FAST Funded Research

Stem cell technology would prove invaluable in finally discovering effective treatments for developmental brain disorders, including Angelman syndrome (AS). In the brain, information of learning and memory is processed and stored in the form of electrical signals transmitted through millions of circuits made of neurons. The supporting glial cells, such as oligodendrocytes, are active partners in such information processing by providing structural support to neurons to speed up the flow of electric signals. In AS, this function of oligodendrocyte falls short in children and young adults, and may slow the brain development and increase seizures. To date, scientists still do not know the cause and hence are unable to come up with a treatment for this dysfunction. This project aims to understand and translate how the unique biology and genetics of AS make a difference in the oligodendrocyte function. Specifically, a few selected compounds identified from a stem cell-based drug screen will be tested for their efficacy by using a mouse model and the induced pluripotent stem cells (iPS cells) donated from AS patients and health individuals. In doing so, we believe this project will significantly contribute to the creation of urgently needed new treatments for AS, helping reduce the heavy disease burden.

Angelman syndrome (AS) results from the loss of functional UBE3A. In the case of large deletions, which account for the majority of all individuals living with AS (~70%), not only is UBE3A deleted but an additional 10 or so surrounding genes are also missing in the 15q11-13 region. Deletion of these genes results in haploinsufficiency that likely contributes to the increased symptom severity of the deletion genotype and adds to the differences between deletion and non-deletion patients. This project will explore a unique therapeutic avenue that aims to upregulate some of the most important critical genes, outside of UBE3A, looking at them both individually and collectively, and then assess them in cellular and rodent models. This project is a collaboration with Dr. Nadav Ahituv at the University of California, San Francisco, Dr. Albert Keung at North Carolina State University, and Dr. Yong-Hui Jiang at Yale University. Dr. Ahituv is a new investigator in Angelman syndrome and a world expert in CRISPR activation, a tool used to upregulate haploinsufficient genes. Drs. Keung and Jiang are leading AS researchers that have been awarded previous funding to create model systems to evaluate therapeutic candidates for AS. Dr. Keung’s work includes the creation of human stem cells with “landing pads” that control the expression of genes within the 15q11-13 region, thus allowing for research in how the loss of certain genes contribute to symptom severity. Dr. Jiang has designed a large deletion AS mouse model, with Ube3a expression intact, to understand how the loss of surrounding genes affect mouse behavior relevant to AS. The overarching hypothesis of this newly funded study aims to understand how upregulating the additional haploinsufficient genes on the paternal allele may result in therapeutic benefit beyond addressing just the loss of UBE3A. This is important for individuals living with AS that have a deletion genotype as it could address the additional genes that contribute to the symptom severity and bring the deletion phenotype (symptoms) closer to that of the nondeletion individuals.

The work being done here is particularly timely. While there have been significant strides in therapeutic design targeting UBE3A for the treatment of AS for all genotypes, these advancements often overlook the other genes within the 15q11-13 locus that likely play a role in the condition's severity in the largest population of AS, deletion. This presents a distinctive chance to invest in a potential therapy that can target essential genes beyond UBE3A, potentially offering an additional treatment option to improve Angelman syndrome management for the largest affected population.

Stem cell technology could prove invaluable in discovering effective treatments for developmental brain disorders, including Angelman syndrome (AS). In the brain, information of learning and memory is processed and stored in the form of electrical signals transmitted through millions of circuits made of neurons. The supporting glial cells, such as oligodendrocytes, are active partners in such information processing by providing structural support to neurons to speed up the flow of electric signals. In AS, this function of oligodendrocyte falls is impacted in children and young adults, and may slow the brain development and increase seizures. This project aims to understand and translate how the unique biology and genetics of AS make a difference in the oligodendrocyte function. Specifically, a few selected compounds identified from a stem cell-based drug screen will be tested for their efficacy by using a mouse model and the induced pluripotent stem cells (iPS cells) donated from AS patients and health individuals.

With a CRISPR/Cas9 genome editing tool, this project aims to restore UBE3A expression from the paternal chromosome via a single treatment approach. The paternal UBE3A allele is silenced by a long noncoding RNA that is antisense to UBE3A. This strategy will unsilence the paternal UBE3A allele, using CRISPR/Cas9 genome editing. This treatment will be delivered through an innovative nonviral chemically modified ribonucleoprotein (RNP) complex (cRNP-Cas9/gRNA) delivery system. It will carry the ready Cas9 protein and single guide RNA in one intrathecal injection to permanently activate the paternal UBE3A expression. The proposed study will not only provide landmark knowledge on non-viral delivery systems of CRISPR/Cas9 gene therapy, but also support the possibility of translational application using this system as a therapeutic approach to treat patients with Angelman syndrome.

The role of the microbiota-gut-brain (MGB) axis in maintaining overall health and well-being is now well established. Host-microbe interactions are paramount for maintaining normal physiology, including the brain and behavior. Nevertheless, mechanisms by which gastrointestinal (GI) microbes communicate with the brain adversely impact a wide variety of behaviors are poorly understood. Since colonization by the gut microbiota begins at birth, and maturation of the GI tract and brain development continue during neonatal life, identifying pathways of establishment of communication between these organs is critical to maintaining health across the lifespan. Disruptions to the MGB axis due to the gut microbiota, mucosal barrier defects, and/or changes in behavior, occur in multiple diseases, including GI diseases (i.e., inflammatory bowel disease) and behavioral disorders (i.e., neurodevelopmental disorders (NDD), intellectual disabilities (ID), and autism spectrum disorders (ASD), epilepsies). 

