UCLA

The urea cycle is essential in terrestrial mammals to detoxify ammonia into urea, and disruptions of this process may lead to neurotoxic ammonia levels and growth deficits. Carbamoyl phosphate synthetase 1 (CPS1) deficiency is caused by a loss of functional CPS1 protein, which catalyzes the initial and rate-limiting step of the urea cycle. Protein-restricted diets and chronic use of ammonia scavengers, the standards of care, are only modestly effective for treating CPS1 deficiency. The only curative option is orthotopic liver transplantation, which is limited by low donor availability and complications from surgery/immunosuppression. To investigate and develop novel therapeutics, we took two separate but converging approaches using either cell- or virus-based approaches. The first approach was to establish induced pluripotent stem cell lines from patients and use them as a platform to test potentially therapeutic genome modifications using the CRISPR/Cas9 system. Genomic addition of human codon optimized CPS1 (hcoCPS1) at the adeno-associated virus (AAV) insertion site, a known euchromatic safe harbor for exogenous genes, failed to reconstitute ureagenesis, demonstrating that further work is needed to improve and optimize this approach. The second approach utilized a split AAV platform to deliver hcoCPS1 to Cps1 deficient mice. hcoCPS1, along with its regulatory elements, is too large to fit into a single AAV capsid; by splitting the entire transgenic cassette in two, we capitalized on the recombinogenic nature of AAVs to reconstitute the full-length transgene in vivo and restore urea production. This approach successfully prevented mortality and reestablished ureagenesis in Cps1 deficient mice. The studies here provide the first insights into clinically translatable gene and cell therapies for CPS1 deficiency patients who have had no access to new treatments for over 40 years.