Search

Abstract Computational analysis of infant movement has significant potential to reveal markers of developmental health. We report two studies employing dynamic analyses of motor kinematics and motor behaviours, which characterise movement at two levels, in 9-month-old infants. We investigate the effect of preterm birth ( 37 weeks of gestation), infants born preterm showed greater permutation entropy in their left ankle and torso movements, but not in right ankle or wrist movements. We did not find effects of preterm birth or emotional context on micro-level behavioural dynamics. Our methodology and findings inform future work using multiscale entropy to study infant development. Dynamic analysis of behaviour is a relatively young field, and applications to emotional self-regulation requires further methodological development.

Abstract Public engagement with research (PEwR) has become increasingly integral to research practices. This paper explores the process and outcomes of a collaborative effort to address the ethical implications of PEwR activities and develop tools to navigate them within the context of a University Medical School. The activities this paper reflects on aimed to establish boundaries between research data collection and PEwR activities, support colleagues in identifying the ethical considerations relevant to their planned activities, and build confidence and capacity among staff to conduct PEwR projects. The development process involved the creation of a taxonomy outlining key terms used in PEwR work, a self-assessment tool to evaluate the need for formal ethical review, and a code of conduct for ethical PEwR. These tools were refined through iterative discussions and feedback from stakeholders, resulting in practical guidance for researchers navigating the ethical complexities of PEwR. Additionally, reflective prompts were developed to guide researchers in planning and conducting engagement activities, addressing a crucial aspect often overlooked in formal ethical review processes. The paper reflects on the broader regulatory landscape and the limitations of existing approval and governance processes, and prompts critical reflection on the compatibility of formal approval processes with the ethos of PEwR. Overall, the paper offers insights and practical guidance for researchers and institutions grappling with ethical considerations in PEwR, contributing to the ongoing conversation surrounding responsible research practices.

Infant attachment is an antecedent of later socioemotional abilities, which can be adversely affected by preterm birth. The structural integrity of amygdalae and hippocampi may subserve attachment in infancy. We aimed to investigate associations between neonatal amygdalae and hippocampi structure and their whole-brain connections and attachment behaviours at nine months of age in a sample of infants enriched for preterm birth. In 133 neonates (median gestational age 32 weeks, range 22.14–42.14), we calculated measures of amygdala and hippocampal structure (volume, fractional anisotropy, mean diffusivity, neurite dispersion index, orientation dispersion index) and structural connectivity, and coded attachment behaviours (distress, fretfulness, attentiveness to caregiver) from responses to the Still-Face Paradigm at nine months. After multiple comparisons correction, there were no significant associations between neonatal amygdala or hippocampal structure and structural connectivity and attachment behaviours: standardised β values − 0.23 to 0.18, adjusted p-values > 0.40. Findings indicate that the neural basis of infant attachment in term and preterm infants is not contingent on the structure or connectivity of the amygdalae and hippocampi in the neonatal period, which implies that it is more widely distributed in early life and or that network specialisation takes place in the months after hospital discharge.

Abstract Background Improving the ways in which routinely-collected mental health data are shared could facilitate substantial advances in research and treatment. However, this process should only be undertaken in partnership with those who provide such data. Despite relatively widespread investigation of public perspectives on health data sharing more generally, there is a lack of research on the views of people with mental illness. Methods Twelve people with lived experience of mental illness took part in semi-structured interviews via online video software. Participants had experience of a broad range of mental health conditions including anxiety, depression, schizophrenia, eating disorders and addiction. Interview questions sought to establish how participants felt about the use of routinely-collected health data for research purposes, covering different types of health data, what health data should be used for, and any concerns around its use. Results Thematic analysis identified four overarching themes: benefits of sharing mental health data, concerns about sharing mental health data, safeguards, and data types. Participants were clear that health data sharing should facilitate improved scientific knowledge and better treatments for mental illness. There were concerns that data misuse could become another way in which individuals and society discriminate against people with mental illness, for example through insurance premiums or employment decisions. Despite this there was a generally positive attitude to sharing mental health data as long as appropriate safeguards were in place. Conclusions There was notable strength of feeling across participants that more should be done to reduce the suffering caused by mental illness, and that this could be partly facilitated by well-managed sharing of health data. The mental health research community could build on this generally positive attitude to mental health data sharing by following rigorous best practice tailored to the specific concerns of people with mental illness.

Abstract Antisense oligomers (AOs) are increasingly being used to modulate RNA splicing in live cells, both for research and for the development of therapeutics. While the most common intended effect of these AOs is to induce skipping of whole exons, rare 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.

Abstract Background Restricted and repetitive behaviours vary greatly across the autism spectrum, and although not all are problematic some can cause distress and interfere with learning and social opportunities. We have, alongside parents, developed a parent group based intervention for families of young children with autism, which aims to offer support to parents and carers; helping them to recognise, understand and learn how to respond to their child’s challenging restricted repetitive behaviours. Methods The study is a clinical and cost-effectiveness, multi-site randomised controlled trial of the Managing Repetitive Behaviours (MRB) parent group intervention versus a psychoeducation parent group Learning About Autism (LAA) (n = 250; 125 intervention/125 psychoeducation; ~ 83/site) for parents of young children aged 3–9 years 11 months with a diagnosis of autism. All analyses will be done under intention-to-treat principle. The primary outcome at 24 weeks will use generalised estimating equation (GEE) to compare proportion of children with improved RRB between the MRB group and the LAA group. The GEE model will account for the clustering of children by parent groups using exchangeable working correlation. All secondary outcomes will be analysed in a similar way using appropriate distribution and link function. The economic evaluation will be conducted from the perspective of both NHS costs and family access to local community services. A ‘within trial’ cost-effectiveness analysis with results reported as the incremental cost per additional child achieving at least the target improvement in CGI-I scale at 24 weeks. Discussion This is an efficacy trial to investigate the clinical and cost-effectiveness of a parent group based intervention designed to help parents understand and manage their child’s challenging RRB. If found to be effective, this intervention has the potential to improve the well-being of children and their families, reduce parental stress, greatly enhance community participation and potential for learning, and improve longer-term outcomes. Trial registration Trial ID: ISRCTN15550611 Date registered: 07/08/2018. Sponsor and Monitor: Cumbria, Northumberland, Tyne and Wear NHS Foundation Trust R&D Manager Lyndsey Dixon, Address: St Nicholas Hospital, Jubliee Road, Gosforth, Newcastle upon Tyne NE3 3XT, lyndsey.dixon@cntw.nhs.uk , Tel: 0191 246 7222

