FAST Funded Research

Our cells use DNA and RNA as instruction manuals to make proteins that our body needs to function. In disorders like Angelman syndrome (AS), errors in DNA or RNA can lead to the wrong instructions. This can result in the body producing too much, too little, or non-functional proteins. This can cause serious health problems. Scientists have developed special treatments called antisense oligonucleotides (ASOs) to help fix these mistakes. ASOs are made by stringing together building blocks called bases. Current drugs that are being developed for AS use specialty bases, chemistries and modifications to improve the drug delivery and penetration to cells. These ASOs in the clinic need to be injected directly into the spinal fluid multiple times a year because they are metabolized and don't last more than a couple of months after the injection. Theoretically, these drugs can be modified through chemical reactions and new building blocks. This team aims to engineer the enzymes to make new building blocks with more efficiency. The learnings may provide researchers with new opportunities for next generation ASO therapies that last longer.

This grant was generously funded by the Maddie's Mission Foundation.

Base editing is a precision gene-editing approach to make targeted and lasting changes to DNA. This project explores the use of base editing to disrupt the Ube3a antisense transcript (Ube3a-ATS), a mechanism that keeps the Ube3a gene turned off in neurons. The goal of this approach is to increase Ube3a expression in the neurons of the brain. To deliver this to neurons, the system must be packaged inside a carrier, and in this project the carrier is a lipid nanoparticles (LNPs). LNPs have been used successfully in the delivery of therapies like mRNA and can be targeted to different organs in the body by modifying their chemical composition. The goal here is to target the brain through various different modifications to the carrier.

Chemically modified divalent silencing RNA’s (di-siRNA’s) are potent oligonucleotide based therapeutics potentially capable of widespread brain distribution and prolonged target gene silencing. The goal of this project is to identify candidate di-siRNA’s that can silence the paternal UBE3A antisense transcript, which typically silences the expression of the paternal UBE3A gene. The goal is that this may allow for re-expression of the paternal UBE3A gene. The team will use pluripotent stem cell-derived neurons from Angelman syndrome (AS) patients and mouse primary neurons to screen and identify human and mouse candidate di-siRNA’s, respectively. Identified mouse candidate di-siRNA’s will then be injected into the brain of the AS mouse model to test their efficacy in re-expressing paternal Ube3a and safety. These studies are critical for determining the feasibility of this approach and, if promising, will support further preclinical testing to potentially develop this technology into a future therapeutic.

Knowing that Angelman Syndrome (AS) is a brain disorder caused by a missing or nonfunctional gene called UBE3A, many treatments attempts are trying to turn on the silent copy of this gene. Some require frequent injections, while others use complicated gene therapies using a virus to carry the payload, which are expensive. This team is developing a new

treatment strategy that works with the body’s natural processes to switch a silent gene back on in a precise and long-lasting way. The hope is that this could lead to a safer, easier, and more effective treatment option for individuals living with AS.

Angelman syndrome (AS) is caused by the loss of UBE3A protein expression in the neurons of the brain. Typically, one copy of the UBE3A gene is missing (deleted) or mutated (maternal), while the other copy of UBE3A (paternal) is functional but silenced, or turned off. This silencing is due to the presence of a long non-coding RNA that is preventing the expression of the UBE3A gene on the fathers copy.

In prior FAST funded work by this laboratory, the team demonstrated that they can make a therapeutic protein that can unsilence paternal UBE3A expression, restoring the functional Ube3a protein in the neurons of the brain and improve the symptoms in a mouse model of AS. This project is taking human neurons to find the right candidates to unsilence the paternal UBE3A gene. The goal is to rapidly screen through many designs of zinc figer proteins to get the one that is highly specific for unsilencing the human UBE3A gene, while not affecting other genes. The prior work in mice show that this could be a potential therapeutic option for AS.

