Full-length ATP7B reconstituted through protein trans -splicing corrects Wilson disease in mice.

Wilson disease (WD) is a genetic disorder of copper homeostasis, caused by deficiency of the copper transporter ATP7B. Gene therapy with recombinant adeno-associated vectors (AAV) holds promises for WD treatment. However, the full-length human gene exceeds the limited AAV cargo capacity, hampering the applicability of AAV in this disease context. To overcome this limitation, we designed a dual AAV vector approach using split intein technology. Split inteins catalyze seamless ligation of two separate polypeptides in a highly specific manner. We selected a DnaE intein from (Npu) that recognizes a specific tripeptide in the human coding sequence. We generated two AAVs expressing either the 5'-half of a codon-optimized human cDNA followed by the N-terminal Npu DnaE intein or the C-terminal Npu DnaE intein followed by the 3'-half of cDNA, under the control of a liver-specific promoter. Intravenous co-injection of the two vectors in wild-type and mice resulted in efficient reconstitution of full-length ATP7B protein in the liver. Moreover, mice treated with intein-ATP7B vectors were protected from liver damage and showed improvements in copper homeostasis. Taken together, these data demonstrate the efficacy of split intein technology to drive the reconstitution of full-length human ATP7B and to rescue copper-mediated liver damage in mice, paving the way to the development of a new gene therapy approach for WD.