EMMI KOKKI INTRAOCULAR NEOVASCULARIZATION -MOUSE MODELS AND GENE THERAPY APPLICATIONS

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1 PUBLICATIONS OF THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences EMMI KOKKI INTRAOCULAR NEOVASCULARIZATION -MOUSE MODELS AND GENE THERAPY APPLICATIONS

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3 Intraocular neovascularization -mouse models and gene therapy applications

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7 EMMI KOKKI Intraocular neovascularization -mouse models and gene therapy applications To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in Tietoteknia auditorium, Kuopio, on Friday, April 5 th, 2019, at 12 noon Publications of the University of Eastern Finland Dissertations in Health Sciences Number 504 Department of Molecular Medicine, A. I. Virtanen Institute for Molecular Sciences, Faculty of Health Sciences, University of Eastern Finland Kuopio 2019

8 Grano Oy Jyväskylä, 2019 Series Editors: Professor Tomi Laitinen, M.D., Ph.D. Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences Professor Hannele Turunen, Ph.D. Department of Nursing Science Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D. Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences Associate Professor (Tenure Track) Tarja Malm, Ph.D. A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences Lecturer Veli-Pekka Ranta, Ph.D. (pharmacy) School of Pharmacy Faculty of Health Sciences Distributor: University of Eastern Finland Kuopio Campus Library P.O.Box 1627 FI Kuopio, Finland ISBN (print): ISBN (pdf): ISSN (print): ISSN (pdf): ISSN-L:

9 III Author s address: Supervisors: A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland KUOPIO FINLAND emmi.kokki@uef.fi Professor Seppo Ylä-Herttuala, M.D., Ph.D. A.I. Virtanen Institute for Molecular Sciences University of Eastern Finland KUOPIO FINLAND Docent Kati Kinnunen, M.D., Ph.D. Department of Ophthalmology Kuopio University Hospital KUOPIO FINLAND Reviewers: Docent Johanna Liinamaa, M.D., Ph.D. Department of Ophthalmology Oulu University Hospital OULU FINLAND Eeva-Marja Sankila, M.D., Ph.D. Department of Ophthalmology Helsinki University Hospital HELSINKI FINLAND Opponent: Docent Hannele Uusitalo-Järvinen, M.D., Ph.D. Department of Ophthalmology University of Tampere TAMPERE FINLAND

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11 V Kokki, Emmi Intraocular neovascularization mouse models and gene therapy applications University of Eastern Finland, Faculty of Health Sciences Publications of the University of Eastern Finland. Dissertations in Health Sciences Number p. ISBN (print): ISBN (pdf): ISSN (print): ISSN (pdf): ISSN-L: ABSTRACT Pathological ocular angiogenesis is a key feature in many vision-impairing diseases such agerelated macular degeneration and diabetic retinopathy. These diseases are common in the elderly and they cause a severe vision impairment. The relevance of vascular endothelial growth factor (VEGF) in ocular neovascularization is well established in both animal models and humans. Thus, anti-angiogenic agents targeting VEGF are the main therapies for these ocular diseases. Although anti-vegf treatments have shown efficacy in improving the morphological outcomes and restoring visual acuity, the need for intraocular injections is frequent. The development of new treatments for neovascular ocular diseases relies on reproducible and reliable pre-clinical models that mimick the pathogenesis of human retinal and subretinal diseases. The aim of the thesis was to generate novel mouse models for both retinal and subretinal neovascularization, to study gene transfer to the mouse eye and to develop anti-vegf gene therapy for subretinal neovascularization. The thesis consists of four projects investigating neovascularization and gene therapy in the eye. The first study compared the biodistribution, transduction efficiency and side effects of adeno-, adeno-associated-, lenti- and baculoviral vectors after intravitreal injection into mouse eye revealing major differences between the four vectors. In the second study, a mouse model expressing human VEGF was developed using the subretinal Cre gene transfer into the eyes of transgenic mouse. We were able to generate a novel animal model for agerelated macular degeneration and subretinal neovascularization. This novel mouse model was used also in the third project that examined anti-angiogenic gene therapy with a lentiviral vector coding anti-human VEGF antibody. In in vitro studies, the anti-human VEGF antibody was demonstrated to mimick bevacizumab (Avastin ). In mice, intravitreal gene delivery with the vector reduced subretinal neovascularization as compared with control injection. The final study explored the effect of vulnerable atherosclerotic plaques on retinal morphology and the vasculature on mice. The results demonstrated that feeding a high-fat diet to transgenic mice with vulnerable plaques was not sufficient to produce spontaneous retinal vessel occlusion and neovascularization in the mouse eye. Overall, the results demonstrate the feasibility of intraocular gene transfer in both inducing and treating subretinal neovascularization. As the viral vectors possess different characteristics after intraocular gene transfer, careful assessments of the gene delivery vehicle and its delivery route are needed. These results will be beneficial in studying the ocular neovascularization and novel therapies in mice in a way towards translating the pre-clinical results into the treatment of humans with vision-threatening disease due neovascularization. National Library of Medicine Classification: QU 107, QU 560, QY 58, WW 140, WW 270 Medical Subject Headings: Eye; Retina; Neovascularization, Pathologic; Angiogenesis Inhibitors; Genetic Therapy; Gene Transfer Techniques; Genetic Vectors; Lentivirus; Vascular Endothelial Growth Factor A; Macular Degeneration; Plaque, Atherosclerotic; Disease Models, Animal; Mice

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13 VII Kokki, Emmi Silmänsisäinen uudissuonitus hiirimallit ja geeniterapiasovellukset Itä-Suomen yliopisto, terveystieteiden tiedekunta Publications of the University of Eastern Finland. Dissertations in Health Sciences Numero s. ISBN (print): ISBN (pdf): ISSN (print): ISSN (pdf): ISSN-L: TIIVISTELMÄ Patologisella silmän uudissuonituksella eli angiogeneesilla on merkittävä osa monissa näkökykyä heikentävissä sairauksissa, kuten silmänpohjan ikärappeumassa ja diabeettisessa retinopatiassa. Nämä silmäsairaudet ovat yleisiä ikääntyvillä ja ne aiheuttavat vakavaa näkökyvyn alenemista. Verisuonen endoteelin kasvutekijän (VEGF) osuus silmän uudissuonituksessa on luotettavasti osoitettu eläinmalleissa ja ihmisillä. Tämän vuoksi silmän uudissuonitusta estävät anti-angiogeeniset terapiat ovat ensisijainen valinta näiden silmäsairauksien hoidossa. Uusien hoitojen kehittäminen silmän uudissuonistusta aiheuttaviin sairauksiin on riippuvainen toistettavista ja luotettavista pre-kliinisistä malleista, jotka mallintavat ihmisen verkkokalvon ja suonikalvon sairauksien patogeneesia. Väitöskirjatutkimuksen tavoitteena oli kehittää uusia eläinmalleja sekä suonikalvon että verkkokalvon uudissuonitusta aiheuttaviin sairauksiin, tutkia silmänsisäistä geeninsiirtoa sekä kehittää anti-angiogeeninen geeniterapia suonikalvon uudissuonituksen hoitoon. Väitöskirja koostuu neljästä osatyöstä, jotka käsittelevät silmän angiogeneesiä ja geeniterapiaa. Ensimmäinen osatyö vertaili neljän eri virusvektorin biodistribuutiota, transfektiotehokkuutta ja haittavaikutuksia lasiaiseen tehdyn geeninsiirron jälkeen adeno-, adeno-associated-, lenti- ja bakulovirusvektoreilla. Työssä havaittiin suuria eroja tutkittujen vektoreiden välillä. Toisessa osatyössä luotiin ihmisen VEGF-proteiinia silmässään ilmentävä hiirimalli subretinaalisen Cre geeninsiirron avulla. Työssä kehitettiin uusi eläinmalli neovaskulaariseen silmänpohjan ikärappeumaan. Hiirimallia käytettiin myös kolmannessa osatyössä, joka tutki anti-angiogeenista geenihoitoa. Työssä luotiin ihmisen VEGF-proteiiniin sitoutuvaa vasta-ainetta ilmentävä lentivirusvektori. In vitro kokeissa tämän VEGF-proteiiniin sitoutuvan vasta-aineen havaittiin vastaavan bevasitsumabia (Avastin ). Eläinkokeissa lasiaiseen tehty geeninsiirto kyseisellä lentivirusvektorilla vähensi uudissuonitusta kontrolli-injektioon verrattuna. Viimeinen työ tutki hauraiden ateroskleroottisten plakkien vaikutusta verkkokalvoon ja sen verisuoniin hiirillä. Tulokset osoittivat, että runsasrasvainen ruokavalio muuntogeenisillä hiirillä ei ole riittävä luomaan spontaaneja verkkokalvon verisuonitukoksia ja uudissuonitusta hiiren silmässä. Väitöskirjatyön tulokset osoittavat, että silmänsisäinen geeninsiirto on hyödyllistä uudissuonituksen luomisessa ja hoidossa. Koska virusvektoreilla on erilaisia ominaisuuksia silmänsisäisessä geeniterapiassa, geenikuljettimien ja annostelureittien huolellinen tutkiminen on tärkeää kehitettäessä uusia hoitoja. Tutkimustulokset ovat hyödyllisiä tutkittaessa silmän uudissuonitusta ja kehitettäessä uusia hoitoja. Tulevaisuudessa näitä prekliinisiä tuloksia voitaneen hyödyntää niiden potilaiden avuksi, jotka ovat vaarassa menettää näkökykynsä uudissuonitusta kehittävien sairauksien vuoksi. Luokitus: QU 107, QU 560, QY 58, WW 140, WW 270 Yleinen Suomalainen asiasanasto: verkkokalvo; angiogeneesi; silmänpohjan ikärappeuma; geenitekniikka; geeniterapia; vektorit (biologia); lentivirukset; kasvutekijät; verisuonet; koe-eläinmallit; hiiret

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15 IX Tule pois sieltä puun alta! Isaac Newtonin luokanvalvoja

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17 XI Acknowledgements This thesis work was carried out in A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland. Many people have helped me within these years either with my PhD studies or simply by making AIVI a great place to do science. For this, I am sincerely grateful for. I have been honored to work with such a talented, inspiring and fun colleagues that many have become my friends. First and foremost I want to thank my main supervisor Professor Seppo Ylä-Herttuala for the opportunity to be part of this gifted research group. Not only do I greatly appreciate the knowledge and guidance, but also I am grateful for your encouragement, positivity and enthusiasm, which have been needed every now and then. I owe my sincere thanks to my other supervisor Docent Kati Kinnunen for advice, support and optimism. I want to acknowledge my co-author Tommi Karttunen for your impact in the projects. It has been great to co-operate with the Department of Ophthalmology in Kuopio University Hospital. I also want to say thanks to co-authors Giedrius Kalesnykas and Laura Alasaarela. I am sincere grateful to the official reviewers of this thesis, Johanna Liinamaa and Eeva-Marja Sankila. Your comments helped to improve this thesis. I want to thank Ewen MacDonald for the linguistic review of this thesis. I want to thank all talented people in AIVI for their help. Pyry Toivanen for all the help with in vitro analyses, Minna Kaikkonen-Määttä for the help with the cloning, Elisa Hytönen for the advice regarding virus work and Svetlana Laidinen for the genotyping and practical issues in the lab. I owe my warmest thanks to Lari Holappa and Tommi Heikura for their help with animal work and other various issues I have encountered. You have always had time to help. Nihay, thank you for your warm support and guidance. I also wish to thank Anna-Kaisa for helping with practical issues regarding preparation of the dissertation. My deepest thanks belong to gifted laboratory technicians and secretaries for assistance. All the past and present researchers have made the group a unique and inspiring place to work in. There are so many people in the group to go to if any help, idea or company for some wine are needed. I could not have wished for any better co-workers. I have had the opportunity to share the office with many great people within the years. Warmest thanks to Venla and Sanna for co-authoring and making the office always a fun place to go to. I am glad to be still in touch with you, Tiina and Aga, even though we do not work together anymore. Especially I owe my greatest thank to dear Taina for all the help within these years. I really appreciate sharing thoughts about work and other aspects of life with you. Taina, Heidi and Laura, I value your knowledge in science and your friendship. When starting in SYH-group with my pro gradu, Laura and Heidi were the first people I got to know there. You made me feel welcome and taught me much about research. It was easy to continue to PhD studies with co-workers like you. Annakaisa, I appreciate your outgoing personality and determination. The clop your heels approaching our office always knows a laugh. I also owe thanks to dear friends outside the work. I have had the privilege to know them already for many years. Friends from more or less from Faculty of Pharmacy; Jenni, Antti, Aki, Saara, Sakke, Ennska and Tatu: trips with Aki-Travels and Retkiriitat and other fun moments are beloved and valued. Dear Maija, thank you for your friendship. Meeting you is always a joy. My oldest friends I have get to known already before school. Anne, Jysky and Höökki, I am so glad that after all these years you still mean the world to me. I am lucky to have you all friends in my life.

18 XII My loving thanks belong to my dear mom, thank you for all that caring and believing in me during these years, I would not have managed through these years without your support and the invaluable help. Finally, Antti. Thank you for love, support and laughter. I cannot wait to start our lives together. Kuopio, March 2019 Emmi Kokki This study was supported by grants from the Finnish Cultural Foundation, the Otto A. Malm Foundation, the Diabetes Research Foundations, the Foundation of Eye and Tissue Bank and the Maud Kuistila Memorial Foundation.

19 XIII List of the original publications This dissertation is based on the following original publications: I II III Kalesnykas G, Kokki E*, Alasaarela L*, Lesch HP, Tuulos T, Kinnunen K, Uusitalo H, Airenne K, Ylä-Herttuala S. Comparative Study of Adeno-associated Virus, Adenovirus, Baculovirus and Lentivirus Vectors for Gene Therapy of the Eyes. Current Gene Therapy 17(3): , Kokki E, Karttunen T, Olsson V, Kinnunen K, Ylä-Herttuala S. Human Vascular Endothelial Growth Factor A165 Expression Induces the Mouse Model of Neovascular Age-Related Macular Degeneration. Genes 9(9): 438, Kokki E, Karttunen T, Toivanen P, Kaikkonen M, Wirth T, Kinnunen K, Ylä- Herttuala S. Lentivirus-mediated intravitreal delivery of anti-vegf antibody in AMD mouse model. Manuscript. IV Kokki E, Karttunen T, Kettunen S, Kinnunen K, De Mayer GRY, Ylä-Herttuala S. Ocular Phenotype of Mice with Impaired Fibrillin-1 Function on Hypercholesterolemic Apolipoprotein E-Deficient Background. Current Trends in Ophthalmology, *Equal contribution The publications were adapted with the permission of the copyright owners.

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21 XV Contents 1 INTRODUCTION REVIEW OF THE LITERATURE The visual pathway and ocular vasculature in man and mouse Anatomy Retina Ocular vasculature Imaging techniques of the eye Retinal and subretinal vascular diseases Retinal vascular diseases Subretinal vascular diseases Connection of subretinal and retinal neovascular diseases Pre-clinical animal models of ocular vascular diseases Angiogenesis and vascular endothelial growth factors in ocular vascularization Vascular endothelial growth factors Vascular endothelial growth factor receptors VEGF in healthy and diseased eye Anti-angiogenic treatments for ocular neovascularization Gene therapy of the eye Ocular gene therapy Gene delivery vectors Adenoviral vectors Adeno-associated viral vectors Lentiviral vectors Baculoviral vectors Non-viral vectors Biodistribution after intraocular gene transfer Gene therapy products Clinical trials of gene therapy AIMS OF THE STUDY MATERIALS AND METHODS In vitro studies Construction of anti-vegf-antibody plasmid Protein production and transduction Binding assays and comparison of anti-vegf antibody to Avastin In vivo studies Experimental animals Viral vectors and gene transfer methods Methods for evaluating in vivo studies Imaging of the eye Histological and immunohistochemical methods Protein and gene expression analyses... 37

22 XVI Clinical chemistry Statistical analyses RESULTS Transduction efficacy and adverse effects of intravitreal delivery of adeno-associated virus, adenovirus, baculovirus and lentivirus vectors (I) Ocular effects and biodistribution of adenoviral Cre inducible human VEGF-A165 expression in the eye of transgenic mice (II) The effects of anti-vegf antibody in vitro and in the mouse eye after gene therapy (III) Lack of retinal vascular occlusion and neovascularization in mice with vulnerable atherosclerotic plaques (IV) DISCUSSION Transduction efficacy of intraocular gene transfer (I-III) Biodistribution and safety after intraocular gene transfer (I-III) Development of retinal neovascularization and AMD-associated subretinal neovascularization (II-IV) The effects of anti-vegf antibody in vitro and after lentivirus-mediated gene therapy (III) CONCLUSIONS EPILOGUE REFERENCES ORIGINAL PUBLICATIONS AND A MANUSCRIPT (I-IV)... 85

23 XVII Abbreviations a Arteriole AAV Adeno-associated virus Ab Antibody Ad Adenovirus ALT Alanine aminotransferase AMD Age-related macular degeneration ApoE Apolipoprotein E BCVA Best corrected visual acuity BRB Blood-retinal barrier BRVO Branch retinal vein occlusion BV Baculovirus BM Bruch's membrane c Concentration cdna Complementary deoxyribonucleic acid CAG CMV enhancer chicken betaactin promoter, rabbit betaglobin splice acceptor site CMV Cytomegalovirus CNV Choroidal neovascularization CRAO Central retinal artery occlusion CRVO Central retinal vein occlusion DAPI 4'-6-Diamidino-2- phenylindole DME Diabetic macular edema DNA Deoxyribonucleic acid DR Diabetic retinopathy ELISA Enzyme linked immune sorbent assay ELM External limiting membrane F Fovea FA Fluorescein angiography FAB Antigen binding fragment Fbn-1 Fibrillin-1 GCL Ganglion cell layer GFP Green fluorescent protein HE Hemotoxylin-eosin HEK293T Human embryonic kidney 293T HIF-1 Hypoxia-inducible factor HIV Human immunodeficiency virus HRP Horseradish peroxidase IL-3 Interleukin 3 ILM Inner limiting membrane IN Inferonasal INL Inner nuclear layer IPL Inner plexiform layer IRS-1 Insulin receptor substrate 1 IS Inner segment IT Inferotemporal LV Lentivirus kb Kilobase kda Kilodalton KO Knockout MAC Membrane attack complex MOI Multiplicity of infection mrna Messenger RNA NeuN Neuronal nuclei NFL Nerve fiber layer NPDR Non-proliferative diabetic retinopathy n.s. Non-significant NVG Neovascular glaucoma OCT Optical coherence tomography OCTA Optical coherence tomography angiography OD Optic disc OIR Oxygen induced retinopathy ONL Outer nuclear layer OPL Outer plexiform layer OS Outer segment PCR Polymerase chain reaction PDR Proliferative diabetic retinopathy PEC Peritoneal exudate cell PEDF Pigment epithelium-derived factor PGK Phosphoglycerate kinase-1 PlGF Placental growth factor PU Particle units qrt-pcr Quantitative reverse transcriptase polymerase chain reaction RNA Ribonucleic acid RPE Retinal pigment epithelium RAO Retinal artery occlusion RAP Retinal angiomatous proliferation ROP Retinopathy of prematurity RVO Retinal vein occlusion s Soluble

24 XVIII SD Spectral-domain shrna Short hairpin RNA sirna Short interfering RNA SN Superonasal SR Subretinal ST Superotemporal STZ Streptozotocin tg Transgenic TMB 3,3,5,5 - Tetramethylbenzidine U/l Unit/litre v Venule VEGF Vascular endothelial growth factor VEGFR Vascular endothelial growth factor receptor VLDL Very low-density lipoprotein

25 1 Introduction Although the prevalence of people with blindness and visual impairment has decreased worldwide, the number has risen due to the increase in the elderly population (Bourne et al., 2013; Flaxman et al., 2017). In addition, the number of people with moderate/severe or complete visual loss due to diabetic retinopathy (DR) and age-related macular degeneration (AMD) has increased and is anticipated to rise further. Ocular pathological angiogenesis i.e. new vessel growth, is a key feature in many common vision-impairing diseases, like DR and AMD (Witmer et al., 2003). There are two major types of ocular neovascularization: retinal and subretinal or choroidal neovascularization (CNV) (Campochiaro, 2015). Retinal neovascularization occurs in ischemic retinopathies such as in retinal vein occlusion (RVO), retinopathy of prematurity (ROP) and most evidently in DR. Subretinal and choroidal neovascularization originate from a break or defect in Bruch s membrane (BM) and it is most commonly found in AMD. Vascular endothelial growth factor (VEGF) plays a crucial role in angiogenesis (Penn et al., 2008). The relevance of VEGF in retinal and choroidal neovascularization has been well established in animal models and in humans. Overexpression of VEGF can result in many ocular complications, such vitreous hemorrhages, macular edema and retinal detachment, leading eventually to vision impairment and even blindness. As VEGF is essential in the pathology of ocular angiogenesis, neutralizing of VEGF is the first line therapy for diseases of retinal and choroidal neovascularization (Amadio et al., 2016). Unfortunately, antiangiogenic treatment is injected intravitreally at monthly or bimonthly, even up to six years (Mrejen et al., 2015) and furthermore, the effect is usually transient (Comparison of Agerelated Macular Degeneration Treatments Trials (CATT) Research Group et al., 2016). In gene therapy, nucleic acids are transferred into somatic cells, resulting in the expression of the therapeutic transgene in the target cells and thus therapeutic effect (Ylä-Herttuala and Alitalo, 2003). Thus, it can be considered presenting an alternative option for continual injections in the treatment of ocular angiogenesis. The eye posseses several anatomical features that make it a great target for gene therapy (Bainbridge et al., 2006; Bennett, 2003; Petit et al., 2016). Transparency of the eye enables accurate application of the viral vectors to specific target sites and allows non-invasive imaging of the eye. The eye is anatomically restricted and compartmentalised, which make it ideal for the precise and targeted delivery of vectors. The small size of the target organ enables efficient transduction using small injection volumes. The eye is a relatively immune privileged site due to the cellular and physical barriers that limits the risk of evoking a strong systemic immune response leading to inflammation and limited transgene expression. Due to the small amounts of vector injected and the anatomical barriers of the eye, the possibility of systemic biodistribution and side effects are minor. In addition, for study purposes, the contralateral eye offers an appropriate control. The route of administration affects highly the intraocular tissues transduced after the injection. In addition, as the selection of the vector has an effect on the target cell transduction and the duration of the transgene expression, we compared adenoviral (Ad), adenoassociated viral (AAV), baculoviral (BV) and lentiviral (LV) vectors after intravitreal injection into the mouse eye. Intraocular gene transfers were used to create a mouse model of neovascular AMD with human VEGF expression in the eye and, subsequently to treat subretinal neovascularization in this mouse model with anti-angiogenic gene therapy. Another mouse model of spontaneous retinal vessel occlusion and neovascularization was also examined. This thesis explores mouse models of both subretinal and retinal neovascularization and intraocular viral vector mediated gene therapy in the development of neovascular mouse models and anti-angiogenic treatments.