Introduction - Despite extensive debate surrounding mental health services in the UK, there is little empirical evidence regarding the views of those who use them. We therefore used data collected as part of a wider survey to examine satisfaction amongst those seeking treatment from mental and physical health services. Methods – An online survey designed with input from people with experience of mental illness was used to measure satisfaction with NHS mental and physical health services at first contact and in the previous 12 months. Results – A total of 2187 people responded. During the 12 months prior to the survey, 526 respondents had sought mental health care and 1379 had sought physical health care. Participants were significantly more satisfied with their most recent contact with mental health services (48.1% very/satisfied) than with their first contact (38.2% very/satisfied). More than 1 in 10 respondents who sought mental health care (11.4%) stated that they received no treatment/support from the NHS, compared to approximately 1 in 20 respondents who sought physical health care (4.6%). Of those who received the mental health treatment they requested (n = 424), most were satisfied or very satisfied with their care (54.7%), although this was lower than the corresponding figure (77.9%) for satisfaction with physical health care received (n = 1190). Conclusion –There was evidence that mental health services are satisfactory for a slim majority of users, but people were generally more satisfied with NHS physical health care. This survey was conducted in the year prior to the coronavirus disease 2019 pandemic. Future research could examine what influences satisfaction with care and whether this picture has changed following the emergence of the pandemic and consequent impact on health service delivery and daily life.

Oligonucleotides and nucleic acid analogues that alter gene expression are now showing therapeutic promise in human disease. Whilst the modification of synthetic nucleic acids to protect against nuclease degradation and to influence drug function is common practice, such modifications may also confer unexpected physicochemical and biological properties. Gapmer mixed-modified and DNA oligonucleotides on a phosphorothioate backbone can bind non-specifically to intracellular proteins to form a variety of toxic inclusions, driven by the phosphorothioate linkages, but also influenced by the oligonucleotide sequence. Recently, the non-antisense or other off-target effects of 2′ O- fully modified phosphorothioate linkage oligonucleotides are becoming better understood. Here, we report chemistry-specific effects of oligonucleotides composed of modified or unmodified bases, with phosphorothioate linkages, on subnuclear organelles and show altered distribution of nuclear proteins, the appearance of highly stable and strikingly structured nuclear inclusions, and disturbed RNA processing in primary human fibroblasts and other cultured cells. Phosphodiester, phosphorodiamidate morpholino oligomers, and annealed complimentary phosphorothioate oligomer duplexes elicited no such consequences. Disruption of subnuclear structures and proteins elicit severe phenotypic disturbances, revealed by transcriptomic analysis of transfected fibroblasts exhibiting such disruption. Our data add to the growing body of evidence of off-target effects of some phosphorothioate nucleic acid drugs in primary cells and suggest alternative approaches to mitigate these effects.

Receiving a diagnosis of autism in adulthood can be a life changing event, impacting identity, relationships, and mental health. A lack of post-diagnostic support has been highlighted by autistic adults, their allies, clinicians, and service providers. It can be a source of distress for autistic adults, reinforcing feelings of social isolation and rejection. Peer support could be a cost-effective, flexible, and sustainable model to provide community-based support for autistic adults. However, there is little research on the value of peer support, despite calls from the autistic community. This qualitative study explored autistic experiences and needs post-diagnosis, identifying specific ways that peer support may benefit them, and exploring the limitations of peer support. Twelve autistic adults who had all received an autism diagnosis in adulthood completed a semi-structured interview focussing on the diagnostic experience, post-diagnostic support needed and provided, engagement with the autistic community, and post-diagnostic peer support. Thematic analysis of interview transcripts resulted in four themes: (1) Mismatch in support needed and provided; (2) Community connection; (3) Flexible and personalised support; and (4) Sustainability. Participants indicated that peer support may be a useful mechanism to support autistic adults’ post-diagnosis and offers unique opportunities not available through other support channels. Though informal peer support exists, it could be more sustainable and effective if well-supported and funded.

Introduction: Severity and disease progression in people with Cystic Fibrosis (CF) is typically dependent on their genotype. One potential therapeutic strategy for people with specific mutations is exon skipping with antisense oligonucleotides (AO). CFTR exon 9 is an in-frame exon and hence the exclusion of this exon would excise only 31 amino acids but not alter the reading frame of the remaining mRNA. Splice mutations 1209 + 1 G > C and 1209 + 2 T > G were documented to cause CFTR exon 9 skipping and these variants were reported to manifest as a milder CF disease, therefore exon 9 skipping could be beneficial for people with class I mutations that affect exon 9 such as p.Trp401X. While the impact of exon 9 skipping on gene expression and cellular pathways can be studied in cells in vitro, trace amount of full-length normal or mutated material could confound the evaluation. To overcome this limitation, the impact of CFTR exon 9 skipping on disease phenotype and severity is more effectively evaluated in a small animal model. It was hypothesised that antisense oligonucleotide-mediated skipping this particular exon could result in a “mild mouse CF phenotype”.Methods:Cftr exon 9 deleted mice were generated using homologous recombination. Survival of homozygous (CftrΔ9/Δ9) and heterozygous (CftrΔ9/+) mice was compared to that of other CF mouse models, and lung and intestinal organ histology examined for any pathologies. Primary airway epithelial cells (pAECs) were harvested from CftrΔ9/Δ9 mice and cultured at the Air Liquid Interface for CFTR functional assessment using Ussing Chamber analysis.Results: A CftrΔ9/Δ9 mouse model presented with intestinal obstructions, and at time of weaning (21 days). CftrΔ9/Δ9 mice had a survival rate of 83% that dropped to 38% by day 50. Histological sections of the small intestine from CftrΔ9/Δ9 mice showed more goblet cells and mucus accumulation than samples from the CftrΔ9/+ littermates. Airway epithelial cell cultures established from CftrΔ9/Δ9 mice were not responsive to forskolin stimulation.Summary: The effect of Cftr exon 9 deletion on Cftr function was assessed and it was determined that the encoded Cftr isoform did not result in a milder “mouse CF disease phenotype,” suggesting that Cftr exon 9 is not dispensable, although further investigation in human CF pAECs would be required to confirm this observation.

