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193 Delivery of Glucocerebrosidase to the Liver and Brain for Treatment of Gaucher''''s Disease by Targeted Uptake Via the LDL Receptor Molecular Therapy �������� ��� ���� ���������������� �������� ����[.]
GENETIC AND METABOLIC DISEASES: PART ONE and few have addressed the issue of which cells within the lung are targeted by gene transfer vectors For Cystic Fibrosis, successful gene therapy is likely to require not only adequate levels of transgene expression but also appropriate distribution of expression in specific cell types We evaluated transgene expression in the murine lung using vectors incorporating the red-shifted variant of green fluorescent protein (EGFP) Six female BALB/c mice were anaesthetised with methoxyfluorane and 300μg of the plasmid pEGFP-N1 (Clontech, Oxford, UK) was administered intranasally by insufflation Six control mice were administered with 300μg of the luciferase expressing plasmid pCIKLux 24 hours after vector administration, mice were sacrificed and lungs were fixed in 4% paraformaldehyde 30 nonconsecutive 7μm cryosections were prepared from each of the five lobes and the trachea of treated animals (180 sections/mouse in total) All sections were examined using a fluorescent microscope with a standard FITC excitation/emission filter set and the number and location of EGFP positive cells was recorded for each mouse Positive cells were identified in all mice treated with pEGFP-N1 (none were visible in negative control animals) with a mean expression of 347±280 positive cells per mouse The majority (79%) of positive cells were located in the epithelium of conducting airways and the remainder (21%) were located in the lung parenchyma To identify positive epithelial cells immunohistochemistry was performed on pEGFP-N1 treated sections using an antibody specific for ciliated epithelial cells (anti-Beta Tubulin IV(BioGenex, San Ramon, USA); 1:100 dilution) and an antibody that binds to CCSP in Clara cells (anti-HUP (DAKO, Ely, UK); 1:100 dilution) Using this technique, 51% of EGFP positive epithelial cells were identified as ciliated cells and 22% as Clara cells In subsequent experiments to compare other vector systems, 80μg of pEGFP-N1 complexed to the cationic lipid GL67 (Genzyme, Framingham, USA) was administered to the lungs of BALB/c mice with pCIKLux/GL67 as a negative control EGFP positive cells were identified in all animals dosed with pEGFP-N1/GL67 with a mean expression of 2,020 ±435 positive cells per mouse (no positive cells were seen in control animals) In contrast to naked DNA the majority (99.4%) of EGFP positive cells were located in the lung parenchyma and only a very small number (0.6%) were identified in the conducting airway epithelium Studies are currently underway to further characterise EGFP positive cell types in these experiments Additional cell specific antibodies have been identified and validated in the mouse lung and will be used in co-localisation studies with EGFP positive cells These data reflect that EGFP expression is a robust first step in the detection and identification of cell types targeted by gene transfer vectors in the mouse lung GENETIC AND METABOLIC DISEASES: PART ONE 192 The Origin and Liver Repopulating Capacity of Murine Oval Cells Xin Wang,1 Mark Foster,1 Muhsen Al-Dhalimy,1 Eric Lagasse,2 Milton Finegold,3 Markus Grompe.1 Molecular & Medical Genetics, Oregon Health & Science University, Portland, OR, United States; 2Stem Cell Inc., Palo Alto, CA, United States; 3Pathology, Texas Children’s Hospital, Houston, TX, United States The appearance of bipotential oval cells in chronic liver injury suggests the existence of hepatocyte progenitor/stem cells To study the origin and properties of this cell population, oval cell proliferation was induced in adult mouse liver by 3,5-diethoxycarbonyl-1, 4dihydrocollidine (DDC) and a method for their isolation was developed Transplantation into fumarylacetoacetate hydrolase (Fah) deficient mice was used to determine their capacity for liver Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy repopulation In competitive repopulation experiments, hepatic oval cells were at least as efficient as differentiated hepatocytes in repopulating the liver In mice with chimeric livers, the oval cells were not derived from hepatocytes but from liver non-parenchymal cells This finding supports a model in which intra-hepatic progenitors differentiate into hepatocytes irreversibly To determine whether oval cells originated from stem cells residing in the bone marrow, bone marrow transplanted wild-type mice were treated with DDC for months and oval cells were then serially transferred into Fah mutants The liver repopulating cells in these secondary transplant recipients lacked the genetic markers