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Targeted, non-viral delivery of gene editing technologies for the treatment of ASL deficiency

A Hangu(1) E Young(1) A Bouchareb(1) J Baruteau(2) A Walker(1)

1:4basebio; 2:UCL Institute of Child Health

Argininosuccinate lyase (ASL) deficiency is a rare genetic disorder caused by a deficit in the ASL enzyme. This deficiency leads to argininosuccinic aciduria, characterised by hyperammonaemia and multi-organ disorder. CRISPR technologies provide an attractive therapeutic approach for liver monogenic diseases now reaching clinical translation stage. We aim to fully restore the function of the deficient gene to improve the long-term outcomes, especially the most severe patients presenting with acute metabolic decompensation as newborn or in infancy, and reduce the overall burden on the healthcare system.

Here, 4basebio’s synthetic, enzymatically produced DNA, and Hermes™ nanoparticles are combined to generate proof-of-concept data on a corrective strategy for ASL deficiency. Hermes™ nanoparticles were deployed to encapsulate Cas9 mRNA or Cas9 protein along with an sgRNA targeting the ASL gene in the human hepatocyte derived Huh7 cell line. The mRNA was produced using 4basebio’s synthetic opDNA™ template. Hermes™ nanoparticles had favourable biophysical characteristics, stability, and achieved high cell viability, with knockout efficiency >95%.

To investigate a gene-integration approach using 4basebio synthetic DNA, we designed an oeDNA™ template encoding a GFP sequence with homology arms flanking the target gene . The co-delivery of template DNA along with the Cas9 mRNA/gRNA encapsulated in Hermes™ nanoparticles resulted in successful integration of the oeDNA template, and GFP expression in vitro.

The present study provides proof-of-concept for the use of Hermes™ particles to deliver RNA or Ribonucleoprotein complex (RNP)  and donor DNA templates to achieve targeted integration, offering a promising approach to treat monogenic diseases such as ASL deficiency. 

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