ATP Binding Cassette Subfamily A Member 3 (ABCA-3) is a lipid transporter protein highly expressed in type-II alveolar (AT-II) cells. Mutations in ABCA3 can result in severe respiratory disease in infants and children. To study ABCA-3 deficiency in vitro, primary AT-II cells would be the cell culture of choice although sample accessibility is limited. Our aim was to investigate the suitability of primary nasal epithelial cells, as a surrogate culture model for AT-II cells, to study ABCA-3 deficiency. Expression of ABCA3, and surfactant protein genes, SFTPB and SFTPC, was detected in primary nasal epithelial cells but at a significantly lower level than in AT-II cells. ABCA-3, SP-B, and SP-C were detected by immunofluorescence microscopy in primary nasal epithelial cells. However, SP-B and SP-C were undetectable in primary nasal epithelial cells using western blotting. Structurally imperfect lamellar bodies were observed in primary nasal epithelial cells using transmission electron microscopy. Functional assessment of the ABCA-3 protein demonstrated that higher concentrations of doxorubicin reduced cell viability in ABCA-3 deficient nasal epithelial cells compared to controls in an assay-dependent manner. Our results indicate that there may be a role for primary nasal epithelial cell cultures to model ABCA-3 deficiency in vitro, although additional cell culture models that more effectively recapitulate the AT-II phenotype may be required.

Abstract Background Cryptic exons are typically characterised as deleterious splicing aberrations caused by deep intronic mutations. However, low‐level splicing of cryptic exons is sometimes observed in the absence of any pathogenic mutation. Five recent reports have described how low‐level splicing of cryptic exons can be modulated by common single‐nucleotide polymorphisms (SNPs), resulting in phenotypic differences amongst different genotypes. Methods We sought to investigate whether additional ‘SNPtic’ exons may exist, and whether these could provide an explanatory mechanism for some of the genotype–phenotype correlations revealed by genome‐wide association studies. We thoroughly searched the literature for reported cryptic exons, cross‐referenced their genomic coordinates against the dbSNP database of common SNPs, then screened out SNPs with no reported phenotype associations. Results This method discovered five probable SNPtic exons in the genes APC, FGB, GHRL, MYPBC3 and OTC. For four of these five exons, we observed that the phenotype associated with the SNP was compatible with the predicted splicing effect of the nucleotide change, whilst the fifth (in GHRL) likely had a more complex splice‐switching effect. Conclusion Application of our search methods could augment the knowledge value of future cryptic exon reports and aid in generating better hypotheses for genome‐wide association studies.

Understanding pre-mRNA splicing is crucial to accurately diagnosing and treating genetic diseases. However, mutations that alter splicing can exert highly diverse effects. Of all the known types of splicing mutations, perhaps the rarest and most difficult to predict are those that activate pseudoexons, sometimes also called cryptic exons. Unlike other splicing mutations that either destroy or redirect existing splice events, pseudoexon mutations appear to create entirely new exons within introns. Since exon definition in vertebrates requires coordinated arrangements of numerous RNA motifs, one might expect that pseudoexons would only arise when rearrangements of intronic DNA create novel exons by chance. Surprisingly, although such mutations do occur, a far more common cause of pseudoexons is deep-intronic single nucleotide variants, raising the question of why these latent exon-like tracts near the mutation sites have not already been purged from the genome by the evolutionary advantage of more efficient splicing. Possible answers may lie in deep intronic splicing processes such as recursive splicing or poison exon splicing. Because these processes utilize intronic motifs that benignly engage with the spliceosome, the regions involved may be more susceptible to exonization than other intronic regions would be. We speculated that a comprehensive study of reported pseudoexons might detect alignments with known deep intronic splice sites and could also permit the characterisation of novel pseudoexon categories. In this report, we present and analyse a catalogue of over 400 published pseudoexon splice events. In addition to confirming prior observations of the most common pseudoexon mutation types, the size of this catalogue also enabled us to suggest new categories for some of the rarer types of pseudoexon mutation. By comparing our catalogue against published datasets of non-canonical splice events, we also found that 15.7% of pseudoexons exhibit some splicing activity at one or both of their splice sites in non-mutant cells. Importantly, this included seven examples of experimentally confirmed recursive splice sites, confirming for the first time a long-suspected link between these two splicing phenomena. These findings have the potential to improve the fidelity of genetic diagnostics and reveal new targets for splice-modulating therapies.

Background: Autistic children show reduced attentional preferences to social stimuli early in development, and these differences have consequences on a range of social domains. One factor that could influence development in those processes is bilingualism. Parents and practitioners frequently voice unfounded concerns that bilingualism could cause delays in autistic children, yet there is little evidence to dispute this idea. While there are studies focusing on the impact of bilingualism on cognition in autistic children, no research has focused on the relationship between bilingualism and social attention. Aims: This study therefore investigated the impact of bilingual exposure on social attention in autistic (n = 33) and neurotypical children (n = 42) aged 6–13 years. Rather than a monolingual/bilingual comparison, participants had varying degrees of bilingual exposure, and exposure was treated as a continuous variable. Participants completed an eye-tracking task measuring visual attention to interacting versus non-interacting human figures. Results: Bilingual exposure did not affect dwell time to interacting or non-interacting figures for the neurotypical or autistic groups. However, there was a three-way interaction between diagnosis, figure type and vocabulary scores on dwell time. Conclusions: Higher vocabulary scores in neurotypical participants was associated with significantly less dwell time to non-interacting stimuli. This is the first study to assess the effects of bilingualism on social attention; here, concerns of bilingualism are not upheld.

Background: Children born preterm (before 37 weeks of gestation) are at risk for several adverse childhood outcomes. Parent priorities for research into these outcomes, and preferences for receiving information about these risks, have not previously been established. Here we report the results of an online survey designed to understand parent priorities for research and their preferences for receiving information about childhood outcomes. Methods: An online survey was circulated through social media and was completed by 148 parents of preterm children between the ages of 0 and 12 years from around the United Kingdom (UK). Survey questions were in the form of rating scale, multiple choice, ranking or open-ended free text questions. Descriptive analysis was applied to the quantitative data. Illustrative quotes were extracted from the qualitative free text data and a subset of these questions were analysed using framework analysis. Results: Parent priorities for research centre around identification of factors which can protect against or improve adverse cognitive or developmental outcomes. The majority of parents would prefer for communication to begin within the first year of the child’s life. Parents reported a knowledge gap among health visitors, early years educators and schools. Conclusions: In order to align with parent preferences, research should prioritise identification of protective factors and the development of effective interventions to improve outcomes. Training for health visitors and educational professionals could improve the experiences of families and children.