of the original bone marrow donor We conclude that hepatic oval cells not originate in bone marrow, but in the liver itself and that they have valuable properties for therapeutic liver repopulation 193 Delivery of Glucocerebrosidase to the Liver and Brain for Treatment of Gaucher’s Disease by Targeted Uptake Via the LDL Receptor Brian J Spencer,1 Inder M Verma.1 Department of Genetics, Salk Institute of Biological Studies, San Diego, CA Gaucher’s disease is an inherited lysosomal storage disease resulting from mutations and loss of activity of glucocerebrosidase Symptoms range from painful ‘bone crisis’ and hepatosplenomegaly to neurological disorders and death Recently enzyme replacement therapy and bone marrow transplants have been used as effective treatments showing that delivery of glucocerebrosidase to only a small population of cells is effective at reducing systemic symptoms; however, no treatment effectively treats the neurological disorders associated with the more severe Type and Type Gaucher’s disease In order to test the potential of gene therapy for Gaucher’s disease, a fusion construct was designed such that the glucocerebrosidase gene was fused at the N-terminus with the LDL receptor-binding domain of ApoB or ApoE, thus targeting the protein for uptake via the LDL receptor and transport to the lysosome These constructs were tested in vitro for their ability to express and secrete enzymatically active glucocerebrosidase enzyme In addition, cultured supernatant from transfected cells was fed to human hepatocytes, HepG2, expressing the LDL receptor Western blot analysis of lysates from these HepG2 cells showed uptake of the enzyme These constructs were then inserted into the 3rd generation lentivirus vector under the control of the murine CMV promoter (mCMV) or the CAG promoter These viruses were delivered intra-venously into 4-6 week old BALB/c mice and or 14 days later, blood and tissue samples were collected and analyzed Serum from animals injected with the CAG glucocerebrosidase viruses showed increased enzyme activity compared to uninjected controls Livers of all mice injected with either the mCMV or CAG glucocerebrosidase constructs contained recombinant enzyme as determined by Western blot In addition, recombinant glucocerebrosidase could be detected in whole brain that was homogenized and subjected to Western blot analysis Since previous reports have shown the lentivirus does not efficiently cross the blood-brain barrier, and an internal GFP expression construct in the S75 GENETIC AND METABOLIC DISEASES: PART ONE virus was detected in the liver but not the brain, it is possible that the liver is functioning as a depot organ for expression of the glucocerebrosidase enzyme which is then able to cross the bloodbrain barrier via the LDL receptor These results suggest a possible treatment for not only the peripheral symptoms of Gaucher’s disease but also the neurological aspect of the disorder more common and clinically relevant human disease Future studies will be designed to further evaluate the potential for systemic delivery of AAV-IDUA in alleviating neurological components of MPS I 194 Correction of Metabolic, Neurologic, and Craniofacial Abnormalities in MPS I Mice Treated at Birth with Adeno-Associated Virus Vector α-L-Iduronidase Gene Transducing the Humanα Dao Pan,1 Roland Gunther,2 Walter C Low,3 Beth E Larson,1 Steven U Walkley,4 Joel L Frandsen,5 Tal Kafri,6 R Scott McIvor,5 Chester B Whitley.1 Department of Pediatrics and Institute of Human Genetics, University of Minnesota, Minneapolis, MN; 2Research Animal Resources, University of Minnesota, Minneapolis, MN; Department of Neuroscience, University of Minnesota, Minneapolis, MN; 4Sidney Weisner Laboratory of Genetics Neurological Disease, Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY; 5Department of Genetics, Cell Biology and Development and Institute of Human Genetics, University of Minnesota, Minneapolis, MN; 6Gene Therapy Center, University of North Carolina, Chapel Hill, NC Seth D Hartung,1 Joel Frandsen,1 Dao Pan,1,3 Brenda Koniar,1 Patrick Graupman,2 Roland Gunther,1 Walter Low,2 Chester B Whitley,1,3 R S McIvor.1 Gene Therapy Program and Institute of Human Genetics, University of Minnesota, Minneapolis, MN; 2Department of Neurosurgery, University of Minnesota, Minneapolis, MN; Department of Pediatrics, University of Minnesota, Minneapolis, MN Murine models of lysosomal storage diseases (LSD) provide an opportunity to evaluate the potential for gene therapy to prevent systemic manifestations of the disease Previously, we demonstrated that adeno-associated virus (AAV) vectors transducing the human iduronidase (IDUA) gene achieved levels of IDUA gene transfer and expression in vitro that were sufficient to mediate intercellular metabolic