Antisense oligonucleotide-mediated therapeutic strategies for neurodegenerative repeat expansion diseases

Over 40 diseases, primarily affecting the nervous system, are caused by expansion of simple repetitive sequences found throughout the human genome, termed repeat expansion diseases. Expansions can occur in coding and non-coding regions of the genome, leading to several proposed mechanisms of disease, accumulation of either toxic RNA or toxic protein, although gain-of-function mechanisms are suggested causes of pathogenesis. Currently, there is no cure nor effective treatment strategy for any repeat expansion diseases. However, for many of these expansion diseases, splice-switching antisense oligonucleotides (AOs) may offer promise as a therapeutic strategy, as these compounds have already demonstrated efficacy in the treatment of other types of genetic disorders. Antisense oligonucleotides are short synthetic nucleic acid analogues, designed to target specific pre-mRNA sequences by reverse-complementary Watson-Crick binding, thereby modifying processing and/or abundance of the transcript and the sequence of the encoded protein. While there are a number of applications for AOs, this study focuses on their utility for preventing translation of toxic protein isoforms, either by altering the target transcript to encode a truncated protein isoform, or by disrupting the reading frame to downregulate endogenous protein production. The first part of this study focused on ameliorating the toxic polyglutamine tract found in the ataxin-3 protein that causes spinocerebellar ataxia type 3 (SCA3). One of nine known polyglutamine disorders, SCA3 is a clinically heterogeneous disease, primarily exemplified by progressive ataxia impairing the speech, balance and gait of affected individuals. SCA3 is caused by expansion of a glutamine-encoding tract located at the 5′ end of the penultimate exon (exon 10) of the ATXN3 gene transcript, resulting in conformational changes in ataxin-3 and a toxic gain-of-function. Here, we describe highly efficient removal of the toxic polyglutamine tract of ataxin-3 in vitro by phosphorodiamidate morpholino oligomers (PMOs). Additionally, these PMOs induced a potentially beneficial downregulation of both the expanded iv and non-expanded protein isoforms. As SCA3 has a typical age of onset in the fourth decade, the observed downregulation could delay age of onset by reducing the amounts of the toxic aggregates. Although we induce downregulation of both isoforms, we believe that the proportion of the truncated protein may be sufficient for overall function of ataxin-3, as some studies have shown ataxin-3 protein to be partially dispensable. Recently, several in vitro and in vivo studies have found that targeted knockdown of transcription elongation factors SUPT4H1, and to a lesser extent SUPT5H, can reduce aggregation of expanded transcripts and protein, and alleviate the disease phenotype in animal models of various expansion diseases. We therefore sought to investigate in vitro, the potential of AO-mediated SUPT4H1 downregulation as a therapeutic strategy. We found that our AOs were able to significantly downregulate SUPT4H1, with minimal changes to the rest of the transcriptome. We then assessed whether this downregulation of SUPT4H1 lead to a reduction in expanded ATXN3 mRNA and/or ATXN3 protein expression, however, unfortunately in the models available and under the current study, no modification to the ATXN3 transcript or protein was observed. This lack of effect may be due to the relatively short, expanded repeat lengths in SCA3 cell lines, and we therefore recommend that future studies assess genes with larger expansions, such as the 100-1000s repeat tracts frequently observed in myotonic dystrophy type 1 (DMPK). In order to create an efficient screening process for finding clinic-ready AOs, it is important to have a detailed understanding of the principles of AO design. We therefore present a comprehensive rationale for efficiently design and in vitro delivery of splice modulating AOs. These approaches and recommendations provide a streamlined methodology for any researcher developing AO therapeutics. The results presented in this thesis indicate that morpholino oligomers will provide superior benefit for the treatment of spinocerebellar ataxia type 3, without the toxic effects that result from other antisense oligomer chemistries. Additionally, AO-induced SUPT4H1 knockdown may yet demonstrate therapeutic v application for a multitude of expansion diseases, pending further investigation into the whole transcriptome effects and in vivo efficacy of this strategy. Lastly, our guidelines for therapeutic AO development should aid other researchers in creating the most efficacious and safe AOs for clinical trials. The work presented in this thesis contributes to the greater body of knowledge about the applications of AOs, as well as the need for reliable and systematic protocols in AO research and interpretation. With ongoing collaboration from our industry partners, Sarepta Therapeutics, there is hope that the work presented here will provide a solid foundation for further research into AO therapeutics for the treatment of neurodegenerative expansion diseases

The behaviors and experiences of the community pharmacy team on the provision of multi-compartment compliance aids.

