INTRAVITREAL DELIVERY OF ADENO-ASSOCIATED VIRAL VECTORS IN DOGS:  MOVING TOWARD MORE EFFECTIVE RETINAL GENE THERAPY By Ryan Francis Boyd           A THESIS  Submitted to  Michigan State University  in partial fulfillment of the requirements  for the degree of    Comparative Medicine and Integrative BiologyMaster of Science  2016    ABSTRACT INTRAVITREAL DELIVERY OF ADENO-ASSOCIATED VIRAL VECTORS IN DOGS:  MOVING TOWARD MORE EFFECTIVE RETINAL GENE THERAPY By Ryan Francis Boyd Delivery of therapeutic transgenes to retinal photoreceptors using adeno-associated virus (AAV) vectors has traditionally required subretinal injection. Recently, retinal transduction efficiency following intravitreal injection (IVT) has been improved in rodent models through use of capsid-mutant AAV vectors; but remains limited in large animal models. Reasons for this inter-species variation remains undefined. The studies described herein compare the performance of newly developed AAV vectors containing capsid amino acid substitutions following IVT in dogs. The ability of these novel vectors to transduce inner and outer retinal cell populations was determined and compared to prior reports in dogs as well as other large animal models. Multiple previously described barriers to IVT retinal gene therapy were evaluated and manipulated to assess their specific role in attenuating the performance of the vectors. The ability of two promoter constructs to restrict reporter transgene expression to photoreceptors was also evaluated. When progressive intraocular inflammation developed during one investigation, the study plan was modified to allow detailed characterization of the etiology as a secondary goal. The collective results provide crucial insight into factors impacting AAV retinal transduction efficiency, and guidance in avoiding adverse effects following IVT administration of vectors in dogs.  Ultimate translation of these findings into the human gene therapy field will provide groundwork for maximizing safety and efficacy of IVT retinal AAV gene therapy for patients affected by both heritable and acquired blinding retinal diseases.  iii          I would like to dedicate this thesis to my family, without their endless support this training program would not have been possible. To my wife Gerri, who put her career on hold to allow me to fulfill my career dream, and whose hours of work in loving and caring for our children and our household enabled me to spend the time necessary to complete this project. To my children, Emily and James, who were somehow understanding of my absence at dinnertime and weekends when work required, they were always able to make me smile no matter how hard the day was. And to my parents, Richard and Ruby, my mother-in-law Ginny, and my late father-in-law Steve, their advice and encouragement always helped me to keep perspective of what matters most, and their sacrifice of time spent together as a family cannot be replaced.  iv  ACKNOWLEDGEMENTS  I would like to express gratitude to my supervisor Dr. Joshua Bartoe for his support, mentorship, and collegiality throughout this training program and research project. I would like to extend thanks to my committee members Drs. Simon Petersen-Jones, András Komáromy, and Ingeborg Langohr, who never hesitated to answer questions and offered constant guidance and instruction for both my scientific and clinical training. I would also like to acknowledge Dr. Dodd Sledge, Dr. Laurence Occelli, Dr. Kristen Gervais, Janice Querbin, Kristin Koehl, and Christine Harman for their assistance and instruction on the laboratory techniques needed to complete this project.  I thank the research team at the University of Florida Departments of Ophthalmology and Pediatrics, in particular Drs. Sanford and Shannon Boye, for producing the vectors used in the project and providing substantial support when unexpected changes needed to be made during the study.     v  TABLE OF CONTENTS  LIST OF TABLES ..................................................................................................................... viii LIST OF FIGURES ..................................................................................................................... ix KEY TO ABBREVIATIONS ...................................................................................................... x CHAPTER 1 .................................................................................................................................. 1 INTRODUCTION ....................................................................................................................... 1 1.1 Retinal Gene Therapy ............................................................................................................ 1 1.1.1 Inherited retinal dystrophies ........................................................................................... 1 1.1.2 The development of retinal gene therapy for LCA type II ............................................. 3 1.1.3 Animal models of retinal dystrophy ............................................................................... 4 1.1.4 Notable advances in gene therapy through the use of animal models ............................ 6 1.1.5 Gene therapy for more prevalent retinal degenerations ................................................. 8 1.2 Adeno-Associated Virus ....................................................................................................... 9 1.2.1 Properties of AAV vectors ............................................................................................. 9 1.2.2 AAV capsid serotypes .................................................................................................. 10 1.2.3 AAV vector size limitations ......................................................................................... 11 1.2.4 AAV cellular binding and transport ............................................................................. 12 1.2.5 Nuclear processing of AAV ......................................................................................... 13 1.2.6 Intracellular barriers to AAV transduction ................................................................... 