Bilingualism is a valuable tool that enriches and facilitates cultural, social and lived experiences for autistic and non-autistic people alike. Research consistently finds no negative effects of bilingualism and highlights the potential for positive effects across cognitive and socio-cultural domains for autistic and non-autistic children. Yet parents of autistic children remain concerned that bilingualism will cause delays in both cognitive and language development and are still frequently advised by practitioners to raise their child monolingually. Evidently, findings from research are not reflected in practice or subsequent advice, and it is essential to identify ways to ensure equal access to additional language learning. We briefly summarise the existing literature on bilingualism and autism, considering perspectives from the bilingual autistic community, and experimental research. We identify the most pertinent barriers to participation for autistic bilingual children in terms of familial, clinical and educational perspectives. We propose novel solutions to promote additional language learning and suggest changes to practice that will contribute to an evidence base for families and practitioners. This commentary makes innovative recommendations at both the individual and societal level to ensure that autistic bilingual people have equal rights and opportunities to language learning and are optimally supported in accessing them.

Stargardt disease (STGD1) is the most common inherited retinal dystrophy and ABCA4 c.546-–10 T>C is the most commonly reported splice mutation. Here, we generated and characterized two induced pluripotent stem cell (iPSC) lines from a STGD1 patient with compound heterozygous mutations in ABCA4 (c.[5461-10 T > C;5603A > T];[4163 T > C;455G > A]). Episomal vectors containing OCT4, SOX2, KLF4, L-MYC, LIN28 and mp53DD were employed to conduct the reprogramming of patient-derived fibroblasts. Both lines had a normal karyotype, displayed iPSC morphology, expressed pluripotency markers and showed trilineage differentiation potential. These lines can provide a powerful platform for further investigating the pathophysiological consequences of mutations in ABCA4.

Mutations in ABCA4 gene are causative for autosomal recessive Stargardt disease (STGD1), the most common inherited retinal dystrophy. Here, we report the generation of an induced pluripotent stem cell (iPSC) line from a STGD1 patient carrying biallelic c.[5461–10T>C;5603A>T];[6077T>C] mutations in the ABCA4 gene. Episomes carrying OCT4, SOX2, KLF4, L-MYC, LIN28 and mp53DD were employed for the reprogramming of patient-derived fibroblasts. This iPSC line expressed comparable pluripotency markers as in a commercially available human iPSC line, displayed normal karyotype and potential for trilineage differentiation, and were negative for both reprogramming episomes and mycoplasma test.

Background: Mental health research is commonly affected by difficulties in recruiting and retaining participants, resulting in findings which are based on a sub-sample of those actually living with mental illness. Increasing the use of Big Data for mental health research, especially routinely-collected data, could improve this situation. However, steps to facilitate this must be enacted in collaboration with those who would provide the data - people with mental health conditions.Methods: We used the Delphi method to create a best practice checklist for mental health data science. Twenty participants with both expertise in data science and personal experience of mental illness worked together over three phases. In Phase 1, participants rated a list of 63 statements and added any statements or topics that were missing. Statements receiving a mean score of 5 or more (out of 7) were retained. These were then combined with the results of a rapid thematic analysis of participants' comments to produce a 14-item draft checklist, with each item split into two components: best practice now and best practice in the future. In Phase 2, participants indicated whether or not each item should remain in the checklist, and items that scored more than 50% endorsement were retained. In Phase 3 participants rated their satisfaction with the final checklist.Results: The final checklist was made up of 14 “best practice” items, with each item covering best practice now and best practice in the future. At the end of the three phases, 85% of participants were (very) satisfied with the two best practice checklists, with no participants expressing dissatisfaction.Conclusions: Increased stakeholder involvement is essential at every stage of mental health data science. The checklist produced through this work represents the views of people with experience of mental illness, and it is hoped that it will be used to facilitate trustworthy and innovative research which is inclusive of a wider range of individuals.

ObjectiveThere is a critical need to establish genetic markers that explain the complex phenotypes and pathogenicity of ALS. This study identified a polymorphism in the Stathmin-2 gene and investigated its association with sporadic ALS (sALS) disease risk, age-of onset and survival duration.MethodsThe candidate CA repeat was systematically analyzed using PCR, Sanger sequencing and high throughput capillary separation for genotyping. Stathmin-2 expression was investigated using RT-PCR in patient olfactory neurosphere-derived (ONS) cells and RNA sequencing in laser-captured spinal motor neurons.ResultsIn a case-control analysis of a combined North American sALS cohort (n = 321) and population control group (n = 332), long/long CA genotypes were significantly associated with disease risk (p = 0.042), and most strongly when one allele was a 24 CA repeat (p = 0.0023). In addition, longer CA allele length was associated with earlier age-of-onset (p = 0.039), and shorter survival duration in bulbar-onset cases (p = 0.006). In an Australian longitudinal sALS cohort (n = 67), ALS functional rating scale scores were significantly lower in carriers of the long/long genotype (p = 0.034). Stathmin-2 mRNA expression was reduced in sporadic patient ONS cells. Additionally, sALS patients and controls exhibited variable expression of Stathmin-2 mRNA according to CA genotype in laser-captured spinal motor neurons.ConclusionsWe report a novel non-coding CA repeat in Stathmin-2 which is associated with sALS disease risk and has disease modifying effects. The potential value of this variant as a disease marker and tool for cohort enrichment in clinical trials warrants further investigation.

Mutations in the USH2A gene are the most common cause of Usher syndrome and autosomal recessive non-syndromic retinitis pigmentosa. Here, we describe the generation of three induced pluripotent stem cell lines from dermal fibroblasts derived from a patient carrying biallelic c.949C > A and c.1256G > T variants in the USH2A gene, using episomal reprogramming plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28, mir302/367 and shRNA targeting TP53. All three lines expressed pluripotency markers, displayed unaltered karyotypes as well as trilineage differentiation potential, and were negative for reprogramming episomes and mycoplasma.

