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A Gene Therapy Approach for PLA2G6-Associated Neurodegeneration

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A Gene Therapy Approach for PLA2G6-Associated Neurodegeneration Novel therapeutic strategies in NBIA: A gene therapy approach for PLA2G6-associated neurodegeneration Sammie Whaler Thesis submitted for the degree of Doctor of Philosophy University College London Department of Pharmacology UCL School of Pharmacy 29-39 Brunswick Square London WC1N 1AX May, 2018 1 I, Sammie Whaler confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Infantile neuroaxonal dystrophy (INAD) is a debilitating, intractable and ultimately lethal neurodegenerative disorder. It is caused by mutations in the PLA2G6 gene that encodes for phospholipase A2. INAD patients present neurodegeneration-associated symptoms between six months and three years of age. Severe spasticity, progressive cognitive decline, and visual impairment typically result in death during the first decade (Morgan et al, 2006). There is no disease-modifying treatment available and palliative care focuses on quality of life. Therefore, there is an overwhelming need to develop novel therapies to treat INAD patients. To create a landscape of the behavioural and pathological deficits, we aim to first conduct an in-depth characterization of the PLA2G6 mouse model developed by Wada et al (2009). Additionally, we aim to develop an AAV-mediated gene therapy approach for the treatment of INAD and conduct a pre-clinical study in the pla2g6-inad mouse model. The objective is to be able to prevent or ameliorate both the central and peripheral nervous system phenotype and improve the lifespan and/or quality of life of the animal. Recombinant adeno-associated virus serotype 9 vector (AAV9) will be used to deliver the therapeutic human PLA2G6 gene to the neonatal pla2g6-inad mouse. The strong neuron specific synapsin-I promoter will drive the human PLA2G6 gene. The efficacy of different administration routes including intracerebroventricular (ICV), intravenous (IV) and a combination of intracerebroventricular (ICV)/ intravenous (IV) and intracerbroventricular (ICV)/intraperioteneal (IP) will be investigated in the pla2g6-inad mouse model. AAV9-hSyn1-hPLA2G6 gene therapy treated pla2g6-inad mice showed an increased lifespan with the largest improvements observed in the animal cohort that received a combined administration of AAV9-hPLA2G6. The significant increase in lifespan 3 supplemented with significant improvements in behavioural tests validates the potential beneficial use of gene therapy for infantile neuroaxonal dystrophy (INAD). 4 Impact statement With the potential to provide an efficient treatment for a wide range of diseases, the clinical impact of gene therapy is a rapidly developing and growing field. INAD is a rare, pediatric neurodegenerative disease with a pathological phenotype that includes the central and peripheral nervous system. There is no cure for INAD and treatment options rely on symptom relief. The lack of treatment options for INAD is an important factor to include when considering the impact of gene therapy for this particular disease. The success of gene therapy is driving a wave of enthuisiam and we are entering an era of gene therapy treatments for a spectrum of diseases, from cancer to diseases of the central nervous system. Gene therapy, including Adeno-Associated Virus (AAV)- based vectors show great potential for therapeutic delivery and have demonstrated great efficiacy in the rescue of a large number of animal models. AAV vectors are also currently being investigated in a large number of clinical trials. The results of this study strongly support that gene therapy has the potential to offer a successful and efficient treatment strategy for INAD. A limiting factor for gene therapy however is GMP vector manufacturing costs. The development of manufacturing processes for recombinant AAV is viewed as complex, time consuming and expensive. The production scale-up is considered to be a significant challenge technically and poses a large obstacle for commercialization. 5 Nevertheless, we are confident that with the advent of new technologies and further progress in scientific knowledge and research, new manufacturing strategies will be developed that will ultimately decrease the cost of gene therapy mediated treatments. 