Development of gene therapies for treatment of neurological disorders

The increasing global prevalence of neurological disorders, their debilitating impact on affected individuals, and the current lack of effective curative treatments for such conditions underscore the critical need for novel therapeutic approaches. Gene therapies offer promising avenues for providing disease-modifying treatments by addressing the genetic underpinnings of disorders. Among these, CRISPR-based genome editing platforms have revolutionized the field of biomedicine by enabling programmable and permanent modifications in the genome. One such genome editing platform is base editing, which is capable of installing targeted modifications with unparalleled efficiency and precision. Base editing has shown great potential for directly modifying underlying disease mechanisms, including the correction of pathogenic mutations associated with genetic disorders and the modulation of RNA splicing, making it a powerful therapeutic tool. In this dissertation, I describe the development of a suite of CRISPR base editing strategies that collectively form a toolbox for treating neurological diseases. In Chapter 2, I employ base editors (BEs) for targeted correction of a pathogenic point mutation in the MeCP2 gene that leads to a neurodevelopmental disorder known as Rett syndrome. In this work, I screened multiple base editing technologies and identified tools to precisely correct the MeCP2-R133C missense mutation in mouse models of Rett syndrome, which significantly improved the symptoms of the disease and improved survival. While mutation-specific correction is a powerful approach, it does not address genetic heterogeneity, particularly in instances where disease-causing mutations occur at low incidence rates across the patient population. As such, transcript-level modification technologies such as exon skipping approaches that prevent the inclusion of target exons in mature mRNA transcripts can be leveraged as effective approaches to modulate progression of diseases where mutational hotspots occur within single exons. In Chapter 3, I demonstrate the potential of BE-induced exon skipping strategies to develop a treatment for Huntington’s disease (HD). HD is caused by an abnormal CAG repeat expansion in the HTT gene, leading to the production of mutant huntingtin (mHTT) protein. A critical pathogenic event in HD is the proteolytic cleavage of mHTT by caspase-6, generating highly toxic N-terminal fragments that drive neurodegeneration. I used BEs to target highly conserved splice acceptor sites in HTT and to generate isoforms resistant to caspase-6 proteolytic cleavage, an approach that attenuated HD-related biomarkers in transgenic mice. Finally, although BE-induced disruption of splice acceptor sequences can promote skipping of some exons, skipping of many other exons remains challenging due to sequence constraints or aberrant splicing outcomes. To overcome these limitations, in Chapter 4, I utilized engineered BE variants to develop novel exon skipping strategies that enable simultaneous editing of specific sequences. This approach achieved improved levels of exon skipping, reduced aberrant splicing, and skipping of exons refractory to splicing. I demonstrated the applicability of our toolbox by targeting APP exon 17, a therapeutic target for Alzheimer’s disease, which modulated the formation of amyloid-beta peptides in vitro and enabled efficient exon skipping in a mouse model of the disease. Together, my studies establish versatile and modular CRISPR base editing strategies as a one-time treatment of neurological disorders. By harnessing the precision of BEs, I demonstrate their efficacy for both reverting specific mutations and inducing exon skipping to modify disease progression in preclinical models. Overall, my findings highlight the potential of BEs as a platform for diverse therapeutic applications, further advancing their clinical translation and laying the groundwork for treatment of a broad range of diseases.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Bioengineering

Grantor

University of Illinois Urbana-Champaign

Year dc:date

2025

Author dc:creator

• Shirguppe, Shraddha Laxmi Nitin

Contributors dc:contributor

• Perez-Pinera, Pablo
• Llano, Daniel
• Underhill, Gregory
• Jee Jang, Min
• Gaj, Thomas

• CRISPR Gene editing
• neurological disorders

Statement dc:rights

• Copyright 2025 Shraddha Laxmi Nitin Shirguppe

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/129693

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F129693