The Double Empathy Problem suggests that communicative difficulties between autistic and non-autistic people are due to bi-directional differences in communicative style and a reciprocal lack of understanding. If true, there should be increased similarity in interaction style, resulting in higher rapport during interactions between pairs of the same neurotype. Here, we provide two empirical tests of rapport, with data revealing whether self- and observer- rated rapport varies depending on the match or mismatch in autism status within a pair. An additional opportunity afforded by these data is to examine the effect of the autism status of the rater on the perceived rapport between matched and mismatched pairs. In Study 1 72 participants were allocated to one of three dyad conditions: autistic pairs (n = 24), non-autistic pairs (n = 24) and mixed pairs (n = 12 autistic; n = 12 non-autistic). Each participant completed three semi-structured interactions with their partner, rating rapport after each interaction. Non-autistic pairs experienced higher self-rated rapport than mixed and autistic pairs, and autistic pairs experienced higher rapport than mixed pairs. In Study 2 (n = 80) autistic and non-autistic observers rated interactional rapport while watching videoed interactions between autistic pairs, non-autistic pairs, and mixed pairs (n = 18, a subset of participants in Study 1). Mixed pairs were rated significantly lower on rapport than autistic and non-autistic pairs, and autistic pairs were rated more highly for rapport than non-autistic pairs. Both autistic and non-autistic observers show similar patterns in how they rate the rapport of autistic, non-autistic, and mixed pairs. In summary, autistic people experience high interactional rapport when interacting with other autistic people, and this is also detected by external observers. Rather than autistic people experiencing low rapport in all contexts, their rapport ratings are influenced by a mismatch of diagnosis. These findings suggest that autistic people possess a distinct mode of social interaction style, rather than demonstrating social skills deficits. These data are considered in terms of their implications for psychological theories of autism, as well as practical impact on educational and clinical practice.

Background: The UK hosts some of the world’s longest-running longitudinal cohort studies, which make repeated observations of their participants and use these data to explore health outcomes. An alternative method for data collection is record linkage; the linking together of electronic health and administrative records. Applied nationally, this could provide unrivalled opportunities to follow a large number of people in perpetuity. However, public attitudes to the use of data in research are currently unclear. Here we report on an event where we collected attitudes towards recent opportunities and controversies within health data science. Methods: The event was attended by ~250 individuals (cohort members and their guests), who had been invited through the offices of their participating cohort studies. There were a series of presentations describing key research results and the audience participated in 15 multiple-choice questions using interactive voting pads. Results: Our participants showed a high level of trust in researchers (87% scoring them 4/5 or 5/5) and doctors (81%); but less trust in commercial companies (35%). They supported the idea of researchers using information from both neonatal blood spots (Guthrie spots) (97% yes) and from electronic health records (95% yes). Our respondents were willing to wear devices like a ’Fit-bit’ (88% agreed) or take a brain scan that might predict later mental illness (73%). However, they were less willing to take a new drug for research purposes (45%). They were keen to encourage others to take part in research; whether that be offering the opportunity to pregnant mothers (97% agreed) or extending invitations to their own children and grandchildren (98%). Conclusions: Our participants were broadly supportive of research access to data, albeit less supportive when commercial interests were involved. Public engagement events that facilitate two-way interactions can influence and support future research and public engagement efforts.

Abstract Background Deletion–insertion (delins) variants in the retina‐specific ATP‐binding cassette transporter gene, subfamily A, member 4 (ABCA4) accounts for

IntroductionPreterm birth is closely associated with altered brain development and is a leading cause of neurodevelopmental, cognitive and behavioural impairments across the life course. We aimed to investigate neuroanatomic variation and adverse outcomes associated with preterm birth by studying a cohort of preterm infants and controls born at term using brain MRI linked to biosamples and clinical, environmental and neuropsychological data.Methods and analysisTheirworld Edinburgh Birth Cohort is a prospective longitudinal cohort study at the University of Edinburgh. We plan to recruit 300 infants born at

The severe childhood disease spinal muscular atrophy (SMA) arises from the homozygous loss of the survival motor neuron 1 gene (SMN1). A homologous gene potentially encoding an identical protein, SMN2 can partially compensate for the loss of SMN1; however, the exclusion of a critical exon in the coding region during mRNA maturation results in insufficient levels of functional protein. The rate of transcription is known to influence the alternative splicing of gene transcripts, with a fast transcription rate correlating to an increase in alternative splicing. Conversely, a slower transcription rate is more likely to result in the inclusion of all exons in the transcript. Targeting SMN2 with antisense oligonucleotides to influence the processing of terminal exon 8 could be a way to slow transcription and induce the inclusion of exon 7. Interestingly, following oligomer treatment of SMA patient fibroblasts, we observed the inclusion of exon 7, as well as intron 7, in the transcript. Because the normal termination codon is located in exon 7, this exon/intron 7-SMN2 transcript should encode the normal protein and only carry a longer 3′ UTR. Further studies showed the extra 3′ UTR length contained a number of regulatory motifs that modify transcript and protein regulation, leading to translational repression of SMN. Although unlikely to provide therapeutic benefit for SMA patients, this novel technique for gene regulation could provide another avenue for the repression of undesirable gene expression in a variety of other diseases.

Children with neurodevelopmental diagnoses often experience discrimination from their peers at school. This may result from a lack of understanding, and intolerance of differences in their thinking, communication and social interactions. Learning About Neurodiversity at School (LEANS) is a teaching programme designed to educate primary school children about the concept of neurodiversity. The LEANS programme was created by a neurodiverse team, using participatory methods. In the current study, we evaluated whether the wider neurodiverse community endorsed the planned design generated by our participatory approach. Respondents (n = 111) rated their support for key elements of the planned LEANS content, via an online survey. Participants were majority neurodivergent (70%), 98% of whom reported moderate-to-high familiarity with neurodiversity concepts. Over 90% of respondents expressed support for the planned content presented, and 73% of respondents endorsed the draft neurodiversity definition provided. A small number of respondents provided open-ended comments giving further detail on their views. Overall, the LEANS programme plan received a high level of support from this independent, neurodiversity-aware sample -- demonstrating the potential of small-group participatory methods to generate wider community support. The completed resource is now available as a free online download.

