1028 Application of AAV Vectors for In Vivo Gene Therapy of Gaucher Disease Molecular Therapy �������� ��� ���� ���������������� �������� ���� ������®������������ �!����� ����"� �������� S396 ���� ���[.]
GENETIC AND METABOLIC DISEASES: PART TWO 1026 Exploring the Use of Ribozymes with Gene Therapy for Phenylketonuria Catherine E Charron,1 Alfred S Lewin,2 Philip J Laipis.1 Biochemistry and Molecular Biology; 2Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Phenylketonuria (PKU) is usually caused by a deficiency of the enzyme phenylalanine hydroxylase (PAH) Even though dietary treatment of PKU has been successful, an alternative is needed Patients, especially adults, often have poor compliance with the unpleasant, socially restrictive and expensive diet Maternal PKU syndrome also requires attention Pregnant PKU mothers even when on diet have difficulty in lowering their blood phenylalanine (phe) levels to a range that is safe for fetuses Children born to PKU mothers have an elevated risk of birth defects, emphasizing the need for alternative therapeutic approaches Based on previous studies, we suggest that overcoming the dominant-negative effects of the endogenous mutant PAH monomers will be needed in order to achieve successful gene therapy for PKU We are exploring the efficiency of hammerhead ribozymes to suppress the synthesis of these mutant monomers Ribozymes catalyze in trans sequence-specific cleavage of RNA Two hammerhead ribozymes were designed to target the mouse phenylalanine hydroxylase (mPAH) message The ribozymes each contain an 11-nucleotide catalytic core attached to a stable hairpin structure flanked by two arms of five or six nucleotides used for target recognition The targets chosen were both AUC based on an analysis of all the possible “NUX” cleaving sites found in the PAH message In vitro analyses of these ribozymes were performed using synthetic RNA ribozymes and short RNA targets A time course analysis revealed that one of the ribozymes (RzI209) cleaved 50% of the 13-nucleotide target by minutes at 5mM MgCl2 in a 30:1 molar ratio of target to ribozyme A second in vitro test showed that RzI209 could cleave the full length PAH RNA target The second ribozyme (RzI94) lacked catalytic activity Ribozyme I209 was cloned into a recombinant Adeno Associated Virus vector (rAAV) The vector contains the same promoter used in our rAAVmPAH construct Transient transfection of both ribozyme and mPAH vectors in HEK-293 cells showed in vivo functionality Using a to molar ratio of mPAH to ribozyme vector, a four-fold drop in PAH enzyme activity was seen in the harvested cells as compared to an mPAH-only transfection control Western blots of these cell extracts confirmed the decrease in PAH protein levels Further in vivo testing of the ribozyme using the mouse BTBR Pahenu2 PKU model is in progress These mice carry a missense mutation at the locus F263S, and closely mimic the human phenotype The ribozyme is targeted to cleave the endogenous message, while a “hardened” functional mPAH gene is introduced simultaneously Functional enzyme formation will be monitored along with blood phe levels 1027 Lentiviral Transduction and Transplantation of Human and Non-Human Primate Hepatocytes in Murine and Simian Models Alexandre Parouchev,1 Marion Andreoletti,1 Ibrahim Dagher,1 Julie Branger,1 Tuan Nguyen,2 Aurore Coulomb,1 Tao Lin,1 Lyes Boudechiche,1 Sylvie Mainot,1 Karen Wersterman,3 Philippe Leboulch,3 Dominique Pariente,4 Dominique Franco,1 Anne Weber.1 INSERM EMI 00-20, Hopital Antoine Beclere, Clamart, France; Department of Genetics and Microbiology, University of Geneva Medical School, Geneva, Switzerland; 3Genetix Pharmaceuticals, Inc., Cambridge, MA; 4Department of Radiology and Pediatry, Hopital Bicetre, Le Kremlin-Bicetre, France Orthotopic liver transplantation (OLT) is currently the only curative therapy for patients with liver failure and life-threatening S396 metabolic disorders However, this procedure is highly costly, complex and limited by a relative lack of liver donors Thus, there is a growing need to develop new approaches for liver insufficiency treatment A number of laboratory animal studies have demonstrated the potential of hepatocyte transplantation (HT) as an alternative to OLT for the treatment of liver failure and inborn disorders of liver-based metabolism HT is a technically simple procedure compared to OLT, percutaneous catheters can be used to transplant cells, and the procedure is “reversible”, because the host liver remains intact Moreover, hepatocytes obtained from one donor can be used for multiple patients Hepatocytes seem also to be an excellent vehicle for ex vivo gene therapy However, the results from the few clinical trials performed on patients with metabolic diseases, either by autologous transplantation of genetically modified hepatocytes, or allotransplantation, have been rather disappointing This highlights the need to develop scale up protocols in large animal models A non-human primate model seems an appropriate choice for the establishment of pre-clinical procedures of hepatic cell transplantation Indeed, the simian hepatic anatomy and vascularization are very similar to that of humans and differ from that