Mammoth Biosciences, a biotechnology company harnessing its proprietary next-generation CRISPR gene editing platform to create potential one-time curative therapies, today announced new preclinical research, published on the preprint server bioRxiv, that establishes proof-of-concept of NanoCas™, the first ultracompact CRISPR system capable of efficient extrahepatic editing when delivered systemically using a single adeno-associated viral (AAV) vector.
Gene editing holds promise for curing genetic diseases, but faces delivery challenges that limit its therapeutic applications. Current methods are restricted to ex vivo approaches or in vivo liver editing. First-generation CRISPR systems, such as Cas9 and Cas12a, are too large for efficient in vivo delivery via a single AAV vector.
This study describes the discovery, engineering and benchmarking of NanoCas – a novel Cas enzyme approximately one third the size of Cas9 – which can be easily accommodated within a single AAV vector, while leaving ample room for additional payload such as regulatory elements, guide RNAs, or non double strand break editing machinery for techniques such as reverse transcriptase editing, base editing, and epigenetic editing. Discovering the NanoCas system required comprehensive screening of naturally occurring CRISPR variants, leading to the functional evaluation of more than 150 candidates, followed by targeted protein engineering to enhance its editing efficiency.
Key takeaways from the study include:
• Editing efficiency matches that of first-generation CRISPR systems: when targeting the PCSK9 gene in mouse liver in vivo, NanoCas showed saturating editing efficiencies of approximately 60%, on par with that of SaCas9, which is about three-fold larger in size. Both CRISPR systems reduced serum PCSK9 protein to undetectable levels
• Robust single AAV editing across multiple muscle tissues: NanoCas demonstrated 10% to 40% editing of the dystrophin gene across the quadricep, calf and heart muscle in a humanized mouse model of Duchenne Muscular Dystrophy (DMD), when delivered via a single AAV vector
• First demonstration of single AAV muscle editing in non-human primates: NanoCas achieved in vivo editing efficiencies of up to 30% when targeting dystrophin in the skeletal muscle of cynomolgus macaques. NanoCas also showed 15% editing across the heart, compared to 10% with SaCas9. Analysis of liver tissue showed minimal off-target editing.