Background - Multi-compartment compliance aids (MCAs) are repackaging systems for solid dosage form medicines. Acknowledging the lack of evidence that MCAs improve adherence or clinical outcomes, the Royal Pharmaceutical Society has expressed concern that MCAs have 'become regarded as a panacea for medicines use'. Objectives - To determine the behaviors and experiences of the community pharmacy team around MCA provision. Methods - A cross-sectional survey was conducted in 26 community pharmacies in the north east of Scotland. Survey items were grouped into: current activities in the provision of MCAs; potential influences on these activities; reports of patient experiences; and demographics. Data were analysed using descriptive and inferential statistics, and content analysis of responses to open questions. Principal component analysis (PCA) was performed on the items of potential influences on activities. Results - Data were collected from 136 community team members (median 4, range1-10 per pharmacy; 32.3% pharmacists). All were involved in some aspect of MCA provision and within the same pharmacy, several different staff positions were commonly involved in the same activity. PCA gave seven components; the lowest scores were obtained for the component of 'others expecting me to provide MCAs'. Participants agreed that GPs, patients and their families, and carers expected them to provide MCAs. Positive experiences of MCA provision were in themes of promoting patient adherence, reducing patient stress and enhancing patient monitoring. Further negative experiences were in of lack of shared patient decision making, worsening adherence and generation of medicines waste, and dealing with changing medicines. MCAs were not always considered to be the most appropriate solution. Conclusion - While community pharmacy teams value MCAs, there may be issues around staff assignment to particular roles, expectations from others and reports of negative patient experiences. A systematic approach to MCA provision and monitoring involving the multidisciplinary health and social care team is warranted

Why health visiting? Examining the potential public health benefits from health visiting practice within a universal service: A narrative review of the literature

INTRODUCTION: There is increasing international interest in universal, health promoting services for pregnancy and the first three years of life and the concept of proportionate universalism. Drawing on a narrative review of literature, this paper explores mechanisms by which such services might contribute to health improvement and reducing health inequalities. OBJECTIVES: Through a narrative review of empirical literature, to identify: DESIGN: The paper draws upon a scoping study and narrative review. REVIEW METHODS: We used three complementary approaches to search the widely dispersed literature: Our key inclusion criterion was information about health visiting practice. We included empirical papers from United Kingdom (UK) from 2004 to February 2012 and older seminal papers identified in search (3), identifying a total of 348 papers for inclusion. A thematic content analysis compared the older (up to 2003) with more recent research (2004 onwards). RESULTS: The analysis revealed health visiting practice as potentially characterized by a particular 'orientation to practice.' This embodied the values, skills and attitudes needed to deliver universal health visiting services through salutogenesis (health creation), person-centredness (human valuing) and viewing the person in situation (human ecology). Research about health visiting actions focuses on home visiting, needs assessment and parent-health visitor relationships. The detailed description of health visitors' skills, attitudes, values, and their application in practice, provides an explanation of how universal provision can potentially help to promote health and shift the social gradient of health inequalities. CONCLUSIONS: Identification of needs across an undifferentiated, universal caseload, combined with an outreach style that enhances uptake of needed services and appropriate health or parenting information, creates opportunities for parents who may otherwise have remained unaware of, or unwilling to engage with such provision. There is a lack of evaluative research about health visiting practice, service organization or universal health visiting as potential mechanisms for promoting health and reducing health inequalities. This paper offers a potential foundation for such research in future

Integrated methylome and phenome study of the circulating proteome reveals markers pertinent to brain health

