In vertebrates, the liver is the central metabolic organ of the body, which carries out an estimated 500 functions that range from general detoxification to protein synthesis, bile production, metabolism of fats, carbohydrates, proteins, bilirubin, vitamin and mineral storage and it even has an immune function. Hepatocytes are considered the professional liver cells, which carry out all these functions. With such a variety of tasks to perform, it is not surprising that more than 400 rare monogenic disorders of hepatic origin have been described. For many of these, liver transplantation remains the only curative strategy, however, this is limited by organ availability and requires lifelong immune suppression. The fact that liver transplantation is curative led to the assumption that the restoration of the expression of the defective gene would result in the resolution of the disease. Thus, liver-directed gene therapy and gene editing strategies have emerged as promising alternatives to transplantation in inherited monogenic liver disorders. We have applied gene supplementation for several metabolic disorders such as Wilson disease, PFIC3 or PKU with very promising results.

Herein, we will present our more recent progresses on the development of AAV mediated CRISPR-Cas9 gene editing approach for the treatment of Primary hyperoxaluria type I. An attractive therapeutic strategy for diseases associated with the accumulation of a toxic metabolite is substrate reduction therapy (SRT). Recently, we demonstrated the therapeutic efficacy of an AAV-CRISPR/Cas9 system to disrupt the Hao1 gene, which encodes GO in a PH1 mouse model. To reduce the likelihood of off-targets, we investigated the administration in PH1 mice of two AAV-CRISPR/Cas9(D10A)-nickases, each with a guide RNA, targeting nearby regions on the opposite strands of Hao1. Simultaneous on-target nicking resulted in undetectable GO levels. Individual nicks were faithfully repaired, as detected by next generation sequencing and normal GO expression, evidencing the inability of a single nickase to generate indels, and therefore reducing the probability of off-target events. Next, we combined the dual nickase-gRNA system into a single vector (all-in-one) allowing us to reduce the effective minimal dose by three-fold in comparison with the administration of the two independent vectors. Furthermore, a single administration in PH1 mice of the all-in-one vector showed a significant therapeutic effect preventing oxalate accumulation.