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

Fabiola Porro,Andrés F Muro,Alessia De Caneva,Simone Vodret,Giulia Bortolussi,Lorena Zentilin,Adi Barzel,Alessandra Iaconcig,Mark A Kay

doi:10.15252/emmm.201707601

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.

Highlights

At post-natal day 2 (P2), P4, P10, and P30 WT pups were transduced with rAAV8-Alb-eGFP (0.7E12 and 1.0E12 vgp/mouse) and sacrificed at P30 (I.P., P2, P4, and P10) and at P45 (i.v., P30) (Fig EV1A)
Histological analysis of liver sections showed that the highest recombination rate was obtained in P4 pups transduced with the higher associated virus (AAV) dose, reaching about 0.14% of hepatocytes (Figs 1B and C, and EV1B and EV2A), with a marked decrease in those injected at P10 and P30
We successfully applied the promoterless gene targeting without nucleases approach to a severe and clinically relevant lethal mouse model of the Crigler-Najjar syndrome type I (CNSI), a liver disease of the newborns

Summary

Introduction

The Crigler-Najjar syndrome type I (CNSI) is a rare monogenic pediatric disease (0.6–1 cases per 106 live births) caused by a deficiency in the liver-specific uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), resulting in severe unconjugated hyperbilirubinemia since birth, with lifelong risk of permanent neurological damage, kernicterus, and death (Crigler & Najjar, 1952; Huang et al, 1970).Current clinical practice consists of intense phototherapy (PT) treatment (12–14 h/day), but it becomes less effective with age, leaving liver transplantation as the only therapeutic option, with all the limitations and risks of the approach (Adam et al, 2012; Fagiuoli et al, 2013).Gene replacement mediated by adeno-associated virus (AAV) is a promising approach (Kay, 2011; Mingozzi & High, 2011; Nathwani et al, 2011). Gene editing with sequence-specific endonucleases (Urnov et al, 2010; Joung & Sander, 2013; Wang et al, 2016) results in the permanent correction of disease-causing mutations. These approaches face a number of significant adverse effects, such as immunogenicity of the endonucleases, off-target cleavage and mutagenesis, and induction of chromosomal aberrations, concerns which are enhanced by the long-term expression of the nucleases (Carroll, 2014). Off-target integration of the transgene and endonuclease vectors bearing potent gene promoters carries the potential of transactivating cancer-related genes (Fu et al, 2013; Chandler et al, 2015)

Methods

Results

Conclusion