Promoterless gene targeting without nucleases rescues lethality of a Crigler‐Najjar syndrome mouse model

Abstract

Crigler‐Najjar syndrome type I (CNSI) is a rare monogenic disease characterized by severe neonatal unconjugated hyperbilirubinemia with a lifelong risk of neurological damage and death. Liver transplantation is the only curative option, which has several limitations and risks. We applied an in vivo gene targeting approach based on the insertion, without the use of nucleases, of a promoterless therapeutic cDNA into the albumin locus of a mouse model reproducing all major features of CNSI. Neonatal transduction with the donor vector resulted in the complete rescue from neonatal lethality, with a therapeutic reduction in plasma bilirubin lasting for at least 12 months, the latest time point analyzed. Mutant mice, which expressed about 5–6% of WT Ugt1a1 levels, showed normal liver histology and motor‐coordination abilities, suggesting no functional liver or brain abnormalities. These results proved that the promoterless gene therapy is applicable for CNSI, providing therapeutic levels of an intracellular ER membrane‐bound enzyme responsible for a lethal liver metabolic disease.

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

“…All rAAV8-treated mice survived, whereas those who received only phototherapy up to P8 died before day 19 ( Figure 4B). Mice treated with only the rAAV8-donor-hUGT1A1 vector presented life-compatible plasma bilirubin levels that were below toxic levels but still too high for a potential clinical application, confirming previous data (20). Importantly, mice treated with both rAAV vectors had plasma bilirubin levels similar to those of WT littermates for all the duration of the experiment ( Figure 4C).…”

“…In the present work, we demonstrated that GeneRide coupled to CRISPR/SaCas9 platform resulted in a substantial improvement of the gene-targeting rate in vivo by fully rescuing a lethal mouse model of the CNSI, with normal cerebellar architecture and plasma bilirubin levels similar to WT for the whole duration of the experiment (10 months). This approach permanently modifies the hepatocyte genomes and, in line with previous results (19,20), recombinant cells were found in small clusters providing strong evidence that the inserted transgene is stably transmitted to daughter cells during hepatocyte proliferation, also suggesting that recombination is an early event that occurs upon AAV transduction.…”

“…Genome targeting of the albumin locus with RNA-guided SaCas9 nuclease. To increase the therapeutic efficacy of liver gene-targeting into the albumin locus, we combined the GeneRide methodology (19,20) with the CRISPR/SaCas9 platform (ref. 28 and Figure 1, A and B).…”

“…We have recently developed a gene-targeting approach (namely "GeneRide") without nucleases based on the in-frame insertion of a promoterless therapeutic cDNA just upstream of the albumin stop codon (19). This strategy resulted in the amelioration of the bleeding diathesis in hemophilia B mice (19) and the rescue of a lethal mouse model of the CNSI (20). Here, we further increased the recombination rate and therapeutic efficacy by combining GeneRide with the CRISPR/SaCas9 platform.…”

“…The targeted allele continues to actively produce albumin and is stably transmitted to daughter cells without loss of genetic information. We have successfully applied the GeneRide strategy to a lethal mouse model of Crigler-Najjar syndrome type I (CNSI), showing the complete rescue of neonatal lethality and cerebellar and behavioral abnormalities (20). However, plasma bilirubin levels, despite being stable, safe, and life-compatible even 12 months after AAV delivery, were not fully normalized, prompting us to improve the gene-targeting rate and the therapeutic potential of the approach.…”