Prenatal delivery of a therapeutic antisense oligonucleotide achieves broad biodistribution in the brain and ameliorates Angelman syndrome phenotype in mice.

Angelman syndrome (AS), an early-onset neurodevelopmental disorder characterized by abnormal gait, intellectual disabilities, and seizures, occurs when the maternal allele of the UBE3A gene is disrupted, since the paternal allele is silenced in neurons by the UBE3A antisense (UBE3A-AS) transcript. Given the importance of early treatment, we hypothesized that prenatal delivery of an antisense oligonucleotide (ASO) would downregulate the murine Ube3a-AS, resulting in increased UBE3A protein and functional rescue. Using a mouse model with a Ube3a-YFP allele that reports on-target ASO activity, we found that in utero, intracranial (IC) injection of the ASO resulted in dose-dependent activation of paternal Ube3a, with broad biodistribution. Accordingly, in utero injection of the ASO in a mouse model of AS also resulted in successful restoration of UBE3A and phenotypic improvements in treated mice on the accelerating rotarod and fear conditioning. Strikingly, even intra-amniotic (IA) injection resulted in systemic biodistribution and high levels of UBE3A reactivation throughout the brain. These findings offer a novel strategy for early treatment of AS using an ASO, with two potential routes of administration in the prenatal window. Beyond AS, successful delivery of a therapeutic ASO into neurons has implications for a clinically feasible prenatal treatment for numerous neurodevelopmental disorders.

Current and emerging treatment options for Angelman syndrome.

INTRODUCTION: Angelman syndrome (AS) is a neurodevelopmental disorder characterized by intellectual disability, limited expressive language, epilepsy, and motor impairment. Angelman syndrome is caused by haploinsufficiency of the UBE3A gene on the maternal copy of chromosome 15. There have been ongoing advances in the understanding of neurological, behavioral, and sleep-based problems and associated treatments for patients with AS. These results along with gene-based therapies entering into clinical development prompted this review. AREAS COVERED: The authors summarize the research basis describing phenomenology of epilepsy and behavioral concerns such as hyperactivity behavior, aggression, self-injury, repetitive behavior, and sleep disorder. The evidence for recent treatment advances in these target symptom domains of concern is reviewed, and the potential for emerging gene therapy treatments is considered. EXPERT OPINION: The prospect for emerging gene therapies means that increasing efforts should be directed toward the early identification of AS implemented equitably. Recent studies emphasize the important role of behavioral therapy in addressing mental health concerns such as aggression and disordered sleep.

An ASO therapy for Angelman syndrome that targets an evolutionarily conserved region at the start of the UBE3A-AS transcript.

Angelman syndrome is a devastating neurogenetic disorder for which there is currently no effective treatment. It is caused by mutations or epimutations affecting the expression or function of the maternally inherited allele of the ubiquitin-protein ligase E3A (UBE3A) gene. The paternal UBE3A allele is imprinted in neurons of the central nervous system (CNS) by the UBE3A antisense (UBE3A-AS) transcript, which represents the distal end of the small nucleolar host gene 14 (SNHG14) transcription unit. Reactivating the expression of the paternal UBE3A allele in the CNS has long been pursued as a therapeutic option for Angelman syndrome. Here, we described the development of an antisense oligonucleotide (ASO) therapy for Angelman syndrome that targets an evolutionarily conserved region demarcating the start of the UBE3A-AS transcript. We designed and chemically optimized gapmer ASOs targeting specific sequences at the start of the human UBE3A-AS transcript. We showed that ASOs targeting this region precisely and efficiently repress the transcription of UBE3A-AS, reactivating the expression of the paternal UBE3A allele in neurotypical and Angelman syndrome induced pluripotent stem cell-derived neurons. We further showed that human-targeted ASOs administered to the CNS of cynomolgus macaques by lumbar intrathecal injection repress UBE3A-AS and reactivate the expression of the paternal UBE3A allele throughout the CNS. These findings support the advancement of this investigational molecular therapy for Angelman syndrome into clinical development (ClinicalTrials.gov, NCT04259281).

