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Marcelo Simon Sola - One of the best experts on this subject based on the ideXlab platform.
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Preclinical Development of an AAV8-hUGT1A1 Vector for the Treatment of Crigler-Najjar Syndrome
2019Co-Authors: Fanny Collaud, Giulia Bortolussi, Severine Charles, Patrice Vidal, Marcelo Simon Sola, Laurence Guianvarc’h, Sem J. Aronson, Thierry Bordet, Philippe Veron, Stephanie RundwasserAbstract:Adeno-associated viruses (AAVs) are among the most efficient vectors for liver gene therapy. Results obtained in the first hemophilia clinical trials demonstrated the long-term efficacy of this approach in humans, showing efficient targeting of hepatocytes with both self-complementary (sc) and single-stranded (ss) AAV vectors. However, to support clinical development of AAV-based gene therapies, efficient and scalable production processes are needed. In an effort to translate to the clinic an approach of AAV-mediated liver gene transfer to treat Crigler-Najjar (CN) Syndrome, we developed an (ss)AAV8 vector carrying the human UDP-glucuronosyltransferase family 1-member A1 (hUGT1A1) transgene under the control of a liver-specific promoter. We compared our construct with similar (sc)AAV8 vectors expressing hUGT1A1, showing comparable potency in vitro and in vivo. Conversely, (ss)AAV8-hUGT1A1 vectors showed superior yields and product homogeneity compared with their (sc) counterpart. We then focused our efforts in the scale-up of a manufacturing process of the clinical product (ss)AAV8-hUGT1A1 based on the triple transfection of HEK293 cells grown in suspension. Large-scale production of this vector had characteristics identical to those of small-scale vectors produced in adherent cells. Preclinical studies in animal models of the disease and a good laboratory practice (GLP) toxicology-biodistribution study were also conducted using large-scale preparations of vectors. These studies demonstrated long-term safety and efficacy of gene transfer with (ss)AAV8-hUGT1A1 in relevant animal models of the disease, thus supporting the clinical translation of this gene therapy approach for the treatment of CN Syndrome. Keywords: AAV vector, Crigler-Najjar Syndrome, UGT1A1, liver gene transfer, long-term safet
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A translationally optimized AAV-UGT1A1 vector drives safe and long-lasting correction of Crigler-Najjar Syndrome
2016Co-Authors: Giuseppe Ronzitti, Giulia Bortolussi, Remco Van Dijk, Fanny Collaud, Severine Charles, Christian Leborgne, Patrice Vidal, Samia Martin, Bernard Gjata, Marcelo Simon SolaAbstract:Crigler-Najjar Syndrome is a severe metabolic disease of the liver due to a reduced activity of the UDP Glucuronosyltransferase 1A1 (UGT1A1) enzyme. In an effort to translate to the clinic an adeno-associated virus vector mediated liver gene transfer approach to treat Crigler-Najjar Syndrome, we developed and optimized a vector expressing the UGT1A1 transgene. For this purpose, we designed and tested in vitro and in vivo multiple codon-optimized UGT1A1 transgene cDNAs. We also optimized noncoding sequences in the transgene expression cassette. Our results indicate that transgene codon-optimization is a strategy that can improve efficacy of gene transfer but needs to be carefully tested in vitro and in vivo. Additionally, while inclusion of introns can enhance gene expression, optimization of these introns, and in particular removal of cryptic ATGs and splice sites, is an important maneuver to enhance safety and efficacy of gene transfer. Finally, using a translationally optimized adeno-associated virus vector expressing the UGT1A1 transgene, we demonstrated rescue of the phenotype of Crigler-Najjar Syndrome in two animal models of the disease, Gunn rats and Ugt1a1-/- mice. We also showed long-term (>1 year) correction of the disease in Gunn rats. These results support further translation of the approach to humans.
Stephen C. Strom - One of the best experts on this subject based on the ideXlab platform.
