CRISPR-based approaches for proteinopathies of the central nervous system

Neurodegenerative proteinopathies, which include Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD), are a class of neurologic disorders characterized by the abnormal deposition of certain proteins within the central nervous system (CNS). Despite their high incidence and devastating effects, these diseases are incurable and conventional therapies provide a limited benefit to patients. Promising therapeutic approaches to alleviate toxic protein aggregation include gene silencing of the major aggregating protein of the disease. While traditional gene silencing approaches have made tremendous advances in lowering the expression of pathogenic proteins, these tools have a transient effect and require re dosing, which can represent a physical burden for patients and can result in periods of reduced efficacy. Our lab has developed genome editing technologies programmed to permanently disable mutant genes known to cause disease that can facilitate single-dose therapies. We have developed CRISPR-based technologies capable of targeting mutant genes characteristic of familial Huntington’s disease and ALS. However, most neurodegenerative disease cases are sporadic. In contrast to hereditary forms, whose genetic roots are widely linked to a specific mutant gene, sporadic cases occur due to a complex combination of multiple environmental, genetic, and lifestyle-related factors. Thus, sporadic cases have few clear factors for therapeutic targeting and remain a longstanding goal of biomedicine. This notwithstanding, the abnormal aggregation of specific proteins remains a major pathologic characteristic of sporadic cases of neurodegenerative diseases. For instance, ~97% of ALS cases have been characterized by inclusions consisting of ubiquitinated and hyper-phosphorylated TAR DNA-binding protein 43 (TDP-43) protein, a finding that has helped to elevate its status as a therapeutic target. However, because of its essential role in transcription regulation and the side effects that arise on its absence, TDP-43 may not be an appropriate target for therapeutic silencing. To this end, various attempts have been undertaken to identify modifiers whose targeting can attenuate the toxic effects induced by TDP-43 aggregation without affecting its underlying abundance. One such modifier is ataxin-2, a protein whose targeting, has been shown to lower TDP-43-associated toxicity. This dissertation focuses on harnessing CRISPR-based technologies to target protein modifiers of pathogenic protein aggregation in the neurodegenerative proteinopathies ALS, FTD and AD. We first describe the use of an RNA-targeting CRISPR effector proteins to lower ataxin-2, a protein modifier of the toxicity elicited by TDP-43, to mitigate pathology in ALS and FTD. Further, we program CRISPR-based genome editing systems to ablate the expression of a G-protein coupled receptor capable of modulating amyloid-beta aggregation to improve memory in a mouse model of Alzheimer’s disease. Finally, we also engineer adeno-associated virus (AAV) vectors, a clinically promising tool capable of delivering CRISPR-based technologies to the nervous system, to target specific cell types associated with disease. Overall, this dissertation develops CRISPR-based gene therapies to target modifiers of toxic protein aggregation with the goal of treating sporadic forms of neurodegenerative proteinopathies including ALS, FTD and AD.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Bioengineering

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2024

Author dc:creator

• Zeballos Castro, Maria Alejandra

Contributors dc:contributor

• Gaj, Thomas
• Murphy, Catherine J
• Perez-Pinera, Pablo
• Underhill, Gregory H

• Crispr
• Genome Editing
• Gene Silencing
• Gene Therapy
• Cns Proteinopathies
• Neurodegenerative Diseases
• Amyotrophic Lateral Sclerosis (als)
• Ataxin-2
• Tar Dna-binding Protein 43 (tdp-43)
• Alzheimer's Disease
• G-protein Coupled Receptor 3 (gpr3)
• Adeno-associated Virus (aav).

Statement dc:rights

• © 2024 M. Alejandra Zeballos C. All rights reserved.

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/124579

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F124579