- Email: [email protected]
896. Complete and Permanent Correction of Hyperbilirubinemia in Newborn Gunn Rats by Lentiviral-Mediated Gene Transfer
INBORN ERRORS OF METABOLISM: LIVER AND PANCREATIC DISEASES performed, rapid, does not required primary culture of hepatocytes, and preserves full cell engraftment potential and in vivo cell functionality. In this study, we used the Gunn rat, which is the model for Crigler-Najjar syndrome type I, a defect in bilirubin UDPglucuronyltransferase (BGT), to evaluate our approach. Methods: We constructed vectors coding for human BGT under control of the E1a ubiquitous promoter. Freshly isolated hepatocytes were transduced in suspension at a multiplicity of infection of 30 for 4 hrs. After washing, twenty millions hepatocytes were immediately transplanted into jaundiced Gunn rats. Results: Transduction efficiency was limited to 30% in Gunn rat hepatocytes. Despite this low level of transduction efficiency, serum bilirubin levels decreased by 30% in rats receiving BGT-transduced hepatocytes. This decline was equivalent to that obtained with transplantation of wild-type hepatocytes. It was sustained for up to 150 days. Accordingly, conjugated bilirubin appeared in the bile. In contrast, in rats receiving untransduced or GFP-transduced hepatocytes, serum bilirubin didn’t decline but rather increased. GFP-positive hepatocytes amounted at most to 1% of rat livers, which is in agreement with the gene transfer efficiency. Noteworthy, these results also suggested an absence of immune response against the transduced hepatocytes, as expected by selective targeting of the hepatocytes using an ex vivo approach, even though transgene expression was driven by a ubiquitous promoter. Conclusion: In contrast to human hepatocytes, adult rodent hepatocytes were lowly transducible by lentiviral vectors. Despite this limitation, we demonstrated the efficacy of ex vivo approach in Gunn rats. Recent clinical trials have shown that uncultured allogeneic hepatocyte transplantation has resulted in metabolic benefits in patients with metabolic liver disorders. Altogether, these data open new perspectives for the treatment of metabolic liver disorders using an ex vivo approach using lentiviral vectors.
896. Complete and Permanent Correction of Hyperbilirubinemia in Newborn Gunn Rats by Lentiviral-Mediated Gene Transfer Tuan huy Nguyen,1 Marta Bellodi-Privato,2 Dominique Aubert,2 Anne Myara,3 Didier Trono,1 Nicolas Ferry.2 1 Department of Microbiology and Molecular Medicine, University of Geneva Medical School, CMU, Geneva, Switzerland; 2 Biothérapies Hépatiques, CIC-INSERM, CHU Hotel Dieu, Nantes, France; 3Service de Biochimie, Hôpital Saint Joseph, Paris, France. Background: Crigler Najjar type 1 disease (CN1) is a rare inherited metabolic disease characterized by complete absence of hepatic UDP-Glucuronosyl transferase (UGT1A1, EC 2.4.1.17). The hallmark of CN1 is a very high level of circulating unconjugated bilirubin that may result in kernicterus. The only curative treatment of CN1 is orthotopic liver transplantation. The Gunn rat is the model for CN1 and many previous studies have attempted to correct hyperbilirubinemia in the Gunn rat using a large array of gene transfer vectors. However in most cases phenotypic correction was transient, mainly because of immune response directed against the transduced cells. Here we show that in vivo neonatal hepatocyte transduction with lentiviral vectors resulted in long-term complete phenotype correction in Gunn rats. Methods: Lentiviral vectors pseudotyped with a VSVG envelop protein and harboring either the human UGT1A1 or the GFP gene as a control were produced by transient transfection and concentrated by ultracentrifugation. Two days-old newborn Gunn rats were injected via the temporal vein with 5x108 transducing units. Bilirubinemia was monitored at 6 weeks and every month thereafter. The presence of bilirubin conjugates was assessed by HPLC in bile
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
samples. The presence of transduced hepatocytes was analysed by PCR and Western blot and by immunohistochemistry in GFPinjected animals. Results: In control GFP-injected animals as well as in non-injected controls, bilirubinemia was elevated at 6 weeks (mean: 76 µM) and remained around 100µM during the whole experimetal follow-up. In contrast in UGT1A1-injected animals bilirubinemia was normal at 6 weeks (3µM) and remained in the normal range during the follow-up (22 weeks as of january 2005). Large amounts of bilirubin mono and di-glucuronides were present in the bile of corrected animals. Finally, PCR and Western blots confirmed the presence and expression of UGT1 in liver. The estimated proportion of transduced hepatocytes was around 10%. Transduced cells were also detected by PCR in extra hepatic tissues. Conclusion This work represents the first demonstration of complete and permanent correction of hyperbilirubinemia in Gunn rats and pave the way for future gene therapy of CN1 using lentiviral vectors.