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27 3 2 Review of the literature 2.1 THE VISUAL PATHWAY AND OCULAR VASCULATURE IN MAN AND MOUSE The eye is a complex system of many specialized tissues integrated into a functional sense organ. Many basic elements of the eye are conserved between species and the eyes are similar between human and mice, although some anatomical differences exist (Figure 1). (Treuting et al., 2012) Anatomy In human and mouse eye the outer layer of the eye provides structural integrity consisting of the transparent cornea anteriorly and the sclera posteriorly. The eye is divided into three compartments. Between the iris and cornea is the anterior chamber and between the iris and lens is a narrow posterior chamber, both chambers filled with aqueous humor providing nutrients to avascular structures in the front of the eye and removing waste products. The third compartment between posterior chamber and the retina is vitreous cavity filled with vitreous gel. In humans, the vitreous occupies the majority of the volume of the eye, whereas in the mouse the large round lens fills the most of the chamber. At the back of the eye is the retina, the complex nerve layer that absorbs the light and converts the visual information to signals transmitted to the brain through the optic nerve. The optic nerve as well as retinal veins and arteries enter the eye at the optic nerve head, which is located in the retina. (Treuting et al., 2012; Vézina, 2012) Figure 1. The gross anatomy of the human and mouse eye. Though there are many structural similarities between the human and mouse eye, there are some anatomical differences in the mouse eye such as the absence of the macula and fovea in the retina and large, round lens. (Treuting et al., 2012) Image modified from (Volland et al., 2015). RPE: Retinal pigment epithelium Retina In both man and mouse, the retina is about 200 µm thick lining at the back of the eye (Adhi et al., 2012; Ferguson et al., 2013). As light enters the eye, it travels through the cornea crossing chambers and lens before impacting the retina wherein the visual impulse is transmitted from photoreceptors to the brain via the optic nerve (Kels et al., 2015).

28 4 The retina consists of several layers of cells, which are the same in humans and mice, although humans with trichromatic vision have three types of cones in the photoreceptor cell layer whereas mice possess only two (Treuting et al., 2012). Nine layers of neural retina process the visual sensation (Figure 2). The retina is composed of three nuclear layers of nerve cell bodies: ganglion cell layer (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL), separated by two plexiform layers of synapses: the inner plexiform layer (IPL) and the outer plexiform layer (OPL). The direct chain from photoreceptors to bipolar cells, ganglion cells and their axons in the nerve fiber layer (NFL) is the major route of visual information flow to the optic nerve and on to the brain for interpretation (Remington, 2005). Other cells of neural retina horizontal cells and amacrine cells modify and integrate the signal before it leaves the eye. In addition to neurons, the retina contains glial cells Müller cells, microglial cells and astrocytes that provide structure and support and play a role in the neural tissue reaction to injury or infection. Müller cells blend between the inner limiting membrane (ILM) and external limiting membrane (ELM). Beneath neural retina is the retinal pigment epithelium (RPE), facing the choroid (Ofri, 2008). The RPE serves as a metabolic interface between the photoreceptors and choroid supplying metabolites and removing waste from the outer retina. The most outstanding difference between the two species is the lack of a macula and fovea in mouse retina (Treuting et al., 2012). A pit in the center of the macula is called the fovea. At the center of the fovea, the retina is avascular and consists primarily of cones (Vézina, 2012). In humans, the macula is responsible for the majority of high-resolution daytime vision. Many disabling human retinal and choroidal disorders express the macular dysfunction. As rodents do not have a macula, the question has arisen about the relevance of those species as animal models in maculopathies like AMD and diabetic macular edema (DME) (Huber et al., 2010). It is not known why macula is more susceptible to degeneration but the phagocytic load on RPE and relative thinness of Bruch s membrane in this area could play a role (Volland et al., 2015). Another hypothesis is related to the high rod and cone density in macular area. Photoreceptors consume more oxygen than any other cell type in the body (Linsenmeier and Padnick Silver, 2000; Provis et al., 2005). Since they are highly dependent on the choroidal oxygen supply, even a minor reduction in blood flow results in oxidative stress, supporting the development of AMD (Provis et al., 2005). A comparison of human and mouse retina has revealed that the central mouse retina models at least the peripheral part of the macula (Volland et al., 2015).

29 5 Figure 2. Retinal layers. A) Neural retina consists of ganglion, amacrine, bipolar and horizontal cells as well as photoreceptors, which transform the light energy into a neural signal (Remington, 2005). Between neural retina and choroid lies RPE supplying metabolites and removing waste from the outer retina (Ofri, 2008). B) Hematoxylin-eosin staining of the retina presenting the same retinal layers. Image B modified from (Volland et al., 2015). BM: Bruch's membrane, ELM: External limiting membrane, GCL: Ganglion cell layer, ILM: Inner limiting membrane, INL: Inner nuclear layer, IPL: Inner plexiform layer, IS: Inner segment, NFL: Nerve fiber layer, ONL: Outer nuclear layer, OPL: Outer plexiform layer, OS: Outer segment, RPE: Retinal pigment epithelium Ocular vasculature Because the retina has the highest metabolic demand of any tissue in the body (Saari, 1987), the ability to regulate blood flow is an essential feature of the mammalian retina (Kur et al., 2012). The retina has a dual vascular supply that is similar in both men and mice (Figure 3) (Treuting et al., 2012). The optic nerve head is the point of entry into the eye for the optic nerve and the major retinal veins and arteries (Vézina, 2012). The optic nerve head i.e. optic disc is located on the globe centrally in mice and nasally in men. In humans, after entering the eye along with the optic nerve, the central retinal artery branches into four arterial arcades near the anterior surface of the retina (Treuting et al., 2012). In mice, the number of retinal arterioles varies, usually ranging from four to six arterioles. The venous drainage follows a similar vascular pattern in the two species. Arterioles, capillaries and venules originating from these inner vessels form the retinal vasculature (Ofri, 2008). The arteries and venules generated from the retinal arteries and veins form an extensive capillary network that spreads throughout the inner retina and is diffusely distributed between the arterial and venous systems (Anand-Apte and Hollyfield, 2010). Nevertheless, there are also some specific areas of the retina that lack capillaries. For example, the retina adjacent to the major arteries and some veins is missing a capillary bed. The central retinal artery and its branches supply the anterior two-thirds of the inner retinal layer with two capillary networks (Remington, 2005; Treuting et al., 2012). The superficial capillary plexus lies immediately beneath the ILM in the NFL or GCL and the deep capillary plexus permeates the INL near the OPL. The outer third of the retina from the OPL is avascular. In humans, there is a zone free from all vessels within the fovea

30 6 (Remington, 2005). The absence of blood vessels in the fovea allows the light to pass through the photoreceptor s outer segment without any obstruction. The photoreceptor layer and the RPE are supplied by the choriocapillaris diffusing through Bruch s membrane. The choroid is a multiplex pigmented vascular network situated underneath the retina (Treuting et al., 2012; Vézina, 2012). Besides providing the blood supply to, it also removes waste from the RPE and photoreceptors. The choroidal vasculature is supplied by the posterior ciliary arteries, and drainage occurs via vortex veins, both penetrating through the sclera (Anand-Apte and Hollyfield, 2010; Treuting et al., 2012). Choroidal vessels are fenestrated and lack a blood-retinal-barrier (BRB) (Penn et al., 2008). Nevertheless, there are barriers in the eye which restrict the transport of substances into the retina (Ofri, 2008). BRB has two components: tight junctions of adjacent RPE cells form the outer part and tight junctions of retinal vascular endothelial cells in the retinal capillary plexus form the inner part (Penn et al., 2008). In addition, Bruch s membrane provides an important part of the BRB (Vézina, 2012). Mouse eye lacks circle of Zinn, an annular artery surrounding the optic nerve (Anand- Apte and Hollyfield, 2010), and lamina cribrosa, a sieve-like opening in the sclera transmitting the optic nerve axons (Ofri, 2008). In human eye, the optic nerve head is supplied by the circle of Zinn by the choroidal arteries (Lieberman et al., 1976). In mouse eye, the optic nerve head derives from branches of the central retinal artery, and none of the capillaries are derived from choroidal vessels (May and Luẗjen-Drecoll, 2002). Figure 3. Retinal vasculature. A) The retinal and choroidal vasculature in human eye. The retinal layers up to the inner nuclear layer are nourished by the retinal arteries, whereas the outer retinal layers are nourished by the choroidal vessels. B) The vasculature in human optic nerve. The central retinal artery and vein enter the eye through the optic nerve. C-D) Distribution of main arteries and veins and location of the optic disc in the retina of human (C) and mouse (D). (Treuting et al., 2012) Images A and B modified from (Kur et al., 2012) and C and D from (Ramos et al., 2013). a: Arteriole, F: Fovea, IN: Inferonasal, IT: Inferotemporal, OD: Optic disc, SN: Superonasal, ST: Superotemporal, v: Venule

31 Imaging techniques of the eye The eye is a unique organ as it can be studied non-invasively to reveal details that in most organs are only visible histopathologically or during invasive surgery (Ofri, 2008). Fluorescein angiography (FA) and optical coherence tomography (OCT) are widely used as diagnostic tools for detecting choroidal and retinal abnormalities in humans (Bhagat et al., 2009; Coleman et al., 2008; Dattilo et al., 2017; Ehlers and Fekrat, 2011) and to some extent in mice (Fischer et al., 2009; McLenachan et al., 2015; Robinson et al., 2012). FA allows the study of blood flow in the retina and choroid. For the imaging, sodium fluorescein is injected intravenously or intraperitoneally in humans or mice, respectively. Seconds after injection, fluorescein enters the ocular circulation. To image the fluorescence, a blue light from camera enters the eye and excites the unbound fluorescein molecules circulating in the retinal and choroidal layers or the ones that have leaked out of the vasculature and stimulates them to emit a longer-wavelength. Both the emitted fluorescence and some degree of reflected blue light from structures that do not contain fluorescein exit the eye and return to the camera. The image formed by the emitted fluorescence is recorded on photographs and/or videos. (American Academy of Ophthalmology, 2018; Arevalo, 2009) After injection, fluorescein enters the ocular circulation via the ophthalmic artery. First during the transit phase, it fills choroidal vessels and then retinal vessels. Choroidal vessels filling is characterized by a patchy choroidal flush, with the lobules often visible. Next, the retinal arteries fill during the arterial phase. The following arteriovenous phase begins with complete filling of the retinal arteries and capillaries and finishes with laminar filling of the retinal veins. Over the next few minutes, a gradual decline in fluorescence is seen as the dye recirculates. In the late phases of the angiography, fluorescein stains the choroid, Bruch s membrane, and the sclera. The larger choroidal vessels are often seen as hypofluorescent areas against this hyperfluorescent background. In healthy tissue, fluorescein stays in the capillaries and does not diffuse through RPE and larger choroidal vessels. Fluorescein can leak out of retinal capillaries into the retina and through RPE into subretinal space only when the capillary endothelium or retinal interstitium is damaged, respectively. This allows the detection of hyperfluorescent lesions. The interpretation of the FA pattern includes autofluorescence by naturally highly reflective substances such as drusen, hypofluorescence of reduced or absent blood flow as a result of a vascular filling defect or blocked fluorescence or hyperfluorescence as an excess of normal fluorescence. OCT, on the other hand, is a computer-assisted optical instrument that generates crosssectional images of ocular structures without the need of any contrast agent (Arevalo et al., 2009). It uses low-coherence light waves to obtain a reflectivity profile of the tissue. A light source is directed onto the imaged tissue and onto an internal reference mirror. Backscattered light from both sources is combined and interference is measured. The relative location of the light backscattered from the imaged tissue is then determined based on the information obtained from the controlled internal reference mirror. OCT can be used to assess retinal thickness, volume and fluid involvement and to identify the location and level of neovascularization intraretinally, subretinally or in the subretinal pigment epithelium (Hee et al., 1996). In addition, it can be used to detect other lesion components such as blood, fluid, drusen, pigment and fibrosis. Optical coherence tomography angiography (OCTA) is a newer and better non-invasive technique for imaging the microvasculature of the retina and choroid without the need for a label dye (Gao et al., 2016). Spectral domain OCT (SD-OCT) uses a high-speed spectrometer to measure light echoes from all time delays, enhancing OCT s capabilities. Improved sensitivity enables upgrade in sampling speed and signal-noise-ratio thus having an advantage of detecting small changes in the morphology of the retinal layers and subretinal space, allowing the precise anatomic detection of structural changes that may correspond to progression or regression of the neovascular lesions. (Regatieri et al., 2011)

32 8 A newer technique, swept-source OCT (SS-OCT) has an improved depth of imaging and scan speed compared with the previous modalities of OCT. The deeper penetration allows the simultaneous detailed visualization of the vitreous, the choroid and the sclera. It has better ability to visualize retinal structures behind dense pre-retinal hemorrhages and sub- RPE pathology such as CNV. Thus, the multimodal approach using SS-OCT is expected to advance the understanding of retinal pathologies such as AMD and diabetic maculopathy. (Kishi, 2016; Lavinsky and Lavinsky, 2016). 2.2 RETINAL AND SUBRETINAL VASCULAR DISEASES Angiogenesis is the formation of new blood vessels from the pre-existing vasculature (Qazi et al., 2009). Ocular neovascularization is a complex pathological disorder associated with many ocular diseases (Campochiaro, 2015). In the eye, neovascularization by angiogenesis into previously avascular tissues often leads to vision impairment. The two types of intraocular neovascularization are retinal and subretinal or choroidal neovascularization. Retinal neovascularization occurs in a group of diseases referred to as ischemic retinopathies. The most common disease of retinal neovascularization is DR. Subretinal and choroidal neovascularization occur in the outer retina originating with the break or defect in BM and it is commonly found in AMD. AMD and DR are responsible for the most severe and moderate vision loss in the developed countries (Bourne et al., 2013; Ojamo, 2017) Retinal vascular diseases In retinal vascular diseases, leakage or neovascularization occurs from retinal vessels (Campochiaro, 2015). Most common retinal vascular diseases are DR, ROP and RVO. A typical feature is that the underlying disease damages the retinal vessels causing them to close and leading to retinal ischemia (Campochiaro, 2013). As a result, neovascularization arises from pre-existing retinal vessels. Neovascularization may originally grow within the retina but over time, the neovessels grow to the retinal surface and into the vitreous. New vessels adhere to the inner surface of the retina and outer surface of the vitreous pulling the retina towards the vitreous and leading to retinal detachment. In addition, leakage of plasma from the permeable new vessels into the vitreous can cause the vitreous gel to degenerate, contract and pull the retina. Rupture of the neovascularization results in vitreous hemorrhage. Both hemorrhage and retinal detachment lead to severe vision loss. In addition, retinal vascular leakage followed by macular edema due to the fluid accumulation in the macular region is a vision-threatening condition. (Antonetti et al., 2012; Campochiaro, 2013) DR is the most common type of retinal vascular disease, increasing globally up to 0.8 million blind and 3.7 million vision impaired in the year 2010 with 27 % and 64 % increase in 20 years, respectively (Leasher et al., 2016). The prevalence of DR is the highest in highincome countries with aging population. Globally, the crude prevalence of blindness in all ages has declined for all causes except for DR (Flaxman et al., 2017). Hyperglycemia damages retinal vessels via variety of biological processes (Boulton and Cai, 2002). DR begins as a non-proliferative diabetic retinopathy (NPDR) (Antonetti et al., 2012; Lechner et al., 2017). Microaneurysms are the initial characteristic of DR, but as the disease progresses, NPDR evolves into moderate and severe forms with exudative changes (hard exudates i.e. lipid deposits), intraretinal hemorrhages, ischemic changes (cotton-wool spots i.e. accumulation of debris with ganglion cell axons), venous dilation and intraretinal microvascular abnormalities. DME is the major cause of vision loss in patients with NPDR (Klaassen et al., 2013). Loss of capillaries near to areas of ischemic retina induces BRB breakdown which in turn leads to leakage of plasma and thus to diffuse edema. Focal edema from leaky microaneurysms represents another pattern of macular edema (Antonetti et al., 2012; Hasegawa et al., 2016; Lee et al., 2016).