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isolated hepatocyte transplantation for crigler najjar Syndrome type 1
2005Co-Authors: Giovanni Ambrosino, Stephen C. Strom, Sergio Varotto, Graziella Guariso, Elisa Franchin, Diego Miotto, Luciana Caenazzo, Stefano M M Basso, Paolo Carraro, Marialuisa ValenteAbstract:Crigler-Najjar Syndrome type 1 (CN1) is an inherited disorder characterized by the absence of hepatic uridine diphosphoglucuronate glucuronosyltransferase (UDPGT), the enzyme responsible for the conjugation and excretion of bilirubin. We performed allogenic hepatocyte transplantation (AHT) in a child with CN1, aiming to improve bilirubin glucuronidation in this condition. A 9-year-old boy with CN1 was prepared with plasmapheresis and immunosuppression with prednisolone and tacrolimus. When a graft was made available, 7.5 × 10 9 hepatocytes were isolated and infused into the portal vein percutaneously. After 2 weeks phenobarbitone was added to promote the enzymatic activity of UDPGT of the transplanted hepatocytes. Nocturnal phototherapy was continued throughout the studied period. Total bilirubin was considered a reliable marker of allogenic cell function. There was no significant variation of vital signs nor complications during the infusion. Mean ± SD bilirubin level was 530 ± 38 µmol/L before and 359 ± 46 µmol/L after AHT (t-test, p < 0.001). However, the introduction of phenobarbitone was followed by a drop of tacrolimus level with increase of alanine aminotransferase (ALT) and increase of bilirubin. After standard treatment of cellular rejection bilirubin fell again but from then on it was maintained at a greater level. After discharge the patient experienced a further increase of bilirubin that returned to predischarge levels after readmission to the hospital. This was interpreted as poor compliance with phototherapy. Only partial correction of clinical jaundice and the poor tolerability to nocturnal phototherapy led the parents to refuse further hepatocyte infusions and request an orthotopic liver transplant. After 24 months the child is well, with good liver function on tacrolimus and prednisolone-based immunosuppression. Isolated AHT, though effective and safe, is not sufficient to correct CN1. Maintenance of adequate immunosuppression and family compliance are the main factors hampering the success of this procedure.
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treatment of the crigler najjar Syndrome type i with hepatocyte transplantation
1998Co-Authors: Jayanta Roy Chowdhury, Timothy C Goertzen, Phyllis Irene Warkentin, Kenneth Dorko, Bernhard V. Sauter, Stuart S. Kaufman, Namita Roy Chowdhury, Stephen C. StromAbstract:Crigler–Najjar Syndrome type I is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia beginning at birth. The Syndrome results from an absence of hepatic uridine diphosphoglucuronate (UDP) glucuronosyltransferase activity, which is essential for the conjugation and excretion of bilirubin. Because of the accumulation of unconjugated bilirubin in plasma, patients are at risk for kernicterus.1 Although phototherapy successfully reduces serum bilirubin levels, patients are again at risk for kernicterus around the time of puberty, when phototherapy becomes less effective.2 The necessary daily duration of phototherapy often approaches 14 to 16 hours. At present, liver transplantation is the only definitive treatment. . . .
Jayanta Roy Chowdhury - One of the best experts on this subject based on the ideXlab platform.
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treatment of the crigler najjar Syndrome type i with hepatocyte transplantation
1998Co-Authors: Jayanta Roy Chowdhury, Timothy C Goertzen, Phyllis Irene Warkentin, Kenneth Dorko, Bernhard V. Sauter, Stuart S. Kaufman, Namita Roy Chowdhury, Stephen C. StromAbstract:Crigler–Najjar Syndrome type I is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia beginning at birth. The Syndrome results from an absence of hepatic uridine diphosphoglucuronate (UDP) glucuronosyltransferase activity, which is essential for the conjugation and excretion of bilirubin. Because of the accumulation of unconjugated bilirubin in plasma, patients are at risk for kernicterus.1 Although phototherapy successfully reduces serum bilirubin levels, patients are again at risk for kernicterus around the time of puberty, when phototherapy becomes less effective.2 The necessary daily duration of phototherapy often approaches 14 to 16 hours. At present, liver transplantation is the only definitive treatment. . . .
