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Treatment of a Mouse Model of ALS by In Vivo Base Editing
Colin K W Lim1, Michael Gapinske1, Alexandra K Brooks1
1Department of Bioengineering, University of Illinois, Urbana, IL 61801, USA.
Abstract:
Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal disorder that can be caused by mutations in the superoxide dismutase 1 (SOD1) gene. Although ALS is currently incurable, CRISPR base editors hold the potential to treat the disease through their ability to create nonsense mutations that can permanently disable the expression of the mutant SOD1 gene. However, the restrictive carrying capacity of adeno-associated virus (AAV) vectors has limited their therapeutic application. In this study, we establish an intein-mediated trans-splicing system that enables in vivo delivery of cytidine base editors (CBEs) consisting of the widely used Cas9 protein from Streptococcus pyogenes. We show that intrathecal injection of dual AAV particles encoding a split-intein CBE engineered to trans-splice and introduce a nonsense-coding substitution into a mutant SOD1 gene prolonged survival and markedly slowed the progression of disease in the G93A-SOD1 mouse model of ALS. Adult animals treated by this split-intein CRISPR base editor had a reduced rate of muscle atrophy, decreased muscle denervation, improved neuromuscular function, and up to 40% fewer SOD1 immunoreactive inclusions at end-stage mice compared to control mice. This work expands the capabilities of single-base editors and demonstrates their potential for gene therapy.
Insights
CRISPR base editors offer new hope for treating Amyotrophic Lateral Sclerosis (ALS) by disabling the mutant SOD1 gene. A novel split-intein system delivered via AAV vectors successfully slowed disease progression in a mouse model.
Area of Science:
- Genetics
- Neuroscience
- Biotechnology
Background:
- Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease often caused by mutations in the superoxide dismutase 1 (SOD1) gene.
- Current treatments for ALS are limited, highlighting the need for innovative therapeutic strategies.
- Adeno-associated virus (AAV) vectors are promising for gene therapy but face limitations due to their small cargo capacity.
Purpose of the Study:
- To develop a novel CRISPR base editing system for in vivo gene therapy of SOD1-linked ALS.
- To overcome the AAV packaging limitations using an intein-mediated trans-splicing approach.
- To evaluate the therapeutic efficacy of this system in a preclinical mouse model of ALS.
Main Methods:
- Engineered a cytidine base editor (CBE) using a split-intein system for trans-splicing within AAV vectors.
- Delivered dual AAV particles encoding the split-intein CBE via intrathecal injection into G93A-SOD1 mice.
- Assessed therapeutic effects by monitoring survival, disease progression, muscle atrophy, denervation, neuromuscular function, and SOD1 inclusions.
Main Results:
- The split-intein CBE system successfully introduced a nonsense mutation into the mutant SOD1 gene in vivo.
- Intrathecal delivery of the dual AAV particles significantly prolonged survival in G93A-SOD1 mice.
- Treated mice exhibited reduced muscle atrophy, decreased denervation, improved neuromuscular function, and fewer SOD1 inclusions.
Conclusions:
- Intein-mediated trans-splicing enables the delivery of larger base editors via AAV vectors for gene therapy.
- This split-intein CRISPR base editing approach demonstrates significant therapeutic potential for SOD1-ALS.
- The study expands the utility of base editors for treating genetic disorders with large gene targets.