Gastrointestinal (GI) complications in children and adults with neurodevelopmental disorders have drawn attention to gaps in understanding their causes and treatment. GI dysfunction is particularly common in individuals with NDDs such as ASD, Phelan-McDermid and Rett syndromes.  GI disorders in these conditions can include gut malformations present at birth (such as pyloric stenosis or Hirschsprung disease) but also functional issues such as feeding problems, gastro-esophageal reflux disease (GERD), cyclic vomiting, delayed gastric emptying, diarrhea, bloating, celiac disease, irritable bowel symptoms, and constipation leading to encopresis, incontinence, and stool impaction. These GI issues may be associated with severe nutritional deficiencies, weight loss, failure to thrive and lack of seizure control. Unfortunately, mechanisms to accurately diagnose GI conditions in specific rare genetic NDDs are limited, and tailored treatments to address them are nonexistent, since clinical trials for IDD populations are rare, to date.

The overall goal of the NHS is to support the Angelman syndrome community by: 1) Seeking answers to questions pertaining to the clinical management and care of children and adults with Angelman syndrome, such as the types and frequency of various medical complications (e.g., constipation, seizures, and sleep disorders), medications that have been used, behavioral challenges, and developmental trajectories, 2) Providing foundational longitudinal data that can be used in the design of clinical trials by pharmaceutical companies that are developing therapies for Angelman syndrome; the longitudinal nature of these data will also allow these companies to assess the "pre-treatment" trajectories of the various developmental skills and hence the potential efficacy of their therapeutic compounds when submitting their data to the FDA to request regulatory approval for these compounds. The current outcome measures, obtained annually through a secure online portal, virtual visits, and in-person evaluations, include assessments of neurodevelopment, behavior, and medical complications, including seizures, sleep, and gastrointestinal issues.

Brain-derived neurotrophic factor (BDNF) is a key protein essential for regulating the cellular processes that underlie learning and memory. Studies show that the deficits in learning observed in a mouse model of Angelman syndrome (AS) stem from reduced BDNF activity. This research project will test a novel class of therapeutics, referred to as Syn compounds, that facilitate learning and restore normal mobility in AS mice. In humans, these compounds are envisaged to facilitate learning, for example, a new visual symbol, and enable the child to retain this information over an extended time until it becomes permanent. Syn compounds will undergo further animal studies to determine safety and ability to treat symptoms (learning and walking) in the Angelman animal models. The results of this project with the AS model mouse will be used to inform treatment of Angelman syndrome in patients.

In the last fifteen years much progress has been made in both understanding the basic genetics of AS and developing tools for treating AS. However, one area that still needs further exploration is the method of delivery for all of the therapies being developed. The brain still remains an extremely challenging organ to target for treatments. Multiple promising technologies have been successful in animal models of AS, but achieving widespread delivery of these therapeutics in a human brain remains elusive and is a major limiting factor in the effectiveness of these therapeutic approaches. The main goal of this grant is to increase delivery options for AS therapeutics by developing novel cell penetrating peptides (CPPs) from viruses that target neuronal cells. These cell penetrating peptides will act as “access codes” through the blood brain barrier allowing for delivery of potential AS therapeutics. Development of neuronal CPPs will bring the AS field closer to a mode of delivery that is minimally invasive, dosage controlled and with potentially a minimal immune response. This has the potential to benefit all modes of therapeutics in AS and also have a broad impact in the entire neurodevelopmental field.

Exciting scientific progress over the past ten years has led to clinical trials to treat Angelman syndrome at its root cause. Three current trials by different groups all use the same mechanism: antisense oligonucleotides that are designed to unsilence the dormant paternal UBE3A gene in the brain. As these trials begin, other promising treatment approaches are also in development. What all clinical trials have in common is the need to reliably measure clinically meaningful improvement as a result of treatment. If individuals with AS improve but this improvement cannot be quantified, a clinical trial will be deemed a failure and can set the community back years. Measurable biomarkers are needed to quantify improvement in clinical trials. Electroencephalography (EEG) meets many of the criteria of a good biomarker: (1) it is safe and non-invasive, (2) differences in AS are measurable and reliable, and (3) the degree of differences in AS is linked to the severity of AS symptoms. Current EEG biomarkers work well in children with AS, but their value wanes with age. With this proposal, we will develop new EEG biomarkers specifically designed for clinical trials in older children, adolescents, and adults with AS. Development of new biomarkers will ensure that improvement can be accurately measured during clinical trials in individuals of all ages.

In the process of developing novel therapeutics for Angelman syndrome (AS) several challenges exist that can hinder the process and speed of treatment discovery. These include 1) AS models in animals do not fully mimic the disease in humans and hence accurate assessment of the effectiveness of a putative treatment is difficult; 2) animal studies are time consuming and expensive: 3) AS is molecularly diverse, and experimental and screening models do not capture a large proportion of patient genotypes; and 4) high throughput sensor platforms for rapid screening of direct therapeutic effects on neuronal function are needed. This work aims to address these key challenges that hinder the development of new therapeutics for AS by developing a method to embed micro-sensors within organoids and use them to measure functional phenotypes of large deletion, mutation, and uni parental disomy AS organoids. Optimization of this sort of high throughput screening platform will be beneficial to rapidly study the efficacy of new emerging therapeutics for AS, and determine specific responses of each distinct AS genotype.

The Emergency Care Consortium will provide a global emergency and urgent care hotline. The hotline will be free of charge, available 24-hours a day and seven days a week, and will enable provider-to-provider consultations to manage urgent issues with the appropriate standards directly related to Angelman syndrome, especially seizures. More than 90% of individuals with Angelman syndrome experience seizures which are often difficult to control with traditional seizure medications. The Emergency Care Consortium will be available for provider-to-provider use in July 2021, with clinical experts that truly understand the nuances unique to Angelman syndrome.

Dr. Keung and his team at North Carolina State University will provide the Angelman syndrome research community a set of cell lines that can be used to efficiently model the biology of ICD and UPD, as well as organoids that model the mosaic genotype. This will aim to help understand any potential impact of gene overexpression in these different genotypes.

