Development of Gene Therapy for the Treatment of Retinal Dystrophies caused by mutations in AIPL1 Mei Hong Tan A thesis submitted for the degree of Doctor of Philosophy 2011 Department of Genetics Institute of Ophthalmology University College London 1 Declaration I, Mei Hong Tan 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. ............................................... DATE: ................................... 2 Abstract Genetic defects in AIPL1 cause a heterogeneous set of clinical conditions depending on the severity of the mutant alleles. Diseases can range from Leber Congenital Amaurosis (LCA), the severest form of early-onset retinal degeneration, to milder forms such as retinitis pigmentosa (RP) and cone-rod dystrophy. There is currently no effective treatment for LCA and inherited retinal dystrophies, which are the commonest cause of childhood blindness. AIPL1 is expressed primarily in retinal photoreceptors and is required for the biosynthesis of photoreceptor phosphodiesterase (PDE). This thesis describes a programme of work that examines the potential and efficacy of gene replacement therapy in the treatment of AIPL1- associated retinal diseases. It centres on the use of recombinant adeno-associated virus for the transfer of murine and human AIPL1 cDNA into photoreceptor cells. AAV-mediated gene replacement was assessed in two genetically engineered mouse models carrying null and hypomorphic alleles, Aipl1 -/- and Aipl1 h/h mice, which simulate retinal degenerations similar to human LCA and RP respectively. Three different rates of photoreceptor degeneration were simulated using the mouse models. To treat the different rates of degeneration, two pseudotypes of AAV (serotype 2 and 8) exhibiting different transduction kinetics were used for gene transfer. Substantial and long term rescue of the disease phenotype was seen as a result of Aipl1 transgene expression mediated by AAV2/2 vector in Aipl1 h/h mice and by AAV2/8 in rapid degenerations in light accelerated Aipl1 h/h mice and in Aipl1 -/- mice. Thus, the results presented in this thesis validates the efficacy of AIPL1 gene replacement using AAV vectors in varying rates of degeneration that reflected the clinical spectrum of disease. This is the first study to report long-term rescue of a photoreceptor-specific defect and to demonstrate effective rescue of rapid photoreceptor degeneration. The development of an efficient therapy depends on the identification of patients and characterisation of disease phenotype. A panel of DNA samples from patients with LCA and early onset severe retinal dystrophy was 3 screened for mutations in the AIPL1 gene. Patients identified with AIPL1- associated disease demonstrated varying severity of disease from LCA to milder form of rod cone dystrophy. Clinical characterisation and imaging of the patients highlighted distinctive features which will direct future identification and molecular screening of patients. Residual retinal integrity and function in young patients and patients with milder phenotype suggests that AIPL1 defects may be amenable to treatment. 4 Publications and conference abstracts MH Tan, AJ Smith, B Pawlyk, Xu Xiaoyun, Liu Xiaoqing, JW Bainbridge, M Basche, J McIntosh, HV Tran, A Nathwani, T Li, RR Ali. Gene therapy for retinitis pigmentosa and Leber congenital amaurosis caused by defects in AIPL1: effective rescue of mouse models of partial and complete Aipl1 deficiency using AAV2/2 and AAV2/8 vectors. Human Molecular Genetics. Jun 2009; Vol18(12): 2099-2114. MH Tan, D MacKay, J Cowing, HV Tran, AJ Smith, , R Henderson, I Russell-Eggitt , AG Robson, AR Webster, GE Holder, RR Ali, AT Moore. Genetics and phenotypes of AIPL1 mutations in recessive inherited retinal degeneration. Manuscript in preparation. Conference abstracts: MH Tan, HV Tran, AJ Smith, , R Henderson, I Russell-Eggitt , AG Robson, AR Webster, GE Holder, RR Ali, AT Moore. Clinical spectrum of patients with AIPL1 mutations in recessive inherited retinal dystrophies. Invest. Ophthalmol. Vis. Sci. 2010. 51: ARVO E-abstract 3087. MH Tan, HV Tran, AJ Smith, R Henderson, P Moradi, I Russel-Eggitt, M Cheetam, AR Webster, RR Ali, AT Moore. Comparative analysis of AIPL1 mutations in recessive inherited retinal dystrophies. Invest. Ophthalmol. Vis. Sci. 2009. 50: E-abstract 4127. MH Tan, AJ Smith, B Pawlyk, T Li, RR Ali Gene replacement therapy leads to photoreceptor survival and restores beta-phosphodiesterase localization to outer segment in AIPL1-related retinal dystrophy.. Invest. Ophthalmol. Vis. Sci. 2008. 49: E-abstract 1130. 