6 Acknowledgements First and foremost, I would like to thank Dr Ahad Rahim for giving me the opportunity to be involved in such an important and exciting project. Thank you for all your guidance and support throughout this project and for making this PhD project such a positive experience for me. I’ve been very lucky to have been a part of such a wonderful group. Thank you to Dr Manju Kurian for all her help and invaluable input towards the success of this project. Thank you to Dr Simon Waddington for always being available to help with injections. The project would not have been able to move forward as quickly as it did without your help. Special thanks to the Michael…I can’t express how grateful I am to you for all the help you have given me over the years. Thank you for never being too busy to help (even though I knew you were) and thank you for always having an answer to my questions. Special thanks also to Dr G who made frustrating days in the lab so much easier. Thank you for your help in and out of the lab. Thank you to Archie for being the best desk neighbour anyone could ask for. Thank you for all your support in and out of the lab. Thank you to Laura for all the help with those tricky westerns. Thank you to Ed, Andrea, Joana, Ola, Kim, Emma, Jonny, Simone, George, Raj, Nat, Jo and Suzy. Thank you to Saul and Laura, who joined our lab and made it even better. Thank you to the King’s crew, Hemanth and Yewande. I’m very grateful for the help that you gave me not only through my MSc but also my PhD. Thank you for always being available for me. Thank you to my family, especially my mum, for all the support and those encouraging words of wisdom. You’re my hero. Thank you to James for tagging me in every mouse/rat video there is and for making me laugh every single day. Thank 7 you to Jacqui and June for being a constant source of support throughout all my PhD successes and failures. Thank you to Erol for all the words of wisdom and always asking after the welfare of my animals. Thank you to Ivy and Archie for always putting a smile on my face. Thank you to Carly for all the help with the formatting amongst other things. Special thanks to Sam for his continued support in every aspect of my life. You are amazing. Thank you to the NBIA funding body, thank you to the patients and to the parents/carers and of course to all the animals that were used for this study. 8 Publications Csányi B., Papandreou A., Cuka S., Rahim AA., Chong K and Kurian MA (2016) Update in Neurodegeneration with Brain Iron Accumulation (NBIA): Advances in Molecular Diagnosis and Treatment Strategies. Journal of Pediatric Neurology, 1304- 2580 9 Table of Content Abstract 3 Impact statement 5 Acknowledgements 7 Publications 9 Abbreviations 19 1. INTRODUCTION 22 1.1 Neurodegeneration with Brain Iron Accumulation 22 1.1.1 Pantothenate kinase-associated neurodegeneration (PKAN) – 23 1.1.2 Mitochondria membrane protein-associated neurodegeneration (MPAN) - 24 1.1.3 Beta-propeller protein-associated neurodegeneration (BPAN) – BPAN – 25 1.1.4 Phospholipase A2-associated neurodegeneration (PLAN) - 25 1.2 Clinical entities of PLAN 27 1.2.1 Atypical neuroaxonal dystrophy (Atypical NAD): 27 1.2.2 Adult-onset dystonia-parkinsonism or Parkinson Disease-14 (PARK14): 28 1.2.3 Infantile Neuroaxonal Dystrophy 31 1.3 Treatment for INAD 36 1.4 Phospholipase A2 36 1.4.1 Cytosolic phospholipase A2 (cPLA2): 38 1.4.2 Secretory phospholipase A2 (sPLA2): 39 1.4.3 Calcium-independent phospholipase A2 (iPLA2β): 40 1.4.4 Calcium independent phospholipase A2 distribution 50 1.4.5 Murine Models of INAD 50 1.4.6 The iPLA2β-/- mouse model 51 1.4.7 Pla2g6-inad mouse model 53 1.5 Gene Therapy 55 1.5.1 Viral Vectors: 56 1.5.2 Adeno-associated Viruses 56 1.5.3 Gene Delivery to the CNS 64 2. METHODS 75 2.1 AAV Construct and Design 75 2.2 Cloning 75 10 2.2.1 Bacterial Transformation 75 2.2.2 Plasmid Purification 76 2.2.3 PCR Amplification and Restriction Enzyme Digest 77 2.2.4 Restriction enzyme digest 79 2.2.5 DNA Electrophoresis 79 2.2.6 DNA Extraction 80 2.2.7 Ligations 80 2.2.8 DNA Sequencing 81 2.3 Tissue Culture 81 2.3.1 Maintenance of cell lines 81 2.3.2 HEK293T Transfection 82 2.3.3 Protein extraction from cell lysate 82 2.3.4 Western Blotting 83 2.4 In vivo experiments 85 2.4.1 Animals: Pla2g6-inad mouse model: 85 2.5 Animal identification and genotyping 86 2.5.1 Toe tattoo ink puncture on neonatal mice 86 2.5.2 Tail clipping on neonatal mice 87 2.5.3 Ear clipping on P14 mice 87 2.5.4 DNA Extraction from Tissue 87 2.6 Genotyping 88 2.6.1 Allele-Specific PCR 88 2.7 Behavioural Testing 90 2.7.1 Rotarod 90 2.7.2 Vertical Pole Test 91 2.7.3 Inverted Screen Test 91 2.7.4 Open Field Test 91 2.8 Vector administration 92 2.8.1 Intracerebroventricular injections into neonatal mice 92 2.8.2 Intravenous injections in neonatal mice 92 2.8.3 Intraperitoneal injections in neonatal mice: 93 2.9 Expression Analysis 94 2.9.1 Double gelatine coating of slides: 94 2.9.2 Tissue Processing 94 2.9.3 Immunohistochemistry 96 2.9.4 Hematoxylin and Eosin (H&E) staining on free-floating sections 101 2.9.5 Immunofluorescence and scanning confocal microscopy 101 2.10 Quantitative Analysis 102 11 2.10.1 Stereology 102 2.10.2 Optical Fractionator 104 2.10.3 Thresholding analysis 105 2.10.4 Qualitative Analysis 106 2.10.5 Statistical Analysis 107 2.10.6 Confocal Microscopy 107 3.
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