897. Recombinant AAV2/8 Vector-Mediated Gene Therapy Corrects Hyperphenylalaninemia in Murine PKU Cary O. Harding,1 Melanie B. Gillingham,1 Kelly Hamman,1 Andrew Bird,2 Dwight Koeberl.2 1 Pediatrics, Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR; 2Pediatrics and Medical Genetics, Duke University, Durham, NC. Novel recombinant adeno-associated virus vectors pseudotyped with serotype 8 capsid (rAAV2/8) have recently shown exciting promise as effective liver-directed gene transfer reagents. We have designed and produced an rAAV2/8 vector containing the mouse phenylalanine hydroxylase (PAH) cDNA under the transcriptional control of a strong liver promoter (LSP) and have administered this vector to hyperphenylalaninemic Pahenu2 mice, a model of human PKU. Our hypothesis was that this vector would produce sufficient hepatocyte transduction frequency and PAH activity to correct blood phenylalanine (Phe) levels in murine PKU. Recombinant AAV2/8 vector was injected via the portal vein into five adult (four male and one female) hyperphenylalaninemic Pahenu2 homozygous mice (preinjection Phe = 2108 ± 102 µM, normal = 145 ± 45 µM) and evaluated the effect upon serum Phe levels weekly. Each animal received 2.5 X 1011 rAAV2/8 vector genomes via portal vein injection. A sixth Pahenu2 mouse (female, preinjection Phe = 3579 µM) received only saline as a control, and serum Phe levels remained elevated in this animal (4174 µM ten weeks following injection). Administration of rAAV2/8 yielded a dramatic and stable reduction in serum Phe to normal levels by 1-2 weeks following injection. At ten weeks following injection, serum Phe levels remained normal (157 ± 32 µM) and were significantly reduced in comparison to pretreatment levels (p < 0.001). Phenylalanine clearance measured after parenteral Phe challenge significantly increased following rAAV2/8 treatment. Hyperphenylalaninemia-associated hypopigmentation was also completely corrected. Liver PAH activity was 6-10% of wild type levels in three mice that underwent liver biopsy at 8-10 weeks postinjection. Vector copy number, integration status of the vector genome, biodistribution, and long-term stability of expression will be evaluated. Recombinant AAV2/8-mediated, liver-directed gene therapy is a promising novel treatment approach for PKU and allied inborn errors of metabolism.
