Split AAV-Mediated Gene Therapy Restores Ureagenesis in a Murine Model of Carbamoyl Phosphate Synthetase 1 Deficiency

Mol Ther. 2020 Jul 8;28(7):1717-1730. doi: 10.1016/j.ymthe.2020.04.011. Epub 2020 Apr 17.


The urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS1) catalyzes the initial step of the urea cycle; bi-allelic mutations typically present with hyperammonemia, vomiting, ataxia, lethargy progressing into coma, and death due to brain edema if ineffectively treated. The enzyme deficiency is particularly difficult to treat; early recognition is essential to minimize injury to the brain. Even under optimal conditions, therapeutic interventions are of limited scope and efficacy, with most patients developing long-term neurologic sequelae. One significant encumberment to gene therapeutic development is the size of the CPS1 cDNA, which, at 4.5 kb, nears the packaging capacity of adeno-associated virus (AAV). Herein we developed a split AAV (sAAV)-based approach, packaging the large transgene and its regulatory cassette into two separate vectors, thereby delivering therapeutic CPS1 by a dual vector system with testing in a murine model of the disorder. Cps1-deficient mice treated with sAAVs survive long-term with markedly improved ammonia levels, diminished dysregulation of circulating amino acids, and increased hepatic CPS1 expression and activity. In response to acute ammonia challenging, sAAV-treated female mice rapidly incorporated nitrogen into urea. This study demonstrates the first proof-of-principle that sAAV-mediated therapy is a viable, potentially clinically translatable approach to CPS1 deficiency, a devastating urea cycle disorder.

Keywords: adeno-associated virus; carbamoyl phosphate synthetase deficiency; gene therapy; hyperammonemia; split AAV; urea cycle disorder; ureagenesis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ammonia / metabolism
  • Animals
  • Carbamoyl-Phosphate Synthase (Ammonia) / genetics*
  • Carbamoyl-Phosphate Synthase I Deficiency Disease / genetics
  • Carbamoyl-Phosphate Synthase I Deficiency Disease / metabolism
  • Carbamoyl-Phosphate Synthase I Deficiency Disease / therapy*
  • DNA Packaging
  • Dependovirus / genetics*
  • Disease Models, Animal
  • Female
  • Genetic Therapy
  • Genetic Vectors / administration & dosage
  • Humans
  • Mice
  • Proof of Concept Study
  • Urea / metabolism*


  • Ammonia
  • Urea
  • CPS1 protein, human
  • Carbamoyl-Phosphate Synthase (Ammonia)