This grant focuses on the development of a microRNA approach as a potential therapeutic for the treatment of Angelman syndrome.  In collaboration with the Gene Therapy Program at the University of Pennsylvania, the latest research plan will build on the milestones already achieved with CRISPR-Cas9/sgRNA interference of the UBE3A-antisense transcript (UBE3A-AS) to develop a novel strategy to suppress UBE3A-AS extension utilizing microRNAs as a potential one-and-done treatment option.

Angelman syndrome (AS), Prader-Willi syndrome (PWS), and Dup15q syndrome are largely caused by aberrant inheritance of imprinting markers at the 15q11-13 locus resulting in disruption of gene regulation. The conditions have different etiologies but share diagnostic options and are targets for emerging therapeutics. The average age of diagnosis ranges from 6 months for infants with PWS to 3 years for children with Dup15q, and much later for cases with nondeletion subtypes of PWS or AS. Existing molecular methodologies can identify the majority of cases of all three conditions by interrogating DNA methylation patterns at the SNRPN/UBE3A locus. Newborn screening (NBS) provides the only population-based strategy to implement such testing broadly and identify infants who could benefit from early disease detection and treatment. However, a prospective population-based pilot study is needed to demonstrate feasibility of large-scale NBS and evaluate screening and follow-up protocols for public health implementation. Early Check, an ongoing voluntary NBS program in North Carolina, provides an opportunity to screen for a variety of rare disorders, provide short- and long-term follow-up services to identified infants, and generate data to inform public policy. This study focuses on the necessary preparatory steps that we are taking to add screening for PWS, AS and Dup15q to the Early Check Program to make prospective screening available to the parents of all ~120,000 newborns in the state.

This work aims to accelerate the publication of four research projects including: 1) Vineland-II longitudinal analyses, 2) Exploration of Quality of Life Assessments, 3) Development of Minimally Clinically Important Difference (MCID) for Bayley and Vineland, and 4) Association of Vineland/Bayley with other outcome measures.

The combination of gene therapy with hematopoietic stem cells (HSC) offers a promising approach for constitutive and life-long delivery of UBE3A to affected cells. By genetically modifying a patient’s own blood stem cells (HSCs), the modified cells can make future cells that are capable of migrating into the central nervous system (CNS) and provide a functional UBE3A copy to deficient neurons throughout the brain through a mechanism called “cross-correction.” This is a novel approach in AS in order to distribute UBE3A to more neurons of the brain than what might be achieved with traditional gene therapy using AAV. With the promising results produced from the previously FAST-funded FT2016-001 grant, the work here aimed to move forward with IND-enabling studies.

Casa Angelman developed a National Genetic Campaign to provide testing for children across Argentina and with assistance from FAST, will be able to bridge that gap for many Argentinian families.

Dr. Jiang will create a novel ~6MB full deletion Angelman syndrome mouse model. As we near a therapeutic aimed at getting a working copy of UBE3A into humans living with Angelman syndrome (AS), this work will contribute to the understanding of the other genes impacted in the largest population of individuals living with AS, those with a large deletion (>70% of individuals with AS), including numerous other genes around UBE3A and how these haploinsufficient genes may be contributing to symptoms of deletion positive individuals.

The patient iPSC derived neurons, and brain organoids (aka mini-brains), become essential tools or systems to test various approaches in human therapeutics, especially for sequence specific neurogenetic disorders (e.g. ASO and CRISPR/Cas9 based therapies, as well as other potential molecular treatments for AS). The major goal of this proposal is to create a high-quality biorepository of all genotypes using human AS patient derived IPSC lines and brain organoids that could be shared with the AS research community both in academic and industrial institutes in order to be able to test various therapeutic compounds and understand effect and tolerability with those compounds.

Dr. Duis used the ActiMyo device(s) in a clinical study to evaluate the tolerability and gait parameters in Angelman syndrome. This device was shown to be well  tolerated and associated with various unique gait parameters when compared to neurotypical age-matched controls.

This is a unique program that is aiming to create a core facility as a resource for developing and evaluating various treatments for Angelman syndrome. The intent is to create a stable infrastructure for the rapid testing of potential therapeutics in various models of Angelman syndrome (AS). The expectation is that academic investigators, and industry, would have access to experts in neurobehavioral testing, molecular biology and epigenetics. Through this model there is also the potential that trainees will develop expertise and interest in Angelman syndrome in an effort to support and breed the next generation of AS focused scientists. Rather than setting up all of the necessary components for animal testing at many different labs or R&D companies, it would be more efficient and higher throughput to have a central resource of experts who have a proven track record of understanding the AS animal models and the genetics and pathophysiology of AS.

Although there are no currently drug approved therapies that address the underlying causes of Angelman syndrome, new molecular therapies that are targeted to specific proteins, RNA, or DNA hold tremendous promise for the future. Past work demonstrated artificial transcription factors (ATFs) that could reactivate paternal Ube3a expression by silencing Ube3a-AS transcription in mouse brain and human cells. Reducing the UBE3A-AS has the collateral effect of reducing other RNAs including SNORD116 and SNORD115. Recently, four different strategies were evaluated to use ATFs to activate paternal UBE3A without affecting SNORD116 and SNORD115 in human cells. We demonstrate positive results for two of these approaches, and are now evaluating additional experiments to test these strategies in rodent models of Angelman syndrome (AS).

This work will develop a rapid gene insertion platform for human pluripotent stem cell and organoid systems that will unlock the ability to rescue the expression of UBE3A and its ten neighboring protein-coding genes lost in Class 1 deletions. These deleted genes contribute to the clinical phenotypes of AS individuals, and developing an experimental platform that can functionally interrogate and screen for their individual roles and responses to putative therapeutics would be important for over 50-70% of AS individuals with a large 15q11-13 deletion. Human cerebral organoids will also be generated from these stem cell lines to analyze changes in neuronal development, function and responses to putative therapeutics.