15 1.3 AAV for Retinal Gene Therapy .......................................................................................... 16 1.3.1 Delivery route of AAV vectors for retinal gene therapy .............................................. 16 1.3.2 Extracellular barriers to intravitreal retinal gene therapy ............................................. 19 1.3.3 Intravitreal AAV for gene therapy in animal models ................................................... 22 1.4 Immune Responses to AAV ................................................................................................ 25 1.4.1 Ocular defense mechanisms ......................................................................................... 25 1.4.2 Humoral response to AAV ........................................................................................... 26 1.4.3 Cell-mediated responses to AAV ................................................................................. 27 1.5 Study Hypotheses and Specific Aims ................................................................................. 28 1.5.1 Safety and efficacy study.............................................................................................. 29 1.5.2 Photoreceptor-targeted Vector Study ........................................................................... 30 1.5.3 Vitrectomy Study.......................................................................................................... 32 CHAPTER 2 ................................................................................................................................ 34 SAFETY AND EFFICACY STUDY ....................................................................................... 34 2.1 Materials and Methods ........................................................................................................ 34 2.1.1 Animals......................................................................................................................... 34 2.1.2 Ophthalmic examinations and retinal imaging ............................................................. 34 2.1.3 Vectors and vector preparation ..................................................................................... 35 2.1.4 Intravitreal injections .................................................................................................... 35 2.1.5 Globe collection............................................................................................................ 37 2.1.6 Immunohistochemistry ................................................................................................. 38 vi  2.1.7 Speed of onset evaluation ............................................................................................. 40 2.1.8 Cellular transduction efficiency ................................................................................... 40 2.1.9 Statistical evaluation ..................................................................................................... 40 2.2 Results and Discussion ........................................................................................................ 40 2.2.1 Ophthalmic examinations ............................................................................................. 40 2.2.2 Speed of GFP expression onset .................................................................................... 41 2.2.3 RGC layer transduction efficiency ............................................................................... 43 2.2.4 Extraocular GFP expression ......................................................................................... 45 CHAPTER 3 ................................................................................................................................ 48 PHOTORECEPTOR-TARGETED VECTOR STUDY ........................................................... 48 3.1 Introduction ......................................................................................................................... 48 3.2 Results and Discussion ........................................................................................................ 50 3.2.1 GFP expression and safety ........................................................................................... 50 3.2.2 AAV vector retinal transduction efficiency ................................................................. 54 3.2.3 AAV vector retinal tropism .......................................................................................... 56 3.2.4 Distribution of photoreceptor transduction .................................................................. 60 3.3 Materials and Methods ........................................................................................................ 63 3.3.1 Animals......................................................................................................................... 63 3.3.2 AAV vectors ................................................................................................................. 64 3.3.3 Intravitreal injections .................................................................................................... 65 3.3.4 Ophthalmic examinations and imaging ........................................................................ 65 3.3.5 Eyecup collection and sectioning ................................................................................. 66 3.3.6 Immunohistochemistry and cell quantification ............................................................ 66 3.3.7 Evaluation of ILM thickness ........................................................................................ 67 3.3.8 Statistical analysis ........................................................................................................ 68 CHAPTER 4 ................................................................................................................................ 69 VITRECTOMY STUDY .......................................................................................................... 69 4.1 Introduction ......................................................................................................................... 