5 Acknowledgement I am truly grateful to my principal supervisor and mentor Professor Robin Ali for his unwavering support, patience, guidance and encouragement. Most of all, I am thankful for his constant positive outlook and understanding of the big picture, which has been a major motivator for the project. I would also like to thank Professor Anthony Moore, my secondary supervisor who has given me invaluable perspective and insight in the clinical discipline. I am also grateful to Alexander Smith for his technical advice and critical appraisal, which was integral to this work. It has been a pleasurable experience and I look forward to working with them again in the future. I would like to acknowledge the generous funding that I have received from the Medical Research Council (MRC, UK) to carry out the work presented in this thesis. Many thanks also to all of the members of staff and colleagues in the Department of Genetics, Institute of Ophthalmology for their help and support. In particular, I would like to thank Emma West and Peter Munro who processed tissue specimens into the beautiful semithin and ultrathin sections respectively that are presented here and Jim Bainbridge for performing the subretinal injections. Last but not least, I am eternally thankful to my parents, family and friends for their love, patience and their belief in me, I could not have done it without them. “Give a man fish and he eats for a day, teach him to fish and he will have food for life” 6 Table of Contents Abstract ........................................................................................ 3 Publications and conference abstracts .................................... 5 Acknowledgement ...................................................................... 6 Figures ……………………………………………………………....13 Tables ......................................................................................... 18 1. Introduction ........................................................................... 19 1.1 Aims and objectives .................................................................................. 22 1.2 The eye – structure and function ............................................................ 22 1.2.1 The retina ............................................................................................. 25 1.2.2 Phototransduction ................................................................................ 32 1.2.3 The visual cycle .................................................................................... 38 1.3 Inherited retinal degeneration ................................................................. 44 1.3.1 Background .......................................................................................... 44 1.3.2.2 Clinical features and genotype-phenotype correlation in LCA ........... 52 1.3.2.3 AIPL1 mutations in inherited retinal dystrophies ................................ 60 1.3.2.4 Molecular genetics of AIPL1 mutations .............................................. 66 1.3.2.5 The phenotype of LCA in patients with AIPL1 mutations .................... 68 1.3.3 Photoreceptor cell death in inherited retinal dystrophies. ..................... 74 1.3.4 Animal models of inherited retinal dystrophies ................................... 82 1.4.1 The eye as a target for gene therapy .................................................. 99 1.4.2 Vectors for gene transfer to the retina .............................................. 100 1.4.2.1 Non-viral methods ............................................................................ 100 1.4.2.2 Adenovirus ....................................................................................... 102 1.4.2.3 Lentiviral vectors ............................................................................. 105 1.4.2.4 Adeno-associated viral vectors ........................................................ 108 1.4.3.1 Gene therapy for treatment of dominant diseases ........................... 120 7 1.4.3.2 Gene replacement therapy for treatment of recessive diseases ..... 122 1.4.3.3 Neuroprotection and anti-apoptotic therapy for treatment of retinal dystrophies ..................................................................................................... 131 1.5 Summary ................................................................................................. 135 2. Materials and methods ....................................................... 137 2.1 Amplification of plasmid DNA in bacteria ............................................. 137 2.1.1 Transformation of competent cells ...................................................... 137 2.1.2 Amplification and recovery of recombinant plasmid DNA ................... 137 2.1.3 Quantification of nucleic acid .............................................................. 138 2.2 DNA analysis