A non-permanent treatment for genetic disorders that result in a missing enzyme has been the focus for many disorders over the last few decades through enzyme replacement therapy (ERT). These types of drugs have been approved by the FDA for various conditions (Pompe disease, MPS, Gaucher’s disease, etc). Angelman syndrome is a disorder that results from a dysfunctional, or missing, UBE3A gene, which results in the lack of a functional UBE3A protein, which is an enzyme. Replacing or activating the missing, or silent, gene, will ultimately result in production of a functional UBE3A enzyme. This leaves the potential for the UBE3A enzyme to be replaced, without the need for genetic manipulation, with the ultimate goal being the same. This grant is to investigate this approach to see if this is a viable option for the treatment of AS and if this enzyme can be replaced, and functional, in neurons of the central nervous system (CNS). ERT is considered well tolerated and has a good safety profile in other conditions. Typically, these proteins are infused intravenously either weekly, biweekly, or monthly, and is often performed at home for these conditions. There is an FDA approved therapy where the enzymes are infused into the CNS through a port in the lumbar spine for intermittent CSF infusions. ERT has not been investigated for AS. Based on data with HSC-Lentiviral delivery of a secreted UBE3A protein, this protein has been shown to be highly effective in the IL2- AS mouse model. This grant will develop an ERT for AS and demonstrate its efficacy using in-vitro cell models, as well as efficacy in AS-ERT in-vivo mouse models. This project will serve as a proof of concept to see if an ERT could achieve rescue of AS phenotypes in mice.

Dr. Segal’s past work on artificial transcription factors (ATFs) showed global paternal gene activation after subcutaneous injection. In bringing this therapeutic platform to the next level for human use it was prudent to evaluate options to find a mechanism of ensuring that we do not inhibit the SNORD116 and SNORD115 RNAs, which are associated with Prader-Willi syndrome. This proposal is to build on the capabilities of epigenetic editing and brain delivery seeking new targeting strategies to protect SNORD116.

The overall objective of the proposal is to evaluate whether insulin-like growth factor-2 (IGF-2) or an IGF-2 receptor ligand L1 (L1) as a potential therapeutic for Angelman syndrome (AS). The principal investigator (PI) proposes to test IGF-2 and L1 on novel phenotypes recently identified in a novel AS rat model. The PI proposes three specific aims: 1) to determine the optimum dosage of IGF-2 and L1 with subcutaneous injection; 2) to determine whether IGF-2 and L1 treatment can reverse gait, associative memory, and working deficits displayed by AS model rats; 3) to determine whether IGF-2 and L1 treatment increase synaptic function and signal transduction in AS model rats.

Previous studies by the Alberini lab at NYU show that IGF-2 and L1 rescue neurological symptoms in a mouse model of Angelman syndrome. Preliminary analyses of the AS rat model indicate that EEG and seizure phenotypes are largely similar to those observed in the AS mouse model and AS patients. The aims of this study are to determine whether IGF-2 or L1 alters EEG activity in the AS rat model, evaluate the effect of IGF-2 or L1 on seizure threshold in AS rat model, determine effect of IGF-2 or L1 on sleep and circadian cycling in AS rat model, and assess whether IGF2 or L1 affects behavioral related EEG activity in AS rat model.

Dr. Scott Dindot is using an AS pig model to develop, test, and validate potential AS therapeutics and to characterize the AS pig. This testing gets us another step closer to robust preclinical phase testing of potential therapeutics for Angelman syndrome and evaluation of novel behavior changes seen in this large animal model.

The objective of this grant is to evaluate the behavioral rescue in a rat model of AS after treatment with a rat specific ASO designed by Dr. Dindot’s lab. FAST’s unique rat model provides the opportunity to investigate more complex behaviors that have been difficult to capture in mice, which include developmental milestones, neonatal calling as communication, juvenile behaviors, acoustic communication and advanced learning and memory. Furthermore, motor dysfunction, communication, and cognitive dysfunction are translatable to humans with AS, and we can leverage this sophisticated model for human translational therapies. In addition, this study will identify if ASO treatment rescued the MRI changes seen in the AS rat like axonal loss and microcephaly.

The use of ASOs to interrupt the paternal UBE3a allele silencing through antisense inhibition is a potential therapeutic approach for treating AS in humans. Here 3 University teams (USF, UC Davis and BCM) collaboratively propose to determine optimal sequence and concentration range for rat ASOs for the rat homologue. This study will allow the determination of a dose of ASOs to be used in a rat model of AS for a dose response and correlate that with in vivo expression and ATS knockdown changing the rat phenotypical behaviors and documenting if AS symptomatic rescue is achieved in an animal model. Expression will be determined from both CNS tissue and CSF fluid evaluation.

The objective of this grant is to evaluate whether delivery of ASO designed to reactivate paternal Ube3a in the AS rat improves the AS-related EEG and seizure phenotype. Preliminary analyses of the AS rat model indicate that EEG and seizure phenotypes are largely similar to those observed in the AS mouse model and AS patients.

Lowering carbohydrate content of the diet and increasing the fat content (through either a ketogenic diet, MCT based diet, modified Atkins diet, or the low glycemic index diet [LGIT]) has been shown to effectively decrease seizure frequency in patients with epilepsy and AS. Specialized medical food formations have been developed to transition patients to low carb, high fat formations, all of which are very high in fat. The medical food under assessment in this study is a specialized high fate formulation containing a ketone known as beta-hydroxybutyrate (BHB). BHB is a natural fat metabolite (ketone) that shifts metabolism away from carbs toward fats. This BHB will be delivered to those affected with AS to those that are on a standard American diet, the LGIT diet or the ketogenic diet. The metabolic sensitivity of AS forms the baseline for patients, but metabolic pathways used are different depending on diet. The hypothesis of the study is that BHB will enhance the effectiveness of dietary intervention by providing fuel substrates that push metabolism away from carbohydrates and toward fat utilization. The specific aims of the study are the following:

1) Assess tolerability of this supplement in patients with AS

2) Assess ketosis in AS patients

3) Assess safety in AS patients

The goals of this project are to evaluate the effects of IGF2 and IGF2RL in AS rat model in domains of: ultrasonic communication, motor function, learning and memory, and gait as well as structural rescue by MRI (e.g. white matter loss).