Characterising associations between the methylome, proteome and phenome may provide insight into biological pathways governing brain health. Here, we report an integrated DNA methylation and phenotypic study of the circulating proteome in relation to brain health. Methylome-wide association studies of 4058 plasma proteins are performed (N = 774), identifying 2928 CpG-protein associations after adjustment for multiple testing. These are independent of known genetic protein quantitative trait loci (pQTLs) and common lifestyle effects. Phenome-wide association studies of each protein are then performed in relation to 15 neurological traits (N = 1,065), identifying 405 associations between the levels of 191 proteins and cognitive scores, brain imaging measures or APOE e4 status. We uncover 35 previously unreported DNA methylation signatures for 17 protein markers of brain health. The epigenetic and proteomic markers we identify are pertinent to understanding and stratifying brain health

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Genomic Relationships, Novel Loci, and Pleiotropic Mechanisms across Eight Psychiatric Disorders

Genetic influences on psychiatric disorders transcend diagnostic boundaries, suggesting substantial pleiotropy of contributing loci. However, the nature and mechanisms of these pleiotropic effects remain unclear. We performed analyses of 232,964 cases and 494,162 controls from genome-wide studies of anorexia nervosa, attention-deficit/hyper-activity disorder, autism spectrum disorder, bipolar disorder, major depression, obsessive-compulsive disorder, schizophrenia, and Tourette syndrome. Genetic correlation analyses revealed a meaningful structure within the eight disorders, identifying three groups of inter-related disorders. Meta-analysis across these eight disorders detected 109 loci associated with at least two psychiatric disorders, including 23 loci with pleiotropic effects on four or more disorders and 11 loci with antagonistic effects on multiple disorders. The pleiotropic loci are located within genes that show heightened expression in the brain throughout the lifespan, beginning prenatally in the second trimester, and play prominent roles in neurodevelopmental processes. These findings have important implications for psychiatric nosology, drug development, and risk prediction.Peer reviewe

Determinants of recovery from post-COVID-19 dyspnoea: analysis of UK prospective cohorts of hospitalised COVID-19 patients and community-based controls

Background The risk factors for recovery from COVID-19 dyspnoea are poorly understood. We investigated determinants of recovery from dyspnoea in adults with COVID-19 and compared these to determinants of recovery from non-COVID-19 dyspnoea. Methods We used data from two prospective cohort studies: PHOSP-COVID (patients hospitalised between March 2020 and April 2021 with COVID-19) and COVIDENCE UK (community cohort studied over the same time period). PHOSP-COVID data were collected during hospitalisation and at 5-month and 1-year follow-up visits. COVIDENCE UK data were obtained through baseline and monthly online questionnaires. Dyspnoea was measured in both cohorts with the Medical Research Council Dyspnoea Scale. We used multivariable logistic regression to identify determinants associated with a reduction in dyspnoea between 5-month and 1-year follow-up. Findings We included 990 PHOSP-COVID and 3309 COVIDENCE UK participants. We observed higher odds of improvement between 5-month and 1-year follow-up among PHOSP-COVID participants who were younger (odds ratio 1.02 per year, 95% CI 1.01–1.03), male (1.54, 1.16–2.04), neither obese nor severely obese (1.82, 1.06–3.13 and 4.19, 2.14–8.19, respectively), had no pre-existing anxiety or depression (1.56, 1.09–2.22) or cardiovascular disease (1.33, 1.00–1.79), and shorter hospital admission (1.01 per day, 1.00–1.02). Similar associations were found in those recovering from non-COVID-19 dyspnoea, excluding age (and length of hospital admission). Interpretation Factors associated with dyspnoea recovery at 1-year post-discharge among patients hospitalised with COVID-19 were similar to those among community controls without COVID-19. Funding PHOSP-COVID is supported by a grant from the MRC-UK Research and Innovation and the Department of Health and Social Care through the National Institute for Health Research (NIHR) rapid response panel to tackle COVID-19. The views expressed in the publication are those of the author(s) and not necessarily those of the National Health Service (NHS), the NIHR or the Department of Health and Social Care. COVIDENCE UK is supported by the UK Research and Innovation, the National Institute for Health Research, and Barts Charity. The views expressed are those of the authors and not necessarily those of the funders

Treatment of anorexia nervosa:A multimethod investigation translating experimental neuroscience into clinical practice