33 9 Proliferative diabetic retinopathy (PDR) is characterized by neovascularization or vitreous hemorrhage (Lechner et al., 2017). Neovascularization and vitreous hemorrhages occur when capillary non-perfusion results in ischemia as the disease progresses. Vessels form on the surface of the retina and grow towards vitreous. These newly formed vessels are abnormal and leaky. The subsequent repeated vitreous hemorrhaging is associated with fibrovascular scar formation, which can contract and lead to retinal detachment. In addition to macular edema, also hemorrhage and retinal detachment can cause vision loss (Antonetti et al., 2012). Contrary to macular edema, vision loss in PDR is often sudden and results in blindness if untreated (Caldwell et al., 2003). RVO is the second most common type of retinal vascular disease after DR with a prevalence rate of % (Laouri et al., 2011) with an estimated 16 million globally affected people (Rogers et al., 2011). The two most common types of RVOs are branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO), with BRVO being about five times more common than CRVO (Laouri et al., 2011; Rogers et al., 2011). Although occlusion in the retinal artery is much less common, with an estimated incidence of one case per people (Rumelt et al., 1999), it is an ophthalmic emergency and the ocular analogue of cerebral stroke leading to acute and severe vision loss (Hayreh and Zimmerman, 2005). In humans, hyperlipidemia and atherosclerosis are highly correlated with retinal vessel occlusions (Ehlers and Fekrat, 2011; Varma et al., 2013). Atherosclerosis damages retinal vessel walls by promoting the possibility of thrombosis and emboli leading to retinal vessel occlusion. Embolism originating from an atherosclerotic plaque in the carotid artery is the most common cause of central retinal artery occlusion (CRAO) (Varma et al., 2013). Arteries affect also occlusions in the veins, as the RVO occurs mainly at the arteriovenous crossing (Ehlers and Fekrat, 2011). Arteriosclerosis leads to mechanical compression of the vein and venous narrowing both of which damage the endothelium and elevate the risk of subsequent thrombus formation in the affected vein. Retinal vein occlusion results in dilated and tortuous veins, intraretinal hemorrhage, retinal edema following retinal vascular leakage and sometimes cotton-wool spots in the portion of the retina drained by the involved vein (Ehlers and Fekrat, 2011). Macular edema, vitreous hemorrhage and neovascularization are associated with vision loss. Retinal or optic disk neovascularization can develop in eyes with non-perfusion and lead to vitreous hemorrhage and/or tractional retinal detachment. The probability of neovascularization is greater in eyes with larger areas of retinal non-perfusion and the degree of retinal ischemia. Larger areas of non-perfusion and the extent of retinal ischemia are also associated with poor visual acuity, with BRVO having a more favorable prognosis than CRVO % of eyes with RVO have reported to develop retinal or optic disc neovascularization (Akiba et al., 1991; Hayreh and Zimmerman, 2012; Tsui et al., 2011), whereas the incidence of optic disc neovascularization following central and branch retinal artery occlusion is much less common (Mason et al., 2015). The third most common type of retinal disease with neovascularization is ROP, a vascular disease affecting immature retinas of preterm neonates. Premature birth leads first to arrest of normal retinal vascular growth as a result of loss of the maternal fetal interaction and hyperoxia compared with the intrauterine environment. Later as the retina matures and metabolic demand increases, the retina becomes hypoxic. This leads to retinal neovascularization and because the new vessels poorly perfuse the retina and are leaky, this in turn results in fibrous scar formation and retinal detachment. (Hellström et al., 2013) Severe ROP often leads to long-term visual loss and may even result in blindness (Cryotherapy for Retinopathy of Prematurity Cooperative Group, 1996; Repka et al., 2011). Most preterm infants have a milder stage of the disease (Zin and Gole, 2013), which regresses without treatment and the retina vascularises rather normally (Ju et al., 2013). Nevertheless, former preterm subjects even with mild ROP have been reported to have abnormal photoreceptor rod function (Hansen et al., 2015, 2010) and ROP with retinal detachment is associated with severe vision impairment (Repka et al., 2011). In Finland, 10 % of very

34 10 preterm infants develop ROP (Rautava et al., 2013). Globally, 10 % of preterm babies who had any stage of ROP became blind or sever visually impaired (Blencowe et al., 2013) Subretinal vascular diseases In subretinal vascular diseases, new vessels grow beneath the retina in the subretinal space (Campochiaro, 2013). There are two types of subretinal neovascularization depending on the origin of the neovessels. The majority of subretinal vascular diseases are considered as CNV diseases as neovascularization sprouts from choroidal vessels through Bruch s membrane and RPE into the subretinal space. Less common type of subretinal neovascularization is retinal angiomatous proliferation (RAP) which originates from the deep capillary bed of the retina and extends through the photoreceptor layer to beneath retina. Both types of subretinal neovascularization exist in AMD patients and they have similar consequences with regard to vision loss. The most common cause of CNV is AMD although there are also rare cases of non-amd related CNV (Heier et al., 2011). AMD is considered the leading cause of vision loss in the developed countries (WHO, 2018) although the latest studies have shown a decrease in the percentage of patients blind or with either impaired vision due AMD (Flaxman et al., 2017). Nevertheless, the number of AMD patients has increased rapidly in the low-income countries (Wong et al., 2014). As the name replies, the prevalence of AMD increases with age, leading to emerging number of all AMD patients in the future up to estimated 288 million in the year RAP constitutes 10 % of cases with neovascular AMD (Daniel et al., 2016). AMD is a highly complex disease with demographic, environmental, and genetic risk factors. The disease affects primarily the photoreceptors, RPE, Bruch s membrane, and choriocapillaris. The pathogenesis of AMD is still poorly understood, but the incidence of the disease increases dramatically with age possibly due to the gradual, cumulative damages to the retina from daily oxidative stress and the accumulation of lipofuscin and other oxidative lipids, leading to loss of normal physiological functions in the RPE cells, often associated with drusen deposition. (Coleman et al., 2008; Ding et al., 2009; Kauppinen et al., 2016) Drusen formation in Bruch s membrane is the first clinical finding of AMD (Coleman et al., 2008). In addition, thickening and abnormal architecture within Bruch s membrane and lipofuscin accumulation in the RPE are early signs of AMD (Ding et al., 2009). The drusen found in AMD patients are most frequently found as clusters within the macular region. Hard drusen, that are relatively common in elderly patients even without AMD or its risk factors, appear clinically as small and yellowish. Soft drusen are more diffuse, paler and larger with blurry edges. They are characteristics of early AMD and a significant risk factor for the development of late stage AMD. The composition of drusen varies between aged but AMD-free and AMD samples (Crabb et al., 2002). Intermediate AMD is characterized by at least one large druse, numerous medium size drusen, or geographic atrophy that does not extend to the center of the macula (Coleman et al., 2008). In the early stages of the disease, the visual loss is generally mild and usually asymptomatic. Possible symptoms are blurred vision, visual scotomas, decreased contrast sensitivity and abnormal dark adaptation. When AMD develops further, it is is subdivided into two forms - dry i.e. atrophic or wet i.e. neovascular (Ding et al., 2009). The two forms of AMD in the late stage of the disease are today equally common (Robman et al., 2015; Rudnicka et al., 2015, 2012) and the number of patients with vision impairment are the same for the two forms of the disease (Colijn et al., 2017). The main clinical characteristic of late stage dry AMD is the appearance of RPE atrophy, usually known as geographic atrophy (Ding et al., 2009). Geographic atrophy is characterized by a sharply demarcated oval area of hypopigmentation and is usually a consequence of RPE cell loss. The loss of RPE cells leads to the gradual degeneration of nearby photoreceptors and may extend to the outer retinal layers, including the outer plexiform and inner nuclear layers. The loss of retinal layers results in thinning of the retina and a gradual visual impairment with central or pericentral visual scotomas (Coleman et al., 2008).

35 11 The other form is wet, neovascular AMD. Although it is mostly characterized by CNV, also RPE detachment, subretinal neovascular fibrous tissue and hard exudates in the macular area are described as findings of AMD (Weinreb et al., 2000). The neovascularization in AMD has two etiologic patterns (Ding, Patel et al., 2009). In the majority cases of AMD, new vessels sprout from the choriocapillaris, penetrate through Bruch s membrane and grow into the subretinal pigment epithelium, the subretinal space or both. The other etiologic pattern of neovascularization is much less frequent than classical AMD (Ding, Patel et al., 2009). This pattern occurs in RAP where new vessels are derived mainly from the retinal circulation instead of choroidal vessels (Yannuzzi et al., 2001). In RAP, neovascularization develops extending outward into the subretinal space, sometimes anastomosing with the choroidderived vessels. CNV is a serious vision-limiting complication in which leaky, abnormal, tortuous vessels and edema disrupt the precise alignment of the photoreceptor cells and the RPE (Miller et al., 2013). Conversely to the insidious vision loss in dry AMD, wet AMD may lead to sudden visual loss within days or weeks resulting from this subretinal hemorrhage or fluid accumulation secondary to CNV (Coleman et al., 2008). CNV may also lead to detachment of the RPE from the choroid which often contributes to the loss of central vision (Zayit-Soudry et al., 2007). When AMD extends, CNV develops as well. Fibrosis occurs as an excessive wound healing response to tissue damage (Wynn, 2007) and when it develops after subretinal hemorrhages, it may lead to permanent scarring (Miller et al., 2013). Furthermore, the subretinal fibrosis formation may lead to local destruction of photoreceptors, RPE, and choroidal vessels leading to permanent dysfunction of the macular visual system (Ishikawa et al., 2016a). In addition, atrophy has been shown to be associated with fibrotic scarring in some patients with previous CNV (Daniel et al., 2014). Atrophy may develop into areas previously occupied by CNV even without the step of subretinal fibrosis (Channa et al., 2015) Connection of subretinal and retinal neovascular diseases The subretinal and retinal neovascular diseases share common features (Figure 4) and risk factors. The diseases are often linked together, as diabetes increases the incidence of other retinal neovascular diseases, like RVO (Klein et al., 2000; Sperduto et al., 1998) and retinal artery occlusion (RAO) (Hayreh et al., 2009). Although some studies have detected a positive correlation between ROP and high glucose levels in infants (Garg et al., 2003), hyperglycemia can not definitely be considered as a risk factor (Au et al., 2015). Although diabetes does not increase the risk of AMD (Chakravarthy et al., 2010; Klein et al., 2004), retinal and subretinal neovascular diseases share common risk factors such as high age, hypertension and smoking (Chakravarthy et al., 2010; Klein et al., 2000, 1998; Varma et al., 2013). Macular edema is a common feature in diabetes but it occurs also in humans during the course of numerous other retinal and subretinal disorders like branch and central retinal vein occlusion and CNV. Macular edema consists of intra- or subretinal fluid accumulation in the macular region resulting from an imbalance between processes governing fluid entry and exit. Independently of its etiology, macular edema may lead to permanent structural damage and severe impairment of central vision as the tissue transparency and light transmission are disturbed. (Daruich et al., 2018) Retinal vascular leakage from loss of function of the BRB and subsequent macular edema are the main causes of visual loss and blindness in these diseases (Klaassen et al., 2013). BRB breakdown or increased capillary hydrostatic pressure leads to increased osmotic pressure of the interstitial compartment due to leakage of plasma solutes. This may result in edema and if the swelling occurs in the macular region, it causes severe loss of central vision (Caldwell et al., 2003). Otherwise, it is asymptomatic.

36 12 Figure 4. Molecular pathogenesis of retinal and choroidal neovascularization. The type of pathogenesis found only in age-related macular degeneration (AMD) are shown in green. BRB: Blood-retinal-barrier, DR: Diabetic retinopathy, ROP: Retinopathy of prematurity, RAO: Retinal artery occlusion, RPE: Retinal pigment epithelium, RVO: Retinal vein occlusion, VEGF: Vascular endothelial growth Pre-clinical animal models of ocular vascular diseases There are numerous pre-clinical models of choroidal and retinal neovascularization (Grossniklaus et al., 2010) out of which the most common ones are summarized in Table 1. Animal models mimick many features of clinical neovascularization but the progression and lesion appearance vary among the models and between species. Mouse models are popular animal models to recapitulate neovascularization but the major limitation in mice is the lack of a macula or a resembling specialized retinal region or vascular pattern (Huber et al., 2010). As ischemia is the main cause of retinal neovascularization, the animal models inducing ischemia include laser and hyperoxia (Grossniklaus et al., 2010). Hyperoxia is exploited in oxygen-induced retinopathy (OIR), which is one of the most widely used mouse models for retinal neovascularization (Liu et al., 2017). For the induction of neovascularization, neonatal mice are exposed to hyperoxia that suppresses the development of the retinal vasculature and leads to regression of the existing immature retinal vessels (Smith et al., 1994). After returning to room air, the relative hypoxia triggers ischemia and hypoxia in the retinal tissue resulting in VEGF overexpression and abnormal neovascularization at the junction between vascularized and avascular areas. The model resembles human ROP. OIR models successfully and reliably reproduce the initial vaso-obliteration phase and a subsequent ischemia- and hypoxia-induced neovascularization phase (Liu et al., 2017), but the expression of pro- and antiangiogenic factors as well as retinal neovascularization vary between different mouse strains (Chan et al., 2005). The ischemic murine OIR model is not only used as an experimental model for ROP, since from the view of pathogenesis, it shares phenotypes with other retinal neovascular diseases, such as DR, retinal vein and artery occlusion (Mi et al., 2014). The OIR model is commonly considered a model for PDR, as the current rodent models of DR reproduce only the early non-proliferative stage of DR (Liu et al., 2017). However, the model lacks systemic diabetic characteristics, and the progression of vascular abnormalities in OIR models is fundamentally different from DR (Lai and Lo, 2013).

37 13 Most mouse models of DR are based on induced or spontaneous hyperglycemia and diabetes or they are models with definite retinal neovascularization. Pharmacologically induced hyperglycemia, such as streptozotocin (STZ) induced type I diabetic mouse model, does not fully simulate the DR disease process (Liu et al., 2017) and expresses only mild retinal neovascularization (Su et al., 2012). Also Akita mice, a mouse model of spontaneous hyperglycemia harbouring an insulin 2 gene mutation, have mostly been reported expressing none or only the early signs of DR (Barber et al., 2005; Chaurasia et al., 2018; Rakoczy et al., 2010; Wisniewska-Kruk et al., 2014) and only rarely microaneurysm, vascular leakage and new vessel formation in OPL (Han et al., 2013). Nevertheless, crossbreeding Akita mice with another genetically modied mouse strain, Kimba mice, leads to PDR phenotype (Rakoczy et al., 2010). Kimba mice are a non-diabetic model of definite retinal neovascularization resulting from the expression of human VEGF (Lai et al., 2005). The model expresses retinal neovascularization but also other retinal vascular changes common to PDR (Rahman et al., 2011; Rakoczy et al., 2010; Tee et al., 2008). Crossbreeding of Akita and Kimba mice has been used to generate the Akimba mouse model combining VEGF expression and hyperglycemia (Rakoczy et al., 2010). In addition to neovascularization, Akimba mice display numerous other changes such as hemorrhage, edema and retinal detachment beginning already in a few weeks of age lasting several months (Chaurasia et al., 2018; Shen et al., 2006). In common with Kimba mice, there is another transgenic (tg) mouse model expressing human VEGF in the retina leading to spontaneous retinal neovascularization (Okamoto et al., 1997; Tobe et al., 1998). Neovascularization in these VEGF-transgenic mice resembles more RAP-like lesions. The model is useful for investigating early VEGF-induced changes in the retina rather than advanced neovascularization (Liu et al., 2017). Another transgenic mouse model of RAP is a mouse with targeted mutations in the very low density lipoprotein receptor (VLDL) gene (Heckenlively et al., 2003; Hu et al., 2008; Li et al., 2007). At an early age, the mice develop at early age neovascular growth originating from the retinal vasculature, which extends into the subretinal space. Nonetheless, there is no transgenic animal model spontaneously expressing retinal artery or vein occlusion. Many of the animal models for retinal vein and artery occlusion have been investigated in larger species than mice (Khayat et al., 2017; Minhas et al., 2012). Induced mouse models of RVA and RVO do not commonly mimick neovascularization but instead exhibit cell death (Ebneter et al., 2015; Gaydar et al., 2011; Martin et al., 2018), VEGF expression (Ebneter et al., 2015; Fuma et al., 2017; Goldenberg-Cohen et al., 2008; Martin et al., 2018) and non-perfusion areas (Ebneter et al., 2015; Martin et al., 2018) which are linked to retinal vessel occlusion. Some mouse models modelling occlusions have shown neovascularization (Uddin et al., 2017; Zhang et al., 2007), hemorrahage (Fuma et al., 2017) and collateral vessel formation (Ebneter et al., 2015). In rodents, retinal branch and central vein and artery occlusions are most commonly induced by laser photocoagulation with a photosensitizer (Khayat et al., 2017; Minhas et al., 2012) as is done also with the mouse models expressing retinal neovascularization (Uddin et al., 2017; Zhang et al., 2007). Rose Bengal dye is injected intravenously and subsequently its toxic effect is potentiated with a laser photocoagulator. The model is not optimal as the laser induces inflammation and destroys the outer retinal layer and photoreceptors (Fuma et al., 2017). Retinal ischemia and vessel occlusion can be induced also by ligating the ophthalmic retinal artery and vein (Joly et al., 2014) or carotid artery (Steele et al., 2008). There are also many models mimicking CNV, but the existing animal models attempting to simulate AMD do not fully replicate the complex clinical, histological and angiographic features of the human disease. Generation of animal models utilizes the key features of CNV, such as damage in Bruch s membrane, VEGF expression or inflammatory cytokines (Grossniklaus et al., 2010). The most common ways to develop animal models of CNV are laser or surgical induction or the use of transgenic mice. CNV models based on overexpression of VEGF are created either by administration of VEGF into animals or by

38 14 using transgenic mice that express VEGF. Nevertheless, the models studied to date indicate that VEGF overexpression alone is insufficient for evoking the development of CNV (Grossniklaus et al., 2010). One of the models in which CNV has been successfully produced is the model of AAV.shRNA.sFLT-1 in which a short hairpin ribonucleic acid (shrna) targets the VEGF receptor-1 (Luo et al., 2014, 2013). The other common types of animal models of CNV are based on laser or mechanically induced breaks in Bruch s membrane (Grossniklaus et al., 2010; Pennesi et al., 2012). The laser photocoagulation with laser spots is the standard method for obtaining CNV as it is relatively rapid to develop, cost-effective and relatively simple (Liu et al., 2017). The development of CNV has been reported after laser induction in C57BL/6J mice (Giani et al., 2011; Kwak et al., 2000; Lambert et al., 2013) as well as in transgenic mice (Combadière et al., 2007; Doyle et al., 2012). The limitation of laser-induced models is that the CNV disappears approximately 1 month after lasering and it does not recapitulate the aging aspect of AMD (Edelman and Castro, 2000; Giani et al., 2011; Ishikawa et al., 2016a). As the retina is partially burned in laser-induced CNV, due to significant damages to the overlying neural retina, anatomic discrepancies exist to a greater degree than in the typical human AMD (Pennesi et al., 2012). Besides laser, a break in Bruch s membrane can also be induced surgically. Subretinal injection of Matrigel has been shown to induce CNV in mice (Li et al., 2011) although it is more often applied in rats (Cao et al., 2010; Shen et al., 1998). Chemotactic cytokine and their receptor knockout (KO) mice have impaired leukocyte migration which leads to an AMD phenotype (Pennesi et al., 2012; Zeiss, 2010). The mice express thickening of Bruch s membrane, subretinal microglia and autofluorescence and lipofuscin granules (Ambati et al., 2003; Combadière et al., 2007; Tuo et al., 2007). CNV has been reported existing spontaneously in around 20 % of the mice (Ambati et al., 2003; Tuo et al., 2007) or only after laser-injury (Combadière et al., 2007). Nevertheless, also contrary results with no ocular findings specific to AMD (Luhmann et al., 2013) or decreased CNV formation after lasering (Luhmann et al., 2009) have been reported. In addition to these knockout mice (Ambati et al., 2003; Combadière et al., 2007; Tuo et al., 2007), also other aged mouse models of AMD have drusen-like and basal laminar-like deposits but the presence of CNV in these models remains remarkably low (Imamura et al., 2006; Malek et al., 2005). In addition to neovascularization, also subretinal fibrosis is a key element of AMD. Although neovascular AMD develops into a cicatricial stage with disciform scar on humans (Bloch et al., 2013; Daniel et al., 2014; Kvanta et al., 1996; Lopez et al., 1996; Miere et al., 2015) as a response to wound healing, only a few mouse models with subretinal fibrosis are available (Ishikawa et al., 2016a). VLDL-mice with RAP-like lesions display also subretinal fibrosis (Hu et al., 2008) but the neovascularization in the model is not choroidal. Laserinduction alone may result in subretinal fibrosis formation (Zhang et al., 2016) but one of the most widely used models of subretinal fibrosis is a murine model utilizing laser photocoagulation and injection of macrophage-rich peritoneal exudate cells (PEC) into the subretinal space (Jo et al., 2011; Yang et al., 2013; Zhang et al., 2013). One major concern is the complexity of the model with multiple steps, making the reproducibility of the model very challenging. The ideal animal model for retinal and choroidal neovascularization would utilize a species that is easily genetically modified, fast and easy to reproduce, accessible and ethical. The model should mimick the course and time of progression of the disease as well as other clinical features. The animal model should be reproducible and surgically operable, and express neovascularization sustainably and with similar pattern as in humans.