Angelman syndrome (AS) results from the loss of functional UBE3A expression. In the case of large deletions, which account for the majority of individuals living with AS (~60-70%), not only is UBE3A deleted but an additional 10 or so surrounding genes are also missing from the maternal allele in the 15q11-13 region. Deletion of these genes results in haploinsufficiency, or half the amount that is generally present, and this can contribute to the increased symptom severity seen in those individuals living with the deletion genotype. This project will explore a unique therapeutic avenue that aims to upregulate some of the most important critical genes in this region, outside of UBE3A, looking at them all individually and collectively. They will be evaluated in both a cellular (in vitro) and rodent model (in vivo). 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 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 both in vitro and in vivo. In addition, Dr. Jiang has designed a 2 large deletion AS mouse models (6MB), one with Ube3a expression intact and one with the entire segment of 6MB missing. These models we hope will help to best understand how the loss of these surrounding genes affect mouse behavior relevant to AS. The overarching goal of this project is to understand how upregulating the additional haploinsufficient genes on the paternal allele may lead to therapeutic benefit beyond addressing just the loss of Ube3a.

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 distinct chance to invest in a potential therapy that can target the few additional essential genes beyond UBE3A, which may one day offer an additional treatment option to improve the lives of those living with a large deletion genotype of AS.

The goal of this proposal is to create a stable infrastructure for the rapid testing of potential therapeutics in human stem cell-derived models of Angelman syndrome (AS) including two-dimensional neurons and three-dimensional cerebral organoids. The evaluation of the cellular entry, neuronal function, and molecular readouts to inform a potential therapeutic, in a high throughput and reliable manner, is highly desirable. This funding is meant to create a stable infrastructure to reliabily test drug candidates for academic researchers and industry. Overall, this can decrease the time and maximize the expertise for all working on AS. This effort also benefits the training and retention of skilled personnel dedicated to AS research, having an even longer-term impact on the Angelman field. Creating a highly synergistic system for FAST to have a rapid, reliable, and interactive resource for testing therapeutic candidates, developing shareable assays and tools, and creating a team of researchers focused on advacing AS drug development, will advance the field.

The overall goal of the Natural History Study (NHS) is to support the Angelman syndrome (AS) community by: 1) seeking answers to questions pertaining to the clinical management and care of children and adults living 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 prospective longitudinal data that can be used in the design of clinical trials by industry who are developing potential therapies for Angelman syndrome by following patients over a period of many years to best understand the trajectory of change naturally, without treatment; 3) to assess outcome measures and biomarkers that are, or may one day be, used in clinical trials to understand how they behave in this population. Supporting the AS NHS allows clinical trial design to be well vetted for functionality, assessment burden, and baseline expectations, which will help to inform later stage drug development for sponsors.

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 finding the best delivery method for all of the therapies being developed. The brain is 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 challenging and could be a 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 could bring the AS field closer to a mode of delivery that is minimally invasive and can potentially be repeated. This has the potential to benefit all therapeutic modalities in AS and have a broad impact in the entire neurodevelopmental field.

Exciting scientific progress over the past ten years has led to clinical trials that have the potential to impact the symptoms of Angelman syndrome at its root cause. What all clinical trials have in common is the need to reliably measure clinically meaningful improvement as a results 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 change in clinical trials and give a clear objective sense of improvement over time. 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 grant, the team is developing 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 a clinical trial in individuals of all ages.

This grant was funded to create and characterize a novel ~6MB full deletion Angelman syndrome mouse model, which is representative of the largest population of individuals living with AS (>70%). This work will contribute to the understanding of the other genes impacted, outside of UBE3A, by the large deletion genotype. As we near a therapeutic aimed at restoring UBE3A into neurons in humans living with Angelman syndrome (AS), our understanding of the role these additional genes play in the symptoms of deletion individuals is critical.

The goal of this grant is to develop an outcome measure to assess caregiver observations in their child’s communication ability in expressive, receptive and pragmatic communication. The goal is to develop a measurement tool that can be used pre-competitively in human clinical trials that can granularly measure a non-verbal patient’s ability to communicate. This tool is designed specifically for individuals living with AS.