A high-fidelity RNA-targeting Cas13 restores paternal Ube3a expression and improves motor functions in Angelman syndrome mice.

Angelman syndrome (AS) is a rare neurodevelopmental disorder caused by loss of function mutations in maternally expressed UBE3A. No gene-specific treatment is available for patients so far. Although intact and transcriptionally active, paternally inherited UBE3A is silenced by elongation of antisense long noncoding RNA UBE3A-ATS in neurons. Here, we demonstrated that RNA targeting of paternal Ube3a-ATS with a high-fidelity CRISPR-Cas13 (hfCas13x.1) system could restore Ube3a expression to similar levels as that of maternal Ube3a in the cultured mouse neurons. Furthermore, injection into lateral ventricles with neuron-specific hSyn1 promoter-driven hfCas13x.1 packaged in adeno-associated virus (AAV-PHP.eb) could restore paternal Ube3a expression in cortex and hippocampus of neonatal AS mice for up to 4 months after treatment. Behavioral tests showed that expression of paternal Ube3a significantly alleviated AS-related symptoms, including obesity and motor function. Our results suggested that hfCas13x.1-mediated suppression of the Ube3a-ATS lncRNA potentially serves as a promising targeted intervention for AS.

Transcriptional reprogramming restores UBE3A brain-wide and rescues behavioral phenotypes in an Angelman syndrome mouse model.

Angelman syndrome (AS) is a neurogenetic disorder caused by the loss of ubiquitin ligase E3A (UBE3A) gene expression in the brain. The UBE3A gene is paternally imprinted in brain neurons. Clinical features of AS are primarily due to the loss of maternally expressed UBE3A in the brain. A healthy copy of paternal UBE3A is present in the brain but is silenced by a long non-coding antisense transcript (UBE3A-ATS). Here, we demonstrate that an artificial transcription factor (ATF-S1K) can silence Ube3a-ATS in an adult mouse model of Angelman syndrome (AS) and restore endogenous physiological expression of paternal Ube3a. A single injection of adeno-associated virus (AAV) expressing ATF-S1K (AAV-S1K) into the tail vein enabled whole-brain transduction and restored UBE3A protein in neurons to ∼25% of wild-type protein. The ATF-S1K treatment was highly specific to the target site with no detectable inflammatory response 5 weeks after AAV-S1K administration. AAV-S1K treatment of AS mice showed behavioral rescue in exploratory locomotion, a task involving gross and fine motor abilities, similar to low ambulation and velocity in AS patients. The specificity and tolerability of a single injection of AAV-S1K therapy for AS demonstrate the use of ATFs as a promising translational approach for AS.

Feasibility of wearable technology for 'real-world' gait analysis in children with Prader-Willi and Angelman syndromes.

BACKGROUND: Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurodevelopmental disorders in need of innovative 'real-world' outcome measures to evaluate treatment effects. Instrumented gait analysis (IGA) using wearable technology offers a potentially feasible solution to measure "real-world' neurological and motor dysfunction in these groups. METHODS: Children (50% female; 6-16 years) diagnosed with PWS (n = 9) and AS (n = 5) completed 'real-world' IGA assessments using the Physilog®5 wearable. PWS participants completed a laboratory assessment and a 'real-world' long walk. The AS group completed 'real-world' caregiver-assisted assessments. Mean and variability results for stride time, cadence, stance percentage (%) and stride length were extracted and compared across three different data reduction protocols. RESULTS: The wearables approach was found to be feasible, with all participants able to complete at least one assessment. This study also demonstrated significant agreement, using Lin's concordance correlation coefficient (CCC), between laboratory and 'real-world' assessments in the PWS group for mean stride length, mean stance % and stance % CV (n = 7, CCC: 0.782-0.847, P = 0.011-0.009). CONCLUSION: 'Real-world' gait analysis using the Physilog®5 wearable was feasible to efficiently assess neurological and motor dysfunction in children affected with PWS and AS.

Improving Gene Therapy for Angelman Syndrome with Secreted Human UBE3A.