39 15 Table 1. Most common experimental retinal and choroidal neovascular mouse models. Mouse model Induction method Disease Hyperoxia induced models OIR 75 % oxygen ROP, DR, occlusion Genetically modified models Akita insulin-2 gene mutation DR Kimba VEGF-A 165 expression under mouse rhodopsin promoter DR Akimba crossbreed of Akita and Kimba DR VEGF-tg VEGF-A 165 expression under bovine rhodopsin promoter RAP chemokine KO chemotactic cytokine or their receptor knockout AMD VLDL very low-density lipoprotein mutation RAP Surgically induced models Matrigel subretinal injection of extracellular matrix proteins AMD AAV.shRNA.sFLT-1 short hairpin RNA induced inhibition of VEGFR-1 AMD STZ induction streptozotocin injection induced pancreatic ß-cell loss DR ligature ligation of artery or vein occlusion PEC inoculation laser + subretinal injection of macrophage-rich peritoneal exudate cells Laser induced models AMD photoactivation dye + light illumination occlusion photocoagulation laser burn AMD AAV: Adeno-associated virus, AMD: Age-related macular degeneration, DR: Diabetic retinopathy, KO: Knockout, OIR: Oxygen induced retinopathy, PEC: Peritoneal exudate cell, RAP: Retinal angiomatous proliferation, ROP: Retinopathy of prematurity, shrna: Short hairpin RNA, STZ: Streptozotocin, tg: Transgenic, VEGF: Vascular endothelial growth factor, VEGFR: Vascular endothelial growth factor receptor, VLDL: Very low-density lipoprotein 2.3 ANGIOGENESIS AND VASCULAR ENDOTHELIAL GROWTH FACTORS IN OCULAR VASCULARIZATION VEGFs are key regulators in physiological and pathological angiogenesis (Ferrara et al., 2003). Angiogenesis is the main mechanism of new vessel formation in adults (Carmeliet, 2000). In angiogenesis new capillaries are formed from the pre-existing vasculature under the control of angiogenic activators and inhibitors (Hanahan and Folkman, 1996; Ribatti, 2005). VEGF-A is the most important angiogenic activator and its overexpression is highly induced in pathological conditions, such as in tumorigenesis and ocular neovascularization (Ferrara et al., 2003). The majority of the studied and in use treatments for neovascular diseases of the eye target VEGF or its receptors (Campochiaro et al., 2016). VEGFs, their receptors and main functions are summarized in Figure Vascular endothelial growth factors VEGF gene family includes placental growth factor (PlGF) and four VEGFs: VEGF-A,-B, -C, and D. In addition to these endogenous VEGF members, viral VEGF homologs (VEGF-E) and snake venom VEGFs (VEGF-F) have been found. VEGF family members have different

40 16 properties in vessel development, angiogenesis, vascular homeostasis and lymphangiogenesis. (Ylä-Herttuala et al., 2007) VEGF-A is the strongest known inducer of vascular permeability (Senger et al., 1983). Later it was found to be also the key mediator of angiogenesis (Leung et al., 1989). VEGF-A is required for embryonic development and in adults it is expressed in all vascularized tissues (Ylä-Herttuala et al., 2007). Low levels of VEGF are needed for the maintenance of general vascular homeostasis and high levels in angiogenic processes in healthy and diseased tissues. Its expression is highly induced by low oxygen via hypoxia-inducible factor -1 (HIF-1) as a response to the metabolic needs of the tissue (Forsythe et al., 1996). The alternative exon splicing of human VEGF-A results mainly in four different isoforms (VEGF121, VEGF165, VEGF189 and VEGF206) with different numbers of amino acids after sequence cleavage (Houck et al., 1991; Tischer et al., 1991). Less frequent splice variants have been also found, such as VEGF145 (Poltorak et al., 1997). Similar to human VEGF, the alternative splicing of mouse VEGF generates isoforms VEGF120, VEGF164, VEGF188 but not the fourth VEGF isoform corresponding to human VEGF206 (Shima et al., 1996). VEGF164/165 is the most prominently expressed isoform in pathological angiogenesis (Nagy et al., 2007). The smallest isoform, VEGF121, is a diffusible protein whereas the longer isoforms, VEGF189 and VEGF206, have a high affinity for heparin and are bound to tissue (Park et al., 1993). Isoform VEGF165 is both soluble and matrix bound (Nagy et al., 2007). It binds to negatively charged matrices but not strongly to heparin. Also anti-angiogenic VEGF165b inhibiting VEGF165- mediated angiogenesis by naturally suppressing tumor growth (Bates et al., 2002) has been discovered (Woolard et al., 2004). VEGF-A being the predominant angiogenic growth factor, the roles of PlGF and VEGF-B are elusive. They are only weak mitogens for endothelial cells and they do not significantly induce acute vascular permeability. Both PlGF and VEGF-B may act by blocking VEGF-A from binding to VEGFR-1 thus making it more available for VEGFR-2 binding. (Ylä-Herttuala et al., 2007) VEGF-C and -D both bind to VEGFR-2 and -3, and are synthesized as long precursor forms having mainly lymphangiogenic activity. The long forms are proteolytically processed into mature forms (indicated by ΔNΔC) having a higher affinity towards VEGFR-2 and thus acting also as angiogenic factors. (Ylä-Herttuala et al., 2007) Vascular endothelial growth factor receptors The VEGF activities are mediated by three receptor tyrosine kinases: VEGF receptor (VEGFR) -1, -2 and -3. VEGFR-1 and -2 are mostly expressed in endothelial cells whereas VEGFR-3 is found in lymphatic endothelial cells. (Ferrara et al., 2003) VEGFR-1 (encoded in humans by FLT1 gene) exists as a decoy receptor and acts as a negative regulator of angiogenesis by preventing VEGF-A binding to VEGFR-2 (Ylä- Herttuala et al., 2007). However, VEGFR-1 activation by PlGF and VEGF-B stimulates vascular growth, although the mitogenic signal in endothelial cells is weak. VEGFR-1 may also form heterodimers with VEGFR-2 (Huang et al., 2001) binding VEGF-A and VEGF- A/PlGF heterodimers in vivo (Cudmore et al., 2012). VEGFR1 2 heterodimer activation is functional in endothelial cells and it negatively regulates both the signalling and cell response of the VEGFR-2 homodimer thus having a role in endothelial cell homeostasis (Cai et al., 2017; Cudmore et al., 2012). The soluble form of VEGFR-1 (svegfr) lacking transmembrane and intracellular parts acts as a decoy receptor negatively regulating angiogenesis by binding to VEGF-A (Kendall and Thomas, 1993) and PlGF (Kendall et al., 1994). In humans, VEGFR-2 is encoded by a gene called KDR or FLK-1 (Ferrara et al., 2003; Ylä- Herttuala et al., 2007). It is the main mediator of angiogenesis by binding to VEGF-A. It has also a role in endothelial cell proliferation and survival, vascular homeostasis and microvascular permeability. VEGFR-2 promotes angiogenesis also by binding mature short forms of VEGF-C and VEGF-D. VEGFR-2 exists also as in a soluble form (Ebos et al., 2004),

41 17 formed by alternative splicing preventing VEGF-C binding to VEGFR-3, thus inhibiting lymphangiogenesis (Ambati et al., 2009). VEGFR-3 (in humans Flt-4) is restricted to lymphatic endothelial cells in adults (Ferrara et al., 2003; Ylä-Herttuala et al., 2007). It mediates lymphangiogenic properties by binding long precursors of VEGF-C and VEGF-D. VEGFR-3 forms heterodimers with VEGFR-2 by VEGF- A, -C and D induced (Alam et al., 2004; Dixelius et al., 2003). VEGFR2-3 heterodimers are involved in the proliferation and migration of lymphatic endothelial cells (Nilsson et al. 2010). Although VEGF-A does not bind VEGFR-3, it may positively mediate angiogenic sprouting through VEGFR2-3 heterodimer formation. Soluble forms of VEGFR-3 have also been detected (Kanefendt et al., 2012; Mouawad et al., 2009). In addition to VEGFRs, some VEGFs interact with a family of co-receptors, the neuropilins (Ferrara et al., 2003). Neuropilins 1 and 2 enhance the ligand-binding affinity of VEGFs to VEGF-receptors thus modulating the biological activities of different VEGFs (Favier et al., 2006; Soker et al., 1998). Figure 5. Vascular endothelial growth factors (VEGF), VEGF-receptors (VEGFR) and their main functions. VEGF binding to the VEGF-receptor leads to formation of receptor homodimers and heterodimers. VEGF-A, VEGF-B, PlGF, VEGF-E, VEGF-F and proteolytically processed mature forms of VEGF-C and VEGF-D (ΔNΔC) are angiogenic while long forms of VEGF-C and VEGF-D are lymphangiogenic. In addition, VEGF-A binding to VEGFR-1 and VEGFR-1-2 heterodimer also prevent activation of VEGFR-2. Neuropilins and soluble VEGFRs are not shown in the figure. Style adapted from the original figure of (Olsson et al., 2006) VEGF in healthy and diseased eye Already in 1948, the presence of a biochemical factor responsible for the growth of capillary blood vessels in fetal development and pathological ocular conditions was postulated (Michaelson, 1948). Aiello et al. were the first to reveal the relationship between VEGF and ocular neovascularization in the human eye (Aiello et al., 1994). Since then VEGF has been implicated in a large number of neovascular ocular diseases. In angiogenesis of the eye, VEGF-A is the most widely studied member of the VEGF family (Witmer et al., 2003). In the healthy human eye, VEGF-A protein levels increase in vitreous

42 18 humor with advancing gestation (Ma et al., 2015) but in adults, its concentration in the eye is low (Cheung et al., 2012; Duh et al., 2004; Funatsu et al., 2003). The studies of VEGF localization in the human retina have reported variable results (Amin et al., 1997; Boulton et al., 1998; Gerhardinger et al., 1998; Kliffen et al., 1997; Matsuoka et al., 2004; Pe er et al., 1998). Nevertheless, VEGF expression in healthy retina is considered low (Boulton et al., 1998; Lopez et al., 1996; Mathews et al., 1997), whereas the increased expression of VEGF-A (Aiello et al., 1994) and soluble receptors (Matsunaga et al., 2008; Motohashi et al., 2018; Noma et al., 2015) are highly associated with various neovascular diseases. Ischemia and a worse status of the disease are correlated with higher VEGF levels (Motohashi et al., 2018; Noma et al., 2015, 2011a, 2011b). Localization of VEGF also in mouse eye has varied between studies, and it has been found in INL, RPE and GCL (Marneros et al., 2005; Pierce et al., 1995; Saint-Geniez et al., 2008, 2006). In AMD patients, VEGF expression is often seen in blood vessels, RPE cells and CNV membranes and fibrovascular membranes (Kliffen et al., 1997; Kvanta et al., 1996; Lopez et al., 1996; Matsuoka et al., 2004). In human eyes with retinal neovascularization, VEGF expression is often seen in blood vessels, GCL as well as in the inner and outer nuclear layers (Boulton et al., 1998; Mathews et al., 1997; Pe er et al., 1998, 1996). Elevated VEGF concentrations in the aqueous and vitreous humor have been detected in patients of ROP (Aiello et al., 1994; Sato et al., 2009), DR (Aiello et al., 1994; Funatsu et al., 2003; Matsunaga et al., 2008; Wells et al., 1996) and RVO (Aiello et al., 1994; Noma et al., 2015, 2006; Yoshimura et al., 2009). In addition to retinal neovascularization, also choroidal vascular diseases are associated with increased levels of intraocular VEGF. CNV of AMD is reported leading to elevated levels of VEGF in the vitreous (Funk et al., 2009; Lip et al., 2001; Motohashi et al., 2018; Tong et al., 2006). In AMD patients, VEGF protein expression is found not only in the vitreous but also in CNV membranes and fibrovascular membranes (Kvanta et al., 1996; Lopez et al., 1996). In mice, VEGFR-2 is detected a few days postnatally and during adulthood throughout the RPE (Ford et al., 2011). In the normal human eye, all three VEGFRs are localised on choriocapillaris, VEGFR-1 on the inner choriocapillaris and other choroidal vessels and VEGFR-2 and -3 on the side of the choriocapillary endothelium facing the RPE cell layer, suggesting a paracrine relation between RPE cells and the choriocapillaris (Blaauwgeers et al., 1999). Although all VEGFRs can be found in the normal human eye, the expression of receptors 2 and 3 are mostly absent or at least sporadic in healthy eyes, whereas VEGFR-1 is in all CD31-positive microvascular structures (Blaauwgeers et al., 1999; Witmer et al., 2002). VEGFR-1 was also expressed outside the vasculature in the choroidal vessels and in the retina in ILM, GCL, IPL, INL and OPL (Witmer et al., 2002). Variable weak granular staining for VEGFR-2 has been observed as well as outside the retinal vasculature in neural and glial elements of the GCL, IPL, INL, OPL, ONL and occasionally in the outer limiting membrane. VEGFR-3 expression outside the vasculature is found in neural elements of the IPL, INL and the inner part of the OPL. The expression of all VEGFRs is increased during pathological ocular angiogenesis (Witmer et al., 2002). In addition, elevated levels of svegfr-1 and -2 have been found in vitreous or aqueous humor of patients with CRVO (Noma et al., 2015, 2011b), BRVO (Noma et al., 2011a; Noma and Mimura, 2013), PDR (Matsunaga et al., 2008) and AMD (Motohashi et al., 2018). An increase in svegfrs have been suggested being secondary to upregulation of transmembrane VEGFRs (Motohashi et al., 2018; Noma et al., 2015). VEGF-A has been shown to correlate positively with svegfr-1 (Matsunaga et al., 2008; Noma et al., 2015), but not with svegfr-2 (Noma et al., 2015, 2011a, 2011b). All svegfrs have been found in the mouse cornea where they have an essential role in keeping the cornea transparent (Ambati et al., 2006, 2009; Mamalis et al., 2013). Retinal hypoxia is the key mediator of retinal neovascular diseases in both animals and humans, resulting in elevated levels of HIF-1 which stimulates expression of VEGF-A and its receptors (Campochiaro, 2013). Hypoxia has been suggested playing a role also in subretinal

43 19 choroidal diseases (Blasiak et al., 2014) although there are substantial differences in the pathogenesis of retinal and subretinal neovascularization (Campochiaro, 2013). Vascular endothelial cell damage in retinal capillaries caused by occlusion or chronic hyperglycemia leads to cell degeneration (Crawford et al., 2009; Ehlers and Fekrat, 2011). Hypoxia and ischemia as a result of the initial microvascular degeneration lead to increased production of VEGF-A in retinal tissues and result in the proliferative phase with neovascularization. Hypoxia may play a role in the development of CNV as well (Witmer et al., 2003). It has been suggested that diffusion of oxygen from the choroid to RPE and retina is decreased due to thickening of Bruch s membrane and the accumulation of lipids. Localized inflammation and death of the RPE causes atrophy leading to dry AMD, while loss of choriocapillaris stimulates RPE ischemia, leading to VEGF production from hypoxic RPE cells and the formation of neovascular AMD (Blasiak et al., 2014; Coleman et al., 2008). Whereas hypoxia plays an important role in the development of occlusions, DR and AMD, hyperoxia is a mediator of ROP (Witmer et al., 2003). Opposite to hypoxia, hyperoxia leads to suppression of oxygen-regulated angiogenic growth factors, particularly VEGF, which in turn causes both obliteration of retinal vessel growth and loss of some existing retinal vessels (Hellström et al., 2013). The poorly vascularized retina becomes hypoxic, resulting in VEGF- A upregulation to even higher levels than in a normally developing retina and stimulation of retinal neovascularization (Witmer et al., 2003). In addition to promoting angiogenesis and neovascularization, VEGF also increases vascular permeability in the retina and causes a breakdown of the BRB in DR, AMD, retinal vein occlusions and ROP (Klaassen et al., 2013; Tarr et al., 2013). In contrast to VEGF-A and VEGFRs, VEGF-B does not seem to play an important role in retinal neovascularization, because mice deficient in VEGF-B have a normal retinal vascular development and no differences in hypoxia-induced retinal neovascularization compared with the wild type mice (Reichelt et al., 2003). In addition, the roles of VEGF-C and D in the retina and neovascularization are less extensively studied. Nevertheless, the expression of VEGF-C and D in the RPE of AMD patients has been discovered (Ikeda et al., 2006). In mice increased expression of VEGF-C and VEGFR-3 is seen around laser induced CNV (Lashkari et al., 2014). They may play a role in ocular angiogenesis instead of lymphangiogenesis, as the presence of a lymphatic-like structure in RPE and choroid in humans and mice remain unclear (Grimaldo et al., 2010; Koina et al., 2015; Nakao et al., 2013) Anti-angiogenic treatments for ocular neovascularization The use of anti-vegf therapies in vascular ocular diseases has had a fundamental impact on improving visual acuity and morphological outcomes (Kim and D Amore, 2012). Intravitreal injection of anti-vegf is the standard treatment for AMD (Kostea silmänpohjan ikärappeuma (AMD): Käypä hoito -suositus, 2016; Solomon et al., 2014) and DME (Diabeettinen retinopatia: Käypä hoito -suositus, 2014; Virgili et al., 2017). In the treatment of central and branch retinal vein occlusion, anti-vegfs have been used additionally to intravitreal steroid and/or laser therapy (Lip et al., 2018) as they are successful in treating macular edema secondary to CRVO and BRVO (Braithwaite et al., 2014; Mitry et al., 2013). In ROP, the safety and efficacy of anti-vegfs in preterm infants remain unclear (Sankar et al., 2018). Intravitreal anti-vegf therapy has been as effective as laser photocoagulation but the fact that the drug can be detected in the serum of infants after treatment has raised questions about its safety (VanderVeen et al., 2017). Currently there are three approved anti-vegf drugs for intraocular vascular diseases. The first one approved in 2004 was pegaptanib (Macugen ), a pegylated modified oligonucleotide, RNA aptamer, which binds with high specificity and affinity to VEGF-A165 inhibiting its activity. It is on the market for neovascular AMD in USA and in some European countries, but no longer in Finland. (Campochiaro et al., 2016; Ng et al., 2006) There are two other intraocular drugs for neovascular AMD, CNV, DME and macular edema secondary to RVO. Aflibercept (Eylea ), also known as VEGF-Trap, is a recombinant

44 20 fusion protein consisting of portions of human VEGFR- 1 and -2 extracellular domains fused to the Fc portion of human IgG1. It is a decoy receptor for all VEGF-A isoforms but also to VEGF-B and PlGF, inhibiting the activation of cognate VEGF receptors. Ranibizumab (Lucentis ) is a humanized recombinant monoclonal antigen binding fragment (Fab) targeted against all human VEGF-A isoforms thus preventing their binding to VEGF receptors 1 and 2. (Campochiaro et al., 2016; Papadopoulos et al., 2012) In addition, cancer-drug bevacizumab (Avastin ) is widely used off-label for the same indications as intravitreal anti-vegf drugs. It is a full-length anti-vegf antibody binding all VEGF-A isoforms (Ferrara et al., 2004) that was studied for intravenous treatment of AMD (Michels et al., 2005; Moshfeghi et al., 2006) and other rare types of CNV (Nguyen et al., 2008). Even though systemic delivery was shown to improve visual acuity and decline retinal thickness without major ocular or systemic adverse events (Michels et al., 2005; Moshfeghi et al., 2006; Nguyen et al., 2008), the clinical studies were discontinued. The use of ranibizumab instead of bevacizumab has been justified for its lack of complement-mediated or cell-dependent cytotoxicity and better choroidal penetrating properties (Ferrara et al., 2006). A single dose of ranibizumab is 40 times more expensive than a single dose of bevacizumab (The CATT Research Group, 2011). The high price of ranibizumab has raised resistance (Sipilä et al., 2018; Ziemssen et al., 2007) as bevacizumab is engineered from the same precursor as ranibizumab binding similarly to all VEGF-A isoforms (Ferrara et al., 2006; Muller et al., 1998). The efficiency of intravitreous bevacizumab in the treatment of AMD has been demonstrated in many clinical studies, making the use reasonable and cost-effective (Ba et al., 2015; Comparison of Age-related Macular Degeneration Treatments Trials (CATT) Research Group et al., 2016). In Finland, the Council of Choices for Health Care in Finland has approved off-label use of bevacizumab for the treatment of AMD (Tuuminen et al., 2017), and it accounts for almost all administered anti- VEGF injections (Kataja et al., 2018). The anti-vegf drugs for intraocular neovascularization and their targets are presented in Figure 6. Figure 6. Anti-angiogenic drugs for intraocular neovascularization. Pegaptanib (Macugen ) is a pegylated RNA aptamer binding to VEGF-A165. Aflibercept (Eylea ) is a recombinant fusion protein consisting of portions of human VEGF receptor 1 and 2 extracellular domains fused to the Fc portion of human IgG1 binding to all VEGF-A isoforms, VEGF-B and placental growth factor (PlGF). Bevacizumab (Avastin ) is a full-length antibody that binds all VEGF-A isoforms. Ranibizumab (Lucentis ) is a humanized recombinant monoclonal antigen binding fragment (Fab) of bevacizumab targeting all human VEGF-A isoforms. (Campochiaro et al., 2016) VEGF: Vascular endothelial growth factor

