In vivo Treatment of a Severe Vascular Disease via a Bespoke CRISPR-Cas9 Base Editor

Nov 28, 2024bioRxiv : the preprint server for biology

Using a Custom Gene Editor to Treat a Serious Blood Vessel Disease in Living Organisms

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Abstract

Delivery of a customized base editor improved survival rates in a murine model of multisystemic smooth muscle dysfunction syndrome (MSMDS).

Pathogenic mutations in the alpha actin isotype 2 gene are linked to severe complications, including stroke and aortic dissection in childhood.
A novel CRISPR-Cas9 enzyme was engineered to specifically target and correct the R179H mutation associated with MSMDS.
A robust screening process identified a precise A-to-G edit with reduced unintended edits compared to traditional editing methods.
The engineered base editor, delivered via a specialized virus, effectively improved systemic health indicators in MSMDS mice.
The approach demonstrates potential for developing targeted therapies for genetic vascular disorders through enhanced gene editing techniques.

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Genetic vascular disorders are prevalent diseases that have diverse etiologies and few treatment options. Pathogenic missense mutations in the alpha actin isotype 2 gene () primarily affect smooth muscle cell (SMC) function and cause multisystemic smooth muscle dysfunction syndrome (MSMDS), a genetic vasculopathy that is associated with stroke, aortic dissection, and death in childhood. Here, we explored genome editing to correct the most common MSMDS-causative mutationR179H. In a first-in-kind approach, we performed mutation-specific protein engineering to develop a bespoke CRISPR-Cas9 enzyme with enhanced on-target activity against the R179H sequence. To directly correct the R179H mutation, we screened dozens of configurations of base editors (comprised of Cas9 enzymes, deaminases, and gRNAs) to develop a highly precise corrective A-to-G edit with minimal deleterious bystander editing that is otherwise prevalent when using wild-type SpCas9 base editors. We then created a murine model of MSMDS that exhibits phenotypes consistent with human patients, including vasculopathy and premature death, to explore thetherapeutic potential of this base editing strategy. Delivery of the customized base editor via an engineered SMC-tropic adeno-associated virus (AAV-PR) vector substantially prolonged survival and rescued systemic phenotypes across the lifespan of MSMDS mice, including in the vasculature, aorta, and brain. Together, our optimization of a customized base editor highlights how bespoke CRISPR-Cas enzymes can enhance on-target correction while minimizing bystander edits, culminating in a precise editing approach that may enable a long-lasting treatment for patients with MSMDS. ACTA2ACTA2in vivo

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In vivo Treatment of a Severe Vascular Disease via a Bespoke CRISPR-Cas9 Base Editor

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