The rare genetic neurodevelopmental disease Angelman syndrome (AS) is caused by the loss of function of UBE3A, a ubiquitin ligase. The disease results in a lifetime of severe symptoms, including intellectual disability and motor impairments for which there are no effective treatments. One avenue of treatment for AS is the use of gene therapy to reintroduce a functional copy of the UBE3A gene. Our group had previously shown that recombinant adeno-associated virus (rAAV) expressing mouse Ube3a could rescue deficits in a mouse model of AS. Here, we expand on this work and show that this approach could be successfully replicated in a second AS model using the human UBE3A gene. Furthermore, we address the challenge of limited vector distribution in the brain by developing a novel modified form of UBE3A. This modified protein, termed STUB, was designed with a secretion signal and a cell-penetrating peptide. This allowed transduced cells to act as factories for the production of UBE3A protein that could be taken up by neighboring non-transduced cells, thus increasing the number of neurons receiving the therapeutic protein. Combining this construct with intracerebroventricular injections to maximize rAAV distribution within the brain, we demonstrate that this novel approach improves the recovery of behavioral and electrophysiological deficits in the AS rat model. More importantly, a comparison of rAAV-STUB to a rAAV expressing the normal human UBE3A gene showed that STUB was a more effective therapeutic. These data suggest that rAAV-STUB is a new potential approach for the treatment of AS.

From first report to clinical trials: a bibliometric overview and visualization of the development of Angelman syndrome research.

Angelman syndrome is a rare neurodevelopmental disorder caused by mutations affecting the chromosomal 15q11-13 region, either by contiguous gene deletions, imprinting defects, uniparental disomy, or mutations in the UBE3A gene itself. Phenotypic abnormalities are driven primarily, but not exclusively (especially in 15q11-13 deletion cases) by loss of expression of the maternally inherited UBE3A gene expression. The disorder was first described in 1965 by the English pediatrician Harry Angelman. Since that first description of three children with Angelman syndrome, there has been extensive research into the genetic, molecular and phenotypic aspects of the disorder. In the last decade, this has resulted in over 100 publications per year. Collectively, this research has led the field to a pivotal point in which restoring UBE3A function by genetic therapies is currently explored in several clinical trials. In this study, we employed a bibliometric approach to review and visualize the development of Angelman syndrome research over the last 50 years. We look into different parameters shaping the progress of the Angelman syndrome research field, including source of funding, publishing journals and international collaborations between research groups. Using a network approach, we map the focus of the research field and how that shifted over time. This overview helps understand the shift of research focus in the field and can provide a comprehensive handbook of Angelman syndrome research development.

The Road to Personalized Therapies: Lessons Learned From Angelman Syndrome.

Angelman syndrome (AS) is a neurogenetic disorder characterized by delays including a severe expressive language delay, motor concerns, ataxia, epilepsy, sleep disturbances, gastrointestinal problems, and characteristic behaviors, including a happy demeanor, hyperactivity, and excitability. The syndrome is one of the first neurodevelopmental disorders with a clear trajectory towards meaningful treatment with approximately 20 companies actively developing targeted therapeutics for AS. Herein, we highlight the historical context of the road to therapeutics and some of the challenges in the field with the potential to impact the success of clinical trials for Angelman syndrome and also have relevance of other neurogenetic developmental disabilities.

A multidisciplinary approach and consensus statement to establish standards of care for Angelman syndrome.