45 21 Systemic reviews have not observed any differences in safety between the anti-vegfs (Campbell et al., 2012; The CATT Research Group, 2011; van der Reis et al., 2011). Despite the BRB, intraocularly administered anti-vegf agents have been found in the systemic circulation (Avery et al., 2014). Aflibercept and bevacizumab were rapidly detected in the bloodstream after intravitreal administration, as neonatal Fc receptor in BRB facilitates the transport of Fc-containig full antibodies from the vitreous into the retinal bloodstream (Kim et al., 2009; Powner et al., 2014). The FcRn receptor is expressed in multiple ocular tissues and in endothelial cells in the retinal and choroidal vasculature in rodent and human eye. Interestingly neonatal Fc receptors are upregulated in the laser-photocoagulated rat retina (Kim et al., 2009). Thus, the elimination in to the bloodstream could be faster in eyes with CNV. Pegaptanib has not been found in the bloodstream after administration of a clinically used dose (Macugen AMD Study Group, 2007) but aflibercept, bevacizumab and ranibizumab have (Avery et al., 2014). Pegaptanib does not either accumulate in blood (Macugen AMD Study Group, 2007), whereas aflibercept and bevacizumab have been shown also to accumulate to serum after multiple doses (Avery et al., 2014). In addition, the systemic exposure after aflibercept and bevacizumab administration is much higher than after ranibizumab administration. Aflibercept and bevacizumab, but not ranibizumab and pegaptanib, have been reported to reducing the levels of systemic VEGF one month after intravitreal injection (Waltl et al., 2018; Wang et al., 2014; Zehetner et al., 2015, 2013). Despite the intermediate serum concentrations of aflibercept, it has the greatest reduction in plasma VEGF levels (Avery et al., 2014). This is most likely because of the higher affinity of aflibercept to VEGF compared with bevacizumab (Papadopoulos et al., 2012). Pegaptanib is given every six weeks until the end of the treatment (European Medicines Agency, 2019). The recommended administration interval for other intravitreal anti-vegf injections is every four weeks for three months after which the treatment is evaluated and can be continued similarly or given an as-needs basis (European Medicines Agency, 2019; Tuuminen et al., 2017), although in AMD and macular edema secondary to RVO it can be less effective than with monthly injections (Schmucker et al., 2015; Scott et al., 2018; The CATT Research Group, 2011). Treatment is continued until maximum visual acuity is achieved and/or there are no changes in visual acuity or other signs and symptoms of the disease under continued treatment. Since especially neovascular AMD and DME frequently affect both eyes (Gangnon et al., 2015; Varma et al., 2015), bilateral treatment is needed (Giocanti-Auregan et al., 2016). Unfortunately, the gained improvement in visual acuity is not usually maintained after finishing the treatment regimen (Comparison of Age-related Macular Degeneration Treatments Trials (CATT) Research Group et al., 2016). 2.4 GENE THERAPY OF THE EYE Approved gene therapy products prove that intraocular neovascularization is a potential gene therapy target (Ginn et al., 2018). Subretinal and intravitreal injections are efficient delivery routes for gene therapy in the eye, but the choice of the vector greatly affects the cells transduced and the duration of the transgene expression (Solinís et al., 2015) Ocular gene therapy Gene therapy can be defined as the transfer of nucleic acids to somatic cells with a resulting therapeutic effect (Ylä-Herttuala and Alitalo, 2003). Intracellular delivery of genetic material results in the production of a therapeutic protein or RNA to either block a harmful or dysfunctional gene or to deliver an advantageous or functional gene or RNA (Solinís et al., 2015). In order to produce the therapeutic protein, gene delivery vehicle or naked deoxyribonucleic acid (DNA) need to enter the cell, release the DNA and target the nucleus (Kootstra and Verma, 2003).

46 22 The eye possesses unique anatomical features that make it an excellent target for gene therapy (Solinís et al., 2015). In pre-clinical studies, the eye is a favorable target, as one eye may be used as an experimental target and the contralateral eye as a control. Considering also the clinical gene therapy, the eye has several advantageous characteristics. Due to the cellular and physical barriers of the eye, it is a relatively immune privileged site, which limits the risk of systemic exposure (Bennett 2003). Nevertheless, there have been an indication of minor immune responses after intraocular gene transfer with various viral vectors in the eye (Auricchio et al., 2001; Haeseleer et al., 2001; Isenmann et al., 2001; Maguire and Bennett, 1997) or in the systemic circulation (Anand et al., 2000; Dudus et al., 1999). As target tissue, the retina is small in size, leading to small amounts of needed therapeutic agent (Petit et al., 2016). An eye is anatomically restricted and divided into compartments, which is ideal for the precise and targeted gene transfer. The transparency of the eye and easy accessibility to different ocular tissues enable accurate application of the viral vectors to specific target sites. With respect to gene therapy of the eye, there are several ocular administration routes with different target cells (Solinís et al., 2015). These are presented in Figure 7. Although intravenous (Zhu et al., 2002) and topical administration (Alqawlaq et al., 2014; Tong et al., 2007) have been explored in gene therapy with non-viral vectors, for potent transduction efficacy intraocular delivery route is used. Intracameral and subconjunctival injections can be used to target cells in the anterior chamber (Oliveira et al., 2017) but intravitreal and subretinal injections are the most common delivery routes to target the posterior part of the eye (Ochakovski et al., 2017). Clinical trials targeting pathological neovascularization use as often the intravitreal as the subretinal delivery route (ClinicalTrials.gov, 2018). Figure 7. Intraocular delivery routes. As genes cannot enter the posterior part of the eye through topical administration, gene transfer is done using intraocular injection. (Solinís et al., 2015) Subretinal injection is a precise way to target the posterior retinal layers and it transduces photoreceptors and RPE cells more efficiently than intravitreal injection (Auricchio et al., 2001; Igarashi et al., 2013; Li et al., 1994; Mori et al., 2002a; Sakamoto et al., 1998; Ueyama et al., 2014). Subretinal injection is technically challenging, but still a clinically viable delivery route (Oliveira et al., 2017). Nevertheless, retinal detachment is a routine feature as the injected volume induces separation of photoreceptors from the RPE layer, although the bleb resolves usually within a few days (Le Meur et al., 2018; MacLaren et al., 2014; Simunovic et al., 2017). In clinical studies, the procedure has been used successfully in gene therapy trials

47 23 and it leads very rarely to long-lasting side effects (Ghazi et al., 2016; Le Meur et al., 2018; MacLaren et al., 2014; Simunovic et al., 2017). The most common adverse event has been retinal thinning. In experiments conducted in mice, the subretinal injections are delivered by the transcorneal route passing through the pupil and lens, by the trans-scleral route through vitreous and by the trans-scleral route through the choroid and Bruch s membrane (Peng et al., 2017). For humans, the subretinal injection requires pars plana vitrectomy (Ochakovski et al., 2017). Vitreous is removed after detaching posterior hyaloid membrane surrounding vitreous body (Lebherz et al., 2008). Then a needle is guided into subretinal space and a small infusion of balanced salt solution is injected to form a bleb. For gene delivery, the same injection channel through neural retina is used to inject the therapeutic agent into the subretinal space. Compared with direct delivery into the subretinal space, the two-step approach offers better access to the correct plane as well as minimizes vector loss but also exposes to surgical complications. Worsening of cataract is a known late complication of pars plana vitrectomy in phakic patients (Feng and Adelman, 2014) and cataract is often an adverse effect in subretinal gene therapy following vitrectomy (Bennett et al., 2016; Constable et al., 2016). Intravitreal injections are directly applicable to patients and are routinely used to administer ophthalmic drugs. Compared with the other intraocular injections, the intravitreal approach may be more efficient route for therapeutic gene delivery as it can potentially expose the entire retina (Petit et al., 2016), although it mostly targets cells in the inner retina (Harvey et al., 2002; Hellström et al., 2009; Igarashi et al., 2013) and anterior chamber (Auricchio et al., 2001; Mori et al., 2002a; Ueyama et al., 2014). Compared with subretinal injection, administration to vitreous is relatively easy and high doses are possible (Solinís et al., 2015). As subretinal injections need to be performed in an operating theater, intravitreous injections are less invasive and less expensive (Ochakovski et al., 2017) Gene delivery vectors The success of gene therapy relies on the efficient delivery of the genetic material to cells, achieving an optimum term gene expression (Ylä-Herttuala and Alitalo, 2003). As only a small amount of naked plasmid DNA will be taken into cells, gene carriers are used to increase the transduction efficiency. Liposomes and polymer complexes improve plasmid delivery to the cytoplasm, although the amount of DNA entering the cytoplasm is low with non-viral vectors. Viral vectors have been widely used because of their superior efficiency, although their potential risks are associated mainly with immunogenicity. Also, potential mutagenesis has promoted the design of non-viral vectors (Solinís et al., 2015). Selection of the vector affects the duration of the transgene expression and cell types transduced in the eye. A variety of viral vectors have been tested for ocular gene therapy, with AAV being the most often used vector in ocular clinical trials (Auricchio et al., 2017) Adenoviral vectors Adenovirus is the most used gene delivery system in all clinical trials (20.5%) although there has been a decline in its use over the past years (Ginn et al., 2018). The main advantages of adenoviral vectors are their high transduction efficiency and insert capacity, ability to induce both dividing and non-dividing cells and easy production with high-titers (Solinís et al., 2015; Verma and Weitzman, 2005). As they are non-integrating vectors, the transduction is transient. Transient expression and high immune responses are limiting factors in ocular gene therapy (Maguire and Bennett, 1997; Reichel et al., 1998). The most common adenoviral vector serotype for therapeutic gene delivery is adenovirus serotype 5 (Ad5) (Solinís et al., 2015; Verma and Weitzman, 2005). Replication deficient AdV vectors are usually generated by deleting early transcription genes E1 and E3 to make vector replication deficient in most cell lines and to increase the cloning capacity (Verma and Weitzman, 2005). Nevertheless, E1-deleted vectors can be propagated in E1 complementing

48 24 cells such as human 293 cell line (Graham et al., 1977). Replication-deficient E1-deleted viruses still elicit cellular and humoral immune responses (Schagen et al., 2004). Additional viral transcription units, E2 and E4, have been deleted to reduce immunogenicity (Verma and Weitzman, 2005). This may need further complementing cell lines in the vector production. In addition, so-called gutless vectors have been created in which almost all viral sequences have been eliminated. These vectors require helper viruses for propagation, thus creating a problem in the purification of the helper-free virus. The immune response eliminates AdV vector transduced cells reducing transgene expression and diminishing the efficacy of vector re-administration. Rapid loss of transgene expression takes place after subretinal re-administration in immunocompetent mice but successful reinjection is possible in immunodeficient mice (Reichel et al., 1998). A rapid decline in transgene expression occurs also in the naïve contralateral eye after repeated intravitreous injection (Gehlbach et al., 2012). The T-cell mediated immune response plays a role in transgene expression in both intravitreal (Maguire and Bennett, 1997) and subretinal injection (Reichel et al., 1998), preventing re-dosing but also affecting the duration of transgene expression (Maguire and Bennett, 1997; Reichel et al., 1998). Nevertheless some studies have reported no difference in transgene expression in immunized and nonimmunized mice after subretinal injection (Bennett et al., 1996; Maguire and Bennett, 1997). Another point of concern especially in intravenous administration is raised by pre-existing immunity to the adenovirus by AdV-specific Abs (Schagen et al., 2004) as many humans have pre-existing immunity to Ad5 (Chirmule et al., 1999). The development of AdV-specific antibodies does not contribute to the elimination of AdV-transduced cells and hence does not affect the transgene expression. However, AdV-specific Abs will bind the AdV vector and thereby prevent cell entry and promote opsonization by macrophages. Consequently, AdV-specific Abs impair the efficacy of adenoviral vector mediated gene therapy (Schagen et al., 2004). The serotype of the adenovirus affects the transgene expression and adenovirus serotype 35 has been shown to result in more successful intraocular gene delivery compared with serotype 5 (Hamilton et al., 2008; Ueyama et al., 2014) Transgene expression after AdV intravitreal injection has been short-term (Hoffman, Maguire and Bennett, 1997; Hamilton et al., 2008). Ad5-mediated transgene expression in mice is detected already a few days after intravitreal injection but is no longer detected after two weeks (Hamilton et al., 2008; Maguire and Bennett, 1997). Some studies have reported transgene expression lasting over three weeks after gene transfer but declining already after few weeks (Gehlbach et al., 2012; Ueyama et al., 2014). In contrast, after subretinal injection, transgene expression has been detected for longer periods (Reichel et al., 1998), even up to two months (Maguire and Bennett, 1997). Subretinal Ad5 mediated gene transfer to mouse eye results mainly in transduction of RPE cells and usually to a lesser extent transduction of photoreceptor cells (Bennett et al., 1996; Mallam et al., 2004; Reichel et al., 1998; Ueyama et al., 2014). In addition to RPE and photoreceptor cells, transgene expression in Müller cells (Bennett et al., 1996; Mallam et al., 2004) and occasional cells in the corneal endothelium and the trabecular meshwork have been reported (Bennett et al., 1996). On the contrary, intravitreal injection leads to transgene expression in the anterior chamber, usually in corneal endothelium, iris and trabecular meshwork (Hamilton et al., 2008; Maguire and Bennett, 1997; Ueyama et al., 2014; Von Seggern et al., 2003) Adeno-associated viral vectors The use of AAVs as viral vectors has increased in the recent years (Ginn et al., 2018) and in ocular clinical gene therapy trials, AAVs have been the most widely used viral vector (ClinicalTrials.gov, 2018). In the eye, AAV serotypes 1-9 have been studied in animal models, with AAV2 being the most often used as it efficiently transduces the retina after subretinal and intravitreal gene delivery (Auricchio et al., 2001; Lebherz et al., 2008; Petrs-Silva et al.,

49 ). AAV2 is also the serotype that is used in clinical trials and gene therapy products for ocular neovascular diseases (Ameri, 2018; Heier et al., 2017). AAVs are small single-stranded DNA viruses, which are able to transduce non-dividing cells and provide long-term transgene expression (Solinís et al., 2015). In the wild-type virus, some integration of virally carried genes into the host genome does occur (Deyle and Russell, 2009; Naso et al., 2017; Verma and Weitzman, 2005). On the contrary, with recombinant AAVs lacking viral DNA, integration of a transgene occurs at a very low frequency. The transgene persists mostly in an extrachromosomal state in episomes in the nucleus of the transduced cells. Because recombinant episomal DNA does not integrate into host genomes, it will eventually be diluted over time leading to the loss of transgene expression. Yet the expression in mice after intravitreal and subretinal injection lasts for several months (Auricchio et al., 2001; Dudus et al., 1999; Lebherz et al., 2008) and even up to one year (Sarra et al., 2002). Transgene expression may be detected already a few days after subretinal injection with increasing levels over time (Lebherz et al., 2008; Sarra et al., 2002). In mice, subretinal injection usually transduces photoreceptors and RPE cells (Auricchio et al., 2001; Dudus et al., 1999; Lebherz et al., 2008; Mori et al., 2002b; Sarra et al., 2002) but occasionally also INL, GCL or Müller cells (Auricchio et al., 2001; Lebherz et al., 2008). Intravitreal injection leads to transgene expression mostly in retinal GCL (Auricchio et al., 2001; Lebherz et al., 2008; Mori et al., 2002b), but also occasionally in Müller cells, cells in the INL and in the anterior chamber (Auricchio et al., 2001; Lebherz et al., 2008). Transgene expression levels are usually lower after intravitreal injection than after subretinal injection (Dudus et al., 1999; Lebherz et al., 2008). The small size of AAV limits the packing capacity allowing the incorporation of packaging cassettes with sizes up to 5 kb (Naso et al., 2017). AAVs are non-replicating viruses requiring coinfection with a helper virus for propagation (Verma and Weitzman, 2005). The next generation vectors consist only of a promoter and a transgene thus preventing the formation of replication competent AAVs during vector production (Kootstra and Verma, 2003). Many different serotypes of AAVs have been identified with different tissue-specific tropisms (Lisowski et al., 2015). AAV serotype 2 (AAV2) is the best characterized and most frequently used serotype (Verma and Weitzman, 2005). Transduction depends not only on serotype but also on the capsid (Bainbridge et al., 2006; Solinís et al., 2015). Capsid proteins can be exchanged with other AAV serotypes leading to hybrid recombinant AAVs in which AAV plasmid is packed within a capsid derived from AAV of a different serotype. This affects transduced cells also in the eye (Auricchio et al., 2001; Lebherz et al., 2008). AAV is non-pathogenic to humans (Verma and Weitzman, 2005). However, AAV vector elicits a strong humoral immune response against the viral capsid, interfering with the readministration. Natural infections also result in a heterogenic prevalence of circulating antibodies, which may inhibit transduction (Chirmule et al., 1999) Lentiviral vectors There is also a trend towards an increasing use of lentiviral vectors in all clinical gene therapy trials (Ginn et al., 2018) and they are the second most widely used viral vector in retinal gene therapy trials (Auricchio et al., 2017). LVs are single stranded, integrating, enveloped RNA retroviruses that possess many properties that make them suitable for ocular gene delivery (Balaggan and Ali, 2012). They can carry large transgenes (> 8 kb) and can achieve stable expression in a broad range of dividing and non-dividing cells without intraocular inflammation. Human immunodeficiency virus type 1 (HIV-1) has been mainly used in LV vector development (Kootstra and Verma, 2003). The major concern about LV vectors is the generation of replication-competent virus (Miyoshi et al., 1998). To increase the safety, the newest LV vectors are produced by splitting the necessary components for virus production into multiple plasmids. Another safety issue about HIV vectors is the possibility of insertional activation of oncogenes by random integration into the host genome. To overcome

50 26 this problem, the viral enhancer and promoter sequences have been deleted in so-called selfinactivating vectors. LV integrates into the genome of the host cell, thus inducing stable transgene expression (Solinís et al., 2015). LV vectors are able to induce efficient and stable transduction in several cells in the anterior and posterior segment of the eye. The vectors produce rapid and sustained transgene expression that can be seen one week after injection lasting several months (Bainbridge et al., 2001) even up to nine months (Auricchio et al., 2001; Yáñez-Muñoz et al., 2006). Injection to the anterior segment of the eye resulted in transgene expression in corneal endothelial cells and the trabecular meshwork (Bainbridge et al., 2001; Lipinski et al., 2014; Yáñez-Muñoz et al., 2006) and occasionally also in the iris (Trittibach et al., 2008). After subretinal injection, the transgene expression is usually detected in RPE cells alone or with photoreceptors at the site of the injection (Auricchio et al., 2001; Bainbridge et al., 2001; Lipinski et al., 2014; Yáñez-Muñoz et al., 2006). With LV vectors, transgene expression in the retinal cells can be achieved also after intravitreal injection (Du et al., 2013; Kostic et al., 2003) although some studies have reported only a minor or non-existent transgene expression (Auricchio et al., 2001; Bainbridge et al., 2001). Depending on the promoters used, the expression is mainly restricted in the RPE cells (Kostic et al., 2003) or in choroid and CNV lesions (Du et al., 2013) Baculoviral vectors BVs are a group of DNA viruses specific to insects but also capable of transducing different types of mammalian cells (Airenne et al., 2013; Chen et al., 2011). BVs have many promising features, such as a large 38 kb insertion capacity allowing for the insertion of multiple genes and regulatory elements, the lack of cytotoxic effects and replication in mammalian cells, the ease of manipulation and production at high titers (Airenne et al., 2013; Bieniossek et al., 2012; Cheshenko et al., 2001). Despite the potential, due to viral inactivation by the serum complement system, only limited success has been achieved in in vivo studies (Hofmann and Strauss, 1998). In addition, as BV does not integrate, the transduction is transient (Airenne et al., 2013). Several strategies for bypassing the complement system have been examined, such as the use of complement inhibitors (Kaikkonen et al., 2010) and direct injection into immune privileged sites (Haeseleer et al., 2001; Tani et al., 2003). Intravitreal injection of transposonbaculovirus hybrid vector into vitreous of a mouse resulted in some transgene expression after two months in the anterior chamber (Turunen et al., 2014). Even with administration into immune-privileged sites, studies with BV have not been able to show transgene expression longer than 14 days in the eye (Haeseleer et al., 2001; Li et al., 2005; Luz-Madrigal et al., 2007) and other organs (Lehtolainen et al., 2002; Sarkis et al., 2000; Tani et al., 2003). The peak of the transgene expression was observed already a few days after the injection. Transgene expression after BV-mediated gene transfer under common cytomegalovirus (CMV) promoter was observed in the anterior chamber and sporadically across retinal layers after intravitreal injection and in the RPE cells around the injection site after subretinal injection (Haeseleer et al., 2001). Intravitreal injection of BV with cell-specific promoters has achieved transgene expression mostly in the ganglion cell layer (Li et al., 2005; Luz-Madrigal et al., 2007) Non-viral vectors Non-viral gene delivery methods exploit the use of chemical delivery methods (most often lipid- and polymer-based systems) or use of physical forces to deliver DNA into target cell (Foldvari et al., 2016; Oliveira et al., 2017). The advantage is the limited immunogenicity and toxicity compared with viral vectors. This enables repeated administration. Non-viral vectors are often used with topical administration when treating ocular diseases. Retinal delivery remains challenging, but it has been used for corneal delivery in clinical trials (Benitez-Del-

