We evaluated nonviral gene delivery into skeletal muscle via femoral artery and great saphenous vein for correction of hyperbilirubinemia in the Gunn rat, the animal model of Crigler-Najjar syndrome type I. A single injection of pDNA expressing hUGT1A1 under the CMV promoter resulted in excretion of bilirubin glucuronides in bile and a significant decrease in serum bilirubin for at least 2 or 4 weeks, respectively. Loss of metabolic effect was associated with a decrease in recombinant protein in muscle, while pDNA and transcript were detectable 4 weeks after gene delivery. Monthly intravenous gene delivery maintained metabolic correction for at least 5 months. Fibrosis around vessels in the arterial group limited the number of successful repeat gene transfer sessions to 3. Animals expressing hUGT1A1 developed anti-hUGT1A1 antibodies and lymphocytic infiltrate in muscle. Immunosuppression abrogated antibody response, ameliorated lymphocytic inflammation, and enhanced metabolic correction but did not prevent a decrease in the amount of recombinant protein. In conclusion, repeated intravenous delivery of pDNA into muscle enables long-term correction of hyperbilirubinemia in the Gunn rat. The procedure is safe and simple, with great clinical potential. Further studies are needed to explain the mechanisms of loss and improve the stability of recombinant hUGT1A1 in muscle.
"However, when we treated in parallel mutant mice with CMV-hUGT1A1 and AAT-hUGT1A1 AAV vectors , plasma bilirubin levels were much higher in CMV- hUGT1A1-treated mice than in AAT-hUGT1A1-treated ones, despite of the 4–6-fold higher muscle expression of hUGT1a1. This result is in line with that observed in previous attempts to treat animal models of CNSI by skeletal muscledirected gene therapy, which have produced partial success, with reduction of plasma bilirubin levels up to 50% of untreated controls, despite moderated to elevated levels of UGT1A1 expression (Danko et al., 2004; Jia and Danko, 2005; Bortolussi et al., 2012; Pastore et al., 2012). These results suggest that one or more steps in the bilirubinconjugation pathway may be limiting or missing in skeletal muscle. "
[Show abstract][Hide abstract] ABSTRACT: Null mutations in the UGT1A1 gene result in Crigler-Najjar syndrome type I (CNSI), characterized by severe hyperbilirubinemia and constant risk of developing neurological damage. Phototherapy treatment lowers plasma bilirubin levels, but its efficacy is limited and liver transplantation is required. To find alternative therapies we applied AAV-liver specific gene therapy to a lethal mouse model of CNSI. We demonstrated that a single neonatal hUGT1A1 gene transfer was successful and the therapeutic effect lasted up to 17 months post-injection. The therapeutic effect was mediated by the presence of transcriptionally active double stranded episomes. We also compared the efficacy of two different gene therapy approaches: liver vs. skeletal muscle transgene expression. We observed that 5%-8% of normal liver expression and activity levels were sufficient to significantly reduce bilirubin levels and maintain lifelong low plasma bilirubin concentration (3.1±1.5 mg/dL). In contrast, skeletal muscle was not able to efficiently lower bilirubin (6.4±2.0 mg/dL), despite 20-30% of hUgt1a1 expression levels, compared to normal liver. We propose that this remarkable difference in gene therapy efficacy could be related to the absence of the Mrp2 and Mrp3 transporters of conjugated bilirubin in muscle. Taken together, our data support the concept that liver is the best organ for efficient and long-term CNSI gene therapy, and suggest that the use of extra-hepatic tissues should be coupled to the presence of bilirubin transporters.
Human Gene Therapy 07/2014; DOI:10.1089/hum.2013.233 · 3.76 Impact Factor
"Therefore, patients with Crigler-Najjar type I are often advised to consider liver transplantation, most frequently in the range of 18 – 25 years of age. Crigler-Najjar syndrome has long been considered a paradigm for developing gene therapies for metabolic liver diseases for several reasons: (a) the underlying defect is well characterized at the biochemical and molecular level; (b) the fraction of corrected hepatocytes required for clinical benefit is small, as deduced from hepatocyte transplantation studies ; (c) the UGT1A1 does not require strict gene regulation for normal activity; (d) an animal model, the Gunn rat, recapitulating the human disease is available; (e) the outcome of the experimental therapies can be easily determined by measuring bilirubin fractions in serum and bile; (f) the UGT1A1 can be produced from skeletal muscle other than liver, its natural production site, and still retain the ability to transform bilirubin into water-soluble derivatives . For these several reasons, Crigler-Najjar syndrome type I is very attractive as a gene therapy disease candidate and its correction has been the goal of several studies using different vector systems including RV, LV, Ad, AAV, and nonviral vectors. "
[Show abstract][Hide abstract] ABSTRACT: The treatment for inborn errors of liver metabolism is based on dietary, drug, and cell therapies (orthotopic liver transplantation). However, significant morbidity and mortality still remain, and alternative strategies are needed. Gene replacement therapy has the potential of providing a definitive cure for patients with these diseases. Significant progress has been made in the pre-clinical arena and achievement of efficacy in different animal models has been reported using multiple gene transfer technologies. This article summarizes the gene transfer strategies being investigated, the pre-clinical data, and the available early clinical results for inborn errors of liver metabolism.
Italian Journal of Pediatrics 02/2008; 34(1):2. DOI:10.1186/1824-7288-34-2 · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: De ziekte van Crigler-Najjar is een ernstige, weinig voorkomende vorm van geelzucht. Kenmerk van de ziekte is ophoping in het lichaam van ongeconjugeerd bilirubine (UCB), een afbraakproduct van rode bloedcellen. Ophoping van UCB kan leiden tot neurologische schade. De gebruikelijke behandeling van de ziekte is fototherapie, waarbij de patiënt dagelijks vele uren onder een soort zonnebank moet doorbrengen. Dit tast het sociale leven van de patiënt erg aan. Bovendien wordt de behandeling met het stijgen van de leeftijd minder effectief. Promovendus Anja Hafkamp deed onderzoek naar een nieuwe behandeling van deze ziekte. Doel was UCB in de darmen te “vangen” door middel van vet. Dit voorkomt heropname van UCB vanuit de darm in het lichaam. Door oraal toe te dienen medicatie werd de vetuitscheiding en daarmee de UCB uitscheiding via de ontlasting bevorderd. Bij patiënten met de ziekte van Crigler-Najjar waren de effecten relatief beperkt. Echter, een subgroep van de patiënten reageerde wél op de behandeling. Nader onderzoek moet duidelijk maken wat de verschillen veroorzaakt. Hafkamp verwacht dat deze nieuwe behandelmethode in de toekomst toegepast kan worden.
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