This project will capitalize on the pilot grant that was funded in 2017 showing that brief, noninvasive, low-cost brain-based measures of learning in children and adults with deletion subtype of AS can complement and extend informant reports of adaptive functioning. Without the need for voluntary behavioral response, these measures are optimal across ages and levels of intellectual and communicative abilities. This project compared auditory evoked brain response potential (ERP) associated with language and learning and memory across individuals of different ages, focusing on the deletion genotype and then moving to other genotypes. A novel behavioral procedure (eye tracking) was used to objectively measure language understanding in persons with AS. Then the relationship was evaluated between these markers of speech and language processing and symptom severity, along with age-related differences. By documenting sensitivity of the objective measures to individual differences among persons with AS it will establish them as effective markers of cognition in AS, suitable for objective measurable biomarkers that could be translated into meaningful outcome measures.

This grant was funded for Dr. Bryce Reeve at Duke University, an expert in clinical outcome measure development.  The goal is to define and label the construct to measure caregiver observations in their child’s ability for expressive, receptive and pragmatic communication and ultimately develop a measurement tool that can be used pre-competitively in human clinical trials to measure a patient’s ability to communicate.

Novel strategies to treat Angelman syndrome are needed as there is currently no transformative corrective therapy, and supportive care only marginally alleviates symptoms. If normal levels of ubiquitin ligase protein E3A (UBE3A) are expressed, regression of pervasive disease pathology and behavioral symptoms is an achievable goal. Advances in rat genetic models, facilitated by FAST, herald a new era of behavioral genetics. Concomitantly, we seized the opportunity, by developing sophisticated assays for the more appropriate model species, to facilitate the discovery of treatments for disorders with social communication and intellectual disability. The goal is to utilize UC Davis’ innovative outcome measures to identify AS relevant functional phenotypes in the Ube3a mutant rat. Future directions will test a variety of therapeutics, by measuring acoustic social communication and touchscreen based learning and memory.

The lack of UBE3A function in neurons of those with AS leads to the accumulation of synaptic proteins that ultimately result in dysregulation of synapse formation and function, also known as a synaptopathy. This group discovered that insulin-like growth factor 2 (IGF-2) has an important role in learning and memory, and can act as a potent memory enhancer through the regulation of synaptic mechanisms. This drug is given subcutaneously and was shown to rescue numerous phenotypes in the mouse model of AS in a pilot study. This study expanded upon this work to evaluate robust phenotypic rescue in the AS mouse and explored the effect at different ages.

This program is designed to support a full pre-clinical plan to develop a novel gene replacement therapy for Angelman syndrome. This program will start with evaluating the natural history of the AS mouse model to fully assess neurobehavioral functional deficits at different ages from 2-6 months of age postpartum. The most robust behaviors will then be focused upon when AAV-Ube3a is used to replace the deficient gene and assess for behavioral rescue. The most commonly known Ube3a isoforms will be evaluated independently to assess for most robust behavioral rescue to determine the best gene therapy candidate. Then the transgenes will be optimized and neurotoxicity evaluated. Once that is completed then NHP feasibility studies will be evaluated to explore the preliminary safety profile for high dose AAV.UBE3A for the human gene therapy candidate which will drive conversation with the FDA in moving this gene therapy forward toward a clinical trial.

Due to the limitations of standardized neurodevelopmental testing in individuals with AS, obtaining an accurate picture of the neurodevelopment profile is challenging. Standardized tests, like the Bayley Score of Infant and Toddler Development-3 (BSID-3), are not adapted to children with special needs, and this results in those individuals being underestimated on a test score. It is recommended that individuals with intellectual disability be evaluated with measures that are better suited for their developmental age. The BSID-3 developers have made allowances within the manual for making accommodations for children with special needs, however, in order to be useful for research studies, these accommodations need to be standardized. While these adaptations have been developed in other languages including the BSID-3 Special needs version, this is available in Dutch and not English. This study, in collaboration with the ABOM, was designed to modify the English version of the BSID-3 to create a special needs edition, and reduce dependence on expressive language and fine motor skills in order to more accurately assess the neurodevelopment of AS individuals.

This is a pilot grant funded by the ABOM in 2017 showing that brief, noninvasive, low-cost brain-based measures of learning in children and adults with deletion subtype of AS can complement and extend informant reports of adaptive functioning. Without the need for voluntary behavioral response, these measures are optimal across ages and levels of intellectual and communicative abilities. There is no need for expressive language to get a sense of the brain activity associated with receptive language.

This program supported further exploration of various ASO candidates for paternal gene activation through antisense technology.

This project funded Agilis Biotherapeutics to work with University of South Florida to establish a novel gene therapy approach to examine the unique delivery methods of AAV in conjunction with a uniquely engineered E6-AP protein that was designed to allow secretion followed by uptake in neurons. This is an opportunity to bypass the limitation of limited neuronal transfection of AAV and distribute more Ube3a into neurons of the brain.

Dr. Beaudet proposed to prepare a series of humanized mouse lines with human DNA insertions of various sizes from 10kb to over 100kb expressing the human UBE3A antisense transcript. All lines will be characterized as to expression of relevant transcripts and for the ability to demonstrate knock-down of the human antisense. One goal of this grant was to make all lines freely and rapidly available to any researchers, academic or corporations working on Angelman syndrome.

The combination of gene therapy with hematopoietic stem cells (HSC) offers a promising approach for constitutive and life-long delivery of Ube3a to affected cells. By genetically modifying a patient’s own blood stem cells (HSCs), the modified cells can make future cells that are capable of migrating into the central nervous system (CNS) and provide a functional Ube3a copy to deficient neurons throughout the brain through a mechanism called “cross-correction.” This is a novel approach in AS in order to distribute Ube3a to more neurons of the brain than what might be achieved with traditional gene therapy using AAV.  This project created a humanized mouse line to accommodate the human UBE3A gene within human blood cells, and then tested behaviors from different ages to evaluate phenotypic rescue.