51 27 Castillo et al., 2016). Some studies have used non-viral vectors in intraocular gene delivery to the mouse eye but the expression was evident only two days in adult mice (Alqawlaq et al., 2014; Dasari et al., 2017; Farjo et al., 2006). The transgene expression in non-viral gene delivery followed the same pattern as in viral vectors: subretinal injection led to expression in RPE and choroid (Dasari et al., 2017) and in some studies also in ONL and ON (Farjo et al., 2006). Intravitreal injection transduced cells in the inner retina and anterior chamber. Transgene positive cells have been detected in GCL, IPL and NFL (Alqawlaq et al., 2014) or in the cornea and trabecular meshwork (Farjo et al., 2006). Occasional expression in the lens after subretinal and intravitreal gene transfer (Farjo et al., 2006) could be caused by needle trauma during the injection Biodistribution after intraocular gene transfer Biodistribution to off-target organs and vector shedding from the injected animal or patient through secretions and excreta are fundamental in identifying and anticipating safety after ocular gene delivery. One important point for patient safety is to avoid germ line transduction. Biodistribution after intravitreal and subretinal injection has been widely evaluated in non-human primates (Seitz et al., 2017). Both delivery route and viral dose had an impact on copy-numbers in off-targets and on the duration of shedding. Compared with subretinal injection, intravitreal injection leads to increased and prolonged shedding in offtarget tissues and blood. The vector was present in several biofluids as well as in different organs. Vector genomes were found in spleen, liver, lymph nodes and blood even as long as 91 days after gene transfer. The vector was not detected in the germline with any of the routes or doses. The study was conducted with AAV8 vector so the results are relevant to vectors with similar tropism, but they do prove the ability of vectors to escape from the eye after intraocular gene transfer. Transgene expression in mice can escape to surrounding tissues, such as the optic nerve (Dudus et al., 1999; Grant et al., 1997; Lebherz et al., 2008; Mallam et al., 2004) and extraocular muscles (Farjo et al., 2006) after delivery of various viral and non-viral vectors. Few positive cells have even been found in the extraocular muscles after topical administration (Alqawlaq et al., 2014). The expression has also been detected in other tissues outside the eye e.g. in brain (Dudus et al., 1999) and liver (Gehlbach et al., 2012). Viral escape from the eye after intraocular application has also been observed in humans. In clinical studies with neovascular AMD, vector DNA has been detected by polymerase chain reaction (PCR) in plasma on the day of injection (Campochiaro et al., 2017). In other clinical studies, vector DNA was detected by PCR in some body fluids but not in the blood up to three to four weeks (Constable et al., 2016; Rakoczy et al., 2015). Therapeutic protein has also been detected in the plasma after non-viral gene delivery. Short interfering RNA (sirna) studies have shown detectable plasma levels after 24 hours and with the highest dose, even as long as seven days following a single intravitreal injection (Nguyen et al., 2012a). Most patients had anti-aav2 antibodies already prior to the gene transfer which elevated in some patients during the study (Heier et al., 2017; Rakoczy et al., 2015). The preexisting antibodies raised the question if it has a negative effect on transgene expression (Heier et al., 2017). Patients with detectable baseline levels of anti-aav2 antibody, failed to show transgene expression in aqueous humor after gene transfer. On the contrary, antibodies against viral vectors have not been detected after subretinal injection of lentivirus (Campochiaro et al., 2017). No systemic adverse events related to the study treatment have been reported by any of the clinical trials Gene therapy products Currently there are five FDA or EMA approved gene therapy products out of which one is for an ocular disease (Ginn et al., 2018). In December 2017 FDA approved voretigene neparvovec-rzyl (Luxturna ) for the treatment of inherited biallelic RPE65 mutationassociated retinal dystrophy (Ameri, 2018). Luxturna is an AAV2-mediated human RPE65

52 28 cdna along with a CMV enhancer and a hybrid chicken β-actin promoter. Subretinal delivery of Luxturna delivers a normal copy of the gene encoding the retinal pigment epithelial 65 kda protein to retinal cells (Georgiadis et al., 2016). Mutations in this RPE65 gene cause dysfunction of rod photoreceptors leading to severely impaired night vision from birth and cone photoreceptor mediated daylight vision loss in early adulthood. The price for the Luxturna gene therapy is $ per eye (Nature America, 2018) Clinical trials of gene therapy In the year 2017, 1.3% (n=34) of gene therapy clinical trials were addressed for ocular diseases (Ginn et al., 2018). Most of these studies are for inherited diseases (Trapani and Auricchio, 2018). The majority of recent gene therapy clinical trials for vascular ocular diseases are aimed at AMD (Table 2) (ClinicalTrials.gov, 2018; Trapani and Auricchio, 2018). Five out of ten clinical studies for intraocular neovascularization target VEGF either directly or through its receptor FLT1. Two of the studies exploit gene delivery of two natural occurring antiangiogenic factors, pigment epithelium-derived factor (PEDF) and endostatin with angiostatin, while the rest target hypoxia-inducible gene, insulin receptor substrate-1 (IRS-1) and membrane attack complex (MAC). Many of these studies are in the earliest stage of clinical trial, phase I, in which the safety and maximum tolerated dose are examined in a small number of individuals (Friedman et al., 1998). Table 2. Completed or active clinical trials for neovascular ocular diseases. Treatment Delivery route DNA carrier Phase Indication Status scd59 intravitreal AAV2 phase I AMD active anti-vegf Ab subretinal AAV8 phase I AMD active oligo against IRS-1 topical no carrier phase II/III CRVO, NVG active 1 sflt1 subretinal AAV2 phase I/II AMD completed 2 sflt1 intravitreal AAV2 phase I AMD completed 3 endostatin-angiostatin subretinal LV phase I AMD completed 4 PEDF intravitreal Ad5 phase I AMD completed 5 sirna against VEGFR-1 intravitreal no carrier phase II AMD terminated sirna against VEGF intravitreal no carrier phase III AMD, DME terminated sirna against RTP801 intravitreal no carrier phase II DME, CNV, DR completed 1 (Lorenz et al., 2017), 2 (Constable et al., 2016), 3 (Heier et al., 2017), 4 (Campochiaro et al., 2017), 5 (Campochiaro et al., 2006). AAV: Adeno-associated virus, Ab: Antibody, Ad: Adenovirus, AMD: Age-related macular degeneration, CNV: Choroidal neovascularization, CRVO: Central retinal vein occlusion, DME: Diabetic macular edema, DR: Diabetic retinopathy, IRS-1: Insulin receptor substrate 1, LV: Lentivirus, NVG: Neovascular glaucoma, PEDF: Pigment epithelium-derived factor, s: Soluble, sirna: Small interfering RNA, VEGF: Vascular endothelial cell growth factor, VEGFR: Vascular endothelial cell growth factor receptor In the fall 2018, there were three active clinical gene therapy trials for neovascular ocular diseases (ClinicalTrials.gov, 2018). Gene therapy with a soluble form of CD59 (scd59) is intended to protect retinal cells by inhibiting the formation of MAC (ClinicalTrials.gov, 2018). CD59 is a glycoprotein expressed in several cell types, such as endothelial cells (Farkas et al., 2002). Normal cells produce CD59 that block MAC, but the trend towards increased MAC expression (Hageman et al., 2005) and reduced expression of CD59 is evident in both atrophic (Ebrahimi et al., 2013) and neovascular AMD patients (Singh et al., 2012). In AMD, the reduced expression of CD59 does not efficiently block MAC formation, leading to the complement cascade upregulation and cell damage and death (ClinicalTrials.gov, 2018). The

53 29 treatment is currently studied in two different clinical trials for both dry AMD with geographic atrophy and wet neovascular AMD. Another active trial using AAV targets VEGF by a single subretinal injection that contains a gene encoding a monoclonal antibody fragment for a soluble anti-vegf protein (ClinicalTrials.gov, 2018). Mouse studies with the gene therapy have shown to reduce subretinal neovascularization and retinal detachment in a short-term study (Liu et al., 2018). The third active clinical trial for vascular ocular diseases is the treatment to prevent neovascular glaucoma (NVG) after ischemic CRVO (ClinicalTrials.gov, 2018; Lorenz et al., 2017). In this on-going phase II/III study of aganirsen, an antisense DNA oligonucleotide is administered topically to patients for 24 weeks daily. A diverse group of therapeutic oligonucleotides, such as small nucleic acids and oligonucleotides are considered as part of oligo-based gene therapeutics (Foldvari et al., 2016). Aganirsen blocks IRS-1 leading to subsequent down-regulation of both angiogenic as well as pro-inflammatory growth factors such as VEGF and tumour necrosis factor (ClinicalTrials.gov, 2018; Lorenz et al., 2017). The treatment is intended to prevent the progression of new vessel formation and consequently to inhibit NVG. It is administered topically as an eye emulsion, aiming to target affected areas in the anterior chamber but also in the retina. Aganirsen eye drops have previously been evaluated in phase II and III clinical trials for inhibition of corneal neovascularization (Cursiefen et al., 2014, 2009). The largest ocular AAV2 clinical trial concluded the gene therapy to be safe but states that further clinical trials would be needed to prove its viability (Constable et al., 2016). In the phase II study for wet AMD, patients that received subretinally sflt-1 gene transfer had more minor ocular adverse effects than the control group (intravitreal ranibizumab injection) and did not demonstrate a significant difference in the best-corrected visual acuity. The phase I study was well tolerated (Rakoczy et al., 2015). Another study using sflt-1, showed gene therapy with low viral doses to be safe (Heier et al., 2017). The study involved AAV2-mediated intravitreal gene transfer. Although the maximum tolerated dose was not detected, the patients receiving the highest dose of viral vector reported the most severe adverse effects and also a decrease in best corrected visual acuity (BCVA) whereas in patients receiving lower doses, the BCVA increased during the study. Only one of the studies used lentiviral vector as the carrier. A completed study with lentiviral equine infectious anemia virus (EIAV) vector expressing endostatin and angiostatin (RetinoStat) evaluated the safety of the treatment after a single subretinal injection. The study reported one serious adverse event related to the procedure and several other ocular adverse effects in the early post-operative period without dose-limiting toxicities. The expression levels of both proteins remained stable in the aqueous humor for up to four years. Nevertheless, in the study with patients with limited visual potential, the treatment did not achieve any beneficial effects in visual acuity nor in anatomical outcomes. (Campochiaro et al., 2017) Furthermore, only one of the studies used adenoviral vector for gene delivery. This is a study of Ad5-mediated gene therapy encoding cdna for human PEDF (Rasmussen et al., 2001). PEDF is a potent inhibitor of angiogenesis found in humans (Holekamp et al., 2002; Ohno-Matsui et al., 2001). It exists naturally in the human eye, but its levels are decreased in diseases characterized by ocular neovascularization like AMD. In a phase I study, AdPEDF was delivered once via intravitreal injection into one eye with the worst visual acuity (Campochiaro et al., 2006). Patients treated with the highest dose, particle units, showed stabilization in CNV lesion size and mild improvement in visual acuity up to one year after injection. Other clinical trials for neovascular ocular diseases have exploited small interfering RNAs (sirna) (Lee et al., 2018). sirna is a double-stranded RNA molecule, bp in length, that mediates silencing of target genes with a complementary sequence (Gavrilov and Saltzman, 2012). Three clinical studies used sirna technology to target intraocular neovascularization.

54 30 sirna against VEGFR-1 has been used to treat AMD, sirnas against VEGF to treat DME and AMD and sirnas against RTP801 to treat DME, AMD and DR. A phase I/II study of intravitreal injection of sirna targeting VEGFR-1 mrna was completed with mild or moderate adverse events without establishing the maximum tolerated dose (Kaiser et al., 2010). Transient improvement in visual acuity and foveal thickness was seen, but the effect decreased or vanished within three months. A phase II study compared sirna treatment with ranibizumab with a poor outcome in best corrected visual acuity (BCVA), lesion size and foveal thickness, and the study was terminated early due to company decision (ClinicalTrials.gov, 2018). In addition, a phase III study with VEGF silencing sirna (bevasiranib) was terminated early. The trial studied intravitreal injection of sirna compared with ranibizumab. Phase I and II studies for AMD and DME have completed, but the results are have not been published. Outcome measures for this terminated phase III study has not been published either. Preliminary results showed an increase in serious and non-serious adverse events in patients treated with bevasiranib together with ranibizumab compared with ranibizumab alone. (ClinicalTrials.gov, 2018) The third clinical trial exploiting sirna technology inhibits the expression of the hypoxiainducible gene RTP801 (Nguyen et al., 2012c). The RTP801 may decrease the downstream response of endothelial cells to VEGF activation. Phase I and II studies for AMD proved the therapy to be safe (Nguyen et al., 2012a, 2012c) but adding sirna against RTP801 did not improve the outcome of the treatment compared with ranibizumab alone (Nguyen et al., 2012c). Detectable plasma levels were found after 24 hours and with the highest dose even after 14 days following single intravitreal injection (Nguyen et al., 2012a). Although an unpublished phase II study for DME, CNV and DR has been completed later (ClinicalTrials.gov, 2018), the first phase II study for DME was terminated based on discontinuation rates (Nguyen et al., 2012b). Discontinuation at early time points was mainly due to adverse events, and after six months, the primary reason for patient withdrawal was lack of efficacy.

55 31 3 Aims of the study The aim of this thesis was to study retinal and subretinal neovascularization in the mouse eye under pathological conditions and after anti-angiogenic gene therapy and to compare different viral vectors for intraocular gene transfer. The specific aims were as follows: I II III IV Compare the transduction efficiency and duration, target site and adverse effects of baculo-, lenti-, adeno-associated- and adenoviral vector gene transfer after intravitreal injection to the mouse eye. Develop a novel mouse model of subretinal neovascularization and age-related macular degeneration expressing human VEGF-A165 in the eye. Create a lentiviral vector expressing α-vegf antibody and use it to treat neovascularization in the mouse eye after a single intravitreal gene transfer. Characterize the effects in the eye of a new transgenic mouse model developing vulnerable atherosclerotic lesions and create a new, spontaneous animal model for retinal vessel occlusion.

56 32

57 33 4 Materials and methods 4.1 IN VITRO STUDIES In vitro methods used in study III are summarized in Table Construction of anti-vegf-antibody plasmid In study III, an LV plasmid encoding anti-human VEGF antibody (LV-α-VEGF) was created by cloning the sequence of heavy and light chains of anti-human VEGF antibody linked by an internal ribosomal entry sites into the pentry-mcs vector. The resulting entry plasmid was cloned into an LV backbone under human phosphoglycerate kinase-1 (hpgk) promoter using the Gateway cloning system and used for protein and viral vector production Protein production and transduction In the in vitro assays, α-vegf protein was produced in human embryonic kidney 293T (HEK293T) cells by calcium phosphate transfection, purified and concentrated. Protein analyses included gel electrophoresis for size determination, and spectrophotometer and protein assay for protein concentration assessment. In the Western blotting, HepG2 cells were transduced with LV-α-VEGF using different multiplicity of infection (MOI) Binding assays and comparison of anti-vegf antibody to Avastin Medium collected from LV-α-VEGF transduced cells was used in Western blotting studies to determine the secretion and compare α-vegf and Avastin. α-vegf protein was compared with Avastin in enzyme-linked immunosorbent assays (ELISA) and BaF3-VEGFR-2 viability assay.

58 34 Table 3. Used in vitro methods. Method Description Protocol DNA cloning Construction of LV-α-VEGF plasmid After restriction, the transgene sequence was ligated into pentry-mcs vector. The resulted entry clone was cloned into the LV vector using the Gateway cloning system and verified by sequencing. Cell transduction Transduction with LV-α-VEGF and medium collection HepG2 cells were transduced with LV-α-VEGF at an increasing multiplicity of infection. Medium was collected on day 4. Western blotting Determination of α-vegf protein expression after transduction Collected media from LV-α-VEGF transduced cells was separated in gel electrophoresis and blotted with anti-mouse HRP-antibody. Western blotting Determination of α-vegf binding to VEGF-A 165 after transduction VEGF-A 165 was separated in gel electrophoresis and collected media from LV-α-VEGF transduced cells or Avastin was used as primary antibody. HRP-conjugated antibodies were used for detection. Protein production, purification and concentration α-vegf protein production and purification After calcium phosphate transfection of HEK293T cells, the purification was done by affinity chromatography and concentrated by ultrafiltration. Final buffer exchange was performed in columns. Protein analyses Determination of α-vegf protein purification and concentration Purified protein samples were separated in gel electrophoresis and stained with protein stain. The concentration was assessed by spectrophotometer and protein assay kit after ultrafiltration and buffer exchange. Binding assay Verifying binding of VEGF-A 165 to α-vegf or Avastin Dilution series of biotinylated VEGF-A 165 was added to the α-vegf or Avastin coated plates. The bound VEGF-A 165 was detected by streptavidin peroxidase and TMB. Competition assay Determination of competitive binding of VEGF-A 165 to soluble VEGFR2-Fc Dilution series of α-vegf or Avastin were preincubated with biotinylated VEGF-A 165 before adding to svegfr-2 Fc coated plates. The bound VEGF-A 165 was detected by streptavidin peroxidase and TMB. BaF3-VEGFR-2 viability assay Determination of biological activity of the α-vegf protein BaF3-VEGFR-2 cells without IL-3 were incubated with VEGF-A 165 and dilution series of α-vegf or Avastin. Cell viability was quantified using colorimetric method. ELISA: Enzyme-linked immunosorbent assay, HEK293T: Human embryonic kidney 293T, HRP: Horseradish peroxidase, IL-3: Interleukin 3, LV: Lentivirus, TMB: 3,3,5,5 -tetramethylbenzidine, VEGF: Vascular endothelial growth factor, VEGFR-2: Vascular endothelial growth factor receptor 2

59 IN VIVO STUDIES Experimental animals All animal experiments were approved by the National Animal Experiment Board of Finland and carried out in accordance with the guidelines of the Finnish Act on Animal Experimentation. Mice were kept in a temperature and humidity controlled environment with a 12 hour light/dark cycle at the National Laboratory Animal Centre in Kuopio. Animals were fed ad libitum. Mice were anesthetized subcutaneously with ketamine (Ketaminol vet 50 mg/ml) and medetomidine (Domitor vet 1 mg/ml). Atipamezole (Antisedan vet 5mg/ml) was used for reversal of the sedatives. Animals were sacrificed with carbon dioxide. Animals and used protocols are summarized in Table 4. Table 4. Used mouse models. Study Strain Model Induction Endpoint I C57BL/6OlaHsd (n=88) Comparison of viral vector gene delivery in the eye Intravitreal injection of saline or baculo-, lenti-, adenoassociated- or adenoviral GFP 3 days, 1, 4, 12 weeks (only AAV and LV) or 24 weeks (only AAV) after intravitreal injection II loxp-stop-vegf- A 165 (n=51) AMD and VEGF-A 165 expression in the eye Subretinal injection of adenoviral Cre or LacZ 2, 6 or 12 weeks after subretinal injection III loxp-stop-vegf- A 165 (n=12) α-vegf antiangiogenic effect in AMD model Intravitreal injection of lentiviral α-vegf or GFP and subretinal injection of adenoviral Cre a week later 3 weeks after intravitreal injection IV ApoE -/- Fbn1 C1039G+/- (n=7) ApoE -/- (n=7) Retinal vessel occlusion High-fat diet 12 weeks after beginning of high-fat diet AAV: Adeno-associated virus, AMD: Age-related macular degeneration, ApoE -/- : Apolipoprotein E deficient, Fbn1 C1039G+/- : Fibrillin-1 C1039G+/- mutation, GFP: Green fluorescent protein, LV: lentivirus, VEGF: Vascular endothelial growth factor Viral vectors and gene transfer methods All the gene transfers were administered subretinally or intravitreally into the mouse eye. Phenylephrine (Oftan Metaoksedrin 100 mg/ml) and tropicamide (Oftan Tropicamid 5 mg/ml) were used to induce mydriasis, oxybuprocaine eye drops (Oftan Obucain 4 mg/ml) as analgesic and carbomer eye gel (Viscotears 2 mg/ml) as a lubricant. 2 µl injections were performed under the microscope using a 34-gauge needle and a 10-µl microsyringe (Hamilton). The subretinal injections were performed transsclerally penetrating through the choroid and Bruch s membrane locating near the papilla. The intravitreal injections were performed through the pars plana. In a comparative study of viral vectors, mice received bilateral injections and both eyes were studied. VEGF-A165 expression in the mouse eye was induced using a single subretinal injection leaving the contralateral eye intact. In anti-angiogenic gene therapy trial, a subretinal injection was administered one week after intravitreal injection. The viral vectors and titers used are presented in Table 5. The source for all viral vectors was A.I. Virtanen Institute.

