Lethality rescue and long-term amelioration of a citrullinemia type I mouse model by neonatal gene-targeting combined to SaCRISPR-Cas9.

Citrullinemia type I is a rare autosomal-recessive disorder caused by deficiency of argininosuccinate synthetase (ASS1). The clinical presentation includes the acute neonatal form, characterized by ammonia and citrulline accumulation in blood, which may lead to encephalopathy, coma, and death, and the milder late-onset form. Current treatments are unsatisfactory, and the only curative treatment is liver transplantation. We permanently modified the hepatocyte genome in lethal citrullinemia mice (Ass1fold/fold) by inserting the ASS1 cDNA into the albumin locus through the delivery of two AAV8 vectors carrying the donor DNA and the CRISPR-Cas9 platform. The neonatal treatment completely rescued mortality ensuring survival up to 5 months of age, with plasma citrulline levels significantly decreased, while plasma ammonia levels remained unchanged. In contrast, neonatal treatment with a liver-directed non-integrative AAV8-AAT-hASS1 vector failed to improve disease parameters. To model late-onset citrullinemia, we dosed postnatal day (P) 30 juvenile animals using the integrative approach, resulting in lifespan improvement and a minor reduction in disease markers. Conversely, treatment with the non-integrative vector completely rescued mortality, reducing plasma ammonia and citrulline to wild-type values. In summary, the integrative approach in neonates is effective, although further improvements are required to fully correct the phenotype. Non-integrative gene therapy application to juvenile mice ensures a stable and very efficient therapeutic effect.

Non-alcoholic fatty liver disease and diabetes mellitus as growing aetiologies of hepatocellular carcinoma.

Obesity-related complications such as non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2D) are well-established risk factors for the development of hepatocellular carcinoma (HCC). This review provides insights into the molecular mechanisms that underlie the role of steatosis, hyperinsulinemia and hepatic inflammation in HCC development and progression. We focus on recent findings linking intracellular pathways and transcription factors that can trigger the reprogramming of hepatic cells. In addition, we highlight the role of enzymes in dysregulated metabolic activity and consequent dysfunctional signalling. Finally, we discuss the potential uses and challenges of novel therapeutic strategies to prevent and treat NAFLD/T2D-associated HCC.

Promoterless Gene Targeting Approach Combined to CRISPR/Cas9 Efficiently Corrects Hemophilia B Phenotype in Neonatal Mice.

Many inborn errors of metabolism require life-long treatments and, in severe conditions involving the liver, organ transplantation remains the only curative treatment. Non-integrative AAV-mediated gene therapy has shown efficacy in adult patients. However, treatment in pediatric or juvenile settings, or in conditions associated with hepatocyte proliferation, may result in rapid loss of episomal viral DNA and thus therapeutic efficacy. Re-administration of the therapeutic vector later in time may not be possible due to the presence of anti-AAV neutralizing antibodies. We have previously shown the permanent rescue of the neonatal lethality of a Crigler-Najjar mouse model by applying an integrative gene-therapy based approach. Here, we targeted the human coagulation factor IX (hFIX) cDNA into a hemophilia B mouse model. Two AAV8 vectors were used: a promoterless vector with two arms of homology for the albumin locus, and a vector carrying the CRISPR/SaCas9 and the sgRNA. Treatment of neonatal P2 wild-type mice resulted in supraphysiological levels of hFIX being stable 10 months after dosing. A single injection of the AAV vectors into neonatal FIX KO mice also resulted in the stable expression of above-normal levels of hFIX, reaching up to 150% of the human levels. Mice subjected to tail clip analysis showed a clotting capacity comparable to wild-type animals, thus demonstrating the rescue of the disease phenotype. Immunohistological analysis revealed clusters of hFIX-positive hepatocytes. When we tested the approach in adult FIX KO mice, we detected hFIX in plasma by ELISA and in the liver by western blot. However, the hFIX levels were not sufficient to significantly ameliorate the bleeding phenotype upon tail clip assay. Experiments conducted using a AAV donor vectors containing the eGFP or the hFIX cDNAs showed a higher recombination rate in P2 mice compared to adult animals. With this study, we demonstrate an alternative gene targeting strategy exploiting the use of the CRISPR/SaCas9 platform that can be potentially applied in the treatment of pediatric patients suffering from hemophilia, also supporting its application to other liver monogenic diseases. For the treatment of adult patients, further studies for the improvement of targeting efficiency are still required.

Coupling AAV-mediated promoterless gene targeting to SaCas9 nuclease to efficiently correct liver metabolic diseases.

Non-integrative AAV-mediated gene therapy in the liver is effective in adult patients, but faces limitations in pediatric settings due to episomal DNA loss during hepatocyte proliferation. Gene targeting is a promising approach by permanently modifying the genome. We previously rescued neonatal lethality in Crigler-Najjar mice by inserting a promoterless human uridine glucuronosyl transferase A1 (UGT1A1) cDNA in exon 14 of the albumin gene, without the use of nucleases. To increase recombination rate and therapeutic efficacy, here we used CRISPR/SaCas9. Neonatal mice were transduced with two AAVs: one expressing the SaCas9 and sgRNA, and one containing a promoterless cDNA flanked by albumin homology regions. Targeting efficiency increased ~26-fold with an eGFP reporter cDNA, reaching up to 24% of eGFP-positive hepatocytes. Next, we fully corrected the diseased phenotype of Crigler-Najjar mice by targeting the hUGT1A1 cDNA. Treated mice had normal plasma bilirubin up to 10 months after administration, hUGT1A1 protein levels were ~6-fold higher than in WT liver, with a 90-fold increase in recombination rate. Liver histology, inflammatory markers, and plasma albumin were normal in treated mice, with no off-targets in predicted sites. Thus, the improved efficacy and reassuring safety profile support the potential application of the proposed approach to other liver diseases.