BACKGROUND: Angelman syndrome (AS) is a rare neurogenetic disorder present in approximately 1/12,000 individuals and characterized by developmental delay, cognitive impairment, motor dysfunction, seizures, gastrointestinal concerns, and abnormal electroencephalographic background. AS is caused by absent expression of the paternally imprinted gene UBE3A in the central nervous system. Disparities in the management of AS are a major problem in preparing for precision therapies and occur even in patients with access to experts and recognized clinics. AS patients receive care based on collective provider experience due to limited evidence-based literature. We present a consensus statement and comprehensive literature review that proposes a standard of care practices for the management of AS at a critical time when therapeutics to alter the natural history of the disease are on the horizon. METHODS: We compiled the key recognized clinical features of AS based on consensus from a team of specialists managing patients with AS. Working groups were established to address each focus area with committees comprised of providers who manage >5 individuals. Committees developed management guidelines for their area of expertise. These were compiled into a final document to provide a framework for standardizing management. Evidence from the medical literature was also comprehensively reviewed. RESULTS: Areas covered by working groups in the consensus document include genetics, developmental medicine, psychology, general health concerns, neurology (including movement disorders), sleep, psychiatry, orthopedics, ophthalmology, communication, early intervention and therapies, and caregiver health. Working groups created frameworks, including flowcharts and tables, to help with quick access for providers. Data from the literature were incorporated to ensure providers had review of experiential versus evidence-based care guidelines. CONCLUSION: Standards of care in the management of AS are keys to ensure optimal care at a critical time when new disease-modifying therapies are emerging. This document is a framework for providers of all familiarity levels.

Sex-dependent influence of postweaning environmental enrichment in Angelman syndrome model mice.

INTRODUCTION: Angelman syndrome (AS) is a rare neurodevelopmental disorder caused by mutation or loss of UBE3A and marked by intellectual disability, ataxia, autism-like symptoms, and other atypical behaviors. One route to treatment may lie in the role that environment plays early in postnatal life. Environmental enrichment (EE) is one manipulation that has shown therapeutic potential in preclinical models of many brain disorders, including neurodevelopmental disorders. Here, we examined whether postweaning EE can rescue behavioral phenotypes in Ube3a maternal deletion mice (AS mice), and whether any improvements are sex-dependent. METHODS: Male and female mice (C57BL/6J Ube3atm1Alb mice and wild-type (WT) littermates; ≥10 mice/group) were randomly assigned to standard housing (SH) or EE at weaning. EE had a larger footprint, a running wheel, and a variety of toys that promoted foraging, burrowing, and climbing. Following 6 weeks of EE, animals were submitted to a battery of tests that reliably elicit behavioral deficits in AS mice, including rotarod, open field, marble burying, and forced swim; weights were also monitored. RESULTS: In male AS-EE mice, we found complete restoration of motor coordination, marble burying, and forced swim behavior to the level of WT-SH mice. We also observed a complete normalization of exploratory distance traveled in the open field, but we found no rescue of vertical behavior or center time. AS-EE mice also had weights comparable to WT-SH mice. Intriguingly, in the female AS-EE mice, we found a failure of EE to rescue the same behavioral deficits relative to female WT-SH mice. CONCLUSIONS: Environmental enrichment is an effective route to correcting the most penetrant phenotypes in male AS mice but not female AS mice. This finding has important implications for the translatability of early behavioral intervention for AS patients, most importantly the potential dependency of treatment response on sex.

Emerging Gene and Small Molecule Therapies for the Neurodevelopmental Disorder Angelman Syndrome.

Angelman syndrome (AS) is a rare (~1:15,000) neurodevelopmental disorder characterized by severe developmental delay and intellectual disability, impaired communication skills, and a high prevalence of seizures, sleep disturbances, ataxia, motor deficits, and microcephaly. AS is caused by loss-of-function of the maternally inherited UBE3A gene. UBE3A is located on chromosome 15q11-13 and is biallelically expressed throughout the body but only maternally expressed in the brain due to an RNA antisense transcript that silences the paternal copy. There is currently no cure for AS, but advancements in small molecule drugs and gene therapies offer a promising approach for the treatment of the disorder. Here, we review AS and how loss-of-function of the maternal UBE3A contributes to the disorder. We also discuss the strengths and limitations of current animal models of AS. Furthermore, we examine potential small molecule drug and gene therapies for the treatment of AS and associated challenges faced by the therapeutic design. Finally, gene therapy offers the opportunity for precision medicine in AS and advancements in the treatment of this disorder can serve as a foundation for other single-gene neurodevelopmental disorders.

Therapies in preclinical and clinical development for Angelman syndrome.

