Extracellular Vesicles Delivered a Functional ARG1 Enzyme and Restored Its Activity in a Mouse Model of ARG1-D...

Arginase 1 (ARG1) deficiency (ARG1-D) is a rare genetic disorder due to loss of ARG1, the final enzyme in the urea cycle. ARG1-D hepatocytes are impaired in converting arginine into urea, resulting in elevated peripheral arginine and ammonia, which leads to progressive neurological symptoms. Current therapeutic strategies mainly focus on managing plasma arginine and ammonia level, but long-term outcomes remain poor. While no approved treatment specific for ARG1-D is available in the United States, a recombinant protein-based enzyme replacement therapy is available in Europe. Recently, extracellular vesicles (EVs) are emerging as a powerful therapeutic vehicle. By using Capricor's StealthX^TM platform, EVs were engineered to express human ARG1 on their surface or encapsulated within. Regardless of their localization on the EV membrane, nanograms of ARG1 carried by EVs were biologically active and able to convert arginine into urea as potent as micrograms of human recombinant ARG1 (rHuArg1). Furthermore, ARG1-encapsulating EVs (STX-Arg1-in) were able to deliver ARG1 intracellularly but not EVs carrying ARG1 on their surface or rHuArg1. STX-Arg1-in EVs were further evaluated in a series of in vivo studies, and the results showed that STX-Arg1-in EVs were non-toxic and able to restore arginase activities in the liver of Arg1^-/- mice, which led to a lowered plasma arginine concentration similar to that in wild-type mice. Most importantly, Arg1-in EVs expanded the lifespan of the lethal neonatal Arg1 deficiency mouse model. Taken together, our data suggested StealthX^TM-engineered STX-Arg1-in EVs have a better safety profile due to the extremely low dosage and have great potential as a novel enzyme replacement strategy for patients suffering from ARG1-D. Significance statement: Intracellular delivery of recombinant protein and improved llifespanare endpoints of successful enzyme replacement therapy for the treatment of ARG1-D. Using the StealthX platform, a fully functional ARG1 enzyme was engineered to be carried inside of the extracellular vesicles, which allowed for the intracellular delivery of ARG1 protein in vitro and in vivo, with an improvement of lifespan in a lethal neonatal mouse model of Arg1 deficiency. More importantly, no toxicity was observed, and efficacy was achieved with a low dose, setting the base for an improved therapeutic approach.