of other large animal models (pig, dog and sheep) We have designed lentiviral vectors expressing the GFP and the human LDL receptor under the control of ubiquitous and hepatospecific promoters to assess the feasibility of ex vivo transduction of human and simian hepatocytes and the functionality of the transgene in vitro and after transplantation in a murine model In parallel, we are developing a protocol for hepatocyte transplantation in macaques (Macacus cynomolgus), in order to augment the cell engraftment The results will be presented and discussed 1028 Application of AAV Vectors for In Vivo Gene Therapy of Gaucher Disease John Marshall,1 Kerry A McEachern,1 Julie A Cavanagh Kyros,1 Jennifer B Nietupski,1 Michael J Lukason,1 Christine Barbon,1 Kristen Taylor,1 Christine Ford,1 John A Barranger,2 Edward H Schuchman,3 Robert J Desnick,3 Scott Lonning,1 Seng H Cheng.1 Gene Transfer Research, Genzyme Corporation, Framingham, MA; 2Human Genetics, University of Pittsburgh, Pittsburgh, PA; Human Genetics, Mount Sinai School of Medicine, New York, NY Gaucher disease is the most common of the more than 40 currently described lysosomal storage diseases It is caused by genetic defects in the lysosomal hydrolase glucocerebrosidase (GC) These defects result in the accumulation of the substrate, glucosylceramide (GL1), in tissue macrophages primarily of the liver and spleen Due to the lack of a transgenic animal model we have used an artificiallyinduced mouse model of Gaucher disease that accumulates exogenous GL-1 in the liver, to demonstrate feasibility of gene therapy Recombinant adenoviral (Ad2/CMV-hGC) and plasmid (pGZBshGC) based gene therapy vectors were evaluated in this induced model by intranasal and intravenous administration respectively The transgene product of these vectors, human GC, normalized the Kupffer cell GL-1 levels, demonstrating the feasibility of in vivo gene therapy of Gaucher disease However, due to the immune stimulatory properties of adenovirus and the administration issues of plasmid DNA, neither of these vectors currently offer a viable treatment for a chronic disease The adeno-associated virus (AAV) gene therapy vector is a feasible alternative, offering both a reduced acute immune response and more persistent transgene expression Preliminary studies in normal mice using an AAV construct (AAV2/ CMV-hGC) administered intravenously, yielded human GC transgene product in the liver (hepatocytes) at about 5% of the normal endogenous level Also, a proportion of this material was secreted, with the amount of circulating hGC at a level found to be therapeutic in the induced model of Gaucher disease Further Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy GENE THERAPY FOR BONE, CONNECTIVE TISSUE AND SKIN TISSUES evaluation of the AAV vector, particularly its ability to deliver hGC to diseased macrophages, will be performed in the Niemann-Pick mouse model This mouse offers a similar profile to Gaucher disease, accumulating sphingolipid in the macrophages of liver, spleen, lung and bone marrow Using histologic and cytometric assays specific for hGC, the potential for AAV derived hGC to be delivered to the target cells can be evaluated Utilizing the Niemann-Pick mouse we hope to continue to optimize the AAV vector and the conditions for its use in gene therapy of Gaucher disease The presenter is a paid employee of Genzyme Corporation 1029 Human Cystathionine b-Synthase (CBS) Gene Transfer Mediated by Recombinant AdenoAssociated Virus Vector Sung-Chul Jung,1 Eun-Sook Park,2 Hyun-Jeong Oh,1 Warren Kruger,3 Jin-Sung Lee.4 Division of Genetic Disease, National Institute of Health, Seoul, Korea; 2Brain Korea 21 Project for Medical Science, Yonsei University, Seoul, Korea; 3Division of Population Science, The Fox Chase Cancer Center, Philadelphia, PA; 4Department of Clinical Genetics, Yonsei University College of Medicine, Seoul, Korea Cystathionine β-synthase (CBS) deficiency in human is the most common cause of Homocystinuria, a rare autosomal recessive disease About 50% of patients with CBS deficiency respond to pyridoxine therapy with a marked reduction in plasma total homocysteine, whereas the remaining subjects respond poorly For the gene therapy of homocystinuria, we constructed human CBS cDNA containing recombinant AAV vector (rAAV-hCBS) We used three kinds of hCBS cDNA, one was normal full length CBS cDNA, two were mutated hCBS cDNA which were deleted or mutated in the region of catalytic domain We used adenovirus-free production system for the production of rAAV-hCBS vectors Three kinds of rAAV hCBS vectors were transduced into NIH3T3 for in vitro evaluation.Western blot analysis using anti-hCBS antibody demonstrated that two rAAV-hCBS vectors expressed high levels of Cytathionine β-synthase enzyme CBS enzyme activities were checked using TLC Enzyme activity of rAAV-hCBS in NIH3T3 was over the CBS enzyme activity of wild type control (nontransduced HepG2 cell) Enzyme activity of normal full length hCBS cDNA containing rAAV vector was higher than point-mutated hCBS cDNA containing rAAV vector It was sufficient to conduct in vivo study to use our rAAV vectors Now, we have used homocystinuria mouse model for in vivo evaluation of our rAAV