Introduction: Angelman syndrome is a rare genetic neurodevelopmental disorder, caused by deficiency or abnormal function of the maternal ubiquitin protein-ligase E3A, known as UBE3A, in the central nervous system. There is no disease-modifying treatment available, but the therapeutic pipeline of Angelman syndrome includes at least 15 different approaches at preclinical or clinical development. In the coming years, several clinical trials will be enrolling patients, which prompted this comprehensive review.Areas covered: We summarize and critically review the different therapeutic approaches. Some approaches attempt to restore the missing or nonfunctional UBE3A protein in the neurons via gene replacement or enzyme replacement therapies. Other therapies aim to induce expression of the normal paternal copy of the UBE3A gene by targeting a long non-coding RNA, the UBE3A-ATS, which interferes with its own expression. Another therapeutic category includes compounds that target molecular pathways and effector proteins known to be involved in Angelman syndrome pathophysiology.Expert opinion: We believe that by 2022-2023, more than five disease-modifying treatments will be simultaneously at clinical testing. However, the are several challenges with regards to safety and efficacy, which need to be addressed. Additionally, there is still a significant unmet need for clinical trial readiness.

Functional rescue in an Angelman syndrome model following treatment with lentivector transduced hematopoietic stem cells.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by impaired communication skills, ataxia, motor and balance deficits, intellectual disabilities, and seizures. The genetic cause of AS is the neuronal loss of UBE3A expression in the brain. A novel approach, described here, is a stem cell gene therapy which uses lentivector-transduced hematopoietic stem and progenitor cells to deliver functional UBE3A to affected cells. We have demonstrated both the prevention and reversal of AS phenotypes upon transplantation and engraftment of human CD34+ cells transduced with a Ube3a lentivector in a novel immunodeficient Ube3amat-/pat+ IL2rg-/y mouse model of AS. A significant improvement in motor and cognitive behavioral assays as well as normalized delta power measured by electroencephalogram was observed in neonates and adults transplanted with the gene modified cells. Human hematopoietic profiles observed in the lymphoid organs by detection of human immune cells were normal. Expression of UBE3A was detected in the brains of the adult treatment group following immunohistochemical staining illustrating engraftment of the gene-modified cells expressing UBE3A in the brain. As demonstrated with our data, this stem cell gene therapy approach offers a promising treatment strategy for AS, not requiring a critical treatment window.

CRISPR/Cas9 directed to the Ube3a antisense transcript improves Angelman syndrome phenotype in mice.

Gene editing holds the potential to correct mutations and cure devastating genetic disorders. The technology has not yet proven efficacious for therapeutic use in CNS diseases with ubiquitous neuronal defects. Angelman syndrome (AS), a severe neurodevelopmental disorder, is caused by a lack of maternal expression of the UBE3A gene. Because of genomic imprinting, only neurons are affected. One therapeutic approach focuses on the intact paternal UBE3A copy in patients with AS that is silenced by an antisense transcript (UBE3A-ATS). We show here that gene editing of Ube3a-ATS in the mouse brain resulted in the formation of base pair insertions/deletions (indels) in neurons and the subsequent unsilencing of the paternal Ube3a allele in neurons, which partially corrected the behavioral phenotype of a murine AS model. This study provides compelling evidence to further investigate editing of the homologous region of the human UBE3A-ATS because this may provide a lasting therapeutic effect for patients with AS.

Emerging Therapies and challenges for individuals with Angelman syndrome.

PURPOSE OF REVIEW: This review describes current understandings in the search for therapies to support children with Angelman syndrome. There is a rapid progression in particular in genetic therapies in this disorder supported by the Angelman community. RECENT FINDINGS: Recent papers shed light on the timing of therapies and novel genetic therapies coming to trial as well as potential therapies still in preclinical phases. Further understanding of UBE3A and its role in neuronal development and plasticity as well as other mechanisms contributing to the Angelman phenotype is offering an opportunity for novel therapeutics. SUMMARY: Greater understanding of the pathophysiology of the different phenotypes will offer an opportunity for novel therapeutics and may well change the course of this disorder over time where previously there has been minimal ability to intervene.