This proposal will investigate the mechanism by which the UBE3A-ATS achieves silencing of UBE3A, which is currently unknown. We hypothesize that UBE3A-ATS silences UBE3A by an epigenetic mechanism that involves the 3′ end of UBE3A-ATS.

This project aims to develop the use of hormones as biomarkers in the screening and diagnosis of Angelman syndrome (AS) (prevalence 1/15000). There is an unequivocal need for an affordable, noninvasive and reliable biomarker that can predict the onset of or diagnose AS. Our novel basic science research work suggests that certain hormone levels are rendered abnormal in the pathobiological course of this disease, thus rendering these hormones as testable screening biomarkers for AS. Thus, quantification of hormone levels, via High Performance Liquid Chromatography (HPLC) or any other validated method, in human blood samples could be used as a screening, as well as a diagnostic tool for AS, and maybe other neurological disorders. To our knowledge, the “use of hormones as biomarkers for screening and diagnosis of AS” is a highly innovative idea with an exciting potential. To accomplish this objective, we have set the following specific aims: 1- To establish, in house, quantification of hormone levels in blood samples of normal humans via HPLC based on an already published method in the literature. 2- To compare hormone levels in AS patients versus their normal siblings. The timeline of this project is summarized as follows: Aim 1 is predicted to be completed the first 3 months of year 1. Aim 2 is predicted to be started in the second month of year 1 and completed by end of year 1. As for the therapeutic significance, the need for a non-invasive routine AS screening test during the first months of infancy and at later age, and the need for an affordable molecularly defined diagnostic test are highly warranted for AS. As for the group of patients that has been identified with AS on clinical basis only, this kind of molecular test is highly necessary. Thus, hormone markers could be instrumental in: 1- Affordably screening and predicting incidence of AS in infants starting day one after birth, and possibly even before birth. 2- Allowing possible prophylactic and protective clinical measures to be taken before onset of disease in cases where test is positive in infants. 3- Opening the door to research related to prophylactic measures that can be taken to prevent or to alleviate onset of symptoms seen in AS. 4- Possibly providing a molecular basis for AS cases that are solely diagnosed on a clinical basis. 5- Possibly presenting a new reliable diagnosis tool for AS.

Loss of the maternally inherited ubiquitin E3A ligase (UBE3A) gene causes Angelman syndrome (AS), a devastating neurological disorder characterized by intellectual disability, ataxia, absent speech, seizures, and a happy disposition [1]. The UBE3A gene is imprinted with maternal-specific expression in the brain and biallelic expression in all other cell types. Consequently, mutations affecting the maternal UBE3A allele cause AS, whereas mutations affecting the paternal allele are non-penetrant. Currently, there are no effective therapies to treat AS patients. There are, however, a number of promising therapeutic strategies recently identified using mouse models of AS. Huang et al. (2011) demonstrated that topoisomerase inhibitors — chemotherapeutic agents used to treat cancer — reactivate the paternal Ube3a allele in the adult mouse brain [2]. Topoisomerase inhibitors are highly toxic, so their use as an AS therapy at this point is unclear. Nevertheless, this study demonstrated that the Ube3a imprint is amenable to pharmacological intervention and may serve as a viable AS therapeutic. Daily et al. (2012) showed that gene therapy improves cognition in adult AS mice, indicating that AS is indeed treatable [3]. There are currently numerous laboratories working to identify therapies for AS, including treatments that have shown promise in mouse models of other conditions (e.g., Rett and Fragile-X syndromes); however, there is a critical need for preclinical models more physiologically similar to humans to validate and evaluate these promising therapeutics. Here, we propose to develop and characterize a pig model of AS. Our long-term goals are to understand the pathogenesis of AS and develop therapies to treat this debilitating condition. Our central hypothesis is that the AS pig model will recapitulate many of the phenotypes characterized by AS, and serve as a final-stage preclinical model for testing promising therapeutics. The objectives of this proposal are to generate a pig model with a loss-of-function mutation in the porcine UBE3A gene and characterize it for AS relevant phenotypes.

This project aims to gather empirical data on how assistive technology can support literacy and communication development in school-aged children with Angelman syndrome, in order to inform ongoing development of effective interventions for this population.  Data gathered from this camp will contribute to the development of reliable evaluation models for students with Angelman syndrome. This camp provides an opportunity to field-test and refine existing assessment tools and to examine the role of assistive technology in more accurately assessing this population. We will evaluate students in the areas of phonological and phonemic awareness, developmental writing, and the use of symbols in communication. We will test the use of existing assessment tools such as early conventional literacy measures (e.g.: the QRI) and emergent measures, such as the Bridge Observational Rating Scales, the Developmental Writing Scales, Developmental Spelling Test, Pragmatics Profile, the Kovach AAC Profile, the Staugler Literacy Rubric, the Hanser 7 point rating scales, and more. Adults will be trained to self-assess their own support behaviors.