69 4.2 Results and Discussion ........................................................................................................ 71 4.2.1 Preliminary safety and dose-range finding study ......................................................... 71 4.2.2 Ophthalmic examinations and GFP fluorescence......................................................... 72 4.2.3 AAV vector retinal transduction efficiency and tropism ............................................. 79 4.2.4 Microscopic evaluation of retinitis ............................................................................... 83 4.2.5 Immune response assays............................................................................................... 85 4.2.6 Biodistribution of AAV vector ..................................................................................... 88 4.3 Materials and Methods ........................................................................................................ 91 4.3.1 Animals......................................................................................................................... 91 4.3.2 Vitrectomy .................................................................................................................... 91 4.3.3 AAV vector .................................................................................................................. 92 4.3.4 Intravitreal injections .................................................................................................... 92 4.3.5 Ophthalmic examinations and imaging ........................................................................ 93 4.3.6 Immune reaction assays ................................................................................................ 93 4.3.7 Eyecup collection and sectioning ................................................................................. 94 4.3.8 Immunohistochemistry and cell quantification ............................................................ 94 4.3.9 AAV Biodistribution analysis ...................................................................................... 96 vii  4.3.10 Statistical analysis ...................................................................................................... 96 CHAPTER 5 ................................................................................................................................ 97 CONCLUSIONS AND FUTURE DIRECTIONS .................................................................... 97 5.1 Photoreceptor-Targeted Vector Study ................................................................................. 97 5.2 Vitrectomy Study .............................................................................................................. 100 5.3 Conclusion ......................................................................................................................... 102 BIBLIOGRAPHY ..................................................................................................................... 103    viii  LIST OF TABLES  Table 1. Inherited retinal dystrophies based on number of causative genes. ................................. 2 Table 2. Non-rodent naturally occurring animal models of inherited retinal dystrophies. ........... 6 Table 3. Binding affinities and retinal cell tropism of different AAV capsid serotypes................ 11 Table 4. Vector dosing arrangement with vector titer dose and timing of euthanasia for safety and efficacy study. ......................................................................................................................... 37 Table 5. Primary and secondary antibodies used for retinal immunohistochemistry on safety and efficacy study. ................................................................................................................................ 39 Table 6. RGC layer transduction efficiency (%) by retinal region (refer to Figure 1) for safety and efficacy study. ......................................................................................................................... 43 Table 7. Intravitreal dosing arrangement and AAV vector descriptions for photoreceptor-targeted vector study. .................................................................................................................... 51 Table 8. Mean photoreceptor transduction and statistical comparison of regions for photoreceptor-targeted vector study. ............................................................................................ 61 Table 9. Primary and secondary antibodies used for retinal immunohistochemistry for photoreceptor-targeted vector study. ............................................................................................ 67 Table 10. Average (peak) retinal ganglion cell layer transduction percentage from pilot safety and efficacy study. ......................................................................................................................... 72 Table 11. Definition of grading scheme for anterior uveitis ........................................................ 72 Table 12. Peripheral blood mononuclear cell (PBMC) stimulation index following challenge with vector-derived antigens. ........................................................................................................ 87 Table 13. Biodistribution of AAV genomes determined by qPCR ................................................ 89 Table 14. Primary and secondary antibodies used for retinal immunohistochemistry on vitrectomy study. ........................................................................................................................... 95    ix  LIST OF FIGURES Figure 1. Diagram depicting the nine eyecup sections embedded from each eye in the safety and efficacy study. ................................................................................................................................ 38 Figure 2. Enhanced in vivo 471 nm-fluorescent light fundoscopic images demonstrating retinal GFP expression. ............................................................................................................................ 