Background: The UK hosts some of the world’s longest-running longitudinal cohort studies, who make repeated observations of their participants and use these data to explore health outcomes. An alternative method for data collection is record linkage; the linking together of electronic health and administrative records. Applied nationally, this could provide unrivalled opportunities to follow a large number of people in perpetuity. However, public attitudes to the use of data in research are currently unclear. Here we report on an event where we collected attitudes towards recent opportunities and controversies within health data science. Methods: The event was attended by ~250 individuals (cohort members and their guests), who had been invited through the offices of their participating cohort studies. There were a series of presentations describing key research results and the audience participated in 15 multiple-choice questions using interactive voting pads. Results: Our participants showed a high level of trust in researchers (87% scoring them 4/5 or 5/5) and doctors (81%); but less trust in commercial companies (35%). They supported the idea of researchers using information from both neonatal blood spots (Guthrie spots) (97% yes) and from electronic health records (95% yes). Our respondents were willing to wear devices like a ’Fit-bit’ (78% agreed) or take a brain scan that might predict later mental illness (73%). However, they were less willing to take a new drug for research purposes (45%). They were keen to encourage others to take part in research; whether that be offering the opportunity to pregnant mothers (97% agreed) or extending invitations to their own children and grandchildren (98%). Conclusions: Our participants were broadly supportive of research access to data, albeit less supportive when commercial interests were involved. Public engagement events that facilitate two-way interactions can influence and support future research and public engagement efforts.

The underlying genetic and molecular mechanisms that drive amyotrophic lateral sclerosis (ALS) remain poorly understood. Structural variants within the genome can play a significant role in neurodegenerative disease risk, such as the repeat expansion in C9orf72 and the tri-nucleotide repeat in ATXN2, both of which are associated with familial and sporadic ALS. Many such structural variants reside in uncharacterized regions of the human genome, and have been under studied. Therefore, characterization of structural variants located in and around genes associated with ALS could provide insight into disease pathogenesis, and lead to the discovery of highly informative genetic tools for stratification in clinical trials. Such genomic variants may provide a deeper understanding of how gene expression can affect disease etiology, disease severity and trajectory, patient response to treatment, and may hold the key to understanding the genetics of sporadic ALS. This article outlines the current understanding of amyotrophic lateral sclerosis genetics and how structural variations may underpin some of the missing heritability of this disease.

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

The scientific landscape surrounding amyotrophic lateral sclerosis (ALS) continues to shift as the number of genes associated with the disease risk and pathogenesis, and the cellular processes involved, continues to grow. Despite decades of intense research and over 50 potentially causative or disease-modifying genes identified, etiology remains unexplained and treatment options remain limited for the majority of ALS patients. Various factors have contributed to the slow progress in understanding and developing therapeutics for this disease. Here, we review the genetic basis of ALS, highlighting factors that have contributed to the elusiveness of genetic heritability. The most commonly mutated ALS-linked genes are reviewed with an emphasis on disease-causing mechanisms. The cellular processes involved in ALS pathogenesis are discussed, with evidence implicating their involvement in ALS summarized. Past and present therapeutic strategies and the benefits and limitations of the model systems available to ALS researchers are discussed with future directions for research that may lead to effective treatment strategies outlined.

Recent approvals of oligonucleotide analogue drugs to alter gene expression have been welcomed by patient communities but not universally supported. These compounds represent a class of drugs that are designed to target a specific gene transcript, and they include a number of chemical entities to evoke different antisense mechanisms, depending upon the disease aetiology. To date, oligonucleotide therapeutics that are in the clinic or at advanced stages of translation target rare diseases, posing challenges to clinical trial design, recruitment and evaluation and requiring new evaluation paradigms. This review discusses the currently available and emerging therapeutics that alter exon selection through an effect on pre-mRNA splicing and explores emerging concerns over safety and efficacy. Although modification of synthetic nucleic acids destined for therapeutic application is common practice to protect against nuclease degradation and to influence drug function, such modifications may also confer unexpected physicochemical and biological properties. Negatively charged oligonucleotides have a strong propensity to bind extra- and intra-cellular proteins, whereas those analogues with a neutral backbone show inefficient cellular uptake but excellent safety profiles. In addition, the potential for incorporation of chemically modified nucleic acid monomers, yielded by nuclease degradation of exogenous oligonucleotides, into biomolecules has been raised and the possibility not entirely discounted. We conclude with a commentary on the ongoing efforts to develop novel antisense compounds and enhance oligonucleotide delivery in order to further improve efficacy and accelerate implementation of antisense therapeutics for human disease.

An increasing body of genetic and imaging research shows that it is becoming possible to forecast the onset of major psychiatric disorders such as depression and schizophrenia before people become ill with ever improving accuracy. Practical issues such as the optimal combination of clinical and biological variables are being addressed, but the application of predictive algorithms to individuals or in routine clinical settings have yet to be tested. The development of predictive methods in mental health comes with substantial ethical questions, including whether people wish to know their level of risk, as well as individual and societal attitudes to the potential adverse effects of data sharing, early diagnosis and treatment, which so far have been largely ignored. Preliminary data suggests that at least some people think predictive research is valuable and would take part in such studies, and some would welcome knowing the results. Future initiatives should systematically assess opinions and attitudes in conjunction with scientific and technical advances.

The literature surrounding the use of antisense oligonucleotides continues to grow, with new disease and mechanistic applications constantly evolving. Furthermore, the discovery and advancement of novel chemistries continues to improve antisense delivery, stability and effectiveness. For each new application, a rational sequence design is recommended for each oligomer, as is chemistry and delivery optimization. To confirm oligomer delivery and antisense activity, a positive control AO sequence with well characterized target-specific effects is recommended. Here, we describe splice-switching antisense oligomer sequences targeting the ubiquitously expressed human and mouse SMN and Smn genes for use as control AOs for this purpose. We report two AO sequences that induce targeted skipping of SMN1/SMN2 exon 7 and two sequences targeting the Smn gene, that induce skipping of exon 5 and exon 7. These antisense sequences proved effective in inducing alternative splicing in both in vitro and in vivo models and are therefore broadly applicable as controls. Not surprisingly, we discovered a number of differences in efficiency of exon removal between the two species, further highlighting the differences in splice regulation between species.

