Targeting Duchenne muscular dystrophy with CRISPR base editors

Winter, Jackson Scott

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https://hdl.handle.net/2142/122231 Copy

Description

Title

Targeting Duchenne muscular dystrophy with CRISPR base editors

Author(s)

Winter, Jackson Scott

Issue Date

2023-12-01

Director of Research (if dissertation) or Advisor (if thesis)

Perez-Pinera, Pablo

Doctoral Committee Chair(s)

Perez-Pinera, Pablo

Committee Member(s)

• Boppart, Marni
• Gaj, Thomas
• Sirk, Shannon

Department of Study

Bioengineering

Discipline

Bioengineering

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• CRISPR
• Base Editors
• Duchenne Muscular Dystrophy
• DMD
• Exon skipping
• AAV
• Gene Editing

Abstract

Duchenne muscular dystrophy (DMD) is a fatal X-linked monogenic disease caused by mutations within the DMD gene which encodes dystrophin. Disruption of dystrophin expression results in the progressive degeneration of muscle tissue, which is characterized by progressive muscle weakening, loss of ambulation, and ultimately death due to respiratory failure or cardiomyopathy. There is currently no cure for DMD, and most therapies merely treat the symptoms of the disease without addressing the underlying molecular cause. Gene therapies that use adeno-associated virus (AAV) to deliver replacement genes have emerged as a promising treatment modality and have earned FDA approval for several genetic diseases. While AAV has proven itself to be a safe and effective gene delivery vehicle, its use in treatments for DMD is complicated by the limited packaging capacity of the virus, which prevents the direct delivery of a full DMD transgene. Several AAV-based therapies that provide a truncated but partially functional copy of the DMD gene are currently in development, but the initial data from clinical trials suggests that they are unlikely to prevent the worst symptoms of the disease and will most likely only slow the progression of the disease. Most cases of DMD are caused by genomic deletions that disrupt the mRNA reading frame, which leads to introduction of a premature stop codon and early truncation of the protein during translation. Accordingly, several therapies have emerged that attempt to induce the targeted skipping of an exon near the deletion to restore the dystrophin reading frame and produce an internally truncated but semi-functional isoform. Traditionally this has been accomplished using synthetic antisense oligonucleotides (ASOs) that block specific splice recognition sequences in mRNA and lead to exclusion of the targeted exon from processed transcripts. While these therapies can be safely administered systemically, they are only transient and require repeated injections to achieve continued restoration of dystrophin expression. Furthermore, they suffer from low tissue distribution and rapid clearance times, which have severely limited their therapeutic effect. Genome editing tools that use programmable nucleases such as CRISPR/Cas9 offer promising solutions to these shortcomings by providing the ability to make precise and permanent changes directly to the DNA. While these tools have been harnessed for targeted exon skipping to treat DMD in multiple animal models, their reliance on the creation of DNA double strand breaks (DSBs) to achieve editing raises a myriad of safety concerns. By contrast, base editors that consist of a deaminase domain tethered to a nickase Cas9, have emerged as a promising gene editing platform that enable targeted nucleotide conversions without the creation of a DSB and can therefore circumvent the deleterious consequences of traditional nuclease-based genome editing tools. Here we outline the development of a novel strategy that uses base editors to mutate the conserved splice sites of target exons to achieve permanent and programmable exon skipping without the creation of a DSB, a technique we termed CRISPR-SKIP. We next describe the creation of a dual-vector strategy that uses trans-splicing inteins to overcome the packaging capacity of AAV, enabling the in-vivo delivery of base editing constructs. Furthermore, we optimized our CRISPR-SKIP strategy for targeted exon skipping of DMD exon 45, an exon that if skipped could treat up to ~9% of DMD patients. After using this technique to restore dystrophin expression in cell culture disease models, we packaged our split base editing system into AAV and delivered it to live mouse models, achieving targeted editing in DNA in-vivo through both local and systemic injections. We next sought to expand the targeting range of CRISPR-SKIP using base editors with engineered deaminase domains and flexible protospacer adjacent motif (PAM) requirements. These efforts led to the targeted editing of DMD exon 51, and exon 53, which if skipped could treat up to ~13% and ~8% of DMD patients respectively, as well as the development of a generalizable framework for optimizing CRISPR-SKIP to target any therapeutic exon of interest. Collectively, these experiments outline a novel therapeutic strategy that could potentially treat up to ~30% of all DMD patients and can be adapted to treat numerous other diseases that are caused by aberrant splicing.

Graduation Semester

2023-12

Type of Resource

Thesis

Copyright and License Information

Copyright 2023 Jackson Winter

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