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961. Delivery of Human Coagulation Factor VIII Gene into Skeletal Muscle Cells Using Lentiviral Vector
961. Delivery of Human Coagulation Factor VIII Gene into Skeletal Muscle Cells Using Lentiviral Vector Hyun Jeong Jcon,' Oak Hee Kim,' Seung Taik Kim,' Tae Keun Oh.' 'Internal Medicine, Chungbuk National University; Cheongju, Chungbuk, Republic 0/ Korea. HemophiliaA is an inherited X-linked lifelong bleeding disorder caused by a deficiency in or functional defect of coagulation factor VIII (FVIII). Gene therapy using lentiviral vector (LV) is a potential therapeutic approach. We investigated the possibility that skeletal muscle cells transduced with lentiviral vectors could produce human coagulation factor VIII resulting in increased in the circulation. A LV containing bacterial LacZ gene as a control or human FVIII gene was intramuscularly administered into a thigh muscle 01'5weeks old Sparague-Dawley rats. The plasma human FVIIl concentration and neutralizing anti-FVIIl antibodies were measured up to 12 weeks in these rats. 1·luman coagulation FVIIl was produced and secreted in vitro following LV carrying FVIII cDNA transduction in the Hela cells. The plasma human FVIIl levels in the rats injected LV carrying FVIII cDNA were elevated and peaked at I week (5.19 ± 0.14 ng/ml, p<0.05) with the levels being maintained for 4 weeks (2.52 ± 0.83 ng/ml, p<0.05) after a single intramuscular injection . The control rats received lentiviral vectors carrying LacZ showed FVIII levels of 0.21 ± 0.05 ng/ml at I week and 0.17 ± 0.08 nglml at 4 weeks , respectively. In the Bethesda assay, neutralizing antibodies were developed only in FVIIl-LV injected rats by 10 weeks, but not in control rats.This study suggested that a single injection of an advanced generation lentiviral vector carrying the human FVIII cDNAresulted in elevated circulating level ofFVIII in the immune competent rat, and this gene transfer approach to the skeletal muscle could be an effective tool in treatment of hemophilia A.
962. Liver-Restricted Administration of Lentiviral Vector during Isolated Hepatic Perfusion Results in Significant Reduction of Plasma Bilirubin Levels in the Gunn Rat
Pascal van del' Wegen,' Gisela aan de Wiel-Ambagtsheer.! Luc J. van del' Laan,' Timo L. ten Hagen,' Bob J. Scholte.' 'Department a/Cell Biology and Genetics, Erasmus MC, Rotterdam, Netherlands; 2Department ofSurgical Oncology, Erasmus MC, Rotterdam, Netherlands; 'Department ofSurgery; Erasmus MC, Rotterdam, Netherlands. Treatment of congenital and acquired liver disease is one of the main issues in the field of gene therapy. Lentiviral vectors have been shown to be effective by intravenous administration in animal models. The Gunn rat is a model for Crigler-Najjar syndrome type I, an inherited disorder ofUDP-glucuronosyltransferase (UGT IA I) characterized by high , neurotoxic plasma bilirubin levels. Recently, we reported that intravenous administration ofa UGTIAI expressing lentiviral vector via the tongue vein stably normalizes plasma bilirubin levels in juvenile Gunn rats for at least 42 weeks after treatment (van del' Wegen et al, 2006). However, systemic delivery of high lentiviral vector dosages is associated with several possible drawbacks. First, activation of the innate immune system may cause inactivation of the vector and toxicity associated with an acute inflammation response. Second , deposition of the vector in non-target cells reduces efficacy and safety in terms of germline transmission and integrational carcinogenesis. To reduce these risks, we investigated the possibility to restrict lentiviral vector deposition to the liver by isolated hepatic perfusion (IHP). We constructed a lentiviral vector LV-ALBUGT that expresses UGTIAI from a liver specific murine albumin promoter/enhancer combination. Adult male Gunn rats >280 g were subjected to oxygenated IHP by cannulation Molecular 'Therapy Volume15.Supplement I, May 2007
Copyright © T he American Socie ty of Gen e Therap y
ofthe hepatic artery and inferior vena cava. At an orthograde perfusion rate of 5 ml/min, a solution containing 5x I0" transducing units (TU) ofLV-ALBUGT, diluted in blood plasma substitute (Eloheas or Gelofusin) was circulated for 10 minutes, followed by a la-minute wash out with fresh plasma substitute. A subgroup of animals was previously depleted from Kuplfercells by intravenous administration of elodronate Iiposomes. After treatment, plasma bilirubin levels decreased significantly (30%) for at Icast eighteen weeks . Kuplfer cell depletion before treatment reduced the vector dosage needed to obtain this result from 1.5x10') to 0.5x Ia')TU. There was no difference in efficiency of vector deposition between a gelatin based (Gelofusin) or starch based (Elohaes) plasma substitute. The liver of treated animals contained approximately 0.1 vector copy per cell as determined by quantitative PCR at eighteen weeks. Determination of p24 antigen and transducing units in the perfusate before and after perfusion showed that only one-third ofthe vector was actually deposited during the l O-minute exposure. Isolated hepatic perfusion can be an appealing alternative for systemic lentiviral vector adm inistration to the liver. IHP is currently used in humans to treat liver carcinomas, and thus could be applied for gene therapy treatment of congenital enzyme deficiency and chronic viral infections. Current experiments in our laboratory inelude the usc of stronger promoters, and modification ofthe perfusion techniques to improve vector deposition.
