïve foals that were at least 14weeks of age. Furthermore, the vaccine candidate was found safe when high doses were injected into rats, with no detectable treatment-related clinical adverse effects. These observations demonstrate that baculovirus-produced rWNV-E can be formulated with aluminum hydroxide to produce a stable and safe vaccine which induces humoral immunity that can protect against WNV infection.

The flavivirus envelope (E) protein mediates cellular attachment and fusion with host cell membranes and is recognized by virus-neutralizing antibodies. We raised antibodies against a broad range of epitopes by immunizing a horse with recombinant West Nile virus (WNV) E protein. To define epitopes recognized by protective antibodies, we selected, by affinity chromatography, immunoglobulins against immobilized linear peptides derived from parts of the E protein. Immunoglobulins binding 9 different peptides from domains I, II, and III of the E protein neutralized WNV in vitro. This indicates that multiple protective epitopes can be found in the E protein. Immunoglobulins recognizing 3 peptides derived from domains I and II of E protein protected mice against a lethal challenge with WNV. These immunoglobulins recognized the E proteins of related flaviviruses, demonstrating that antibodies targeting specific E protein epitopes could be developed for prevention and treatment of multiple flavivirus infections.

Becker (BMD) and Duchenne muscular dystrophy (DMD) are allelic disorders arising from mutations in the dystrophin gene. In-frame mutations lead to the milder BMD while out-of-frame mutations disrupt the reading frame and lead to the severe DMD with lack of dystrophin. A therapeutic strategy for skipping specific exons in dystrophin and restoring the open reading frame has been successfully applied in DMD; this “converts” the out-of-frame deletion in DMD to BMD-like in-frame deletion. Micro RNAs (miRs) are small RNA sequences that regulate gene expression post-transcriptionally.

The rising prevalence of obesity and type 2 diabetes is a global challenge. A possible mechanism linking insulin resistance and weight gain would be attenuation of insulin-evoked responses in brain areas relevant to eating in systemic insulin resistance. We measured brain glucose metabolism, using [(18)F]fluorodeoxyglucose positron emission tomography, in seven insulin-sensitive (homeostasis model assessment of insulin resistance [HOMA-IR] = 1.3) and seven insulin-resistant (HOMA-IR = 6.3) men, during suppression of endogenous insulin by somatostatin, with and without an insulin infusion that elevated insulin to 24.6 +/- 5.2 and 23.2 +/- 5.8 mU/l (P = 0.76), concentrations similar to fasting levels of the resistant subjects and approximately threefold above those of the insulin-sensitive subjects. Insulin-evoked change in global cerebral metabolic rate for glucose was reduced in insulin resistance (+7 vs. +17.4%, P = 0.033). Insulin was associated with increased metabolism in ventral striatum and prefrontal cortex and with decreased metabolism in right amygdala/hippocampus and cerebellar vermis (P0.001), relative to global brain. Insulin's effect was less in ventral striatum and prefrontal cortex in the insulin-resistant subjects (mean +/- SD for right ventral striatum 3.2 +/- 3.9 vs. 7.7 +/- 1.7, P = 0.017). We conclude that brain insulin resistance exists in peripheral insulin resistance, especially in regions subserving appetite and reward. Diminishing the link be-tween control of food intake and energy balance may contribute to development of obesity in insulin resistance.