60 36 Table 5. Viral vectors used. Mice received 2 µl injection once (studies I-II) or twice (intravitreal injection and subretinal) (study III). Study Vector and transgene Promoter Titer Injection route I AdGFP CMV pfu/ml intravitreal injection AAV2-eGFP CMV TU/ml intravitreal injection LV-GFP hpgk TU/ml intravitreal injection BV-GFP CAG pfu/ml intravitreal injection II AdCre CMV pfu/ml subretinal injection AdLacZ CMV pfu/ml subretinal injection III AdCre CMV pfu/ml subretinal injection LV-α-VEGF hpgk 1.11x10 9 TU/ml intravitreal injection LV-GFP hpgk 1.11x10 9 TU/ml intravitreal injection Ad: Adenovirus, AAV2: Adeno-associated virus serotype 2, BV: Baculovirus, CAG: CMV enhancer chicken beta-actin promoter, rabbit beta-globin splice acceptor site, CMV: Cytomegalovirus, egfp: Enhanced green fluorescent protein, hpgk: Human phosphoglycerate kinase-1, LV: Lentivirus, pfu: Plaque-forming unit, TU: Transducing unit, VEGF: Vascular endothelial growth factor 4.3 METHODS FOR EVALUATING IN VIVO STUDIES Imaging of the eye In studies II-IV, SD-OCT and FA were examined with Heidelberg SD-OCT and with Heidelberg Eye Explorer version Mydriatic Oftan Tropicamid drops were used to dilate the pupil and the eye was lubricated. Anesthesized mice were placed on a custom made surface and standard human settings in the programme were used, only altering the focus up to +40 D. OCT pictures were taken with 30 lens. In FA imaging, 0.2 ml of 2 % fluorescein (Fluorescite 100 mg/ml) was injected intraperitoneally into the mice and FA was made with a wide angle 55 lens. Images were taken before the injections and sacrification Histological and immunohistochemical methods Tissue samples were immersed first in 4 % paraformaldehyde for overnight and then in sucrose. In studies II, III and IV samples for histology were immersion fixed in 4 % paraformaldehyde overnight and embedded in paraffin before cutting the eyes into 4 µm serial histologic sections. In study I, the eyes were embedded in an optimal cutting temperature compound and sectioned at 7 µm using a cryostat. For whole-mount analysis, the retinas were removed, rinsed with PBS after fixation in 4 % paraformaldehyde and processed for immunohistochemistry or studied for the presence of green fluorescent protein (GFP). Fluorescent antibodies were used as secondary antibodies and sections were counterstained using 4'-6-diamidino-2-phenylindole (DAPI), except for the detection of VEGF in study II, peroxidase substrate and Methyl Green as a nuclear counterstain were used. Samples were used for GFP detection or stained according to Table 6.

61 37 Table 6. Stainings performed. Antibody Target Source Study α-sma smooth muscle cells in vessel walls Sigma IV β-gal LacZ endoced beta-galactosidase EMD Millipore II Brn3a retinal ganglion cells EMD Millipore I, IV CD34 vessel endothelial cells and hematopoietic progenitor cells Hycult Biotech I-IV GFAP glial cell activation in astrocytes and Müller cells Dako I-II, IV GFP green fluorescent protein Santa Cruz III NeuN retinal ganglion and amacrine cells EMD Millipore I VEGF-A vascular endothelial growth factor A Abcam II-IV F4/80 mature tissue macrophages Abcam (I) Bio-Rad (II-IV) I-IV Stain Target Source Study Hemotoxylin-eosin nucleus and cytoplasmic structures in-house II-IV PicroSirius Red collagen I and III Abcam II-IV TUNEL DNA fragmentation in apoptotic cells Roche (I) Trevigen (II-IV) I-IV α-sma: Alpha smooth muscle actin, β-gal: Beta-galactosidase, GFAP: Glial fibrillary acidic protein, NeuN: Neuronal nuclei, TUNEL: Transferase-mediated dutp nick end-labeling, VEGF: Vascular endothelial growth factor Protein and gene expression analyses ELISA and quantitative reverse transcriptase polymerase chain reaction (qrt-pcr) were used to study anti-gfp antibodies, protein and gene expression of the transgene in the eye and off-target organs. Blood samples and tissue homogenates were used for ELISA. Retina, optic nerve, liver, lung, spleen, forebrain and kidney were used for copy number assay. Liver and whole eye were used to determine VEGF-A165 mrna expression levels. The samples were studied according Table 7.

62 38 Table 7. Protein and gene expression analyses. Assay Target tissue Description Source Study ELISA serum presence of anti-gfp antibodies in-house I ELISA plasma, lung, liver VEGF-A 165 protein levels R&D Systems II ELISA plasma VEGF-A 165 protein levels R&D Systems III Quantitative PCR retina, optic nerve, liver, lung, spleen, forebrain, kidney copy number of GFP Applied Biosystems I Quantitative PCR eye, liver VEGF-A 165 mrna expression Applied Biosystems II Quantitative PCR liver VEGF-A 165 mrna expression Applied Biosystems III ELISA: enzyme-linked immunosorbent assay, GFP: green fluorescent protein, PCR: polymerase chain reaction, VEGF: vascular endothelial growth factor Clinical chemistry In study I, blood samples were analysed for alkaline phosphatase (ALP), alanine aminotransferase (ALT), creatinine (CREA) and C-reactive protein (CRP). The serum samples were analysed by using standard photometric, immunoturbidometric and colorimetric assays and reagents on the Konelab 20 XTi clinical chemistry analyzer Statistical analyses Used statistical test are presented in Table 8. GraphPad Prism, v was used in all studies and in addition SPSS, v. 19 in study I. A value of P 0.05 was considered statistically significant. Table 8. Used statistical analyses. Name Bonferroni s multiple comparison test Mann-Whitney U test One-way analysis of variance (ANOVA) Two-way analysis of variance (ANOVA) Study I, II I, III I, IV II

63 39 5 Results 5.1 TRANSDUCTION EFFICACY AND ADVERSE EFFECTS OF INTRAVITREAL DELIVERY OF ADENO-ASSOCIATED VIRUS, ADENOVIRUS, BACULOVIRUS AND LENTIVIRUS VECTORS (I) After intravitreal gene delivery, the transduction efficiency and duration, target site and adverse effects of the four vectors carrying GFP were examined at different time points. Some eyes were naïve or injected with saline. Figure 8 shows GFP expression in the eye. AdV vector showed transgene expression mostly in the cells in the anterior chamber and occasionally in INL three and seven days after injection whereas only single cells were GFP-positive one month after gene transfer. Transgene expression was almost non-existent in the BV group as only few positive cells were detected three days after injection in only one out of ten studied eyes. LV injection resulted in stronger GFP expression at earlier time points and occasional expression three months after gene transfer. The target site of positive cells varied between mice, some showed transgene expression in the anterior chamber and others in different retinal layers. AAV showed the strongest GFP expression increasing significantly from seven days to three months after the gene transfer. Transgene expression seven days and one month after injection was found widely in different cells in the inner and outer layers of the retina with some positive cells also in the anterior chamber one month post injection. A great number of positive cells were still found three and six months after gene transfer, even though the number of GFP positive cells in whole-mounts slightly declined in six months after injection. AAV-injected mice were the only ones presenting GFP expression in the optic nerve. Wholemounts displayed also that some cells from the RGCL with GFP positive somas had GFP expression in their axons. GFP copy number analysis revealed biodistribution outside the eye only in the optic nerve in AdV, LV and AAV injected mice but not in the liver, lung, spleen, forebrain or kidney. Besides AAV-mediated GFP expression in the optic nerve, single GFP positive cells were located also in the extraocular muscles surrounding the eye after AdV and LV injections. A series of immunohistochemical staining with blood value analyses were performed to evaluate local and systemic adverse effects (Figure 9). The AdV vector resulted in the strongest systemic and ocular immune response. The number of macrophages in the retina and serum concentration of anti-gfp IgG was the highest in that group. In contrast, in situ detection of DNA fragmentation and glial cell activation was detected in all virus-injected eyes but also in saline-injected eyes. A few GFAP positive astrocytes with altered morphology were seen in whole-mount staining, but this was not specific to any particular viral vector. None of the studied blood chemistry screen substances differed between naïve control and virus injected mice. Table 9 presents the found differences between the groups.

64 40 Figure 8. GFP expression in the eye after different viral vectors. A) GFP-positive cells (green) were detected in the anterior chamber and in RPE after AdV vector injection, retina and optic nerve after AAV vector injection and either in the anterior chamber or in the retina and extraocular muscles after LV vector injection. B) GFP positive distribution in the whole-mounts of the eyes after administration of AAV vector. In the graphical representation, each small black ellipse represents a GFP expressing profile in the GCL or INL. Some cells from the GCL with GFP positive somas (arrow) also had GFP expression in their axons (arrowheads). Scale bar is 100 µm (A) and 50 µm (B). AdV: Adenovirus, AAV: Adeno-associated virus, GFP: Green fluorescent protein, GCL: Ganglion cell layer, INL: Inner nuclear layer, LV: Lentivirus

65 41 Figure 9. Local adverse effects in the eye and systemic effects after intravitreal injection. A) F4/80 positive macrophages (arrows), B) apoptotic cells (arrows) and C) GFAP immunoreactivity in Müller cells (arrowheads) in the retina of AdV and saline injected eyes. D) Anti-GFP IgG concentration in the plasma. E) Blood chemistry screen. Scale bar in is 10 µm (A), 20 µm (B) and 50 µm (C). *P<0.05, ***P< AAV: Adeno-associated virus, AdV: Adenovirus, ALP: Alkaline phosphatase, ALT: Alanine aminotransferase, BV: Baculovirus, CREA: Creatinine, CRP: C-reactive protein, GFAP: Glial fibrillary acidic protein, GFP: Green fluorescent protein, INL: Inner nuclear layer, IPL: Inner plexiform layer, LV: Lentivirus, NFL: Nerve fiber layer, ONL: Outer nuclear layer, OPL: Outer plexiform layer, RGCL: Retinal ganglion cell layer, U/l: Unit/litre, TUNEL: Transferasemediated dutp nick end-labeling

66 42 Table 9. Summary of differences after intravitreal injection with viral vectors. Assay BV AdV LV AAV GFP expression non-existent in all except one eye mediate expression in anterior chamber and INL early time points mediate expression in anterior chamber and occasionally in INL and RPE up to 1 month, few positive cells at 3 months strong expression in the retina and mediate in RPE up to 1 month, up to 6 months strong expression in RGCL and INL GFP copy number (copy number/ 100 ng DNA) retina: optic nerve: <30 retina: > optic nerve: retina: > optic nerve: retina: optic nerve: Macrophage recruitment (number of F4/80 positive cells/ section) 3 and 7 days: > 60 3 and 7 days: > 60 3 and 7 days: and 3 months: < 10 3 and 7 days: , 3 and 6 months: < 10 Anti-GFP antibodies 7 days: 3.49 ng/ml 1 month: ng/ml 7 days: ng/ml 1 month: ng/ml 7 days: 5.35 ng/ml 1 month: ng/ml 7 days: 2.30 ng/ml 1 month: 6.03 ng/ml AAV: Adeno-associated virus, AdV: Adenovirus, BV: Baculovirus, GFP: Green fluorescent protein, GCL: Ganglion cell layer, INL: Inner nuclear layer, LV: Lentivirus, RGCL: Retinal ganglion cell layer, RPE: Retinal pigment epithelium 5.2 OCULAR EFFECTS AND BIODISTRIBUTION OF ADENOVIRAL CRE INDUCIBLE HUMAN VEGF-A165 EXPRESSION IN THE EYE OF TRANSGENIC MICE (II) Subretinal injection of AdCre in transgenic mice with a loxp-stop inactivated VEGF-A165 expression cassette was used to study VEGF-A165 expression and its effects in the eye. Cre gene delivery excises the STOP cassette leading to expression of VEGF-A165. Mice were studied for up to 12 weeks. Most changes were seen two weeks after injection. Cre injection resulted in vascular and retinal changes compared with LacZ control (Figure 10). Two weeks after Cre gene transfer, increased retinal thickness with large or diffuse hyperfluorescence was seen in OCT and FA imaging, respectively. Histological analyses also showed subretinal swelling in the Cre injected mice. At 12 weeks, the hyperfluoresence diminished and hypofluorescent areas were found in the atrophic retinas. Both Cre and LacZ injected mice displayed some photoreceptor and ONL atrophy at the site of the injection. Nevertheless, at later time points, there were no changes in retinal thickness in Cre or LacZ injected mice compared with each other or intact eyes. In addition to the vascular changes seen in FA, the CD34 positive capillary area in the retina and subretinal membrane was significantly larger two weeks post Cre injection. Neovascular area developed into fibrovascular membrane in 12 weeks. This was also seen in collagen positive Sirius Red staining.

67 43 After Cre gene delivery, immunohistochemical staining showed additional GFAP immunoreactivity in Müller cells, F4/80 positive macrophages and autofluorescent drusenlike deposits which were seen mostly in the subretinal space of the RPE, subretinal neovascular membrane, and atrophic outer nuclear layer, and occasionally in the GCL. Glial cell activation and macrophage recruitment were also present in some LacZ injected mice, but to a lesser extent. In contrast, the number of apoptotic cells were increased in both groups two weeks after injection with a major reduction in later time points. Apoptotic cells were mainly seen at the site of the injection in the subretinal membrane and in the outer retinal layers. β-gal staining showed that AdLacZ injected mice resulted in transgene expression mostly in ONL and two weeks after gene transfer. Figure 10. Vascular and retinal changes after adenoviral mediated Cre and LacZ gene transfer. A) Retinal thickness, B) retinal and subretinal capillary area and C) fibrotic scarring in the eye after subretinal injection. n.s.: Non-significant *P 0.05, **P VEGF-A165 protein and mrna expression in Cre injected mice were studied at different time points (Figure 11). VEGF-A165 expression was studied by qpcr in the pooled samples of whole eyes two and six weeks after Cre gene delivery. Besides the eye, transgene mrna expression was also detected in the liver. Detectable levels of VEGF-A165 protein were also found in the plasma, liver and lung homogenates in some of the mice. VEGF-A165 protein in the plasma samples were found in 75 % of Cre-injected mice two weeks after the injection and in one mouse 12 weeks after the injection. 50 % of liver and one lung homogenate samples showed detectable levels of the protein. There was a correlation between the results in ELISA and qpcr analyse. The mice with the highest mrna expression in the liver had also the highest protein levels in the plasma and liver homogenates. Only one mouse had

68 44 detectable levels of VEGF-A165 protein in the lung homogenate. This mouse had also exhibited remarkably high mrna and protein levels in other assays. No signs of systemic toxicity were observed in the major organs after Cre injection. Figure 11. VEGF-A165 mrna and protein expression in the eye and off-targets. A) VEGF-A165 mrna expression in the pooled whole eye and B) liver. C) Protein expression in the plasma, D) liver and E) lung. The same symbol represents the same mouse.

69 THE EFFECTS OF ANTI-VEGF ANTIBODY IN VITRO AND IN THE MOUSE EYE AFTER GENE THERAPY (III) To study anti-angiogenic gene therapy in the AMD mouse model, an LV vector encoding α- VEGF antibody was cloned. LV-α-VEGF was first compared with Avastin in vitro. Western blotting from medium of HepG2 transduced cells showed that the protein was produced and secreted. When using medium as a primary antibody, α-vegf bound to VEGF-A165 similarly as Avastin. For ELISA and BaF3-VEGFR-2 cell assay, protein was produced by transfection. Ligand-dependent BaF3-cell proliferation showed that the biological activities of the α-vegf protein and Avastin were the same. Both antibodies prevented VEGF-A165 binding to BaF3- VEGFR-2 cells and hence cell growth and survival. When studied using ELISA method, α- VEGF protein and Avastin had almost identical binding profiles. Binding to VEGF-A165 as well as the competition binding curve were similar between the two antibodies. The results from in vitro studies are presented in Figure 12. Figure 12. A) α-vegf protein compared with Avastin in vitro in Western blot, B) in the BaF3- VEGFR-2 viability assay, C) in the binding assay to VEGF-A165 and D) in the VEGFR-2 competition binding assay. Ab: Antibody, c: Concentration, kda: Kilodalton, MOI: Multiplicity of infection Transgenic mice with Cre inducible VEGF-A165 expression were used to determine if LV-α- VEGF gene therapy could inhibit subretinal neovascularization in AMD mouse model. Seven days after intravitreal LV-α-VEGF gene delivery, Cre was injected subretinally to induce the earlier developed mouse model of AMD (II). Three weeks after intravitreal injection, there were no differences in hyperfluorescence or in retinal thicknesses between LV-α-VEGF and LV-GFP control injected mice in FA and OCT imaging. Although some mice in both groups did show some signs of increased retinal thickness three weeks after intravitreal gene delivery, the change was not statistically

70 46 significant when compared with retinal thickness before the injections. Detected hyperfluorescence in most eyes was restricted to small area. A series of immunohistochemical staining were performed to examine the effects of LVα-VEGF gene therapy. A few CD34-positive endothelial cells were seen in subretinal membrane of LV-α-VEGF injected mice, whereas LV-GFP resulted in a large neovascular membrane. The detected capillary area was smaller in LV-α-VEGF than in LV-GFP treated mice. The larger neovascular membrane of the control group was also visible in hematoxylineosin staining. The detected fibrovascular areas were ± 9492 µm 2 and ± µm 2 in LV-α-VEGF and LV-GFP injected mice, respectively. Nevertheless, the the difference was not statistically significant. Retinal and vascular changes are shown in Figure 13. LV-GFP injected mice presented more often subretinal drusen-like autofluorescent deposits at the site of the injection and partial loss of photoreceptors. In addition, F4/80 positive macrophages were found in subretinal neovascular membranes after LV-GFP injection more often compared with LV-α-VEGF group. There was no statistical difference between the groups with respect to the number of apoptotic cells in the retina.

71 47 Figure 13. Vascular and retinal changes after lentiviral mediated α-vegf (LV-α-VEGF) and control LV-GFP injection. A) Fluorescein angiography (FA) and spectral domain optical coherence tomography (SD-OCT) images three weeks after intravitreal injection. B) Hyperfluoresence and retinal thickness measured from FA and SD-OCT images. C) Retinal and subretinal capillary area and fibrotic scarring measured from immunohistochemical staining. *P GFP: Green fluorescent protein Figure 14 shows protein and mrna expression of VEGF-A165. ELISA showed detectable levels of VEGF-A165 in plasma samples of two LV-GFP injected mice whereas it was not detected in any mice in the LV-α-VEGF group. According to the RT-qPCR assay, mice that had received LV-GFP gene therapy had around three times higher VEGF-A165 expression levels in the liver when compared with LV-α-VEGF injected mice.

72 LACK OF RETINAL VASCULAR OCCLUSION AND NEOVASCULARIZATION IN MICE WITH VULNERABLE ATHEROSCLEROTIC PLAQUES (IV) Transgenic mice with an elastic fiber mutation in the fibrillin-1 (Fbn-1) gene and apolipoprotein E (ApoE) deficiency express vulnerable atherosclerotic plaques that have been associated with strokes. The effects of these vulnerable plaques in the mouse eye were studied 12 weeks after high-fat Western diet. Neither mice with Fbn-1 mutation and ApoE deficiency (ApoE -/- Fbn1 C1039G+/- ) nor ApoE deficient (ApoE -/- ) control mice without the mutation showed any hyper- or hypofluorescence in FA. Furthermore, no vascular or choroidal changes in the retinal thickness or morphology before or after the high-fat diet were detected in OCT (Figure 14). Figure 14. Fluorescein angiography (FA) and spectral domain optical coherence tomography (SD- OCT) images of mice with fibrillin-1 mutation and apolipoprotein E deficiency (ApoE -/- Fbn1 C1039G+/- ) and (ApoE -/- ) with apolipoprotein E deficiency control mice before and after 12-week high-fat diet. Histology and immunohistochemistry either did not reveal abnormalities in the retina (Figure 15). One of the seven control mice had positive staining for collagens I and III in the eye and GFAP immunoreactivity in Müller cells was found in a total of five mice from both groups. Otherwise, immunohistochemical staining did not show anomalous morphology in choroidal or retinal vessels or in the ganglion cells. There were no signs of abnormal VEGF expression, fibrotic scarring or immune reactivity in either of the groups.