963. Complete Phenotype Correction in Female PKU Mice by Repeated Administrations of Murine PAH cDNA Using a Genome-Targeted phiBT1 Bacteriophage Integration System Li Chen; Savio L. C. Woo.' 'Gene and Cell Medicine, Mount Sinai School ofMedicine, New York, N}:
Phenylketonuria (PKU) is a metabolic disorder secondary to a hepatic deficiency of phenylalanine hydroxylase (PAH) that predisposes affected children to develop severe and irreversible mental retardation. We have previously reported the complete and permanent correction ofthe hyperphenylalaniemic and hypopigmentation phenotypes in male PKU mice after genome-targeted delivery of murine PAH cDNA in a phiBTI bacteriophage integration system. The treatment, however, led to only partial correction ofthe disease phenotypes in female PKU mice. The sexual dimorphism was secondary to a lower level oftetrahydrobiopterin, which is an obligate co-factor in the phenylalanine hydroxylation reaction that becomes rate limiting in the genetically reconstituted hepatocytes of the female PKU mice. In this paper we show that sequential administration of GFP- and RFP-expressing cassettes in the phiBTl integration system led to distinct and non-overlapping populations ofgreen and red fluorescent hepatocytes in vivo. 10 weekly administrations ofa plasmid co-expressing SEAP and lacZ in the phiBT I vector system led to an 8-10 fold higher level of serum SEAP as well as 10-15 times more lacZ positive hepatocytes than mice receiving a single administration of the vector. Importantly, there was no apparent liver pathology in mice even after 10 consecutive administrations ofthe phiBT I vectors system. Indeed the hyperphenylalaniemic and hypopigmentation phenotypes of female PKU mice were corrected completely after JO weekly administrations of murine (lAH cDNA in the phiBTl vector system. Importantly, there was no apparent liver pathology in mice even after 10 consecutive administrations ofthe phiBTl integration system. The results indicate that repeated administrations oftransgenes in the phiBTl integration system can lead to their genome-targeted integration in a diverse population of hepatocytes that results in the elevation of transgene expression levels in a cumulative manner, which can be utilized to overcome the limitation of insufficient transgene expression due to low genome integration frequencies in a gene therapy paradigm for metabolic disorders. S367
962. Liver-Restricted Administration of Lentiviral Vector during Isolated Hepatic Perfusion Results in Significant Reduction of Plasma Bilirubin Levels in the Gunn Rat
Pascal van del' Wegen,' Gisela aan de Wiel-Ambagtsheer.! Luc J. van del' Laan,' Timo L. ten Hagen,' Bob J. Scholte.' 'Department a/Cell Biology and Genetics, Erasmus MC, Rotterdam, Netherlands; 2Department ofSurgical Oncology, Erasmus MC, Rotterdam, Netherlands; 'Department ofSurgery; Erasmus MC, Rotterdam, Netherlands. Treatment of congenital and acquired liver disease is one of the main issues in the field of gene therapy. Lentiviral vectors have been shown to be effective by intravenous administration in animal models. The Gunn rat is a model for Crigler-Najjar syndrome type I, an inherited disorder ofUDP-glucuronosyltransferase (UGT IA I) characterized by high , neurotoxic plasma bilirubin levels. Recently, we reported that intravenous administration ofa UGTIAI expressing lentiviral vector via the tongue vein stably normalizes plasma bilirubin levels in juvenile Gunn rats for at least 42 weeks after treatment (van del' Wegen et al, 2006). However, systemic delivery of high lentiviral vector dosages is associated with several possible drawbacks. First, activation of the innate immune system may cause inactivation of the vector and toxicity associated with an acute inflammation response. Second , deposition of the vector in non-target cells reduces efficacy and safety in terms of germline transmission and integrational carcinogenesis. To reduce these risks, we investigated the possibility to restrict lentiviral vector deposition to the liver by isolated hepatic perfusion (IHP). We constructed a lentiviral vector LV-ALBUGT that expresses UGTIAI from a liver specific murine albumin promoter/enhancer combination. Adult male Gunn rats >280 g were subjected to oxygenated IHP by cannulation Molecular 'Therapy Volume15.Supplement I, May 2007