42 Figure 3. Immunohistochemical labeling of retinal cryosections with the antibody NeuN demonstrating regional variation in transduction efficiency observed with both vectors............ 44 Figure 4. GFP expression within the optic nerves of dogs euthanized at 8 weeks post-IVT. ....... 46 Figure 5. GFP expression within the LGN and superior colliculus of dogs euthanized at 8 weeks post-IVT......................................................................................................................................... 47 Figure 6. Photoreceptor transduction efficiency following IVT of AAV2 (quad Y-F + T-V) IRBP and AAV2 (quad Y-F) IRBP. ......................................................................................................... 52 Figure 7.  Localization of maximal photoreceptor transduction following IVT of AAV2 (quad Y-F + T-V) IRBP and AAV2 (quad Y-F) IRBP. ................................................................................... 53 Figure 8.  Photoreceptor-specific transduction following IVT of AAV2 (quad Y-F) IRBP and AAV2 (quad Y-F + T-V) IRBP. ..................................................................................................... 58 Figure 9. Retinal transduction following intravitreal injection of AAV2 (quad Y-F) GNAT2/IRBP and AAV2 (quad Y-F + T-V) GNAT2/IRBP. ................................................................................. 59 Figure 10. Assessment of regional retinal inner limiting membrane thickness. ........................... 62 Figure 11. Comparison of ganglion cell layer transduction efficiency of non-vitrectomized and vitrectomized eyes following IVT of AAV2 (triple Y-F + T-V)...................................................... 74 Figure 12. Demonstration of retinitis following IVT of AAV2 (triple Y-F + T-V). ....................... 77 Figure 13. Comparison of GFP fluorescence in the anterior segments of vitrectomized and non-vitrectomized eyes. ........................................................................................................................ 78 Figure 14. Regional variation observed in non-vitrectomized eyes of all three dogs................... 81 Figure 15. Transduction of all retinal layers following IVT of AAV2 (triple Y-F + T-V), driven by the constitutive chicken ................................................................................................................. 82 Figure 16. Immunohistochemical characterization of the mononuclear cell populations in eyes with retinitis. ................................................................................................................................. 85   x  KEY TO ABBREVIATIONS    4',6-diamidino-2-phen DAPI Adeno-associated virus...... AAV Age-related macular  AMD Anterior chamber-associated immune d  APCs Brain-derived neurotr BDNF - CBA Ciliary neurotro CNTF Cluster of diffe CD Confocal scanning la  Glial fibril Green fluorescent pro   Inherited retinal dystrop....... IRD Inner limiti Inner nuc Interphotoreceptor retinoid- Intravitreal injection..IVT Inverted  xi  Lateral  Leber congenital amaurosis. LCA  Neutralizi  O Paraformaldehyde... Peripheral blood  Phenylala Phospha Phytohemagglutinin Protein kinase C alpha Quantitative PCR Retinal ganglion cell..... RGC Retinal pigment epithelium  Threonine Tyrosine Valine Vascular endothelial growth factor VEGF Viral protein... VP  1  CHAPTER 1 INTRODUCTION 1.1 Retinal Gene Therapy 1.1.1 Inherited retinal dystrophies Inherited retinal dystrophies (IRDs) are a significant cause of blindness in humans, affecting 1 in 2,000 people worldwide.1, 2  IRDs produce visual impairment as a result of inherited gene mutations that affect normal photoreceptor and retinal pigment epithelium structure and/or function.3  A typical classification scheme divides IRDs based on the principally affected cell population, with groups including rod-cone dystrophies, cone-rod dystrophies, cone dystrophies, and retinal pigmented epithelium dystrophies.4  IRDs are often genetically heterogeneous, with mutations in several genes responsible for versions of a single described clinical disease (Table 1).  In addition, different mutations in a single gene can be responsible for phenotypic changes observed for distinct clinical disease categories.  One example is the gene peripherin/RDS, in which unique mutations can produce rod-cone dystrophies, cone-rod dystrophies, or cone dystrophies.2 IRDs can present with a wide variety of phenotypes, considerable differences in the age of disease onset and rate of progression.5  They can affect vision at birth due to abnormal retinal development, or can result in dysfunction or degeneration of the retina following normal development.6  For example, Leber congenital amaurosis (LCA) type 2, caused by a mutation in RPE65, is typically characterized by a functional level of visual acuity and normal retinal architecture in younger patients.  It exhibits slow disease progression, often not resulting in significant visual impairment and degenerate photoreceptor morphology until the third decade of 2  life.  In contrast, a mutation in AIPL1 causing LCA type 4 results in severe visual impairment at birth and rapid disease progression.3 Table 1. Inherited retinal dystrophies based on number of causative genes. (modified from https://sph.uth.edu/RetNet; (current as of October 30, 2015))   Disease Group Mode of Inheritance Genes Retinitis Pigmentosa Recessive, Dominant, X-linked 56 Syndromic/systemic Diseases with Retinopathy Recessive, Dominant, X-linked 55 Cone and Cone-rod dystrophies Recessive, Dominant, X-linked 19 Bardet-Biedl Syndrome Recessive 17 Usher syndrome Recessive 13 Congenital Stationary Night Blindness Recessive, Dominant, X-linked 13 Macular Degeneration Recessive, Dominant 12 Leber Congenital Amaurosis Recessive, Dominant 12 Optic Atrophy Recessive, Dominant, X-linked 4 Other Retinopathies Recessive, Dominant, X-linked 39  Retinitis pigmentosa, a group of progressive rod-cone photoreceptor degenerations, is the most common IRD and has a variable regional prevalence from 1:3,500-4,000 people in the US and Europe to as high as 1:930 people in Southern India.7, 8    Examples of the occurrence rates of other IRDs include