Gene Therapy in Patients with the Crigler-Najjar Syndrome.

BACKGROUND: Patients with the Crigler-Najjar syndrome lack the enzyme uridine diphosphoglucuronate glucuronosyltransferase 1A1 (UGT1A1), the absence of which leads to severe unconjugated hyperbilirubinemia that can cause irreversible neurologic injury and death. Prolonged, daily phototherapy partially controls the jaundice, but the only definitive cure is liver transplantation. METHODS: We report the results of the dose-escalation portion of a phase 1-2 study evaluating the safety and efficacy of a single intravenous infusion of an adeno-associated virus serotype 8 vector encoding UGT1A1 in patients with the Crigler-Najjar syndrome that was being treated with phototherapy. Five patients received a single infusion of the gene construct (GNT0003): two received 2×1012 vector genomes (vg) per kilogram of body weight, and three received 5×1012 vg per kilogram. The primary end points were measures of safety and efficacy; efficacy was defined as a serum bilirubin level of 300 µmol per liter or lower measured at 17 weeks, 1 week after discontinuation of phototherapy. RESULTS: No serious adverse events were reported. The most common adverse events were headache and alterations in liver-enzyme levels. Alanine aminotransferase increased to levels above the upper limit of the normal range in four patients, a finding potentially related to an immune response against the infused vector; these patients were treated with a course of glucocorticoids. By week 16, serum bilirubin levels in patients who received the lower dose of GNT0003 exceeded 300 µmol per liter. The patients who received the higher dose had bilirubin levels below 300 µmol per liter in the absence of phototherapy at the end of follow-up (mean [±SD] baseline bilirubin level, 351±56 µmol per liter; mean level at the final follow-up visit [week 78 in two patients and week 80 in the other], 149±33 µmol per liter). CONCLUSIONS: No serious adverse events were reported in patients treated with the gene-therapy vector GNT0003 in this small study. Patients who received the higher dose had a decrease in bilirubin levels and were not receiving phototherapy at least 78 weeks after vector administration. (Funded by Genethon and others; ClinicalTrials.gov number, NCT03466463.).

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

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 <1011 GC/mouse, total bilirubin levels returned to those seen in phototherapy-rescued UGT1 KO mice. Mice injected with vector at 1011 or 3 × 1011 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 × 1012 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.

Functions and transcriptional regulation of adult human hepatic UDP-glucuronosyl-transferases (UGTs): mechanisms responsible for interindividual variation of UGT levels.

Ten out of 19 UDP-glucuronosyltransferases (UGTs) are substantially expressed in adult human liver (>1% of total UGTs); 5 UGT1 isoforms (UGT1A1, 1A3, 1A4, 1A6 and 1A9) and 5 UGT2 family members (UGT2B4, 2B7, 2B10, 2B15 and 2B17) (Izukawa et al. [11]). Surprisingly, UGT2B4 and UGT2B10 mRNA were found to be abundant in human liver suggesting an underestimated role of the liver in detoxification of their major substrates, bile acids and eicosanoids. Among factors responsible for high interindividual variation of hepatic UGT levels (genetic diversity including polymorphisms and splice variants, regulation by liver-enriched transcription factors such as HNF1 and HNF4, and ligand-activated transcription factors) nuclear receptors (PXR, CAR, PPARalpha, etc.), and the Ah receptor are discussed. Unraveling the mechanisms responsible for interindividual variation of UGT expression will be beneficial for drug therapy but still remains a major challenge.

Long-term reduction of jaundice in Gunn rats by nonviral liver-targeted delivery of Sleeping Beauty transposon.

UNLABELLED: Asialoglycoprotein receptor (ASGPR)-mediated endocytosis has been used to target genes to hepatocytes in vivo. However, the level and duration of transgene expression have been low because of lysosomal translocation and degradation of the DNA and lack of its integration into the host genome. In this study we packaged the DNA of interest in proteoliposomes containing the fusogenic galactose-terminated F-glycoprotein of the Sendai virus (FPL) for targeted delivery to hepatocytes. After the FPL binds to ASGPR on the hepatocyte surface, fusogenic activity of the F-protein delivers the DNA into the cytosol, bypassing the endosomal pathway. For transgene integration we designed plasmids containing one transcription unit expressing the Sleeping Beauty transposase (SB) and another expressing human uridinediphosphoglucuronate glucuronosyltransferase-1A1 (pSB-hUGT1A1). The latter was flanked by inverted/direct repeats that are substrates of SB. In cell culture, FPL-mediated delivery of the E. coli beta-galactosidase gene (LacZ) resulted in transduction of ASGPR-positive cells (rat hepatocytes or Hepa1 cell line), but not of ASGPR-negative 293 cells. Intravenous injection of the FPL-entrapped pSB-hUGT1A1 (4-8 microg/day, 1-4 doses) into UGT1A1-deficient hyperbilirubinemic Gunn rats (model of Crigler-Najjar syndrome type 1) resulted in hUGT1A1 expression in 5%-10% of hepatocytes, but not in other cell types. Serum bilirubin levels declined by 30% +/- 4% in 2 weeks and remained at that level throughout the 7-month study duration. With histidine containing FPL, serum bilirubin was reduced by 40% +/- 5%, and bilirubin glucuronides were excreted into bile. No antibodies were detectable in the recipient rats against the F-protein or human UGT1A1. CONCLUSION: FPL is an efficient hepatocyte-targeted gene delivery platform in vivo that warrants further exploration toward clinical application.

