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.

Identification of UBE3A Protein in CSF and Extracellular Space of the Hippocampus Suggest a Potential Novel Function in Synaptic Plasticity.

Disruptions to the maternally inherited allele UBE3A, encoding for an E3 ubiquitin ligase, leads to the manifestation of Angelman Syndrome (AS). While this disorder is rare, the symptoms are severe and lifelong including but not limited to: intractable seizures, abnormal EEG's, ataxic gait, lack of speech, and most notably an abnormally happy demeanor with easily provoked laughter. Currently, little is known about the neurophysiological underpinnings of UBE3A leading to such globally severe phenotypes. Utilizing the newest AS rat model, comprised of a full UBE3A deletion, we aimed to elucidate novel mechanistic actions and potential therapeutic targets. This report demonstrates for the first time that catalytically active UBE3A protein is detectable within cerebrospinal fluid (CSF) of wild type rats but distinctly absent in AS rat CSF. Microdialysis within the rat hippocampus also showed that UBE3A protein is located in the interstitial fluid of wild type rat brains but absent in AS animals. This protein maintains catalytic activity and appears to be regulated in a dynamic activity-dependent manner. LAY SUMMARY: Angelman syndrome (AS) is a rare genetic disorder caused by the loss of the UBE3A gene within the central nervous system. Although we have identified the gene responsible for AS, we still have a long way to go to fully understand its function in vivo. Here we report that UBE3A is present within normal cerebrospinal fluid (CSF) but distinctly absent in AS CSF. Furthermore, we demonstrate that UBE3A is secreted and that this may occur in a dynamic activity-dependent fashion. Extracellular UBE3A maintained its ubiquitinating activity, thus suggesting that UBE3A may have a novel role outside of neurons. Autism Res 2021, 14: 645-655. © 2021 International Society for Autism Research and Wiley Periodicals LLC.

Generation of a Novel Rat Model of Angelman Syndrome with a Complete Ube3a Gene Deletion.

Angelman syndrome (AS) is a rare genetic disorder characterized by severe intellectual disability, seizures, lack of speech, and ataxia. The gene responsible for AS was identified as Ube3a and it encodes for E6AP, an E3 ubiquitin ligase. Currently, there is very little known about E6AP's mechanism of action in vivo or how the lack of this protein in neurons may contribute to the AS phenotype. Elucidating the mechanistic action of E6AP would enhance our understanding of AS and drive current research into new avenues that could lead to novel therapeutic approaches that target E6AP's various functions. To facilitate the study of AS, we have generated a novel rat model in which we deleted the rat Ube3a gene using CRISPR. The AS rat phenotypically mirrors human AS with loss of Ube3a expression in the brain and deficits in motor coordination as well as learning and memory. This model offers a new avenue for the study of AS. Autism Res 2020, 13: 397-409. © 2020 International Society for Autism Research,Wiley Periodicals, Inc. LAY SUMMARY: Angelman syndrome (AS) is a rare genetic disorder characterized by severe intellectual disability, seizures, difficulty speaking, and ataxia. The gene responsible for AS was identified as UBE3A, yet very little is known about its function in vivo or how the lack of this protein in neurons may contribute to the AS phenotype. To facilitate the study of AS, we have generated a novel rat model in which we deleted the rat Ube3a gene using CRISPR. The AS rat mirrors human AS with loss of Ube3a expression in the brain and deficits in motor coordination as well as learning and memory. This model offers a new avenue for the study of AS.

Voltammetric Study of Some 3-Aryl-quinoxaline-2-carbonitrile 1,4-di-N-oxide Derivatives with Anti-Tumor Activities.

The electrochemical properties of twenty 3-aryl-quinoxaline-2-carbonitrile 1,4-di-N-oxide derivatives with varying degrees of cytotoxic activity were investigated in dimethylformamide (DMF) using cyclic voltammetry and first derivative cyclic voltammetry. With one exception, the first reduction of these compounds was found to be reversible or quasireversible and is attributed to reduction of the N-oxide moiety to form a radical anion. The second reduction of the diazine ring was found to be irreversible. Compounds containing a nitro group on the 3-phenyl ring also exhibited a reduction process that may be attributed to that group. There was good correlation between molecular structure and reduction potential, with reduction being facilitated by an enhanced net positive charge at the electroactive site created by electron withdrawing substituents. Additionally, the reduction potential was calculated using two common basis sets, 6-31g and lanl2dz, for five of the test molecules. There was a strong correlation between the computational data and the experimental data, with the exception of the derivative containing the nitro functionality. No relationship between the experimentally measured reduction potentials and reported cytotoxic activities was evident upon comparison of the data.