Stargardt disease with a prevalence of 1:10,000, juvenile retinoschisis at 1:15,000-30,000, achromatopsia at 1:30,000, and LCA at 1:33,000.4, 6  As of October 30, 2015, mutations in 240 genes have been identified as causative for known IRDs (https://sph.uth.edu/RetNet). Treatment of IRDs must be tailored to the specific gene and mutation type involved, depending on whether it prevents functional protein production or results in the creation of an abnormal protein that disrupts normal cell function.9  Common methods include gene augmentation 3  strategies in the case of autosomal recessive mutations that deliver a copy of the wild-type gene to the affected cell population, leading to endogenous production of the deficient protein.10  Autosomal dominant mutations that cause a toxic gain-of-function (production of protein with harmful effects) or dominant negative (non-functional protein inhibits normal protein assembly or delivery) effects present a more challenging scenario.  In these diseases, silencing of the mutant mRNA transcript is often required in addition to gene augmentation.11, 12 1.1.2 The development of retinal gene therapy for LCA type II In 1997, Gu et al. described a mutation in RPE65 that causes LCA type II, which affects 1 in 1,000,000 newborns.13, 14  This led to the development in 1998 of a genetically engineered mouse model with a null Rpe65 mutation, which exhibited the disease phenotype of LCA type II, characterized by absent rod photoreceptor function during scotopic ERG assessment.15   This mouse model revealed that a deficiency in the metabolism of all-trans-retinyl ester to 11-cis-retinol at the level of the retinal pigment epithelium causes this photoreceptor dysfunction.15  In 1999, Veske et al. described that a naturally occurring LCA-like disease in Briard dogs was caused by a mutation in Rpe65.16  Through the use of the Rpe65-/-  animal models, groundbreaking studies occurred reporting rescue of visual function in the mouse following oral supplementation of 9-cis-retinal, and in the dog following subretinal delivery of a recombinant adeno-associated virus (AAV) vector carrying normal dog Rpe65 cDNA.17-19   Further evaluation in the canine Rpe65-/- model suggested use of subretinal AAV vectors for delivery of RPE65 cDNA would be a safe and sustainable therapeutic option for LCA type II in humans.20-22  In 2007, phase I/II clinical studies were undertaken at the Scheie Eye Institute of the University of Pennsylvania, the University College London, and the University of Florida 4  (UF)/Sh.  All three trials utilized AAV to deliver the normal RPE65 gene for treatment of LCA type II, and all reported subretinally-delivered AAV resulted in no significant adverse effects. 23-25  Clinical response to treatment was variable between and within the three studies, likely attributable to the advanced stage of disease in the patients enrolled, who were between 17 and 26 years of age.25  Additional evaluations in a larger patient population including children as young as eight years of age suggested intervention earlier in the disease process would be more effective at improving visual function.26, 27  The successes observed in these preliminary studies have led to the first ever phase III gene therapy trial in the US, at the of LCA type II (http://www.clinicaltrials.gov), with a plan to commercialize and market the treatment for public use in the future (http://www.sparktx.com/pipeline/inherited-retinal-dystrophies). The development of safe and effective AAV retinal gene therapy for such a rare disease is pivotal to the advancement of gene therapy for other more prevalent diseases. 1.1.3 Animal models of retinal dystrophy The successes experienced in preclinical trials on Rpe65-/- dogs have led to continued use of other animal models of IRDs for preclinical gene therapy evaluations.  Following the identification of a causative gene mutation in humans, transgenic rodent strains can often be engineered to express the same mutation, allowing further characterization of disease mechanisms and providing animals that serve as subjects in preliminary therapeutic evaluations.  A search of the database at The Jackson Laboratory, a leading provider of transgenic mouse strains, reveals that they carry 34 live mouse strains of Retinitis Pigmentosa alone (http://jaxmice.jax.org).  The abundance of these rodent models allows the cost-effective evaluation of a wide variety of novel therapies, which is of utmost importance to the future 5  development of effective treatments for the currently incurable IRDs.5  Although rodents serve as an essential preliminary model for therapeutic efficacy evaluations, their comparative retinal anatomy to humans is limited most notably in globe size and the proportion of rod to cone photoreceptors.  The small size of rodent eyes and their larger lens:globe volume also make it very difficult to perform the exact surgical procedures required for delivery of gene therapy vectors into the human eye.28  The rodent retina is characterized by average rod:cone photoreceptor density ratios of approximately 35:1 across the entire retina, compared to humans with an average density of 20:1, with a peak in the periphery at 30:1 and a rod-absent central foveal region.29, 30  Larger animals such as dogs, non-human primates, and pigs offer a more comparative globe size, with rod:cone photoreceptor distributions closer to that of humans.  