Copyright © T he American Socie ty of Gen e Therap y
ofthe hepatic artery and inferior vena cava. At an orthograde perfusion rate of 5 ml/min, a solution containing 5x I0" transducing units (TU) ofLV-ALBUGT, diluted in blood plasma substitute (Eloheas or Gelofusin) was circulated for 10 minutes, followed by a la-minute wash out with fresh plasma substitute. A subgroup of animals was previously depleted from Kuplfercells by intravenous administration of elodronate Iiposomes. After treatment, plasma bilirubin levels decreased significantly (30%) for at Icast eighteen weeks . Kuplfer cell depletion before treatment reduced the vector dosage needed to obtain this result from 1.5x10') to 0.5x Ia')TU. There was no difference in efficiency of vector deposition between a gelatin based (Gelofusin) or starch based (Elohaes) plasma substitute. The liver of treated animals contained approximately 0.1 vector copy per cell as determined by quantitative PCR at eighteen weeks. Determination of p24 antigen and transducing units in the perfusate before and after perfusion showed that only one-third ofthe vector was actually deposited during the l O-minute exposure. Isolated hepatic perfusion can be an appealing alternative for systemic lentiviral vector adm inistration to the liver. IHP is currently used in humans to treat liver carcinomas, and thus could be applied for gene therapy treatment of congenital enzyme deficiency and chronic viral infections. Current experiments in our laboratory inelude the usc of stronger promoters, and modification ofthe perfusion techniques to improve vector deposition.
963. Complete Phenotype Correction in Female PKU Mice by Repeated Administrations of Murine PAH cDNA Using a Genome-Targeted phiBT1 Bacteriophage Integration System Li Chen; Savio L. C. Woo.' 'Gene and Cell Medicine, Mount Sinai School ofMedicine, New York, N}:
Phenylketonuria (PKU) is a metabolic disorder secondary to a hepatic deficiency of phenylalanine hydroxylase (PAH) that predisposes affected children to develop severe and irreversible mental retardation. We have previously reported the complete and permanent correction ofthe hyperphenylalaniemic and hypopigmentation phenotypes in male PKU mice after genome-targeted delivery of murine PAH cDNA in a phiBTI bacteriophage integration system. The treatment, however, led to only partial correction ofthe disease phenotypes in female PKU mice. The sexual dimorphism was secondary to a lower level oftetrahydrobiopterin, which is an obligate co-factor in the phenylalanine hydroxylation reaction that becomes rate limiting in the genetically reconstituted hepatocytes of the female PKU mice. In this paper we show that sequential administration of GFP- and RFP-expressing cassettes in the phiBTl integration system led to distinct and non-overlapping populations ofgreen and red fluorescent hepatocytes in vivo. 10 weekly administrations ofa plasmid co-expressing SEAP and lacZ in the phiBT I vector system led to an 8-10 fold higher level of serum SEAP as well as 10-15 times more lacZ positive hepatocytes than mice receiving a single administration of the vector. Importantly, there was no apparent liver pathology in mice even after 10 consecutive administrations ofthe phiBT I vectors system. Indeed the hyperphenylalaniemic and hypopigmentation phenotypes of female PKU mice were corrected completely after JO weekly administrations of murine (lAH cDNA in the phiBTl vector system. Importantly, there was no apparent liver pathology in mice even after 10 consecutive administrations ofthe phiBTl integration system. The results indicate that repeated administrations oftransgenes in the phiBTl integration system can lead to their genome-targeted integration in a diverse population of hepatocytes that results in the elevation of transgene expression levels in a cumulative manner, which can be utilized to overcome the limitation of insufficient transgene expression due to low genome integration frequencies in a gene therapy paradigm for metabolic disorders. S367