Successful treatment of UGT1A1 deficiency in a rat model of Crigler-Najjar disease by intravenous administration of a liver-specific lentiviral vector.

Treatment of congenital and acquired liver disease is one of the main issues in the field of gene therapy. Self-inactivating lentiviral vectors have several potential advantages over alternative systems. We have constructed a self-inactivating lentiviral vector (LV-ALBUGT) that expresses the human bilirubin UDP-glucuronosyltransferase (UGT1A1) from a liver-specific promoter. UGT1A1 is involved in the clearance of heme metabolites in the liver. This enzyme is deficient in Crigler-Najjar disease, a recessive inherited disorder in humans characterized by chronic severe jaundice, i.e., high plasma bilirubin levels. Gunn rats suffer from the same defect and are used as an animal model of this disease. We have treated juvenile Gunn rats by single intravenous injection with the LV-ALBUGT vector. Over 1 year after treatment with the highest dose (5 x 10(8) transducing units), we observed a stable reduction of bilirubin levels to near normal levels and normal secretion of bilirubin conjugates in the bile, in contrast to untreated animals. In situ hybridization showed expression of the therapeutic gene in more than 30% of liver parenchymal cells. Thus, we demonstrate stable and complete clinical remission of a congenital metabolic liver disease in an animal model, after systemic administration of a therapeutic lentiviral vector.

Delivery of a vector encoding mouse hyaluronan synthase 2 via a crosslinked hyaluronan film.

We have developed a crosslinked hyaluronic acid (HA) film with DNA incorporated within its structure and have characterized this system for its efficacy in sustained transferring of a vector encoding mouse hyaluronan synthase 2 (Has2). Analysis of the DNA release kinetics indicated that the HA films degraded when treated with hyaluronidase and that they released DNA over a prolonged period of time. Gel electrophoresis revealed that this DNA was intact and immunohistochemical analysis verified the transfection capabilities of DNA release samples. The ability of released DNA encoding Has2 to promote HA synthesis was confirmed by quantifying the amount of HA produced by COS-1 cells that were transfected with release samples. The intended future application of the HA films is in prevention of post-operative peritoneal adhesions. In addition to serving as a physical barrier, the film would function as a vehicle for sustained delivery of DNA encoding Has2, which would promote the synthesis of HA in transfected tissues.

Identification of human UDP-glucuronosyltransferase enzyme(s) responsible for the glucuronidation of 3-hydroxydesloratadine.

Desloratadine is a non-sedating antihistamine recently approved for the treatment of seasonal allergic rhinitis. The major metabolite of desloratadine in human plasma and urine is the glucuronide conjugate of 3-hydroxydesloratadine. 3-Hydroxydesloratadine-glucuronide is also the major in vitro metabolite of 3-hydroxydesloratadine formed by incubation of 3-hydroxydesloratadine with human liver microsomes supplemented with uridine 5'-diphosphate-glucuronic acid (UDPGA). The metabolite structure was confirmed by LC-MS and LC-MS/MS. Out of ten recombinant human UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A3, UGT1A8 and UGT2B15 exhibited catalytic activity with respect to the formation of 3-hydroxydesloratadine-glucuronide. Inhibition studies with known inhibitors of UGT (diclofenac, flunitrazepam and bilirubin) confirmed the involvement of UGT1A1, UGT1A3 and UGT2B15 in the formation of 3-hydroxydesloratadine-glucuronide. The results from this study demonstrated that the in vitro formation of 3-hydroxydesloratadine-glucuronide from 3-hydroxydesloratadine was mediated via UGT1A1, UGT1A3 and UGT2B15 in human liver.