We report the generation of the human iPSC line LEIi008-A from a patient with retinitis pigmentosa-11 caused by a dominant nonsense mutation in the PRPF31 gene (NM_015629.3:c.1205C > A p.(Ser402Ter)). A second line, LEIi009-A, was generated from a related non-penetrant carrier of the same mutation with no retinal disease. Reprogramming of patient dermal fibroblasts using episomal plasmids containing OCT4, SOX2, KLF4, L-MYC, LIN28, shRNA for p53 and mir302/367 microRNA generated cell lines displaying pluripotent stem cell marker expression, a normal karyotype and the capability to differentiate into the three germ layer lineages.Resource tableUnlabelled TableUnique stem cell lines identifierLEIi008-ALEIi009-AAlternative names of stem cell lines1093ips4 (LEIi008-A)1374ips1 (LEIi009-A)InstitutionLions Eye Institute, Nedlands, Western Australia, AustraliaContact information of distributorSamuel McLenachan: smclenachan@lei.org.auFred Chen: fredchen@lei.org.auType of cell linesiPSCOriginHumanCell SourceDermal fibroblastsClonalityClonalMethod of reprogrammingEpisomalMultiline rationaleUnaffected mother and affected son carrying same dominant PRPF31 mutationGene modificationYesType of modificationHereditaryAssociated diseaseRetinitis Pigmentosa 11Gene/locusPRPF31/19q13.42Method of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock dateLEIi008-A: 18/12/17; LEIi009-A: 17/11/17Cell line repository/bankhttps://hpscreg.eu/cell-line/LEIi008-Ahttps://hpscreg.eu/cell-line/LEIi009-AEthical approvalHuman Research Ethics Office, University of Western Australia (RA/4/1/7916)

One of the crucial aspects of screening antisense oligonucleotides destined for therapeutic application is confidence that the antisense oligomer is delivered efficiently into cultured cells. Efficient delivery is particularly vital for antisense phosphorodiamidate morpholino oligomers, which have a neutral backbone, and are known to show poor gymnotic uptake. Here, we report several methods to deliver these oligomers into cultured cells. Although 4D-Nucleofector™ or Neon™ electroporation systems provide efficient delivery and use lower amounts of phosphorodiamidate morpholino oligomer, both systems are costly. We show that some readily available transfection reagents can be used to deliver phosphorodiamidate morpholino oligomers as efficiently as the electroporation systems. Among the transfection reagents tested, we recommend Lipofectamine 3000™ for delivering phosphorodiamidate morpholino oligomers into fibroblasts and Lipofectamine 3000™ or Lipofectamine 2000™ for myoblasts/myotubes. We also provide optimal programs for nucleofection into various cell lines using the P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza), as well as antisense oligomers that redirect expression of ubiquitously expressed genes that may be used as positive treatments for human and murine cell transfections.

Steve D Wilton, Sue FletcherCentre for Neuromuscular and Neurological Disorders, University of Western Australia, Crawley, Perth, WA, AustraliaAbstract: The identification of dystrophin and the causative role of mutations in this gene in Duchenne and Becker muscular dystrophies (D/BMD) was expected to lead to timely development of effective therapies. Despite over 20 years of research, corticosteroids remain the only available pharmacological treatment for DMD, although significant benefits and extended life have resulted from advances in the clinical care and management of DMD individuals. Effective treatment of DMD will require dystrophin restitution in skeletal, cardiac, and smooth muscles and nonmuscle tissues; however, modulation of muscle loss and regeneration has the potential to play an important role in altering the natural history of DMD, particularly in combination with other treatments. Emerging biological, molecular, and small molecule therapeutics are showing promise in ameliorating this devastating disease, and it is anticipated that regulatory environments will need to display some flexibility in order to accommodate the new treatment paradigms.Keywords: Duchenne muscular dystrophy, molecular therapeutics, small molecules

Duchenne and Becker muscular dystrophy severity depends upon the nature and location of the DMD gene lesion and generally correlates with the dystrophin open reading frame. However, there are striking exceptions where an in-frame genomic deletion leads to severe pathology or protein-truncating mutations (nonsense or frame-shifting indels) manifest as mild disease. Exceptions to the dystrophin reading frame rule are usually resolved after molecular diagnosis on muscle RNA. We report a moderate/severe Becker muscular dystrophy patient with an in-frame genomic deletion of DMD exon 5. This mutation has been reported by others as resulting in Duchenne or Intermediate muscular dystrophy, and the loss of this in-frame exon in one patient led to multiple splicing events, including omission of exon 6, that disrupts the open reading frame and is consistent with a severe phenotype. The patient described has a deletion of dystrophin exon 5 that does not compromise recognition of exon 6, and although the deletion does not disrupt the reading frame, his clinical presentation is more severe than would be expected for classical Becker muscular dystrophy. We suggest that the dystrophin isoform lacking the actin-binding sequence encoded by exon 5 is compromised, reflected by the phenotype resulting from induction of this dystrophin isoform in mouse muscle in vivo. Hence, exon skipping to address DMD-causing mutations within DMD exon 5 may not yield an isoform that confers marked clinical benefit. Additional studies will be required to determine whether multi-exon skipping strategies could yield more functional dystrophin isoforms, since some BMD patients with larger in-frame deletions in this region have been reported with mild phenotypes.

Inherited retinal diseases are an extremely diverse group of genetically and phenotypically heterogeneous conditions characterized by variable maturation of retinal development, impairment of photoreceptor cell function and gradual loss of photoreceptor cells and vision. Significant progress has been made over the last two decades in identifying the many genes implicated in inherited retinal diseases and developing novel therapies to address the underlying genetic defects. Approximately one-quarter of exonic mutations related to human inherited diseases are likely to induce aberrant splicing products, providing opportunities for the development of novel therapeutics that target splicing processes. The feasibility of antisense oligomer mediated splice intervention to treat inherited diseases has been demonstrated in vitro, in vivo and in clinical trials. In this review, we will discuss therapeutic approaches to treat inherited retinal disease, including strategies to correct splicing and modify exon selection at the level of pre-mRNA. The challenges of clinical translation of this class of emerging therapeutics will also be discussed.

