2011 ASF Funded Research
Principle Investigator: Benjamin Philpot, PhD
Institution: University of North Carolina at Chapel Hill
Chapel Hill, NC
Study Title: Preclinical testing of a candidate Angelman syndrome therapeutic
$200,000 – 2-years
The core symptoms of Angelman syndrome are caused by the loss of a single gene – UBE3A. Most genes are biallelically expressed, meaning cells express gene copies you inherit from your mother and father. In fact, UBE3A is biallelically expressed throughout the body with the notable exception of the brain. For still unknown reasons, only the maternal copy of UBE3A is expressed in the brain while the paternal copy lies dormant. As a result, mutation or deletion of the maternal copy is sufficient to cause Angelman syndrome (AS), even though they have a functional (but dormant) paternal copy of UBE3A. This suggests it might be possible to treat AS by “awakening” the intact paternal UBE3A allele. In collaboration with the labs of Dr. Mark Zylka and Dr. Bryan Roth, both at the University of North Carolina, our labs made the exciting discovery that certain small molecule compounds can indeed awaken the paternal UBE3A allele in a mouse model of Angelman syndrome. This raises the exciting possibility that these small molecule compounds could be used as Angelman syndrome therapeutics. To begin testing this idea, we will perform preclinical studies of our lead compound in a mouse model of Angelman syndrome. These studies will help us determine if this compound can be pursued for clinical studies in humans.
Principle Investigator: Mark Zylka, PhD
Institution: University of North Carolina at Chapel Hill
Chapel Hill, NC
Study Title: Molecular mechanisms and biomarkers of a candidate Angelman syndrome therapeutic
$200,000 – 2-years
The core symptoms of Angelman syndrome are caused by the loss of a single gene – UBE3A. Most genes are biallelically expressed, meaning cells express genes you inherit from your mother and father. In fact, UBE3A is biallelically expressed throughout the body with the notable exception of the brain. For still unknown reasons, only the maternal copy of UBE3A is expressed in the brain while the paternal copy lies dormant. As a result, mutation or deletion of the maternal copy is sufficient to cause Angelman syndrome (AS), even though everyone also has a functional (but dormant) paternal copy of UBE3A. This suggests it might be possible to treat AS by “awakening” the intact paternal UBE3A allele. In collaboration with the labs of Dr. Ben Philpot and Dr. Bryan Roth, both at the University of North Carolina, our labs made the exciting discovery that certain small molecule compounds can indeed awaken the paternal UBE3A allele in a mouse model of Angelman syndrome. This raises the exciting possibility that these small molecule compounds could be used as Angelman syndrome therapeutics. To begin testing this idea, we will determine how these drugs mechanistically regulate Ube3a expression and identify biomarkers that could be used as clinical indicators of drug efficacy.
Principle Investigator: Arthur Beaudet, MD, PhD
Institution: Baylor College of Medicine
Study Title: The role of antisense RNA Ube3a-ATS in Ube3a imprinting and Angelman syndrome
$100,000 – 1-year
Angelman syndrome is caused by deficiency of UBE3A. Unlike ordinary autosomal genes, it is subject to genomic imprinting with expression only from maternal chromosome. It is unknown how such imprinted status is established, since no differential DNA methylation was found to be associated with the UBE3A locus. On the other hand, an antisense non-coding RNA named UBE3A-ATS overlapping UBE3A locus was identified with mono-allelic expression from paternal chromosome. Extensive negative association between Ube3a-ATS and Ube3a was reported from both mice and human studies. In this proposal, we hypothesize that Ube3a-ATS directly mediates paternal Ube3a silencing by inhibiting its transcription elongation. To test this, we have generated a mouse model with truncated Ube3a-ATS expression. Studying of this mouse model will reveal the role of Ube3a-ATS in Ube3a imprinting and Angelman syndrome. Binding of transcription initiation complex to Ube3a promoter was found to be equal on both paternal and maternal chromosomes. To test if paternal Ube3a silencing is caused by failure of transcription elongation, nascent RNAs immunoprecipitated (RNA-IP) with RNA polymerase II will be analyzed. Finally, profiles of histone modifications of Ube3a will be studied by ChIP (chromatin-immunoprecipitation)-chip, to further expand our knowledge about Ube3a imprinting. Overall, we propose to study the role of antisense RNA Ube3a-ATS in Ube3a imprinting and their interaction using mouse models. UBE3A-ATS may be developed as new therapeutic target for inactivation in Angelman syndrome to reactivate silenced paternal UBE3A, and our research will provide unique insight into the relevant regulatory mechanisms.
Principle Investigator: Michael Greenberg, PhD
Institution: President and Fellows of Harvard College, Harvard Medical School
Study Title: Validation of Arc and Ephexin5 as Novel Therapeutic Targets for the Treatment of Angelman Syndrome
$200,000 – 2-years
While it has been appreciated for some time that loss-of-function mutations in the UBE3A gene are the primary causative factor for Angelman syndrome (AS), the precise mechanisms by which the loss of Ube3A in the nervous system gives rise to the spectrum of cognitive and behavioral features characteristic of this disorder remain unclear. This gap in knowledge has significantly limited the scope of clearly defined therapeutic strategies for AS. Moreover, therapeutic interventions based on the reactivation of mutated genes such as UBE3A have historically proven extremely challenging to implement. Thus, identification of the molecular mechanisms by which the loss of Ube3A function gives rise to the neurophysiological and cognitive dysfunction associated with AS will provide important insight into AS etiology and open new potential therapeutic avenues to combat AS.