Cas9 gene therapy for Angelman syndrome traps Ube3a-ATS long non-coding RNA.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by a mutation or deletion of the maternally inherited UBE3A allele. In neurons, the paternally inherited UBE3A allele is silenced in cis by a long non-coding RNA called UBE3A-ATS. Here, as part of a systematic screen, we found that Cas9 can be used to activate ('unsilence') paternal Ube3a in cultured mouse and human neurons when targeted to Snord115 genes, which are small nucleolar RNAs that are clustered in the 3' region of Ube3a-ATS. A short Cas9 variant and guide RNA that target about 75 Snord115 genes were packaged into an adeno-associated virus and administered to a mouse model of AS during the embryonic and early postnatal stages, when the therapeutic benefit of restoring Ube3a is predicted to be greatest1,2. This early treatment unsilenced paternal Ube3a throughout the brain for at least 17 months and rescued anatomical and behavioural phenotypes in AS mice. Genomic integration of the adeno-associated virus vector into Cas9 target sites caused premature termination of Ube3a-ATS at the vector-derived polyA cassette, or when integrated in the reverse orientation, by transcriptional collision with the vector-derived Cas9 transcript. Our study shows that targeted genomic integration of a gene therapy vector can restore the function of paternally inherited UBE3A throughout life, providing a path towards a disease-modifying treatment for a syndromic neurodevelopmental disorder.

Characterizing spine issues: If offers novel therapeutics to Angelman syndrome.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by severe mental retardation, microcephaly, speech impairment, frequent epilepsy, EEG abnormalities, ataxic movements, tongue protrusion, bursts of laughter, sleep abruptions, and hyperactivity. AS results from loss of function of the imprinted UBE3A (ubiquitin-protein ligase E3A) gene on chromosome 15q11-q13, including a mutation on the maternal allele of Ube3a, a large deletion of the maternally inherited chromosomal region 15q11-13, paternal uniparental disomy of chromosome 15q11-13, or an imprinting defect. The Ube3a maternal deleted mouse model recaptured the major phenotypes of AS patients include seizure, learning and memory impairments, sleep disturbance, and motor problems. Owing to the activity-dependent structural and functional plasticity, dendritic spines are believed as the basic subcellular compartment for learning and memory and the sites where LTP and LTD are induced. Defects of spine formation and dynamics are common among several neurodevelopmental disorders and neuropsychiatric disorders including AS and reflect the underlying synaptopathology, which drives clinically relevant behavioral deficits. This review will summarize the impaired spine density, morphology, and synaptic plasticity in AS and propose that future explorations on spine dynamics and synaptic plasticity may help develop novel interventions and therapy for neurodevelopmental disorders like AS.

Parent perceptions, beliefs, and fears around genetic treatments and cures for children with Angelman syndrome.

Genetic therapies have shown recent promise in alleviating some of the cognitive issues associated with some genetic disorders; however, these therapies may come with significant health and socio-ethical concerns, particularly when they involve child participants. Little is known about what parents of children with genetic disorders think about genetic therapies, or about their knowledge of how genetic-based therapy might treat their child's symptoms. Forty-two parents of children with Angelman syndrome (AS) and 27 parents of a mixed etiology comparison group completed an online survey reporting on their perceptions of, and priorities for, genetic therapy. Almost all parents of children with AS (95%) and the comparison group (89%) agreed that treatments aiming to reduce symptoms associated with their child's syndrome were positive. However, significantly more parents of children with AS (95%) than the comparison group (56%) felt that genetic treatment trials aiming to "cure" their child should be a research priority. AS parent priorities for the focus of clinical trials were neurology/seizures, communication skills, and motor skills/mobility. For the comparison group, the priorities were IQ, immune response, and expressive speech. Parents of both groups did not want treatments to change their child's personality or their happiness. Global assumptions cannot be made about targets for therapy between syndromes, about parental understanding of genetics, or about research evidence across syndromes. This study highlights the need for true family and patient engagement in all stages of the research design and treatment evaluation.