Angelman syndrome (AS) is a complex genetic disorder that affects the nervous system. AS affects an estimated 1 in 12,000 to 20,000 individuals. Characteristic features of AS include developmental delay or intellectual disability, severe speech impairment, seizures, small head size, and problems with movement and balance. Deregulation of the expression of a protein called E6 Associated Protein (E6-AP) is tightly associated with AS. E6-AP has been described to have two functions: the first is a function that mainly leads to the degradation of other proteins, and the second, first described by us, is to activate the DNA transcription of other genes through steroid hormones and their receptors like Estrogen Receptor alpha. Up till now, most of the published studies have examined the role of the first function of E6-AP in the development of AS and it is not known if the second function of E6-AP plays a role in the pathology of AS. In this grant application, we will examine the role of the transcriptional coactivation function E6-AP through Estrogen Receptor alpha signaling in the development of AS. We have taken an interdisciplinary approach by connecting the steroid hormone-signaling field with neuroscience, from which we expect to find exciting results that would hugely impact future research on both fields. This will provide us with valuable information on the currently unidentified downstream effectors of E6-AP. Identification of pathways that are transcriptionally regulated by E6-AP would also lead to the development of novel targeted molecular therapies for AS. Our novel proposed hypothesis is based on extensive evidence indicating that E6-AP has two important biological roles, which the research community has failed to connect: 1) E6-AP is a known coactivator for Estrogen Receptor and 2) E6-AP is a gene associated with AS. Innovation, in this grant application, is mainly presented by focusing on the transcriptional co-activator function of E6-AP through Estrogen Receptor. We believe that the combination of such an innovative idea with practical standard methodologies will greatly contribute to the feasibility of the proposed studies. This project will provide a prime source for the discovery of new molecular pathways and their role in neuropathology of AS. Accomplishing the proposed studies in this grant will not only provide new insights into the mechanism of action of E6-AP and ER in AS but will also provide promising new therapeutic targets and venues for AS patients.

The Segal Lab at the UC Davis Genome Center has created a potential treatment of Angelman syndrome by creating an injectable protein that allows the brain to produce the protein Ube3a. The lack of Ube3a has been determined to be the root cause of AS and there are indications that if it is restored, some alleviation of symptoms may be possible.  Significant progress has been made in a mouse model of Angelman syndrome, and future work will extend these efforts to rat models as well as human cells. This Grant-in-Aid will enable equipment upgrades to be purchased to overcome a critical current limitation in visualizing the RNA and protein levels of Ube3a in the brain following treatment.  Since the Segal Lab has specialized in creation of targeted gene approaches to various molecular studies, these upgrades will allow the group to more rapidly evaluate test results and determine the effectiveness of the proposed treatment.

Angelman syndrome (AS) is a devastating disorder characterized by severe intellectual disability, absence of speech, abnormal gait, seizures, and inappropriate laughter. Loss of function or loss of expression of the maternal, but not paternal, UBE3A allele results in AS due to genomic imprinting of the gene in the brain. The mechanisms regulating genomic imprinting of UBE3A remain poorly understood. To address these important questions, our laboratory has initiated a number of molecular, epigenetic and genetic studies to identify factors regulating genomic imprinting of Ube3a in the brain. In our preliminary studies, we have found that Ube3a is expressed from both paternal and maternal alleles in neural stem cells (NSC) within the hippocampus of mice. Differentiation of these stem cells leads to repression of the paternal allele in neurons, but not in astrocytes. In specific aim 1, we will utilize RNA interference technology to identify epigenetic modifiers initiating and maintaining repression of the paternal Ube3a allele in neurons. Results from this study will provide valuable insight into the fundamental mechanisms regulating genomic imprinting of Ube3a in the brain and may provide the foundation for therapeutic strategies aimed at reactivating the paternal UBE3A allele in AS patients.

Angelman syndrome (AS) is a rare genetic disorder that presents with seizure, ataxia and cognitive impairment, and is genetically and biochemically associated with other cognitive disorders such as autism and Fragile X syndrome (Clayton-Smith and Pembrey 1992; Curia, Papouin et al. 2009). There is currently no known treatment of AS.  AS arises through the genetic or biochemical disruption of the maternal UBE3A allele, which when coupled with typical epigenetic silencing of the paternal UBE3A allele, gives rise to an absence of Ube3a protein in the CNS (Kaplan, Wharton et al.1987; Buiting, Saitoh et al.1995; Matsuura, Sutcliffe et a. 1997; Gustin, Bichell et al. 2010). Our research suggests that intervention in adult AS mice has the potential to rescue the cognitive and behavioral phenotypes prevalent in the AS mouse model (Daily, Nash et al. 2011). The current proposal is focused on short- to long-term development of potential therapeutics and identification of novel therapeutic targets for the treatment of Angelman syndrome.  A particular emphasis is made in three area of research: a) identifying drug precursors that can activate the silenced paternal Ube3a allele; b) evaluating pre-existing therapeutic agents of efficacy in improving behavioral, cognitive and synaptic plasticity defects and reducing seizure/epilepsy propensity; and c) determination of new molecular targets for AS intervention. These aims will be performed using the Ube3a maternal deficient mouse model of Angelman syndrome.  Concurrently, we will engineer a rat model of the disorder in order to complement this mouse model and propel therapeutic development efforts for AS forward.

Angelman syndrome is a rare genetic disorder characterized by phenotypic traits such as global developmental delay, ataxia and seizure. Children diagnosed with AS display a behavioral profile consisting of a happy demeanor with easily provoked laughter and hyperactivity. Building on our previous FAST-TRAC studies, the objective of this study is to examine the efficacy of minocycline to improve the cognitive and behavioral deficits of the Angelman syndrome participant. Currently, the only treatment available for AS patients consists of supportive care including seizure control and behavioral therapy. Our laboratory has collected data indicating improved motor function and cognition after the administration of MC as well as enhanced synaptic function. Further, previously published data suggests the administration of MC to children with other genetically based neurologic disorders (e.g.Fragile X syndrome) dramatically improved dendritic spine morphology and behavioral performance. Taken together, we posit, children treated with MC will experience the same beneficial effects observed in previous studies and the AS mouse model.

This award supports the work of Christina Castellana FAST Postdoctoral Fellow Jason J. Yi, Ph.D., by providing funds to cover supplies and service costs associated with the project.

The previous FAST-TRAC award to Dr. Weeber identified minocycline as a potential therapeutic for Angelman syndrome; reversing deficits of learning, memory, and motor skills in the AS mouse model.  This award provides additional funds to repeat these studies and increase the number of mice analyzed and add additional testing methods for testing the efficacy of minocycline in this model.  These studies will form the basis of the application for IRB approval for a potential clinical trial of minocycline in patients with AS.