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kernicterus in an adult who is heterozygous for crigler najjar Syndrome and homozygous for gilbert type genetic defect
1997Co-Authors: Naga Chalasani, Namita Roy Chowdhury, Jayanta Roy Chowdhury, Thomas D BoyerAbstract:Gilbert Syndrome is a common genetic disorder associated with mild unconjugated hyperbilirubinemia and no clinical illness. In contrast, Crigler-Najjar Syndrome types I and II are rare genetic disorders associated with severe unconjugated hyperbilirubinemia and a life-long risk of kernicterus. Patients with Gilbert Syndrome have low levels of a normal form of uridinediphosphoglucuronate glucuronosyltransferase because of a defect in the promoter region of both alleles, whereas patients with Crigler-Najjar Syndrome are homozygous for a defect that yields an abnormal form of the enzyme that has limited or no activity. This case report describes a young adult with Crigler-Najjar Syndrome type II in whom kernicterus developed after a laparoscopic cholecystectomy. The development of kernicterus was the result of a largely preventable series of events that lead to an increase in the free fraction of his serum bilirubin. Analysis of his genetic defect showed that he was homozygous for the mutation associated with Gilbert Syndrome and heterozygous for a second mutation in the open reading frame of one allele of the bilirubin uridinediphosphoglucuronate glucuronosyltransferase gene. The combined defect leads to severe hyperbilirubinemia and shows how seemingly benign genetic defects, when combined, can cause serious clinical disease.
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the genetic basis of the reduced expression of bilirubin udp glucuronosyltransferase 1 in gilbert s Syndrome
1995Co-Authors: Piter J. Bosma, Jayanta Roy Chowdhury, Conny T Bakker, Shailaja Gantla, Anita De Boer, Ben A Oostra, D Lindhout, Guido N J Tytgat, Peter L M Jansen, Ronald Oude P J ElferinkAbstract:Background People with Gilbert's Syndrome have mild, chronic unconjugated hyperbilirubinemia in the absence of liver disease or overt hemolysis. Hepatic glucuronidating activity, essential for efficient biliary excretion of bilirubin, is reduced to about 30 percent of normal. Methods We sequenced the coding and promoter regions of the gene for bilirubin UDP-glucuronosyltransferase 1 (bilirubin/uridine diphosphoglucuronate-glucuronosyltransferase 1) — the only enzyme that contributes substantially to bilirubin glucuronidation — in 10 unrelated patients with Gilbert's Syndrome, 16 members of a kindred with a history of Crigler–Najjar Syndrome type II, and 55 normal subjects. Results The coding region of the gene for the enzyme was normal in the 10 patients with Gilbert's Syndrome. These patients were homozygous for two extra bases (TA) in the TATAA element of the 5' promoter region of the gene (A(TA)7TAA rather than the normal A(TA)6TAA). The presence of the longer TATAA element resulted in the reduced expr...
R.d. Hughes - One of the best experts on this subject based on the ideXlab platform.
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liver after hepatocyte transplantation for liver based metabolic disorders in children
2008Co-Authors: Alberto Quaglia, Ragai R Mitry, Sharon C Lehec, Stephen Devereaux, Alexander S Knisely, Mohamed Rela, Nigel Heaton, R.d. Hughes, Julie Richards, Bernard PortmannAbstract:There are limited data regarding donor hepatocyte engraftment into recipient liver after human hepatocyte transplantation (HHTx). We reviewed the explant livers of seven children with metabolic disorders [ornithine-transcarbamylase deficiency (one), coagulation factor VII deficiency (three), Crigler-Najjar Syndrome (one), progressive familial intrahepatic cholestasis type 2 (PFIC-2) deficiency (two)] who received allograft hepatocytes by intraportal infusion with improvement in phenotype, although all later underwent liver transplantation (LT). Immunohistochemistry for bile salt export protein (BSEP) in the PFIC-2 patients and genetic typing following laser capture microdissection (LCM) of liver cells in the others were used to identify donor hepatocytes in recipient explant livers. Explant livers usually showed a preserved lobular architecture. In one patient, hepatocytes were identified inside portal vein thrombi. No donor hepatocytes in liver cell plates were identified immunohistochemically or by gene...
