Exon skipping therapy for Duchenne Muscular Dystrophy with CRISPR base editors

Duchenne muscular dystrophy is an X-linked monogenic disease caused by mutations in the dystrophin gene (DMD) and characterized by progressive muscle weakness leading to cardiomyopathy and respiratory failure, significantly decreasing life expectancy. Since the current standard of care for Duchenne muscular dystrophy focuses solely on the symptoms of the disease, there is a dire need for novel treatment modalities that can correct the underlying genetic mutations. One such technology is gene therapy, which has been utilized for treating Duchenne muscular dystrophy by delivering heterologous genes, altering mRNA splicing or directly modifying the genome with CRISPR-Cas gene editing systems. Gene editing represents a particularly promising approach that can permanently restore native expression of corrected dystrophin following a single administration of the therapeutic transgenes. Since CRISPR-Cas editing systems that rely on double-strand DNA breaks have been shown to create potentially deleterious chromosomal aberrations, CRISPR base editing technologies are emerging as safer and more precise alternatives. CRISPR base editors have enormous potential for directly correcting point mutations, the most common type of disease-causing genetic variant, as well as for gene knockout. However, this technology has not previously been applied to the most common form of mutation causing Duchenne muscular dystrophy: deletions that shift the reading frame and introduce premature stop codons to the genetic sequence. Additionally, the large size of the genes encoding base editors has previously prevented their delivery via AdenoAssociated Virus (AAV), the most promising gene delivery vector to date. This dissertation first covers the development of CRISPR-SKIP, our strategy using CRISPR base editors to correct frameshift deletions of exons through the skipping of neighboring exons. We next adapt a method of protein trans-splicing to base editing technology, to enable the expression of a single large base editor protein from two genes small enough to deliver by AAV. This technology has great potential to move base editing technology towards the clinic, as we demonstrate with the in vivo knock out of the human SOD1 gene, mutations in which cause Amyotrophic Lateral Sclerosis. Finally, we apply CRISPR-SKIP to correct a frameshift deletion responsible for Duchenne muscular dystrophy and achieve in vivo editing of the same disease-correcting genomic site in mice.

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

2022

Author dc:creator

• Gapinske, Michael Paul

Contributors dc:contributor

• Perez-Pinera, Pablo
• Gaj, Thomas
• Smith, Andrew M
• Song, Jun S

• CRISPR
• adenine base editors
• cytidine base editors
• exon skipping
• Duchenne Muscular Dystrophy
• ALS
• SOD1
• DMD

Statement dc:rights

• Copyright 2022 Michael Gapinske

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/115531

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F115531