We have recently identified two neuronal proteins, termed Arc and Ephexin5, that act as downstream targets of Ube3A. In the absence of Ube3A activity, elevated levels of these two proteins accumulate in neurons, and reduction of the levels of these factors in Ube3A-deficient neuronal cultures is sufficient to reverse specific cellular deficits associated with AS. Building on these findings, we propose to determine the extent to which elevated Arc or Ephexin5 expression contributes to the neurophysiological and cognitive dysfunction associated with AS in the context of a well-characterized mouse model of the disorder. Implication of one or more these factors in specific aspects of AS pathology will provide new opportunities for therapeutic intervention. It is thus our hope that the proposed experiments will provide a better understanding of Ube3A function, give insight into the etiology of AS, and provide new opportunities for the development of therapeutic strategies to treat this devastating disorder.
Principle Investigator: Eric Levine, PhD
Institution: University of Connecticut Health Center
Study Title: Pathophysiology in a human stem cell model of Angelman syndrome
$120,000 – 2-years
Angelman syndrome (AS) is a neurogenetic developmental disorder characterized by intellectual disability, motor ataxia, absent speech, and seizures. AS results from deletion of a specific gene normally expressed in the brain. In order to develop appropriate treatments, it is necessary to understand the cellular and molecular consequences of this gene deletion. Several mouse models of AS have been developed to address these questions, but until recently it has not been possible to examine the functional properties of human brain cells at the cellular and molecular level. The discovery of genomic reprogramming of human skin cells into induced pluripotent stem cells (iPSCs) provides a novel way to model human diseases with complex genetics. By reprogramming skin cells obtained from patient samples, cell lines can be isolated that have the potential to develop into functional brain cells. We have recently succeeded in reprogramming skin samples from AS patients, as well as age-matched control subjects, into iPSCs, and then differentiated the iPSCs into functional brain cells that maintain the specific genetic profile that is seen in AS patients. The proposed research will use these patient-specific cell lines to explore the underlying physiological and morphological defects in the brain cells of AS patients. Establishment of a human cell culture model of AS provides a unique opportunity to understand the cellular mechanisms that underlie the behavioral and developmental deficits observed in AS patients. This model may also be valuable for identifying novel targets for drug discovery and for screening potential therapeutics aimed at ameliorating and/or curing the seizures, movement disorders, and language and cognitive impairments in Angelman syndrome.
Principle Investigator: Ype Elgersma, PhD
Institution: Erasmus University Medical Center/ ENCORE expertise center for neurodevelopmental disorders
Study Title: An inducible mouse model for Angelman Syndrome: follow up
$92,144 – 1-year
It is currently not clear to what extend the UBE3A gene (the gene causing AS) plays a role in brain development (i.e. hard wiring of the brain), and to what extend it is required for normal brain function (i.e. neuronal plasticity), after brain maturation. Moreover, when considering a therapy which can alleviate the neurological symptoms of AS, it is essential that we know if, and to what extend, the neurological symptoms are reversible once the diagnosis is made. To phrase this question simply: “Imagine if we had solved AS, and developed the best possible drug (e.g. repair the UBE3A gene in all cells). How successful would such a treatment be for a 10 years old child?” The truth is that we have absolutely no clue! This knowledge is of critical importance since it ultimately determines whether effective treatment of the disease is ever going to be feasible. Moreover, pharmaceutical industry requires such a proof of principle before they are willing to screen for candidate drugs.
With previous support from the ASF, French and Italian AS parent organizations, we have engineered a mouse (Ube3ainducible) in which the UBE3A gene (responsible for causing AS) is non-functional when born. However, by just a simple injection at any desired age, we can now restore UBE3a gene function in every cell of the mouse, including the brain. Hence, this represents the ultimate ‘cure’.
We now propose to restore gene function at various ages, and assess the phenotype of these genetically cured mice, using various behavioral and electrophysiological assays. This will address to what extend the UBE3A gene is involved in brain development, and to what extend we can reverse the symptoms once brain development has taken place.
Although not offering a specific therapeutic intervention for patients, this work provides pivotal proof of principle that a putative mechanism-based treatment is still effective after the onset of the neurological symptoms. This knowledge is essential for future trials, aimed at alleviating the symptoms of AS. In addition, this project will give us valuable information in understanding the role of UBE3A in brain development and/or in mature brain function.
Principal Investigator: Scott Dindot, PhD
Institution: Texas A & M University
College Station, TX
Study Title: Examining rescue of neurological deficits in Angelman syndrome mice by expression of the E6-AP isoforms.
$84,011 – 1 year
Efforts are underway to develop a cure for Angelman syndrome (AS), including reactivation of the paternal UBE3A allele in the brain, drug therapies targeting pathways dysregulated by loss of E6-AP, and gene therapy. In this study, we propose to investigate whether gene therapy is a viable therapeutic option for individuals with AS and to examine the function of the E6-AP isoforms. We will use live animal testing of brain function and cell morphological studies in a mouse model of AS to determine if gene therapy can ameliorate the neurological deficits exhibited by these mice. This proposal will examine two unresolved questions regarding gene therapy in AS. First, we will examine whether re-introduction of UBE3A expression into the brain of post-adolescent and adult mice rescues abnormal neuronal morphology, reduces seizures, enhances learning and memory, and improves motor-coordination. Second, we will examine the efficacy of the 3 E6-AP isoforms to improve brain function in AS mice with the intent of identifying a particular isoform that can be used in gene therapy studies.