CRISPR/Cas9 technology offers novel approaches for the development of new therapies for many unmet clinical needs, including a significant number of inherited monogenic diseases. However, in vivo correction of disease-causing genes is still inefficient, especially for those diseases without selective advantage for corrected cells. We reasoned that substrate reduction therapies (SRT) targeting non-essential enzymes could provide an attractive alternative. Here we evaluate the therapeutic efficacy of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I (PH1), a rare inborn dysfunction in glyoxylate metabolism that results in excessive hepatic oxalate production causing end-stage renal disease. A single systemic administration of an AAV8-CRISPR/Cas9 vector targeting glycolate oxidase, prevents oxalate overproduction and kidney damage, with no signs of toxicity in Agxt1−/− mice. Our results reveal that CRISPR/Cas9-mediated SRT represents a promising therapeutic option for PH1 that can be potentially applied to other metabolic diseases caused by the accumulation of toxic metabolites.

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.

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