The dog retina has a central density of 22.8:1, and a peripheral density of 41.4:1.31  The monkey retina has an average density similar to that of humans at 20:1, and also has a rod-absent fovea.32  The pig retina is cone-rich, with an average density of 8:1, with variations from a central density of 3:1 to a peripheral density of 16:1.33 The development of transgenic large animal models is both time and cost prohibitive, but has been accomplished by Petters et al., who developed a genetically-engineered pig model of Retinitis Pigmentosa in 1997.34  Fortunately, a number of naturally occurring large animal models of inherited retinal dystrophy exist, and have played important roles in proof-of-concept studies for retinal gene therapy (Table 2).12, 35-37     6  Table 2. Non-rodent naturally occurring animal models of inherited retinal dystrophies. (adapted from Baehr and Frederick 35; Steiger et al. 38; and Petersen-Jones and Komaromy 37) Animal Model Human IRD Gene Alaskan Malamute dog Achromatopsia Cngb3 German Shorthaired Pointer dog Achromatopsia Cngb3 German Shepherd dog Achromatopsia Cnga3 rcd3 Cardigan Welsh Corgi dog Retinitis Pigmentosa Pde6a rcd1 Irish Setter dog Retinitis Pigmentosa Pde6b Sloughi dog Retinitis Pigmentosa Pde6b American Staffordshire terrier Retinitis Pigmentosa Pde6b English Mastiff dog Retinitis Pigmentosa Rho Papillon dog Retinitis Pigmentosa Cngb1 Collie dog Retinitis Pigmentosa C1orf36 Norwegian Elkhound dog Retinitis Pigmentosa Stk38l Golden Retriever dog Retinitis Pigmentosa Slc4A3 Schapendoes dog Retinitis Pigmentosa Ccdc66 Basenji dog Retinitis Pigmentosa Sag Gordon Setter dog Retinitis Pigmentosa C2orf71 Multiple dog breeds Retinitis Pigmentosa Prcd XLPRA1 Siberian Husky dog Retinitis Pigmentosa Rpgr XLPRA2 Mongrel dog Retinitis Pigmentosa Rpgr rdAc cat Leber congenital amaurosis Cep290 Swedish Briard-Beagle dog Leber congenital amaurosis Rpe65 rd chicken Leber congenital amaurosis Gucy2e Dachshund dog Cone-rod Dystrophy Rpgrip1 Dachshund dog Cone-rod Dystrophy Nphp4 Glen of Imaal Terrier dog Cone-rod Dystrophy Adam9 Pit Bull Terrier dog Cone-rod Dystrophy Iqcb1 Mastiff dog breeds Best Macular Dystrophy Best 1 Coton de Tulear dog Best Macular Dystrophy Best 1 Lapponian Herder dog Best Macular Dystrophy Best 1   1.1.4 Notable advances in gene therapy through the use of animal models Park et al. utilized the Rs1-KO mouse model of human X-linked juvenile retinoschisis to establish both structural and functional retinal rescue using an AAV vector expressing 7  retinoschisin.39  This study demonstrated two important advancements in retinal gene therapy.  First, the AAV vector administered intravitreally to Rs1-KO mice was able to efficiently transduce the cells of the outer retina, a capability not observed in wild-type mice.  This potential was enhanced when the vector was administered at 8 weeks of age compared to 7 months of age.  Second, the authors used a retinoschisin promoter to induce retina-specific gene expression, an important safety consideration when evaluating viral vectors with potential exposure to off-target tissues.39  This concept of retina cell-specific gene therapy was supported by the work of Komaromy et al. in 2010, who demonstrated functional vision rescue in both the missense and null CNGB3 mutation canine models of achromatopsia.40  They showed that use of a long variant of the human red cone opsin promoter was able to specifically target long/medium-wavelength absorbing cones in canine retinas.  Following subretinal administration of the AAV vector, previously deficient cone photoreceptor function and photopic vision were restored. In 2011, Millington-Ward et al. reported encouraging results from a study evaluating gene therapy in the P347S mouse model of rhodopsin-linked autosomal dominant retinitis pigmentosa.41  This treatment involved subretinal coinjection of two different AAV vectors, one expressing an RNA interference-based rhodopsin suppressor, and the other expressing a rhodopsin replacement gene resistant to degradation by the RNA interference suppressor.  The authors observed improvements in both electroretinographic and histological evaluations following therapy.  There is great clinical significance in this animal model work, because it demonstrated a mutation-independent method for treatment of a human condition with over 100 dominant causative mutations.14  8  1.1.5 Gene therapy for more prevalent retinal degenerations  Gene therapy can potentially provide benefit in more prevalent retinal diseases including glaucoma, diabetic retinopathy, and age-related macular degeneration (AMD), which have likely have a combination of genetic, life-style, and/or environmental etiologies.7  The approach to treatment of these conditions does not necessarily involve the correction of a specific gene mutation; instead the focus is on delivery of substances that enhance retinal neuronal survival or inhibit disease progression.  For example, delivery of neurotrophic growth factors to the inner retina of animal models of glaucoma has resulted in promising results.  In a rat optic nerve transection model, Hellstrom et al. showed that increased retinal ganglion cell (RGC) survival and axonal regeneration occurs with intravitreal administration of an AAV vector expressing ciliary neurotrophic factor (CNTF) in combination with short-term pharmacologic support with exogenous CNTF and a cyclic adenosine monophosphate analog.42  In 2012, Ren et al. explored the use of simultaneous IVT of an AAV vector expressing brain-derived neurotrophic factor (BDNF) and a bolus of recombinant BDNF protein following experimental intraocular pressure elevation in rats.43  The authors reported enhanced RGC survival, superior visual function measured by visual evoked potentials, and improved maintenance of visual acuity measured by a choice water maze test. Therapies for diabetic retinopathy and AMD are centered on the suppression of vascular endothelial growth factor (VEGF), a regulator of angiogenesis that is implicated in the uncontrolled neovascularization that characterizes both diseases.44  Lukason et al. described intravitreal administration of an AAV vector encoding the chimeric anti-VEGF protein sFLT01 in both non-human primates and mice.45  When the retinas of the animal models were subjected 9  to laser-induced choroidal neovascularization following AAV administration, a significant reduction in the neovascular response was noted in the eyes receiving AAV-sFLT01.45 1.2 Adeno-Associated Virus 1.2.1 Properties of AAV vectors Adeno-associated virus is a member of the Dependovirus genus of the Parvoviridae family.46  Viral structure includes an icosahedral protein capsid approximately 25 nm in diameter, which houses the single-stranded 4.7 kb DNA genome.47, 48  The genome consists of a rep gene encoding proteins needed for viral replication, a cap gene encoding capsid proteins, and inverted terminal repeats (ITRs) flanking the two genes.49  There are twelve