AAV8 Gene Therapy Rescues the Newborn Phenotype of a Mouse Model of Crigler–Najjar

Abstract

Adeno-associated viral (AAV) vectors can target the liver, making them an attractive platform for gene therapy approaches that require the correction of hepatocytes. Crigler-Najjar syndrome is an autosomal recessive disorder of bilirubin metabolism that occurs when the liver's uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) enzyme activity is partially or completely absent. This syndrome is characterized by elevated bilirubin levels in the blood. An AAV8 vector was developed expressing a codon-optimized human version of UGT1A1 from a liver-specific promoter. High doses of the vector rescued neonatal lethality in newborn UGT1 knockout (KO) mice, which serve as a model of Crigler-Najjar syndrome, and significantly increased survival from 5 to 270 days. Newborn UGT1 KO mice treated with AAV had serum total bilirubin levels that were 5.7 times higher than the levels seen in heterozygous and wild-type mice, likely due to dilution of vector genome copies (GC) in the liver resulting from a proliferation of hepatocytes during growth of the animal. The elevation in serum total bilirubin levels in adult UGT1 KO mice depended on the AAV8 vector dose. At doses <10 GC/mouse, total bilirubin levels returned to those seen in phototherapy-rescued UGT1 KO mice. Mice injected with vector at 10 or 3 × 10 GC/mouse had sustained reduced total bilirubin levels throughout the duration of the study. When an AAV8 vector was re-administered in mice with elevated total bilirubin levels, serum total bilirubin levels decreased to wild-type levels (0.1-0.3 mg/dL) in mice that received a vector dose of 3 × 10 GC/kg. Therefore, a low-level and likely transient decrease in serum total bilirubin during the first days of life is necessary for rescuing the lethal phenotype present in the neonatal UGT1 KO mouse. Furthermore, it was possible to ablate the elevated total bilirubin levels in adult mice by re-administering an AAV8 vector.

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

“…The CNSI mice closely mimic the main features of the human syndrome, with neonatal severe unconjugated hyperbilirubinemia and early neonatal lethality (29,30,38). Although nonintegrative AAV-mediated gene therapy is very effective in rescuing the phenotype in adult CNSI animals (39,40), it requires high doses of liver-specific rAAV8 episomal vectors expressing the UGT1A1 transgene when administered to newborn mutant mice (13,41,42). However, therapeutic efficacy decreases over time and a second administration is necessary to achieve full correction in the long term (41,42).…”

“…Although nonintegrative AAV-mediated gene therapy is very effective in rescuing the phenotype in adult CNSI animals (39,40), it requires high doses of liver-specific rAAV8 episomal vectors expressing the UGT1A1 transgene when administered to newborn mutant mice (13,41,42). However, therapeutic efficacy decreases over time and a second administration is necessary to achieve full correction in the long term (41,42). Similarly, neonatal AAV delivery also results in loss of therapeutic efficacy in other liver diseases, such as OTCD (12), underlining the limits of nonintegrative AAV-mediated gene therapy for severe liver disorders with neonatal and pediatric onset.…”

Structure of the AAVhu.37 capsid by cryoelectron microscopy

“…While hepatic gene transfer can induce immunity to the transferred gene (Dobrzynski et al, 2006), hepatic gene transfer with AAV can induce immune tolerance (Mount et al, 2002;Mingozzi et al, 2003;Breous et al, 2009Breous et al, , 2011, even when the gene transfer is not systemic (Martino et al, 2009). There is an effect of the serotype on the immune tolerance level (Greig et al, 2017), and AAVhu.37 has favorable tolerance and expression effects (Greig et al, 2018). This study has been further extended into an ongoing clinical trial (ClinicalTrials.gov NCT03588299; https://clinicaltrials.…”

“…The clade E serotype AAVhu.37 efficiently transduces the liver in mouse models and nonhuman primates (NHP) after intravenous injection (Wang, Wang et al, 2010;Greig et al, 2018). When injected directly into mouse brain, AAVhu.37 also transduces the central nervous system (CNS;Cearley et al, 2008).…”

“…Although the BBB-penetrating capacity of AAVhu.37 has not been assayed in published work, it is likely that the structural motifs in AAVhu.37 that are responsible for BBB penetration would be identical to those in AAVrh.39 and AAVrh.10. In a preclinical study for the treatment of hemophilia A, macaques administered with AAVhu.37 carrying a functional human blood coagulation factor VIII (hFVIII) gene linked to E03.TTR (liver-specific) promoter exhibited high levels of hFVIII circulating in the blood (Greig et al, 2018). While hepatic gene transfer can induce immunity to the transferred gene (Dobrzynski et al, 2006), hepatic gene transfer with AAV can induce immune tolerance (Mount et al, 2002;Mingozzi et al, 2003;Breous et al, 2009Breous et al, , 2011, even when the gene transfer is not systemic (Martino et al, 2009).…”