rAAV-based gene therapy for molybdenum cofactor deficiency type B Doctoral thesis In partial fulfilment of the requirements for the degree "Doctor rerum naturalium (Dr. rer. nat.)" in the Molecular Medicine Study Program at the Georg-August University Göttingen submitted by Joanna Jakubiczka-Smorag born in Czestochowa, Poland Göttingen, 2015 Members of the Thesis Committee: Supervisor Prof. Dr. rer. nat. Peter Burfeind Institute of Human Genetics, University Medical Centre Göttingen Second member of the Thesis Committee Prof. Dr. rer. nat. Peter Schu Department of Cellular Biochemistry, University Medical Centre Göttingen Third member of the Thesis Committee Prof. Dr. med. Wolfgang Brück Institute of Neuropathology, University Medical Centre Göttingen Date of Disputation: 03.06.2015 AFFIDAVIT Herewith I declare that my doctoral thesis entitled: "rAAV-based gene therapy for molybdenum cofactor deficiency type B" has been written independently with no other sources and aids than quoted. Göttingen, 24.04.2015 ………………………………….. TABLE OF CONTENTS TABLE OF CONTENTS TABLE OF CONTENTS ........................................................................................................................... I LIST OF ABBREVIATIONS .................................................................................................................... VI 1 INTRODUCTION .......................................................................................................................... 1 1.1 Molybdenum cofactor (MoCo) deficiency ............................................................................ 1 1.2 Molybdenum cofactor (MoCo) ............................................................................................. 2 1.3 Characteristics of the MOCS2 gene ....................................................................................... 5 1.3.1 Genomic structure of the human MOCS2 and mouse Mocs2 genes ......................... 5 1.3.2 MOCS2 expression pattern......................................................................................... 7 1.4 MoCo-dependent enzymes ................................................................................................... 7 1.4.1 Aldehyde oxidase (AO) ............................................................................................... 7 1.4.2 Xanthine oxidoreductase (XOR) ................................................................................. 9 1.4.3 Sulphite oxidase (SO)................................................................................................ 11 1.4.4 Mitochondrial amidoxime reducing component (mARC) ........................................ 12 1.5 Substitution therapy for Molybdenum Cofactor Deficiency Type A ................................... 13 1.6 Gene therapy ....................................................................................................................... 13 1.6.1 Adeno-associated virus (AAV) .................................................................................. 15 1.6.2 Recombinant adeno-associated virus (AAV)-based gene therapy ........................... 18 1.7 AIMS .................................................................................................................................... 19 2 MATERIALS AND METHODS ..................................................................................................... 21 2.1 MATERIALS .......................................................................................................................... 21 2.1.1 Chemicals and reagents ........................................................................................... 21 2.1.2 Biochemical enzymes ............................................................................................... 24 2.1.3 Ready-to-use reaction systems ................................................................................ 24 2.1.4 Plasmids .................................................................................................................... 25 2.1.5 Usage ware ............................................................................................................... 25 2.1.6 Technical equipment ................................................................................................ 26 2.1.7 Solutions ................................................................................................................... 28 2.1.8 Culture media antibiotics and agar plates ............................................................... 30 2.1.8.1 Culture media for bacteria ............................................................................... 30 2.1.8.2 Culture media for eukaryotic cells ................................................................... 31 2.1.8.3 Antibiotics ......................................................................................................... 31 2.1.8.4 IPTG/X-Gal plates ............................................................................................. 31 2.1.9 Biological material .................................................................................................... 32 I TABLE OF CONTENTS 2.1.9.1 Bacterial strains ................................................................................................ 32 2.1.9.2 Eukaryotic cell lines .......................................................................................... 32 2.1.9.3 Mouse strains ................................................................................................... 32 2.1.10 Synthetic DNA oligonucleotides ............................................................................... 32 2.1.10.1 Vector-specific primer ...................................................................................... 32 2.1.10.2 Human-specific primers ................................................................................... 33 2.1.10.3 Mouse-specific primers .................................................................................... 33 2.1.10.4 Genotyping primers .......................................................................................... 34 2.1.10.5 cDNA synthesis primers .................................................................................... 35 2.1.11 Antibodies ................................................................................................................ 35 2.1.11.1 Primary antibodies ........................................................................................... 35 2.1.11.2 Secondary antibodies ....................................................................................... 35 2.1.12 Databases ................................................................................................................. 36 2.1.13 Statistical methods ................................................................................................... 37 2.1.14 Sterilisation of solutions and equipment ................................................................. 37 2.2 METHODS ............................................................................................................................ 37 2.2.1 Isolation and purification of nucleic acids ................................................................ 37 2.2.1.1 Minipreparation of plasmid DNA ..................................................................... 37 2.2.1.1.1 Preparation of bacterial glycerol stocks ...................................................... 38 2.2.1.2 Large-scale preparation of Endotoxin-free plasmid DNA using the Qiagen Maxi Kit .......................................................................................................................... 38 2.2.1.3 Isolation of genomic DNA ................................................................................. 38 2.2.1.3.1 Isolation of genomic DNA from tissue samples........................................... 38 2.2.1.3.2 Isolation of genomic DNA from cells ........................................................... 39 2.2.1.3.3 Isolation of genomic DNA from sperm ........................................................ 39 2.2.1.3.4 Isolation of genomic DNA from recombinant adeno-associated virus (rAAV) ..................................................................................................................... 40 2.2.1.4 Isolation of total RNA from tissue samples ...................................................... 40 2.2.1.5 Determination of nucleic acid concentration................................................... 40 2.2.1.6 Cloning techniques ........................................................................................... 40 2.2.1.6.1 Cleavage of DNA with restriction endonucleases ....................................... 40 2.2.1.6.2 Isolation of DNA fragments from agarose gels using the QIAquick Gel Extraction Kit (Qiagen) .................................................................................................... 41 2.2.1.6.3 Dephosphorylation of plasmid DNA ............................................................ 41 II TABLE OF CONTENTS 2.2.1.6.4 Ligation of DNA fragments .......................................................................... 41 2.2.1.6.5 TA-Cloning ................................................................................................... 42 2.2.1.6.6 Transformation of competent bacteria (Hanahan, 1983) ........................... 42 2.2.1.7 Gel electrophoresis .........................................................................................