cross-correction A recently developed mouse model of mucopolysaccharidosis type I (MPS I, generously provided by Dr E Neufeld) has allowed us to extend our previous in vitro work to the in vivo setting A recombinant adeno-associated virus (AAV) vector vTRCA1, (containing the human IDUA cDNA under transcriptional regulation of the CMV early enhancer and β–actin promoter) was constructed and 1x1010 particles were injected intravenously into day - old MPS I mice Measurements beginning month after vector injection showed that high levels of IDUA activity were present in the plasma of vTRCA1–treated animals (mean –4–fold above heterozygous control levels) that persisted for the month duration of the study This level of IDUA activity was sufficient to prevent excessive urinary GAG excretion and to normalize body weights relative to untreated MPS I mice during the study period Five months after vector injection, high levels of IDUA activity were found in the heart and lung of vTRCA1-treated MPS I (-/-) animals, with of the evaluated animals also demonstrating IDUA activity in at least region of the brain Overall gene transfer was determined to be low, with the highest IDUA cDNA copy numbers (0.94 to 0.06 % genome equivalents) detected in the heart and lung Toluidine blue–stained liver sections indicated that systemic IDUA expression was sufficient in vTRCA1–treated MPS I (-/-) animals to prevent the accumulation of lysosomal storage material in hepatocytes and resident Kupfer cells These animals also demonstrated correction of cerebellar parenchymal and hippocampal perivascular histopathology compared to control– treated MPS I (-/-) animals In measurements of facial width parameters by computerized tomography (CT), mean values for month old vTRCA1-treated animals indicated that systemic IDUA activity was sufficient to normalize bony changes of the disease In an open field test, vTRCA1-treated animals exhibited a significant reduction in total squares covered and a trend of normalization in rearing events compared to control–treated MPS I (-/-) animals, demonstrating that AAV-IDUA vector treatment improved habituation in these animals We conclude that AAV-mediated transduction of the IDUA gene in newborn MPS I (-/-) mice was sufficient to have a major curative impact on several of the most important parameters of the disease These results extend the previous observations of others in the MPS VII model to MPS I, a S76 195 Metabolic and Neuropathologic Correction of Hurler Syndrome by a Single Intravenous Injection of Lentiviral Vector to Newborn Mice Local injection of HIV-based lentiviral vectors to tissues has produced regional gene transfer and expression; however, intravenous administration for systemic treatment has not been evaluated Mucopolysaccharidosis type I is a lysosomal storage resulting from deficiency of lysosomal α-L-iduronidase enzyme activity As illustrated in these studies, the murine knockout mouse is a true model of the human disease, Hurler syndrome The mouse exhibits the metabolic defects, dysmorphology and learning deficits analogous to those of children with the most severe clinical form of this disorder Based on our original studies of the biodistribution of VSV-G pseudotyped lentivirus (Mol Therapy 1:S140, 2000; Mol Therapy 6:19, 2000), we sought to evaluate the response of Hurler syndrome to such a vector expressing high levels of enzyme from the α-L-iduronidase cDNA Following a single injection at birth (1 or x 10 TU/mouse), plasma α-L-iduronidase levels (0.2- to 30fold of normal levels) were greatly increased and remained stable throughout the 100-day study period In of 10 mice, hepatic α-Liduronidase enzyme levels were markedly higher than normal (4- to 17-fold) Substantial levels of α-L-iduronidase enzyme were also found in spleen (1-10% of normal), heart (1-6%), brain (0.7-3.5%) and peripheral blood leukocytes (0.8-5%) High levels of IDUA transgene were detected in liver (1-115%, i.e., - 115 copies of IDUA 100 genome equivalents), heart (0.05 – 23%) and spleen (0.06 - 4.1%) Systemic metabolic correction was indicated by the complete or partial absence of storage pathology in the liver, spleen, heart, lung, kidney, brain, ovary and bone marrow in all treated mice In mice with high circulating α-L-iduronidase levels, pathologic GM2- and GM3-ganglioside were markedly reduced in the brain This effect was inversely related to the level of enzyme, and those individuals with highest enzyme levels had brain morphology indistinguishable from normal Moreover, all individuals had normal facial appearance without the cranial dysmorphology seen in untreated mice with Hurler syndrome, including those mice that had the lowest liver IDUA transgene and plasma α-L-iduronidase Notably, the cognitive deficit has been significantly improved (p