Therapeutic efficacy of small molecules to treat channelopathies A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2019 Jingshu Liu School of Biological Sciences Division of Evolution and Genomic Sciences Table of Contents List of Figures ................................................................................................................... 8 List of Tables ................................................................................................................... 11 Abstract ........................................................................................................................... 12 Declaration ...................................................................................................................... 13 Copyright statement ........................................................................................................ 13 Acknowledgement ........................................................................................................... 14 Abbreviations .................................................................................................................. 15 1 Chapter 1: Introduction ............................................................................................ 19 1.1 Human eye ........................................................................................................ 19 1.1.1 Retina ............................................................................................................ 20 1.1.2 Neural retina ................................................................................................. 20 1.1.2.1 The retinal pigment epithelium (RPE) .................................................. 21 1.1.3 Inherited retinal disorders (IRDs) ................................................................. 21 1.1.3.1 Genetics of IRDs ................................................................................... 22 1.1.3.2 Therapeutic strategies for IRDs ............................................................. 22 1.1.3.2.1 Gene therapy ................................................................................... 23 1.1.3.2.2 Cell-based therapy ........................................................................... 23 1.1.3.2.3 Retinal prostheses ............................................................................ 24 1.1.3.2.4 Pharmacotherapy ............................................................................. 24 1.2 Bestrophin-1 ..................................................................................................... 25 1.2.1 Human BEST1 gene ...................................................................................... 25 1.2.2 Structure of bestrophin-1 .............................................................................. 25 1.2.3 Function of bestrophin-1 ............................................................................... 29 1.2.4 Bestrophinopathy .......................................................................................... 29 1.2.4.1 Best vitelliform macular dystrophy (BVMD) ....................................... 30 1.2.4.1.1 Phenotypes and genotypes of BVMD ............................................. 30 1.2.4.1.2 Features of BVMD-related bestrophin-1 ......................................... 31 1 1.2.4.2 Autosomal recessive bestrophinopathy (ARB) ..................................... 31 1.2.4.2.1 Phenotypes and genotypes of ARB ................................................. 31 1.2.4.2.2 Features of ARB-related bestrophin-1 ............................................ 32 1.2.4.3 Autosomal dominant vitreoretinochoroidopathy (ADVIRC) ................ 33 1.2.4.3.1 Phenotypes and genotypes of ADVIRC .......................................... 33 1.2.4.3.2 Features of ADVIRC-related bestrophin-1 ..................................... 33 1.2.5 Cell models of bestrophinopathy .................................................................. 34 1.2.6 Animal models of bestrophinopathy ............................................................. 34 1.2.6.1 Rat model ............................................................................................... 34 1.2.6.2 Mouse model ......................................................................................... 35 1.2.6.3 Canine model ......................................................................................... 36 1.3 Protein folding and proteostasis ....................................................................... 36 1.3.1 Protein folding in the endoplasmic reticulum (ER) ...................................... 37 1.3.1.1 Translocation ......................................................................................... 37 1.3.1.2 Molecular chaperones ............................................................................ 38 1.3.1.3 Disulfide bond formation ...................................................................... 39 1.3.1.4 N-linked glycosylation .......................................................................... 39 1.3.1.5 ER to Golgi export ................................................................................. 41 1.3.2 Protein modification in the Golgi apparatus ................................................. 41 1.3.3 Protein quality control system ...................................................................... 42 1.3.3.1 Unfolded protein response (UPR) ......................................................... 42 1.3.3.2 ER associated degradation (ERAD) ...................................................... 43 1.3.3.3 Protein quality control beyond the ER .................................................. 45 1.3.3.4 Autophagy ............................................................................................. 45 1.3.4 Protein misfolding diseases .......................................................................... 46 1.3.5 Small molecule treatment for protein misfolding diseases ........................... 47 2 Chapter 2: Materials and Methods ........................................................................... 51 2.1 Suppliers ........................................................................................................... 51 2 2.2 Antibodies ......................................................................................................... 51 2.3 Buffers and solutions ........................................................................................ 52 2.4 Bacteriological procedures ............................................................................... 53 2.4.1 Preparation of Luria Bertani (LB) broth and LB agar .................................. 53 2.4.2 Bacterial culture growth ................................................................................ 53 2.5 Nucleic acid procedures ................................................................................... 54 2.5.1 RNA extraction ............................................................................................. 54 2.5.2 Reverse transcription polymerase chain reaction (RT-PCR) ........................ 54 2.5.3 Real-Time quantitative PCR (qRT-PCR) ..................................................... 55 2.5.4 Plasmid DNA preparation ............................................................................. 56 2.5.5 Restriction enzyme digests ........................................................................... 56 2.5.6 DNA purification .......................................................................................... 56 2.5.7 Agarose gel electrophoresis .......................................................................... 57 2.5.8 Ethanol precipitation of DNA ....................................................................... 57 2.5.9 Site directed mutagenesis (SDM) ................................................................. 57 2.5.10 DNA sequencing ....................................................................................... 58 2.6 Tissue culture procedures ................................................................................. 59 2.6.1 Cell Culture ................................................................................................... 59 2.6.1.1 Human embryonic kidney 293T (HEK293T) cell ................................. 59 2.6.1.2 Madin-Darby canine kidney type II (MDCKII) cell ............................. 59 2.6.1.3 Chinese hamster ovary (CHO-K1) cell ................................................. 60 2.6.1.4 Human iPSC-retinal pigment epithelium (iPSC-RPE) cells ................. 60 2.6.1.4.1 Generation of iPSC .......................................................................... 60 2.6.1.4.2 Differentiation of iPSC into RPE .................................................... 61 2.6.2 Transient transfection ................................................................................... 62 2.6.2.1 HEK293T cells ...................................................................................... 62 2.6.2.2 CHO-K1 cells ........................................................................................ 63 2.6.3 Stable transfection ......................................................................................... 63 3 2.6.3.1 Stable transfection of BEST1 in MDCKII cells .................................... 64 2.6.3.2 Stable transfection of EYFP H148Q/I152L in MDCKII cells .............. 65 2.6.4