S347
896. Complete and Permanent Correction of Hyperbilirubinemia in Newborn Gunn Rats by Lentiviral-Mediated Gene Transfer Tuan huy Nguyen,1 Marta Bellodi-Privato,2 Dominique Aubert,2 Anne Myara,3 Didier Trono,1 Nicolas Ferry.2 1 Department of Microbiology and Molecular Medicine, University of Geneva Medical School, CMU, Geneva, Switzerland; 2 Biothérapies Hépatiques, CIC-INSERM, CHU Hotel Dieu, Nantes, France; 3Service de Biochimie, Hôpital Saint Joseph, Paris, France. Background: Crigler Najjar type 1 disease (CN1) is a rare inherited metabolic disease characterized by complete absence of hepatic UDP-Glucuronosyl transferase (UGT1A1, EC 2.4.1.17). The hallmark of CN1 is a very high level of circulating unconjugated bilirubin that may result in kernicterus. The only curative treatment of CN1 is orthotopic liver transplantation. The Gunn rat is the model for CN1 and many previous studies have attempted to correct hyperbilirubinemia in the Gunn rat using a large array of gene transfer vectors. However in most cases phenotypic correction was transient, mainly because of immune response directed against the transduced cells. Here we show that in vivo neonatal hepatocyte transduction with lentiviral vectors resulted in long-term complete phenotype correction in Gunn rats. Methods: Lentiviral vectors pseudotyped with a VSVG envelop protein and harboring either the human UGT1A1 or the GFP gene as a control were produced by transient transfection and concentrated by ultracentrifugation. Two days-old newborn Gunn rats were injected via the temporal vein with 5x108 transducing units. Bilirubinemia was monitored at 6 weeks and every month thereafter. The presence of bilirubin conjugates was assessed by HPLC in bile
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
samples. The presence of transduced hepatocytes was analysed by PCR and Western blot and by immunohistochemistry in GFPinjected animals. Results: In control GFP-injected animals as well as in non-injected controls, bilirubinemia was elevated at 6 weeks (mean: 76 µM) and remained around 100µM during the whole experimetal follow-up. In contrast in UGT1A1-injected animals bilirubinemia was normal at 6 weeks (3µM) and remained in the normal range during the follow-up (22 weeks as of january 2005). Large amounts of bilirubin mono and di-glucuronides were present in the bile of corrected animals. Finally, PCR and Western blots confirmed the presence and expression of UGT1 in liver. The estimated proportion of transduced hepatocytes was around 10%. Transduced cells were also detected by PCR in extra hepatic tissues. Conclusion This work represents the first demonstration of complete and permanent correction of hyperbilirubinemia in Gunn rats and pave the way for future gene therapy of CN1 using lentiviral vectors.
897. Recombinant AAV2/8 Vector-Mediated Gene Therapy Corrects Hyperphenylalaninemia in Murine PKU Cary O. Harding,1 Melanie B. Gillingham,1 Kelly Hamman,1 Andrew Bird,2 Dwight Koeberl.2 1 Pediatrics, Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR; 2Pediatrics and Medical Genetics, Duke University, Durham, NC. Novel recombinant adeno-associated virus vectors pseudotyped with serotype 8 capsid (rAAV2/8) have recently shown exciting promise as effective liver-directed gene transfer reagents. We have designed and produced an rAAV2/8 vector containing the mouse phenylalanine hydroxylase (PAH) cDNA under the transcriptional control of a strong liver promoter (LSP) and have administered this vector to hyperphenylalaninemic Pahenu2 mice, a model of human PKU. Our hypothesis was that this vector would produce sufficient hepatocyte transduction frequency and PAH activity to correct blood phenylalanine (Phe) levels in murine PKU. Recombinant AAV2/8 vector was injected via the portal vein into five adult (four male and one female) hyperphenylalaninemic Pahenu2 homozygous mice (preinjection Phe = 2108 ± 102 µM, normal = 145 ± 45 µM) and evaluated the effect upon serum Phe levels weekly. Each animal received 2.5 X 1011 rAAV2/8 vector genomes via portal vein injection. A sixth Pahenu2 mouse (female, preinjection Phe = 3579 µM) received only saline as a control, and serum Phe levels remained elevated in this animal (4174 µM ten weeks following injection). Administration of rAAV2/8 yielded a dramatic and stable reduction in serum Phe to normal levels by 1-2 weeks following injection. At ten weeks following injection, serum Phe levels remained normal (157 ± 32 µM) and were significantly reduced in comparison to pretreatment levels (p < 0.001). Phenylalanine clearance measured after parenteral Phe challenge significantly increased following rAAV2/8 treatment. Hyperphenylalaninemia-associated hypopigmentation was also completely corrected. Liver PAH activity was 6-10% of wild type levels in three mice that underwent liver biopsy at 8-10 weeks postinjection. Vector copy number, integration status of the vector genome, biodistribution, and long-term stability of expression will be evaluated. Recombinant AAV2/8-mediated, liver-directed gene therapy is a promising novel treatment approach for PKU and allied inborn errors of metabolism.
S347