Graduation Year

2022

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Kevin Nash, Ph.D.

Committee Member

Paula Bickford, Ph.D.

Committee Member

Javier Cuevas, Ph.D.

Committee Member

Danielle Gulick, Ph.D.

Committee Member

Edwin Weeber, Ph.D.

Keywords

rAAV, gene threrapy, neurodevelopmental disease

Abstract

Angelman syndrome (AS) is a rare genetic neurodevelopmental disease caused by interruption of the function of the ubiquitin ligase UBE3A gene. Patients experience an early life onset of symptoms which include pronounced intellectual disability, drastic speech impairmentor inability to speak, severe movement disorders, seizures, aggression, disruptive sleep impairment, and unusual personality traits. Estimates of prevalence show that approximately 1:15000 births are affected.

The disease is severe, and the burden can be devastating to the patients and the caregivers that they require for the duration of their relatively normal lifespan. Current treatments for the disease are lacking. Many are expensive and time consuming and result in minimal benefit. The few treatments that are effective only address single symptoms and do not work for all patients. Investigations into new and effective treatments are crucial for patients and their families.

One challenge to development of effective treatments for AS patients is the lack of a full understanding of the role of UBE3A in the central nervous system. Much has been learned about the role of UBE3A in the central nervous system, but the picture is far from complete. Investigations into the mechanism so far have been unable to pinpoint the specific effects that result in the development of AS arising from loss of function of UBE3A in neurons. Work has been done that demonstrates a wide range of effects of UBE3A loss. UBE3A affects multiple subcellular locations and multiple pathways in the cell. It also has been shown to exert these effects through direct actions through ubiquitination as well as indirect actions through regulation of transcription levels.

Due to the wide variety of effects, we believed that a more global picture of the effects of UBE3A loss is needed. To this end we present an examination of network and pathway changes in an AS mouse model. The results add to the global picture of UBE3A loss. This study used Ingenuity Pathway Analysis (IPA) (Qiagen) in conjunction with RNA-seq data to identify pathways and biofunctions highly affected by the loss of Ube3a in these animals. The results give further evidence that UBE3A is important to the function of synapses based on the identification of multiple pathways involved in synaptic function as dysregulated in AS. These data could be further investigated for potential drug targets and offer outcome measures to be explored with current potential therapeutics.

So far, the treatments available for patients living with AS are quite limited, and the fact that none of the treatments target the underlying etiology means that none are truly disease modifying. To address the lack of treatment several groups have early-stage investigations into disease modifying therapeutics. These mainly fall into one of two categories: unsilencing of the paternal allele or gene replacement therapy. While unsilencing strategies show some promise in AS models, based on our expertise, our group is primarily interested in the alternative approach of gene replacement therapy by using recombinant adeno-associated virus (rAAV). Recent advances in gene replacement therapy have been made, including the approval of treatments for two diseases using rAAV.

UBE3A is expressed as three different isoforms in humans and evidence shows differences in localization and function amongst the isoforms. To create an effective gene replacement therapy, it is necessary to determine if a single isoform can be used or if more than one is required. We investigated each of the human isoforms independently in AS animal models. Our results demonstrate that human UBE3A isoform 1 can improve deficits in animal models and thus should be considered the best candidate for therapeutic use. Isoforms 2 and 3

failed to demonstrate either significant rescue or expression in vivo. This led us to choose isoform 1 for the remaining experiments.

Potential for toxicity due to supraphysiological levels of the UBE3A protein has been a considerable concern to the AS field at the onset of discussions of a gene therapy approach. Evidence from a related disorder, chromosome 15q11.2-q13.1 duplication syndrome (Dup15q) underscores the need for investigating this possibility. We show here that injection of rAAV expressing the human UBE3A gene in wildtype Sprague-Dawley rats, which have normal levels of Ube3a expression, does not create any abnormal cognitive or behavioral deficits, nor did it affect hippocampal electrophysiology. Thus, we concluded that expression of hUBE3A with this vector does not appear to deleteriously alter normal neuronal function.

Together these studies show that human isoform 1 can be used to rescue the AS phenotype and that use of this vector does not elicit negative effects in wild type animals. This is the first effective use of the human UBE3A gene in a rAAV as an AS treatment. Based on this, we used human isoform 1 for further investigations.

Although there is great promise in using rAAV as a gene replacement therapy, there are roadblocks to the success of these therapies. One roadblock to using rAAV as a therapeutic in the human brain is the limited ability to affect a large number of cells, while minimizing the invasiveness of the procedure. To address this issue, we proposed a combination of two concepts to increase the efficacy of our gene therapy approach. We combined intracerebroventricular (ICV) injections, which can give increased vector distribution, with a novel secreted human UBE3A construct we term STUB. Our data shows that STUB, along with ICV injection, is superior to the native non-secreted hUBE3A vector in rescuing behavioral and electrophysiological deficits in the recently created Ube3a deletion rat model of AS.

Together the results presented here move the search for an effective treatment for AS forward. The results reveal additional targets for investigations through a global look at Ube3aloss. Our data also show that there is a strong potential for an effective therapeutic based on the use of rAAV with intracerebroventricular injections and a secreted form of the UBE3A protein.

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