14. Galc Activity 94

Open Research Online The Open University’s repository of research publications and other research outputs Ex Vivo Gene Therapy Approaches for the Treatment of Globoid Cell Leukodystrophy Thesis How to cite: Visigalli, Ilaria (2009). Ex Vivo Gene Therapy Approaches for the Treatment of Globoid Cell Leukodystrophy. PhD thesis The Open University. For guidance on citations see FAQs. c 2009 The Author https://creativecommons.org/licenses/by-nc-nd/4.0/ Version: Version of Record Link(s) to article on publisher’s website: http://dx.doi.org/doi:10.21954/ou.ro.0000f210 Copyright and Moral Rights for the articles on this site are retained by the individual authors and/or other copyright owners. For more information on Open Research Online’s data policy on reuse of materials please consult the policies page. oro.open.ac.uk Ilaria Visigalli EX VIVO GENE THERAPY APPROACHES FOR THE TREATMENT OF GLOBOID CELL LEUKODYSTROPHY PhD thesis in fulfillment of the requirements of the Open University for the degree of Doctor of Philosophy in Molecular and Cellular Biology 2009 Director of studies External Supervisor Prof. Luigi Naldini Prof. Adrian Thrasher Vita-Salute San Raffaele University Telethon Institute for Gene Therapy (TIGET) DIBIT, San Raffaele Scientific Institute, Milan, Italy ProQuest Number: 13837650 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 13837650 Published by ProQuest LLC(2019). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 ABSTRACT Globoid cell leukodystrophy (GLD) is a rare lysosomal storage disorder (LSD) due to the deficiency of the lysosomal enzyme Galactocerebrosidase (GALC). The enzymatic deficiency results in intracellular storage of undegraded metabolites in the nervous system, leading to progressive dysmyelination. We are testing the feasibility and efficacy of a gene therapy strategy based on hematopoietic stem/progenitor cells (HSPC) and lentiviral vectors (LV) in the murine model of GLD. Differently from what observed with other lysosomal enzymes, GALC gene transfer and expression in HSPC causes apoptosis and functional impairment of the transduced cells due to an inbalance of the intracellular content in bioactive sphingolipids consequent to de novo enzyme expression. Differentiated cells of the myeloid and lymphoid lineages are not affected by GALC expression, suggesting a unique sensitivity of HSPC to enzyme toxicity. To overcome this issue, we explored strategies aimed at de-targeting vector expression from HSPC, while permitting GALC over-expression in differentiated cells. The first approach aims at a transcriptional regulation of GALC expression by the microglia/macrophage specific promoter CD lib . The second strategy exploits endogenous microRNAs for post-transcriptional regulation of GALC expression. The use of LV containing the target sequence of miRNA126, expressed only in HSPC, allows expressing GALC only in the differentiated progeny of transduced HSPC. Although these two approaches proved to be very promising in protecting HSPC from enzyme toxicity bothin vitro and in vivo in heterozygous GLD mice, gene-corrected HSPC failed to repopulate lethally irradiated homozygous GLD mice. This poor result might be explained by the existence of a niche defect in homozygous GLD mice, which might hamper the engraftment of HSPC. 2 LIST OF CONTENTS ABSTRACT 2 LIST OF CONTENTS 3 INTRODUCTION 9 1. GLOBOID CELL LEUKODYSTROPHY 10 1.1 Lysosomal Storage Disorders 10 1.2 Globoid Cell Leukodystrophy: clinical manifestation 11 1.3 GALC: the gene and the enzyme 12 1.4 Pathogenetic mechanism 14 1.4.1 Accumulation of Psychosine 14 1.4.2 Toxic effects of Psychosine storage 15 1.5 Diagnosis 17 1.5.1 Clinical features that suggest an LSD 17 1.5.2 Diagnostic instrumental evaluation of GLD 19 1.5.3 Enzymatic activity and genetic diagnosis 19 1.6 Animal models of GLD 23 1.7 Treatment of LSDs 26 1.7.1 Cross correction 26 1.7.3 Hematopoietic Stem Cell Transplantation 30 1.7.4 Substrate reduction therapy 39 2. GENE THERAPY 42 2.1 Non-viral vectors 44 2.2 Viral vectors 45 2.2.1 Oncoretroviruses and derived vectors. 47 2.2.2 Spumaviruses and derived vectors. 