As an E3 ubiquitin ligase, UBE3A catalyzes a reaction to label certain proteins in the cell so that they are targeted for degradation. Mutations that abolish this activity are sufficient to cause Angelman syndrome (AS). Typically, ubiquitin ligases recognize several substrates, and numerous substrates of UBE3A have been proposed. Thus, UBE3A likely regulates an ensemble of proteins that contribute to AS pathogenesis, and therefore, targeting individual components of this group for AS therapies may not be sufficient to alleviate neurological deficits associated with AS. Increasing evidence suggests that abnormal variations in the quantity of UBE3A itself may be at the heart of neurodevelopmental diseases. UBE3A resides within chromosome 15q11-13, a heavily imprinted genomic region associated with a variety of neurodevelopmental disorders. In the brain, UBE3A is only expressed from the chromosome inherited from the mother. Children who inherit a maternal chromosome carrying a deletion in 15q11-13 develop AS, whereas the majority of children who inherit a maternal chromosome carrying duplications in 15q11-13 develop Autism spectrum disorder. Therefore, understanding the factors that control the cellular quantities of UBE3A may provide an effective strategy for the design of AS therapeutics. My preliminary work has found that UBE3A stability is subject to regulation by enzymes that act upstream of UBE3A. This identifies for the first time biological mechanisms within cell that function to maintain cellular UBE3A quantities. I have proposed a system of experiments that investigates how UBE3A misregulation leads to learning defects in individuals with AS. My approach will use recent advances in biosensor development as well as sophisticated microscopy techniques to visualize directly in the brain how loss of UBE3A function perturbs normal events that occur during learning and memory at synapses. Moreover, these experiments will simultaneously allow me to identify pharmacological targets for AS therapy, and test how manipulation of these targets affects the properties of neurons lacking UBE3A. I expect that these experiments will provide insight into previously unknown mechanisms of AS pathogenesis and provide novel targets for the development of therapeutic strategies in AS. The ultimate goals of these studies is to (1) identify the molecular causes of AS by understanding UBE3A regulation and (2) using this information, develop potential pharmacological strategies for AS treatment.

There is a growing consensus in the scientific community that believes that a treatment for Angelman syndrome (AS) is not just possible, but very probable. However, the lack of known therapeutic targets at the cellular level that underlies the mechanisms of AS has hampered the development of therapeutic strategies. Coupled with the laborious and timely task of obtaining FDA approval once a therapeutic strategy is found, it quickly becomes evident that a treatment for AS is years or maybe even decades away. With these roadblocks in mind, this proposal tries to circumvent both of these deficiencies in regard to successfully and responsibly developing a therapeutic strategy for the treatment of AS. In this regard, this proposal does not focus, necessarily, on understanding mechanisms of AS but rather treating AS. Two main concepts were taken into consideration when I developed this proposal: shortening the time to elucidate the underlying mechanisms of AS and shortening the amount of time to have a therapeutic strategy FDA approved. This was accomplished in two ways; 1) Use pharmacological agents that are known to have correlates to counter the molecular or cognitive deficiencies involved with AS. 2) Use pharmacological agents that are already FDA approved for use in humans and have an established treatment regimen. The use of these two strategies will significantly reduce the amount of time from experimental testing, to preclinical evaluation to a working and publicly available treatment for AS. To test the validity of these compounds, the AS mouse model will be used and four compounds tested and treated AS mice compared to wild-type mice at the levels of 1) Degree of cognitive enhancement 2) Rectification a biological and genetic abnormalities 3) Increases in neuronal connectivity and neuronal efficiency. It is my hope that one of these compounds will have a positive effect on one or more of these aspects that underlie AS. Furthermore, any and all positive results will prompt a full preclinical evaluation of the compound(s) and could potentially lead to the development of an effective AS therapeutic strategy.

There is mounting evidence to suggest that a treatment for Angelman syndrome is not just possible, but probable. The lack of known molecular targets associated with AS has hampered the development of specific therapeutics. However, a recent surge of potential therapeutics for other disorders associated with cognitive disruption has begun to be used in human clinical trials. The molecular modes of action for many of these new therapeutic agents have correlates to counter the molecular defects observed in AS. Thus, this proposal seeks to determine the effectiveness of compounds that are FDA approved and currently being used in clinical trials on the well-established AS mouse model. We propose to look at 4 of these compounds at the level of: 1) Degree of cognitive enhancement. 2) Rectification of a biological and genetic abnormality. 3) Increase in synaptic function and/or plasticity. It is our hope that these compounds will have a positive effect on one or more of these aspects. Furthermore, any positive results will prompt a full preclinical evaluation of the compound(s) and may lead to the development of an effective AS therapeutic.

Summary: Angelman syndrome (AS) is a neurodevelopmental disorder with genetic causes. It has been observed that altered gene interactions within different areas of the brain give rise to the symptoms characteristic of AS patients. The focus of the study will be to investigate and discover how genes interact with each other in the area of the brain known as the hippocampus. This brain area is involved in learning, and it is of major significance and relevance to the pathology of AS. We will study tissue from the hippocampus area of the brain from normal and AS subjects. By identifying which gene interactions are dysregulated in the case of AS subjects as compared with normal subjects, we will be able to find a number of possible approaches to intervene and steer the responsible gene interactions toward a normal state. We think that some of those approaches will have the potential to lead to therapeutic treatment for AS patients. We have developed a biomarker platform technology that is capable of identifying not only genes that play a significant role in a given disease but also how those genes are interconnected, how they influence each other, and in what way their networks and overall function differ from the normal state. By applying our technology to the area of AS we think that we will be able to 1) identify the altered gene networks responsible for AS in the hippocampus and 2) find possible targets for therapeutic development to influence those altered gene networks toward a normal pattern.