Quality of life is often relatively lowered in families of children with additional needs, and this may be particularly the case where additional needs are accompanied by an autism spectrum disorder (ASD). Here we explore the effects of diagnostic status specifically, comparing families with children with an ASD diagnosis with others who a) have additional needs but no signs of ASD; and b) have additional needs and signs of ASD but no diagnosis. Mothers (n = 76) of children with additional needs completed standardised questionnaires about quality of life, stress, service provision, child behaviour and presence and severity of ASD traits. In addition, a group of mothers of typically developing young people (n = 17) completed standardised questionnaires on individual and family quality of life and on the behaviour of their son or daughter. Mothers of typically developing young people had significantly higher individual and family quality of life scores than each of the three other groups. Increased severity of ASD was associated with increased maternal stress, which in turn was associated with decreased family and maternal quality of life. The group reporting the lowest quality of life and the highest stress were the mothers of individuals with signs of ASD but no diagnosis. This pattern did not seem to be explained by lack of access to services, or rates of intellectual disability or challenging behaviour in this sub‐group. The finding that poor quality of life and high stress was most apparent in the sub‐group of mothers with children who had signs of ASD but did not have a diagnosis of ASD suggests that an interesting topic for further investigation is whether receipt of a diagnosis itself can positively influence quality of life and levels of maternal stress.

We sought to use splice-switching antisense oligonucleotides to produce a model of accelerated ageing by enhancing expression of progerin, translated from a mis-spliced lamin A gene (LMNA) transcript in human myogenic cells. The progerin transcript (LMNA Δ150) lacks the last 150 bases of exon 11, and is translated into a truncated protein associated with the severe premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS). HGPS arises from de novo mutations that activate a cryptic splice site in exon 11 of LMNA and result in progerin accumulation in tissues of mesodermal origin. Progerin has also been proposed to play a role in the 'natural' ageing process in tissues. We sought to test this hypothesis by producing a model of accelerated muscle ageing in human myogenic cells. A panel of splice-switching antisense oligonucleotides were designed to anneal across exon 11 of the LMNA pre-mRNA, and these compounds were transfected into primary human myogenic cells. RT-PCR showed that the majority of oligonucleotides were able to modify LMNA transcript processing. Oligonucleotides that annealed within the 150 base region of exon 11 that is missing in the progerin transcript, as well as those that targeted the normal exon 11 donor site induced the LMNA Δ150 transcript, but most oligonucleotides also generated variable levels of LMNA transcript missing the entire exon 11. Upon evaluation of different oligomer chemistries, the morpholino phosphorodiamidate oligonucleotides were found to be more efficient than the equivalent sequences prepared as oligonucleotides with 2'-O-methyl modified bases on a phosphorothioate backbone. The morpholino oligonucleotides induced nuclear localised progerin, demonstrated by immunostaining, and morphological nuclear changes typical of HGPS cells. We show that it is possible to induce progerin expression in myogenic cells using splice-switching oligonucleotides to redirect splicing of LMNA. This may offer a model to investigate the role of progerin in premature muscle ageing.

Muscles in Duchenne dystrophy patients are characterized by the absence of dystrophin, yet transverse sections show a small percentage of fibers (termed "revertant fibers") positive for dystrophin expression. This phenomenon, whose biological bases have not been fully elucidated, is present also in the murine and canine models of DMD and can confound the evaluation of therapeutic approaches. We analyzed 11 different muscles in a cohort of 40 mdx mice, the most commonly model used in pre-clinical studies, belonging to four age groups; such number of animals allowed us to perform solid ANOVA statistical analysis. We assessed the average number of dystrophin-positive fibers, both absolute and normalized for muscle size, and the correlation between their formation and the ageing process. Our results indicate that various muscles develop different numbers of revertant fibers, with different time trends; besides, they suggest that the biological mechanism(s) behind dystrophin re-expression might not be limited to the early development phases but could actually continue during adulthood. Importantly, such finding was seen also in cardiac muscle, a fact that does not fit into the current hypothesis of the clonal origin of "revertant" myonuclei from satellite cells. This work represents the largest, statistically significant analysis of revertant fibers in mdx mice so far, which can now be used as a reference point for improving the evaluation of therapeutic approaches for DMD. At the same time, it provides new clues about the formation of revertant fibers/cardiomyocytes in dystrophic skeletal and cardiac muscle.

Spinal muscular atrophy (SMA) is caused by loss of the Survival Motor Neuron 1 (SMN1) gene, resulting in reduced SMN protein. Humans possess the additional SMN2 gene (or genes) that does produce low level of full length SMN, but cannot adequately compensate for loss of SMN1 due to aberrant splicing. The majority of SMN2 gene transcripts lack exon 7 and the resultant SMNΔ7 mRNA is translated into an unstable and non-functional protein. Splice intervention therapies to promote exon 7 retention and increase amounts of full-length SMN2 transcript offer great potential as a treatment for SMA patients. Several splice silencing motifs in SMN2 have been identified as potential targets for antisense oligonucleotide mediated splice modification. A strong splice silencer is located downstream of exon 7 in SMN2 intron 7. Antisense oligonucleotides targeting this motif promoted SMN2 exon 7 retention in the mature SMN2 transcripts, with increased SMN expression detected in SMA fibroblasts. We report here systematic optimisation of phosphorodiamidate morpholino oligonucleotides (PMO) that promote exon 7 retention to levels that rescued the phenotype in a severe mouse model of SMA after intracerebroventricular delivery. Furthermore, the PMO gives the longest survival reported to date after a single dosing by ICV.

Protein-truncating mutations in the dystrophin gene lead to the progressive muscle wasting disorder Duchenne muscular dystrophy, whereas in-frame deletions typically manifest as the milder allelic condition, Becker muscular dystrophy. Antisense oligomer-induced exon skipping can modify dystrophin gene expression so that a disease-associated dystrophin pre-mRNA is processed into a Becker muscular dystrophy-like mature transcript. Despite genomic deletions that may encompass hundreds of kilobases of the gene, some dystrophin mutations appear “leaky”, and low levels of high molecular weight, and presumably semi-functional, dystrophin are produced. A likely causative mechanism is endogenous exon skipping, and Duchenne individuals with higher baseline levels of dystrophin may respond more efficiently to the administration of splice-switching antisense oligomers. We optimized excision of exons 8 and 9 in normal human myoblasts, and evaluated several oligomers in cells from eight Duchenne muscular dystrophy patients with deletions in a known “leaky” region of the dystrophin gene. Inter-patient variation in response to antisense oligomer induced skipping in vitro appeared minimal. We describe oligomers targeting exon 8, that unequivocally increase dystrophin above baseline in vitro, and propose that patients with leaky mutations are ideally suited for participation in antisense oligomer mediated splice-switching clinical studies.