48 2.2.3 Lentiviruses and derived vectors 49 2.2.3.1 Lentivirus life cycle 50 2.2.3.2 Lentiviral vectors 52 2.3 Gene transfer into hematopoietic stem cells 55 2.4 Safety of integrating vectors 57 2.4.1 Adverse events in gene therapy 58 2.4.2 Insertional mutagenesis 58 2.4.3 Pattern of integration 60 3 3. GENE THERAPY FOR LSDS 61 3.1 In vivo gene therapy for LSDs 61 3.1.1 Systemic vector administration 61 3.1.2 Direct CNS gene delivery 63 3.2 Ex vivo gene therapy for LSDs 64 3.2.1 HSC-based ex vivo gene therapy 64 3.2.2 Neural stem cells-based ex vivo gene therapy 66 3.3 Gene therapy for GLD 67 AIM OF THE WORK 71 METHODS 73 1. REAGENTS AND SUPPLIERS 74 1.1 Chemicals 74 1.2 Enzymes and buffers 74 1.3 Bacterial strains 74 1.4 Cell culture 74 2. PLASMIDS 75 3. TRANSFORMATION OF COMPETENT BACTERIA AND PLASMID PREPARATION 76 4. VECTOR PRODUCTION 77 4.1 Transfection 77 4.2 Vector particle amount determination 78 4.3 End-point titration 79 4.4 Infectivity of the vector stocks 80 5. CELL LINES 81 6. MICE STUDIES 81 6.1 Genotyping of FVB/twi mice 82 6.1.1 DNA extraction 82 6.1.2 Amplification of GALC sequence by PCR 83 6.1.3 EcoRV restriction analysis 83 7. HAEMATOPOIETIC STEM/PROGENITOR CELL (HSPC) ENRICHMENT 84 4 7.1 Human HSPC (hHSPC) 84 7.2 Murine HSPC (mHSPC) 85 7.3 Isolation of Seal- murine hematopoietic progenitors 86 8. ISOLATION OF HUMAN LYMPHOCYTES 86 8.1 T lymphocytes 86 8.2 B lymphocytes 87 9. PRIMARY CULTURES 87 9.1 Isolation of human monocytes 87 9.2 Isolation of murine peritoneal macrophages 88 9.3 Isolation of murine microglia 88 9.4 Isolation of murine oligodendrocytes 89 10. TRANSDUCTION 89 10.1 Human HSPC 89 10.1.1 CD34+ cells from CB 89 10.1.2 CD34+cells from BM 90 10.2 Murine HSPC 90 10.3 Seal- murine hematopoitic progenitors 90 10.4 U-937 91 10.5 T lymphocytes 91 10.6 B lymphocytes 91 10.7 Microglia 91 10.8 Oligodendrocytes 92 10.9 Monocytes 92 10.10 Macrophages 92 11. COLONY FORMING CELL (CFC) ASSAY 92 12. HSCTIN MICE 93 12.1 HSCT in murine models of GLD 93 12.2 HSCT in Rag2yc mice 93 13. ANTI-APOPTOTIC TREATMENT 93 5 14. GALC ACTIVITY 94 14.1 GALC activity assay, method A 94 14.2 GALC activity assay, method B 94 15. ANALYSIS OF SPHINGOLIPIDS ON CELL EXTRACTS 95 15.1 Internal Standards for Mass Spectrometry 95 15.2 Experimental Procedures 95 16. DETECTION OF APOPTOSIS 97 16.2 Annexin V staining 97 16.2 TUNEL assay 98 17. GENOMIC DNA EXTRACTION AND REAL-TIME QUANTITATIVE-PCR (Q- PCR) ANALYSIS 99 17.1 Genomic DNA extraction 99 17.2 Real-time Q-PCR 100 18. FLOW CYTOMETRY 102 18.1 Expression titer 102 18.2 Analysis of Rag2yc mice receiving HSCT 102 19. IMMUNOFLUORESCENCE 103 20. WESTERNvBLOT 105 RESULTS 106 1. FORCED GALC EXPRESSION IN HSPC 107 2. IMPAIRED IN VITRO FUNCTION OF GALC EXPRESSING HSPC UPON LV- MEDIATED GALC EXPRESSION 108 2.1 Impaired in vitro function of murine HSPC 108 2.2 Impaired in vitro function of human HSPC 109 3. IMPAIRED IN VIVO FUNCTION OF HSPC UPON LV-MEDIATED GALC EXPRESSION 111 3.1 Engraftment failure of GALC.LV transduced cells in twi mice 111 6 3.2 Engraftment failure in milder murine models 113 3.2 GALC.LV transduced hHSPC repopulate Rag2yc mice 114 4. APOPTOSIS OF GALC EXPRESSING MURINE AND HUMAN HSPC 116 5. IGF1 RESCUES GALC EXPRESSING HSPC FROM APOPTOSIS AND FUNCTIONAL IMPAIRMENT 117 6. INVESTIGATING THE MECHANISM OF GALC DE NOVO EXPRESSION TOXICITY 119 6.1 Apoptosis of GALC expressing HSPC is associated to accumulation of bioactive GALC product 119 6.2 Cathepsin D and PAR-4 are not involved in apoptosis of the transduced HSPC 120 7. SENSITIVITY TO GALC EXPRESSION TOXICITY IS DEPENDENT ON DIFFERENTIATION AND CELL LINEAGE 122 7.1 Monocytes, macrophages and microglia are not sensitive to GALC expression toxicity 122 7.2 Human T and B lymphocytes are not sensitive to GALC expression toxicity 123 7.3 Primary oligodendroglia are resistant to GALC-induced apoptosis 124 8. REGULATION OF GALC EXPRESSION 125 8.1 Trancriptional regulation of GALC expression 125 8.1.1 In vitro regulation of GALC expression by the CD 1 lb promoter 125 8.1.2 In vivo regulation of GALC expression by the CD 1 lb promoter 126 8.2 Post-transcriptional regulation of GALC expression 127 8.2.1 In vitro regulation on GALC expression by miRNA126 128 8.2.2 In vivo regulation on GALC expression by miRNA126 128 DISCUSSION 154 1. TOXICITY OF GALC DE NOVO EXPRESSION 155 2. TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL REGULATION OF GALC EXPRESSION FOR SAFE AND EFFICACIOUS GLD GENE THERAPY162 CONCLUDING REMARKS 165 ACKNOWLEDGEMENTS 167 BIBLIOGRAPHY 168 ABBREVIATIONS 201 7 1.

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