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hepatocyte transplantation for liver based metabolic disorders
2006Co-Authors: Ragai R Mitry, Anil Dhawan, R.d. HughesAbstract:Hepatocyte transplantation is being investigated as an alternative to orthotopic liver transplantation in patients with liver-based metabolic disorders. The progress made in this field to date is reviewed. Protocols have been developed using collagenase perfusion to isolate human hepatocytes from unused donor liver tissue. Hepatocytes with a high viability can often be obtained and can be cryopreserved for later use, though with loss of function on thawing. For clinical use, hepatocytes must be prepared in clean GMP conditions with cells meeting criteria of function and lack of microbial contamination before patient use. Hepatocytes are infused intraportally into the patient's liver, where a proportion of cells will engraft and replace the deficient metabolic function without the need for major surgery. Twenty patients have now received hepatocyte transplantation, including eight children at King's College Hospital. There was a range of aetiologies of liver disease: familial hypercholesterolaemia, Crigler-Najjar Syndrome type 1, urea cycle defects, infantile Refsum disease, glycogen storage disease type Ia, inherited factor VII deficiency and progressive familial intrahepatic cholestasis type 2. Clinical improvement and partial correction of the metabolic abnormality was observed in most cases. Considerable progress has been made in developing the technique, but hepatocyte transplantation is limited by the available supply of liver tissue. Hepatocytes derived from stem cells could provide alternative sources of cells in the future.
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hepatocyte transplantation for metabolic liver disease uk experience
2005Co-Authors: R.d. Hughes, Ragai R Mitry, Anil DhawanAbstract:For end-stage liver disease and liver-based metabolic conditions the accepted treatment is liver transplantation. With developments in surgical techniques and immunosuppressive drug therapy the survival of patients and grafts is now good. In the conventional procedure the patient's whole liver is replaced with a liver obtained from a braindead or living donor. When the donor liver is too big it can be reduced to a compatible size; and, recently, split-liver procedures have been performed whereby the right lobe is transplanted into an adult and the smaller left lobe into a child,1 thus increasing the effective donor pool. Another important advance in surgical technique is the use of auxiliary liver transplantation for patients with acute liver failure and certain liver-based metabolic defects such as Crigler–Najjar Syndrome type I, urea cycle defects, and familial hypercholesterolaemia. In this procedure, part of the patient's liver, often the left lobe, is replaced with part of a donor liver. In a patient with acute liver failure this leaves open the possibility of regeneration of the native liver, in which case immunosuppression can be stopped and the graft allowed to atrophy; and in a patient with a metabolic disorder the native liver will be available for future gene therapy. The results of auxiliary liver transplantation in man2 have supported observations in animals that small amounts of liver tissue can provide sufficient function to correct an underlying metabolic defect. This finding was a spur to work on hepatocyte transplantation for such disorders. The aim is to repopulate the liver with donor hepatocytes, injected either directly into the liver or into the spleen, from which they migrate to the liver. If the technique proves successful, hepatocyte transplantation offers several potential advantages. In terms of supply, there is the possibility of using cells from livers that are unsuitable for conventional transplantation because of steatosis or trauma. The patient does not have to undergo major surgery; moreover, in metabolic conditions the native liver provides a safety net in case of failure. One of the most important advantages is the availability of the liver as a target organ for gene therapy when this becomes a clinical reality. Experience of hepatocyte transplantation has been gained in patients with acute liver failure3,4 and metabolic liver diseases such as Crigler–Najjar Syndrome type I,5 glycogen storage disease type 1a,6 and urea cycle defects.7 The background to this work has been described elsewhere.8–11 The current paper discusses the sources of hepatocytes, the isolation process, preclinical studies and clinical experience in the UK, especially in the treatment of liver-based inborn errors of metabolism.