different naturally occurring AAV serotypes known to infect humans, with over 100 additional variants identified but not yet serologically characterized.50, 51  The viruses are unable to replicate within an infected host in the absence of a helper virus, which could be a herpesvirus, papillomavirus, or adenovirus.52  Following helper virus co-infection of an AAV-infected cell, proteins produced by the helper virus activate AAV rep promoters, upregulating expression of the Rep proteins that then activate AAV replication at a highly efficient rate.53  AAV is considered nonpathogenic, and has not been associated with disease even when present in immunocompromised individuals.54  The virus is ubiquitous within the human population, with prevalence of neutralizing antibodies to AAV serotype 2 most recently reported as being as high as 72% in a study conducted in France.55 AAV vectors are able to transduce post-mitotic cells resulting in stable transgene expression with low immunogenicity, an important consideration when evaluating safety and therapeutic potential.49, 56, 57  Another attractive property of AAV vectors when considering safety for retinal 10  gene therapy is they rarely integrate into the host genome, predominantly existing in an episomal state.46, 49, 53, 58  1.2.2 AAV capsid serotypes The AAV capsid is made up of an icosahedral arrangement of 60 copies of three viral proteins (VPs), with VP3 making up 90% of the capsid protein content.59  These three proteins are produced as a result of alternative splicing of the cap gene encoded within the AAV genome.60, 61  Twelve capsid serotype variants exist, with the structure of serotypes 1-9 described in greatest detail.  Each has an affinity for a specific primary glycosaminoglycan receptor, which in turn results in differences in tissue tropism between capsid serotypes (Table 3).50  Binding to this receptor is determined both by the topography of the capsid surface as well as the amino acid composition of surface-exposed regions.62-66  The surface-exposed amino acids, in particular tyrosine, serine, threonine, and lysine, are also targets for phosphorylation within host cells that can lead to proteasomal degradation of the virus.67-69  These capsid amino acids are popular targets for rational site-directed mutagenesis during recombinant vector development, which has been shown to both increase transduction efficiency of the vector as well as decrease recognition of the vector by the host immune system.68-70 One reason AAV vectors are the leading option for retinal gene therapy is they, unlike other viral vectors, have exhibited the ability to transduce all retinal cell populations.71  The ability to package a therapeutic transgene flanked by AAV2 ITRs into the capsid of other serotypes allows for the development of vectors that are tailored toward preferential targeting of certain retinal cell populations.72  For example, AAV capsid serotype 2 (AAV2) is able to transduce only the retinal pigment epithelium (RPE) and photoreceptors following subretinal delivery, while AAV8 11  and AAV9 are able to transduce inner retinal cell populations as well as RPE and photoreceptors.4  Incorporation of various promoters into the vector genome and manipulation of the capsid amino acid structure can drive even more stringent cell-specificity of AAV vectors.48   Table 3. Binding affinities and retinal cell tropism of different AAV capsid serotypes. (modified from Nonnemacher, et al.49) Serotype Glycan receptor Co-receptor Retinal Tropism AAV1 N-linked sialic acid Unknown Retinal pigmented epithelium73 AAV2 Heparan sulfate proteoglycan Fibroblast growth factor receptor 1, Hepatocyte factor growth receptor, Laminin, CD9 tetraspanin Retinal pigmented epithelium, Photoreceptors, Müller cells, RGCs 73 AAV3 Heparan sulfate proteoglycan Fibroblast growth factor receptor 1, Hepatocyte factor growth receptor, Laminin Photoreceptors, RGCs74 AAV4 O-linked sialic acid Unknown Amacrine cells, Müller cells74 AAV5 N-linked sialic acid Platelet-derived growth factor receptor Retinal pigmented epithelium, Photoreceptors, Müller cells73 AAV6 N-linked sialic acid, Heparan sulfate proteoglycan Epidermal growth factor receptor Amacrine cells, Müller cells, Bipolar cells, RGCs74 AAV7 Unknown Unknown Retinal pigmented epithelium, Photoreceptors73 AAV8 Unknown Laminin Retinal pigmented epithelium, Photoreceptors, Müller cells, RGCs73 AAV9 N-linked galactose Laminin Retinal pigmented epithelium, Müller cells73  1.2.3 AAV vector size limitations Size limitations exist when utilizing AAV vectors for retinal gene therapy.  Production of recombinant AAV vectors involves replacement of the rep and cap genes with the transgene of 12  interest, while retaining the flanking ITRs.49  The DNA genome of wild-type AAV is only 4.7 kb in size, and the transgene carrying capacity of manufactured recombinant vectors is limited by capsid serotype, with the largest being 8.9 kb.10, 75  This significantly impacts the ability to deliver large therapeutic transgenes, for example the ABCA4 gene responsible for Stargardt macular dystrophy.  It also restricts the use of large promoter sequences to drive cell-specific transgene expression, such as the photoreceptor targeted PR2.1 promoter, which would provide an additional safety measure by limiting off-target expression.11, 76  Transgene DNA can be constructed in a single-stranded manner, or in a self-complimentary sequence with complimentary inverted sequences separated by a mutated termination site.77  Advantages of the self-complimentary arrangement include improved vector efficiency and a more rapid transduction speed, while the main disadvantage is loss of roughly half of transgene carrying capacity of the vector.77, 78  Recent studies have successfully utilized hybrid vector methods that allow the delivery of one therapeutic transgene split between two different vectors.  Each vector must then transduce the same cell to achieve reassembly of the entire transgene via recombination of the two shorter DNA segments within the host cell nucleus.79, 80   1.2.4 AAV cellular binding and transport In order to transduce a target cell population, AAV vectors must first recognize and bind to surface receptors on the cell membrane.  