73 49 Figure 15. Histology and immunohistochemistry of mice with fibrillin-1 mutation and apolipoprotein E deficiency (ApoE -/- Fbn1 C1039G+/- ). A) Hematoxylin-eosin (HE), B) Sirius Red and immunohistochemical staining (C-H) after 12-week high-fat diet. C) Smooth muscle cells and D) endothelial cells in superficial capillary plexus (arrow), deep capillary plexus (arrowhead) and choroid (*). E) Brn3a-positive retinal ganglion cells. F) Macrophages in the ciliary body (arrow) and in the iris (arrowhead). G) Glial fibrillary acidic protein (GFAP) activation in astrocytes (arrow) and Müller cells (arrowhead). H) VEGF expression in the ganglion cell layer (arrow) and in the inner segment of photoreceptors and outer nuclear layer (arrowhead). Scale bar is 100 µm. α- SMA: Alpha smooth muscle actin, VEGF: Vascular endothelial growth factor

74 50

75 51 6 Discussion 6.1 TRANSDUCTION EFFICACY OF INTRAOCULAR GENE TRANSFER (I-III) The type and number of transduced cells as well as the duration of the expression significantly determine the efficacy of gene transfer. Besides the vector and promoter, also the injection route affects the transduction (Solinís et al., 2015). Study I revealed major differences in the cells transduced, the transduction efficiency and the duration of transgene expression after intravitreal gene delivery, thus making the choice of vector dependent on the disease to be treated. In clinical settings, gene therapy is often the most useful for conditions requiring long-term treatment. AAV is indeed the most often used viral vector in ocular clinical gene therapy trials (ClinicalTrials.gov, 2018). We also showed superiority of AAV in terms of the number of transduced cells and in the duration of transgene expression. LV mediated GFP expression under phosphoglycerate kinase-1 (PGK) promoter after intravitreal injection was examined in studies I and III. In study I, LV vector gene delivery achieved mediate level transgene expression in the retina lasting up to three months. LV has also been used in an anti-angiogenic clinical trial, although only one of the gene therapy trials for neovascular ocular diseases exploited LV vector (Campochiaro et al., 2017). We also evaluated transgene expression after subretinal injection in study II. AdLacZ resulted in transgene expression mostly in ONL. This differs from the situation after intravitreal injection in which the transgene was expressed mainly in the cells of the anterior chamber (Study I). Also other studies have shown transduction of cells in outer layers after subretinal injection (Bennett et al., 1996; Mallam et al., 2004; Reichel et al., 1998; Ueyama et al., 2014) and expression in the anterior chamber after intravitreal injection (Hamilton et al., 2008; Maguire and Bennett, 1997; Ueyama et al., 2014; Von Seggern et al., 2003). In mice, transgene expression is transient after AdV injection (Hamilton et al., 2008; Maguire and Bennett, 1997; Ueyama et al., 2014), although the duration of expression is longer after subretinal injection (Maguire and Bennett, 1997), possibly due to less intense immune responses (Bennett et al., 1996; Maguire and Bennett, 1997). In clinical trials, one study has used Ad5-mediated PEDF gene therapy for the treatment of AMD (Campochiaro et al., 2006). Transgene expression was not determined but the CNV lesion size had stabilized by three months after intravitreal injection. The AdV vector in clinical trial was also partial E4-deleted (Campochiaro et al., 2006) whereas the vector in study I was only E1 and E3 deleted. It is known that the E4 modification decreases vector toxicity and inflammation in vivo (Christ et al., 2000) and may result in longer and more stable transgene expression (Dedieu et al., 1997; Gao et al., 1996; Ji et al., 1999). Transgene expression after intravitreal gene delivery with BV vector, was almost nonexistent. However, the same construct resulted in an efficient gene transfer in rabbit eye after intravitreal injection (Kinnunen et al., 2009). This could be due to the difference in transduction efficiency between the species, although also the amount of BV was much higher in the previous study than in study I. It is crucial that the transduction efficacy of the used viral vector construct is determined as this is a major factor on the route to successful gene therapy. The challenge in pre-clinical gene therapy studies is that many other factors in addition to vector and delivery route, affect the transduction efficacy. For example, age (Harvey et al., 2002; Kostic et al., 2003), strain (Gupta et al., 2001), injury of the retina (Balaggan et al., 2006) and genotype of the injected mice are all factors that influence the result (Calame et al., 2011; Liang et al., 2001; Maguire and Bennett, 1997).

76 BIODISTRIBUTION AND SAFETY AFTER INTRAOCULAR GENE TRANSFER (I-III) Biodistribution outside the eye, into plasma and other organs is important in evaluating the safety of intraocular gene delivery. The safety of the gene transfer can be assessed by determining the side effects in the eye, in off-target organs and in blood values. Gene transfer has been shown to result in systemic antibody-mediated immunity in humans (Constable et al., 2016; Heier et al., 2017). We also detected an increase of serum anti-gfp IgG concentration in study I, although copy number analysis showed GFP expression only in the retina and optic nerve. Various viral and non-viral vectors in mice have also showed transgene escape into optic nerve (Dudus et al., 1999; Grant et al., 1997; Lebherz et al., 2008; Mallam et al., 2004). GFP expression in the optic nerve was also seen in cryosections of AAV injected mice in study I. This is presumably due to transgene-positive ganglion cells projecting axon fibers to the optic nerve (Chadderton et al., 2013; Grant et al., 1997). Indeed, we detected some ganglion cells with GFP positive somas that also had GFP in their axons. In addition, single GFP positive cells were located in the extraocular muscles surrounding the AdV and LV injected eye in study I, as was seen after subretinal injection of GFP nanoparticles (Farjo et al., 2006). In studies II and III, mrna VEGF-A165 expression was seen in the liver of Cre injected mice revealing viral escape from the eye to the bloodstream. AdV is well-known to move quickly with remarkable amounts passing from the bloodstream to the liver through the Kupffer cells (Xu et al., 2005). Also others have reported transgene expression several weeks after subretinal and intravitreal injection in off-target organs, such as the mouse brain (Dudus et al., 1999) and liver (Gehlbach et al., 2012). In addition, ELISA showed VEGF-A165 protein expression in the plasma and tissue homogenates in some mice. No signs of overexpression of VEGF-A165 were seen during studies II and III, contrary to what has been found after intravenous Cre injection (Leppänen et al., 2006). In study I, there were no changes in clinical chemistry screen values in any viral groups compared with non-injected mice indicating the absence of systemic toxicity. Clinical gene therapy trials for intraocular neovascularization have not reported study-related side effects outside the eye even though detectable anti-viral antibodies (Constable et al., 2016; Heier et al., 2017) and RNA sequences of the transgene (Campochiaro et al., 2017) and sirna (Nguyen et al., 2012a) have been detected in the blood. All injections in studies I-III caused a mild but transient injury leading to minor changes in morphology and biomarkers of the retina. AdV gene transfer is known to induce an immune response (Schagen et al., 2004), and the cell-mediated immune response after intraocular gene delivery is stronger after AdV injection than with AAV or LV (Bennett, 2003). Macrophages are the main effector cells of innate immune responses in the central nervous system and blood-derived macrophages are recruited to the retina from the circulation in response to injury, infection, or trauma (Caicedo et al., 2005). The presence of macrophage was studied by detecting F4/80-immunoreactive cells in studies I-III. In all studies, the number of macrophages was the greatest at the earlier time points. Despite the poor transduction efficiency, also the BV transduced retinas showed as many F4/80 positive cells as AdV in three days and seven days after gene transfer (Study I). GFAP is normally expressed only in astrocytes in the mouse retina (Goldman, 2014). In response to retinal injury or photoreceptor degeneration, however, GFAP gene transcription is strongly activated in the Müller cells. All injected retinas, also only saline injected, showed glial cell activation in Müller cells in addition to normal expression in astrocytes in the NFL in studies I and II. Similarly to macrophage recruitment, expression was highest at the early time points and considerably declined thereafter. The Müller glia response to retinal injury can be either protective or detrimental to retinal function (Goldman, 2014). This injury response is referred to as reactive gliosis. Depending on the severity of damage, this response may include Müller glia proliferation. The reactive gliosis can be beneficial to neurons by protecting them from cell death, however, prolonged gliosis is detrimental because it

77 53 interferes with retinal homeostasis and impairs the ability of Müller glia to support retinal neurons and therefore often leads to neurodegeneration. Furthermore, the deposition of cell masses as a consequence of proliferative gliosis hampers normal retina function. Terminal deoxynucleotidyl transferase-mediated dutp nick end-labeling (TUNEL) detects the degradation of nuclear DNA into nucleosomal units by the terminal deoxynucleotidyl transferase (TdT)-mediated addition of labeled deoxyuridine triphosphate nucleotides (dutps) at the 3 -OH end of DNA strand breaks (Nagata, 2000). Extensive genomic DNA fragmentation is a hallmark of apoptotic cell death but the method detects also necrotic cells and cells in the process of undergoing DNA repair (Elmore, 2007). In situ detection of DNA fragmentation was detected in all virus and saline injected eyes, but not in intact eyes in study I. DNA fragmentation was evident mainly at the earlier time points with only a few TUNEL-positive nuclei in LV and AAV injected eyes one month after gene delivery. In addition, in study II, a marked increase in TUNEL staining was detected two weeks after Ad subretinal injection with a marked decrease at the later time points. 6.3 DEVELOPMENT OF RETINAL NEOVASCULARIZATION AND AMD- ASSOCIATED SUBRETINAL NEOVASCULARIZATION (II-IV) In studies II and III, subretinal neovascularization was detected already two weeks after subretinal AdCre injection in the transgenic mice with a loxp-stop inactivated VEGF-A165 expression cassette. Cre gene delivery results in the production of Cre recombinase enzyme that recombines two DNA recognition sites called loxp (Nagy, 2000). Cre recombinase removes the stop sequence between loxp sites allowing the transgene expression in cells where Cre is active. This allows long VEGF-A165 expression even after AdV gene delivery (Leppänen et al., 2006). Ocular neovascularization is a complex pathological disorder associated with many ocular diseases (Campochiaro, 2015). As the eye is easily reached and transparent organ, the retina and ocular vasculature can be studied non-invasively in living organisms (Ofri, 2008) by using FA and OCT imaging (American Academy of Ophthalmology, 2018; Arevalo et al., 2009). We used high-speed spectrometer SD-OCT with improved sensitivity and as well as FA imaging at the beginning of the experiment and then at different time points in studies II-IV to detect vascular and retinal morphologies. When we compared the imaging results with the basic histological staining and capillary area, it was concluded that the changes seen in a living mouse via imaging methods correlate well with findings seen in samples taken post-mortem. This encourages the use of clinical methods to be exploited in animal studies, thus making it possible to study the development of the neovascularization and real-life blood flow in the mouse eye. The imaging of the mouse is fast, painless and non-invasive, only FA does entail the intraperitoneal injection of a clinically used contrast agent. As each mouse can be imaged several times during the study, fewer animals need to be involved. FA and OCT were used in studies II and III to detect subretinal neovascularization and in study IV to detect retinal neovascularization. As the transparency of the cornea and lens is essential for OCT and FA, a few mice could not be imaged due dry cornea or cataract. OCT and FA changes in the retina and its vasculature tell about edema, blood flow and permeability of the vessels in the eye. The hallmark of AMD is nevertheless the formation of new blood vessels underneath the normally avascular retina. CD34 antibody binding to the vascular endothelium was used to analyze the capillary area subretinally and in the retina. Both studies II and III showed subretinal neovascular membrane common to AMD. Conversely, the transgenic mice with a Fbn-1 mutation and ApoE deficiency did not present an excessive vasculature in the retina in study IV. In RVO and RVA retinal and optic disc neovascularization occurs in around 15 % of patients (Hayreh and Zimmerman, 2012; Mason et al., 2015). Furthermore, only few mouse models of induced RVO show neovascularization (Uddin et al., 2017; Zhang et al., 2007).

78 54 In study II, when the capillary area decreased, the fibrotic area increased at the same site. Control LacZ resulted in only faint Sirius Red positive area indicating that the fibrosis was not a consequence of the subretinal injection but truly represented the Cre induced VEGF- A165 expression. In AMD patients as well, the subretinal fibrosis leads to the formation of an abnormal vascular network with both active and inactive fibrotic choroidal neovessels (Miere et al., 2015). AMD may develop a disciform scar as a response to neovascularization-related wound healing (Ishikawa et al., 2016a). In study II, the development of a fibrovascular membrane reflects a rapid progression of AMD whereas in humans, it takes years to develop (Daniel et al., 2018; Sarks et al., 2006). In AMD patients, the subretinal fibrosis is often seen as part of the atrophic region in the retina (Channa et al., 2015; Daniel et al., 2018). Similarly to the situation in our mouse models, atrophy of RPE and photoreceptor cells occurs in areas of regressed CNV (Channa et al., 2015; Sarks et al., 2006). Although subretinal fibrosis can have a significant impact on a patient s vision, especially if located in the subfoveal area (Channa et al., 2015; Daniel et al., 2018), there are only few AMD mouse models with subretinal fibrosis (Ishikawa et al., 2016a, 2016b). Retinal thickness and morphology, as well as the retinal vasculature were unchanged showing no signs of retinal vascular occlusion or retinal neovascularization after 12-week high-fat diet in transgenic mice with vulnerable plaques (Study IV). However, in humans hyperlipidemia and atherosclerosis are highly correlated with retinal vessel occlusions affecting the occurrence of thrombosis and emboli (Ehlers and Fekrat, 2011; Varma et al., 2013). Nevertheless, at present there is no animal model spontaneously expressing retinal artery or vein occlusion but instead they are induced using either surgery or laser (Khayat et al., 2017; Minhas et al., 2012). Studies II and III found evidence also of other biochemical markers common to AMD mostly at the site of the neovascular membrane. As inflammation has a significant role in AMD, it is not surprising that macrophages participate in early and advanced AMD (Cherepanoff et al., 2010; Skeie and Mullins, 2009). Macrophages express several cytokines and growth factors, including VEGF (Grossniklaus et al., 2002), thus possibly promoting the growth of CNV. In studies II and III, we detected an increased number of F4/80 positive macrophages that have been shown to be present in CNV in mice also in other studies (Nagai et al., 2014; Yang et al., 2016). F4/80 antibody stains both blood-derived macrophages and microglia, a type of specialized tissue resident macrophage in the retina (Caicedo et al., 2005). A previous mouse study has shown that macrophages infiltrating CNV are recruited from the blood. We found that F4/80 positive cells were detected surrounding autofluorescent deposits at later time points. In humans, the presence of extracellular deposits is associated with macrophage recruitment (Cherepanoff et al., 2010) and it is thought to have a role in the removal of drusen and other waste products (Forrester, 2003). Detected autofluoresence is likely caused by lipofuscin yellow-brown pigment granules composed of lipid containing residues of lysosomal digestion (Danis et al., 2015). The accumulation of lipofuscin into macrophages and microglia may interfere with their ability to clear debris and thus it could contribute to the pathogenic mechanisms of AMD (Lei et al., 2012; Xu et al., 2008), although the accumulation of lipofuscin increases with age also in healthy humans (Danis et al., 2015) and mice (Xu et al., 2008). In study II, GFAP staining was detected also in Müller cells in the majority of Cre injected mice. The finding is the same as is seen in AMD patients with geographic atrophy (Wu et al., 2003). 6.4 THE EFFECTS OF ANTI-VEGF ANTIBODY IN VITRO AND AFTER LENTIVIRUS-MEDIATED GENE THERAPY (III) Inhibition of AMD-associated subretinal neovascularization was studied after intravitreal gene therapy. In these experiments, the sequence of murine antibody A.4.6.1, (Kim et al., 1992), a former version of humanized bevacizumab (Avastin ) (Presta et al., 1997) was

79 55 cloned into the LV backbone. Therefore, it was expected that the protein produced from plasmid encoding the α-vegf antibody would display an almost identical binding profile to VEGF-A165 than Avastin in Western Blotting, ELISA and cell based assay. The cancer-drug Avastin is frequently administered as off-label use to treat neovascular AMD (The CATT Research Group, 2011). Thus, we studied intravitreal injection of LV-α- VEGF in the treatment of Cre induced neovascular AMD. Decreased capillary and subretinal membrane area, as well as a trend towards a smaller fibrotic area were seen in LV-α-VEGF group compared with LV-GFP control group. Nevertheless, there were no changes in retinal thickness nor hyperfluorescence. LV vectors are known to induce sustained transgene expression (Auricchio et al., 2001), that is still seen seven days after intravitreal gene transfer (Kostic et al., 2003), whereas adenovirus induces rapid transgene expression (Hoffman et al., 1997). Thus, the LV vector was injected one week before induction of VEGF-A165 expression and neovascularization with AdCre gene transfer. The mice were examined two weeks after Cre injection, as the maximum neovascularization had seen at that point in study II. Nevertheless, the effect of LV-α-VEGF to suppress neovascularization at later time points would be clinically relevant. As α-vegf showed almost identical binding profiles and biological activity compared with Avastin, the transgene is considered suitable for anti-angiogenic gene therapy. Avastin has been studied in AAV-mediated genetic delivery for ocular neovascularization with positive results have been reported in the suppression of neovascularization for up to six months (Mao et al., 2011), thus choosing another vector could result in a more beneficial outcome. In addition, Avastin could serve a proper control also in vivo.

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81 57 7 Conclusions Based on this thesis, the following conclusions can be made: I II III IV Transduction efficiency and transgene expression duration in mouse retinal tissues and side effects vary between adeno-, baculo-, adeno-associated and lentiviral vectors after intravitreal gene transfer. Baculoviral gene transfer results in the development of only single GFP-positive cells in the mouse retina only three days after injection. Adenoviral vector injection leads to transient gene expression and the strongest immune reaction. After lentiviral and adenoassociated viral vector gene transfer, transgene expression lasts for several months. The values of a blood chemistry screen do not differ between virus injected and naïve mice. The control injection evokes some adverse effects such as glial cell activation and in situ detection of DNA fragmentation are seen at the same level in saline and virus injected mice. Subretinal adenoviral Cre gene delivery to transgenic mice with a loxp-stop inactivated human VEGF-A165 expression cassette results in VEGF-A165 expression in the eye and choroidal neovascularization. Neovascularization develops into the fibrovascular membrane within 12 weeks. Besides neovascularization, mice show other age-related macular degeneration related findings such as increased retinal thickness, hyperfluorescence and drusen-like deposits. Control LacZ-injection causes minor outer retinal layer atrophy. Lentiviral vector coding α-vegf antibody acts similarly as bevacizumab in VEGF-A165 binding and biological activity assays. Intravitreal gene transfer decreases the capillary area at three weeks after injection. There is also a trend towards a smaller fibrovascular membrane and reduced VEGF-A165 expression, although no difference after control injection and lentiviral α-vegf antibody injection in retinal thickness or in hyperfluorescence is detected. 12-week high-fat diet is not sufficient to induce retinal vessel occlusion in mice with a fibrillin-1 mutation and apolipoprotein E deficiency. The mice do not show signs of ocular abnormalities in either histology or imaging. Furthermore, these mice do not show evidence of anomalous morphology in their vasculature or ganglion cells or any other types of immunohistochemical alterations. The thesis studies are summarized in Figure 16. In conclusion, intraocular gene transfer in the mouse eye provided information about different viral vectors. Novel mouse models for intraocular neovascularization were assessed. We showed that atherosclerosis and vulnerable plaques are not sufficient to induce spontaneous retinal vessel occlusion in the mouse eye. A novel, clinically relevant mouse model was developed for neovascular AMD.

82 58 Additionally, a lentivirus mediated antibody-based anti-angiogenic gene therapy was created. Although it reduced neovascularization in the mouse s eye and further studies are needed to optimize the treatment. Nevertheless, intraocular viral vector mediated gene delivery was shown present a suitable method for creating neovascular mouse models and for testing novel anti-angiogenic therapies for pathological ocular conditions. Figure 16. Summary of the thesis. Intravitreal gene delivery resulted in GFP expression in the normal mouse retina and elsewhere in the eye. Subretinal gene delivery into mouse eye led to VEGF-A165 expression and subretinal neovascularization, which was reduced with anti-vegf antibody expressing gene therapy. Vulnerable atherosclerotic plaques did not cause retinal neovascularization in the mouse eye. Parts of the image from (Friedlander, 2007). GFP: Green fluorescent protein, RPE: Retinal pigment epithelium