Development of a gene targeting strategy for liver metabolic diseases using the Crispr/Cas9 platform

Many inborn errors of metabolism require life-long treatments and, in severe conditions, organ transplantation remains the only curative treatment. Non-integrative AAV-mediated gene therapy in the liver has shown to be efficient in different clinical trials of adult patients, but treatment in pediatric or juvenile settings may result in the rapid loss of episomal viral DNA associated with hepatocyte duplication during liver growth. Gene targeting approaches have shown potential clinical applications in the treatment at pediatric stages, as the therapeutic transgene is permanently integrated into the genome, ensuring long-term therapeutic efficiency. This PhD project focuses on two liver disorders: citrullinemia type I and hemophilia B. Citrullinemia type I (CTLN1) is a severe liver monogenic urea cycle disorder caused by deficient activity of the ASS1 gene, leading to the accumulation of blood ammonia and citrulline. Hemophilia B instead, is a bleeding disorder, caused by the non-functionality of the coagulation factor 9 (FIX), produced by hepatocytes, leading to a deficient clotting activity and frequent spontaneous bleeding episodes. Both disorders require life-long treatments and, in the case of CTLN1, liver transplantation remains the only permanent cure. We previously developed a gene therapy strategy targeting a therapeutic cDNA into the albumin locus, using the Crispr/SaCas9 platform. We applied this treatment to CTLN1 neonate mice (ASS1fold/fold). A single injection completely rescued phenotype lethality, decreasing plasma citrulline levels, although without reaching wild-type values. We then treated juvenile (P30) ASS1fold/fold mice with an episomal hASS1 AAV8 vector (non-integrative) or with the gene targeting strategy applied previously to neonate mice. The animals treated with the non-integrative gene therapy vector showed a complete rescue of the diseased phenotype, with plasma ammonia and citrulline levels similar to wild-type values up to 3 months post-administration, while mice treated with the gene targeting strategy had an increase in their lifespan. In the second part of this work, we targeted the human coagulation factor IX (FIX) cDNA into the genome of a mouse model of hemophilia B. In this case, a single injection of the AAV vectors into neonate FIX KO mice led to long-term and stable expression of above-normal hFIX levels. Treated mutant mice were subjected to tail clip analysis in which their coagulation activity was comparable to wild-type animals, demonstrating the complete normalization of the phenotype. However, in adult FIX KO mice the targeting rate was less efficient and it did not lead to a correction of the coagulation activity. Altogether, our data demonstrate that a gene-targeting strategy is very promising in the neonatal treatment of disorders where a smaller correction rate is enough to restore the phenotype or to considerably improve the phenotype in disorders where higher levels of correction are required to restore liver functionality. Non-integrative gene therapy may be applied to CTLN1 juvenile mice ensuring a stable therapeutic effect. Gene targeting strategies on adults are still worth further developing, as the non-integrative approach may lose its efficiency associated with the natural rate of hepatocyte duplication and to liver disease conditions resulting in hepatocyte duplication.