vectors GENE THERAPY FOR BONE, CONNECTIVE TISSUE AND SKIN TISSUES κ 1030 Potential Therapeutic Application of NFκ B Decoy Oligodeoxynucleotides on Joint Destruction of Collagen-Induced Arthritis in Cynomolgus Monkeys Tetsuya Tomita,1 Hideo Hashimoto,1 Ryuichi Morishita,2 Yasuo Kunugiza,1 Naruya Tomita,3 Atsuo Waki,4 Shoko Kuroda,4 Yasufumi Kaneda,2 Hideki Yoshikawa.1 Orthopaedic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka, Japan; 2Gene Therapy Science, Osaka University Graduate School of Medecine, 2-2 Yamada-oka, Suita, Osaka, Japan; 3General Medicine, Osaka University Hospital, Suita, Osaka, Japan; 4Preclinical Research, Anges MG Inc., 1-8-31 Midorigaoka, Ikeda, Osaka Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial proliferation The transcription factorMolecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy κB (NFκB) plays a pivotal role in the coordinated transactivation of inflammatory cytokine and adhesion molecule genes, whose activation has been postulated to be involved in destructive changes of articular cartilage and bone in arthritic joints Recently transfection of double-stranded oligodeoxynucleotides (ODN) (= decoy) as a new powerful tool in a new class of anti-gene strategy for gene therapy has been reported The purpose is to evaluate the preclinical therapeutic efficiency and safety of NFκB decoy ODN using CIA in cynomolgus monkey Male cynomolgus monkeys weighting between 2.0 and 2.5 kg were immunized intradermally mg of bovine type II collagen in complete Freunds’ adjuvant Five monkeys were given intraarticular injection of NFκB decoy ODN into bilateral wrist every weeks from day up to 11 weeks Blood samples and x-rays of wrist joints were collected every weeks, starting from just before the first immunization Bilateral wrist joints of all animals were fixed with 4% paraformaldehyde and decalcified with EDTA and prepared for staining with HE To study the effect of NFκB decoy ODN adminstration on in vivo protein expression of TNFα and IL-1β in the joints, the joint synovium was harvested at 35 days, homogenized and centrifuged The amount of TNFα and IL-1β in the supernatant was determined using ELISA kit Radiographic examination showed marked suppression of joint destruction with NFκB decoy ODN treated group compared with untreated or scrambled (SD) group The histologic examination showed hyperplasia of synovitis with severe destruction of articular cartilage and bone in untreated group and SD group respectively The average arthritis score was 0.38±0.50 in NFκB group, and 2.25±1.28 in untreated group and 2.38±1.09 in SD group respectively The average expression level of TNFα and IL-1β in the synovium per joint was 7.8 pg±2.5 and 27.6 U±13.0 (Untreated group), 6.0 pg±2.7 and 37.4 U±11.3 (SD group), 1.4 pg±0.3 and 2.0 U±1.6 (NFκB group) respectively During observation period, except inflammatory parameters, no obvious abnormal change in biochemical parameters suggesting adverse events due to administration of ODN was recognized One of the most important and serious clinical problems in RA patients is progressive joint destruction In this study we demonstrated that in vivo intraarticular administration of NFκB decoy suppressed expression levels of IL-1β and TNFα in the joint, and joint destruction in cynomolgus monkeys CIA model as a local therapy We also demonstrated that consecutive intraarticular injection of NFκB decoy ODN suppressed the severity of joint destruction, and therefore a decoy strategy against NFκB is promising effects can be expected in RA patients based on the results of this preclinical study 1031 Development of a Self-Inactivating, Tet-On Retroviral Vector Expressing BMP4 That Promotes Regulated Bone Formation and Bone Healing Hairong Peng,1 Arvydas Usas,1 Brian Gearhart,2 Brett Young,1 Johnny Huard.1,2 Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, United States; 2Orthopaedic Surgery, Children’s Hospital of Pittsburgh, Pittsburgh, PA A retrovirus-based, regulatable gene expression system would have great potential application in a variety of gene therapy techniques However, the ideal design for such a vector system has not been rigorously studied The aim of this study was to develop a self-inactivating, retroviral-based vector system that would enable doxycycline-regulated, therapeutic gene expression in stem cells We have systematically studied the effects of different regulatory elements in the 3’ LTR upon gene expression in our efforts to develop a self-inactivating vector optimal for regulated gene expression This analysis has revealed that an intact U3 region is essential for high S397 ... containing rAAV vector It was sufficient to conduct in vivo study to use our rAAV vectors Now, we have used homocystinuria mouse model for in vivo evaluation of our rAAV vectors GENE THERAPY FOR. .. mutated in the region of catalytic domain We used adenovirus-free production system for the production of rAAV-hCBS vectors Three kinds of rAAV hCBS vectors were transduced into NIH3T3 for in vitro... decalcified with EDTA and prepared for staining with HE To study the effect of NFκB decoy ODN adminstration on in vivo protein expression of TNFα and IL-1β in the joints, the joint synovium was harvested