Anil Dhawan - One of the best experts on this subject based on the ideXlab platform.
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hepatocyte transplantation for liver based metabolic disorders
2006Co-Authors: Ragai R Mitry, Anil Dhawan, R.d. HughesAbstract:Hepatocyte transplantation is being investigated as an alternative to orthotopic liver transplantation in patients with liver-based metabolic disorders. The progress made in this field to date is reviewed. Protocols have been developed using collagenase perfusion to isolate human hepatocytes from unused donor liver tissue. Hepatocytes with a high viability can often be obtained and can be cryopreserved for later use, though with loss of function on thawing. For clinical use, hepatocytes must be prepared in clean GMP conditions with cells meeting criteria of function and lack of microbial contamination before patient use. Hepatocytes are infused intraportally into the patient's liver, where a proportion of cells will engraft and replace the deficient metabolic function without the need for major surgery. Twenty patients have now received hepatocyte transplantation, including eight children at King's College Hospital. There was a range of aetiologies of liver disease: familial hypercholesterolaemia, Crigler-Najjar Syndrome type 1, urea cycle defects, infantile Refsum disease, glycogen storage disease type Ia, inherited factor VII deficiency and progressive familial intrahepatic cholestasis type 2. Clinical improvement and partial correction of the metabolic abnormality was observed in most cases. Considerable progress has been made in developing the technique, but hepatocyte transplantation is limited by the available supply of liver tissue. Hepatocytes derived from stem cells could provide alternative sources of cells in the future.
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hepatocyte transplantation for metabolic liver disease uk experience
2005Co-Authors: R.d. Hughes, Ragai R Mitry, Anil DhawanAbstract:For end-stage liver disease and liver-based metabolic conditions the accepted treatment is liver transplantation. With developments in surgical techniques and immunosuppressive drug therapy the survival of patients and grafts is now good. In the conventional procedure the patient's whole liver is replaced with a liver obtained from a braindead or living donor. When the donor liver is too big it can be reduced to a compatible size; and, recently, split-liver procedures have been performed whereby the right lobe is transplanted into an adult and the smaller left lobe into a child,1 thus increasing the effective donor pool. Another important advance in surgical technique is the use of auxiliary liver transplantation for patients with acute liver failure and certain liver-based metabolic defects such as Crigler–Najjar Syndrome type I, urea cycle defects, and familial hypercholesterolaemia. In this procedure, part of the patient's liver, often the left lobe, is replaced with part of a donor liver. In a patient with acute liver failure this leaves open the possibility of regeneration of the native liver, in which case immunosuppression can be stopped and the graft allowed to atrophy; and in a patient with a metabolic disorder the native liver will be available for future gene therapy. The results of auxiliary liver transplantation in man2 have supported observations in animals that small amounts of liver tissue can provide sufficient function to correct an underlying metabolic defect. This finding was a spur to work on hepatocyte transplantation for such disorders. The aim is to repopulate the liver with donor hepatocytes, injected either directly into the liver or into the spleen, from which they migrate to the liver. If the technique proves successful, hepatocyte transplantation offers several potential advantages. In terms of supply, there is the possibility of using cells from livers that are unsuitable for conventional transplantation because of steatosis or trauma. The patient does not have to undergo major surgery; moreover, in metabolic conditions the native liver provides a safety net in case of failure. One of the most important advantages is the availability of the liver as a target organ for gene therapy when this becomes a clinical reality. Experience of hepatocyte transplantation has been gained in patients with acute liver failure3,4 and metabolic liver diseases such as Crigler–Najjar Syndrome type I,5 glycogen storage disease type 1a,6 and urea cycle defects.7 The background to this work has been described elsewhere.8–11 The current paper discusses the sources of hepatocytes, the isolation process, preclinical studies and clinical experience in the UK, especially in the treatment of liver-based inborn errors of metabolism.