Background Anorexia nervosa (AN) is a severe psychiatric condition and evidence on how to best treat it is limited. Objectives This programme consists of seven integrated work packages (WPs) and aims to develop and test disseminable and cost-effective treatments to optimise management for people with AN across all stages of illness. Methods WP1a used surveys, focus groups and a pre–post trial to develop and evaluate a training programme for school staff on eating disorders (EDs). WP1b used a randomised controlled trial (RCT) [International Standard Randomised Controlled Trial Number (ISRCTN) 42594993] to evaluate a prevention programme for EDs in schools. WP2a evaluated an inpatient treatment for AN using case reports, interviews and a quasi-experimental trial. WP2b used a RCT (ISRCTN67720902) to evaluate two outpatient psychological therapies for AN. WP3 used a RCT (ISRCTN06149665) to evaluate an intervention for carers of inpatients with AN. WP4 used actimetry, self-report and endocrine assessment to examine physical activity (PA) in AN. WP5 conducted a RCT (ISRCTN18274621) of an e-mail-guided relapse prevention programme for inpatients with AN. WP6 analysed cohort data to examine the effects of maternal EDs on fertility and their children’s diet and growth. WP7a examined clinical case notes to explore how access to specialist ED services affects care pathways and user experiences. Finally, WP7b used data from this programme and the British Cohort Study (1970) to identify the costs of services used by people with AN and to estimate annual costs of AN for England. Results WP1a: a brief training programme improved knowledge, attitudes and confidence of school staff in managing EDs in school. WP1b: a teacher-delivered intervention was feasible and improved risk factors for EDs in adolescent girls. WP2a: both psychological therapies improved outcomes in outpatients with AN similarly, but patients preferred one of the treatments. WP2b: the inpatient treatment (Cognitive Remediation and Emotional Skills Training) was acceptable with perceived benefits by patients, but showed no benefits compared with treatment as usual (TAU). WP3: compared with TAU, the carer intervention improved a range of patient and carer outcomes, including carer burden and patient ED symptomatology. WP4: drive to exercise is tied to ED pathology and a desire to improve mood in AN patients. PA was not increased in these patients. WP5: compared with TAU, the e-mail-guided relapse prevention programme resulted in higher body mass index and lower distress in patients at 12 months after discharge. WP6: women with an ED had impaired fertility and their children had altered dietary and growth patterns compared with the children of women without an ED. WP7a: direct access to specialist ED services was associated with higher referral rates, lower admission rates, greater consistency of care and user satisfaction. WP7b: the annual costs of AN in England are estimated at between £45M and £230M for 2011. Conclusions This programme has produced evidence to inform future intervention development and has developed interventions that can be disseminated to improve outcomes for individuals with AN. Directions for future research include RCTs with longer-term outcomes and sufficient power to examine mediators and moderators of change. Trial registration Current Controlled Trials ISRCTN42594993, ISRCTN67720902, ISRCTN06149665 and ISRCTN18274621

Para-infectious brain injury in COVID-19 persists at follow-up despite attenuated cytokine and autoantibody responses

To understand neurological complications of COVID-19 better both acutely and for recovery, we measured markers of brain injury, inflammatory mediators, and autoantibodies in 203 hospitalised participants; 111 with acute sera (1–11 days post-admission) and 92 convalescent sera (56 with COVID-19-associated neurological diagnoses). Here we show that compared to 60 uninfected controls, tTau, GFAP, NfL, and UCH-L1 are increased with COVID-19 infection at acute timepoints and NfL and GFAP are significantly higher in participants with neurological complications. Inflammatory mediators (IL-6, IL-12p40, HGF, M-CSF, CCL2, and IL-1RA) are associated with both altered consciousness and markers of brain injury. Autoantibodies are more common in COVID-19 than controls and some (including against MYL7, UCH-L1, and GRIN3B) are more frequent with altered consciousness. Additionally, convalescent participants with neurological complications show elevated GFAP and NfL, unrelated to attenuated systemic inflammatory mediators and to autoantibody responses. Overall, neurological complications of COVID-19 are associated with evidence of neuroglial injury in both acute and late disease and these correlate with dysregulated innate and adaptive immune responses acutely