Liver Expression of a MiniATP7B Gene Results in Long‐Term Restoration of Copper Homeostasis in a Wilson Disease Model in Mice

Abstract

Gene therapy with an adeno‐associated vector (AAV) serotype 8 encoding the human ATPase copper‐transporting beta polypeptide (ATP7B) complementary DNA (cDNA; AAV8‐ATP7B) is able to provide long‐term copper metabolism correction in 6‐week‐old male Wilson disease (WD) mice. However, the size of the genome (5.2 kilobases [kb]) surpasses the optimal packaging capacity of the vector, which resulted in low‐yield production; in addition, further analyses in WD female mice and in animals with a more advanced disease revealed reduced therapeutic efficacy, as compared to younger males. To improve efficacy of the treatment, an optimized shorter AAV vector was generated, in which four out of six metal‐binding domains (MBDs) were deleted from the ATP7B coding sequence, giving rise to the miniATP7B protein (Δ57‐486‐ATP7B). In contrast to AAV8‐ATP7B, AAV8‐miniATP7B could be produced at high titers and was able to restore copper homeostasis in 6‐ and 12‐week‐old male and female WD mice. In addition, a recently developed synthetic AAV vector, AAVAnc80, carrying the miniATP7B gene was similarly effective at preventing liver damage, restoring copper homeostasis, and improving survival 1 year after treatment. Transduction of approximately 20% of hepatocytes was sufficient to normalize copper homeostasis, suggesting that corrected hepatocytes are acting as a sink to eliminate excess of copper. Importantly, administration of AAVAnc80‐miniATP7B was safe in healthy mice and did not result in copper deficiency. Conclusion: In summary, gene therapy using an optimized therapeutic cassette in different AAV systems provides long‐term correction of copper metabolism regardless of sex or stage of disease in a clinically relevant WD mouse model. These results pave the way for the implementation of gene therapy in WD patients.

Systemic deletion of Atp7b modifies the hepatocytes’ response to copper overload in the mouse models of Wilson disease

“…Previous characterization of animal models for WD uncovered important aspects of disease pathogenesis, including involvement of mitochondria 15,16,29 , autophagy 30 , the role of nuclear receptors 10,11 , along with a significant impact of Cu overload on Zn balance 31 . These studies stimulated search for new therapeutic modalities, including small molecule drugs, cell replacement therapies, and gene therapies 10,29,32,33 . Hepatocyte-directed ATP7B gene transfer has recently shown significant promise in improving liver morphology and function in Atp7b −/− mouse models of WD [33][34][35] .…”

“…These studies stimulated search for new therapeutic modalities, including small molecule drugs, cell replacement therapies, and gene therapies 10,29,32,33 . Hepatocyte-directed ATP7B gene transfer has recently shown significant promise in improving liver morphology and function in Atp7b −/− mouse models of WD [33][34][35] . In future studies, it would be important to show whether the Atp7b deficit is corrected in hepatocytes alone or also in other non-parenchymal cells when the liver function is restored.…”

Therapeutic strategies in Wilson disease: pathophysiology and mode of action

“…Indeed the observations of neurologic improvement after liver transplantation are ambiguous (53). Sophisticated gene therapy approaches for replacement of the mutated ATP7B were successful in animal models of Wilson disease (54)(55)(56)(57)(58)(59)(60). This is another promising approach which should be explored in human Wilson disease.…”

Recent advances in Wilson disease

“…• A shorter vector coding for a miniATP7B protein was shown to be effective in achieving long-term copper hemostasis in mouse model (20) Cell therapy…”

“…However, the large size of ATP7B cDNA affected the vector's cloning capacity for production of efficient gene transfer. To overcome this limitation, a shorter vector coding for a miniATP7B protein was generated that was effective in achieving long-term copper hemostasis in this mouse model of WD (20). Based on these studies, AAV gene transfer for the genetic correction of WD in humans is being planned.…”