Seisenberger et al. used a single-virus fluorescent labeling technique to map the interaction of individual viral particles with the cell membrane of cultured cells.  They found that AAV viruses touch the cell membrane an average of 4.4 times prior to cell entry or diffusion away from the cell.  They also reported that the penetration efficiency was 13%, suggesting that the cell membrane poses a significant barrier to AAV transduction.81  Following attachment to the cell membrane, AAV vectors are internalized by the 13  process of endocytosis.  This predominantly occurs through the formation of pits coated by the protein clathrin, although clathrin-independent endocytic processes can also occur.58  Internalization occurs very rapidly, at an average rate of 64ms.81   The formation of an endosome during internalization is a necessary process for AAV-mediated transduction of the host cell, as demonstrated by Sonntag et al.82  They showed that direct injection of AAV viral particles into the cytoplasm of host cells reduced transduction rate 100-fold.  This suggests that capsid processing must occur during endosomal transport to expose trafficking domains present on the viral capsid.  Capsid processing occurs in the Golgi apparatus and endoplasmic reticulum, where exposure to a lower pH environment and enzymatic proteins within the cellular compartments causes maturation of the viral capsid to prepare it for nuclear entry.49 1.2.5 Nuclear processing of AAV It is thought that intact AAV viral particles are taken up into the host cell nucleus, although this mechanism is poorly understood.49  Injection of anti-capsid antibodies into the host cell nucleus significantly inhibits transduction, signifying that an intact capsid is necessary for nuclear entry and genome delivery.82  Once inside the nucleus, capsid uncoating must occur prior to replication of the viral genome.  This is a rate-limiting step in AAV transduction of the host cell, and is variable dependent on the capsid serotype.83  Single-stranded AAV2 genomes packaged within an AAV2 capsid remain encapsidated within the host nucleus of mouse hepatocytes for as long as 6 weeks, while those packaged within AAV6 and AAV8 capsids undergo uncoating much more efficiently.83  This results in a higher overall transduction rate for capsids which undergo more rapid uncoating, due to the higher number of single-stranded genomes available for 14  annealing to each other to produce a more stable double-stranded DNA sequence, avoiding targeted degradation of the single-stranded sequence.83  The rate of viral uncoating is both serotype- and cell-specific, as AAV2 capsids uncoat faster in cardiac cells than AAV6 capsids, opposite that described in hepatocytes.84 Vectors carrying self-complimentary AAV genomes are more efficient at transducing cells than those with single-stranded genomes due to the fact that self-complimentary genomes become stable double-stranded sequences immediately after uncoating, without the need for a second complimentary single-stranded sequence or host cell DNA polymerases.83 Wild-type AAV has the ability to integrate its genome into the host cell genome at a specific gene-dense site on chromosome 19, termed AAVS1, containing the gene MBS85.53  The genome inserts in a manner that does not disrupt functional expression of the MBS85 gene.85  The removal of the portion of the AAV genome encoding Rep proteins, which is a standard practice during recombinant AAV vector production, prevents this site-specific integration from occurring.46, 57, 85, 86  Instead, they exist predominantly as episomal concantamers, or loops of double-stranded DNA that exist outside of the host cell genome but that still contribute to transduction.58  Recombinant AAV genomes are still capable of integration, albeit at a much lower rate than wild-type AAV, with published integration rates of between 0.06 and 0.2 vector genomes/cell in hepatocytes, and rates hypothesized to be even lower in non-dividing cells.58, 87  Despite these low integration rates, random viral genome integration in host cells has the potential to cause insertional mutagenesis, in which the integration of viral DNA results in disruption of an essential host gene and risks transformation of the host into a tumor-forming cell.10  Neonatal mice injected intravenously with an AAV2 vector produced hepatocellular adenocarcinoma at rates as high as 56%, compared to 8.3% of non-treated control mice.88 The 15  presence of this risk warrants the effort toward development of vectors that target the specific cell population of interest, to avoid unnecessary risk to non-target tissues. 1.2.6 Intracellular barriers to AAV transduction Prior to reaching the host cell nucleus, AAV particles are transported within endosomal compartments to the Golgi apparatus or endoplasmic reticulum, which then release an AAV particle with a fully processed capsid capable of nuclear import.49  During the endosomal transport of the virus, phosphorylation of capsid tyrosine and serine/threonine amino acid residues can occur.89, 90  This targets the virus for ubiquitination and subsequent proteasomal degradation within the host cell cytoplasm, which can significantly impact transduction efficiency.67  This host cell defense mechanism has been demonstrated not only in AAV2, but also in serotypes 5 and 8.91, 92 Novel vector engineering methodologies have been implemented in order to bypass the cellular proteasomal degradation pathways.  In 2008, Zhong et al. demonstrated that by substituting one of the surface-exposed tyrosine residues on an AAV2 vector with phenylalanine, they achieved a 30-fold increase in transduction efficiency compared to wild-type AAV2 in vivo.70  This occurred at 10-fold lower dose of mutant AAV2 than the wild-type AAV2.  The replacement of capsid tyrosine residues allows the vectors to evade phosphorylation, preventing ubiquitination and proteasome digestion of the vector.  A recent study has exhibited a similar trend when substituting AAV2 capsid serine residues with valine, achieving up to 20-fold increases in transduction efficiency.93  This effect is not limited to AAV2, as Petrs-Silva et al. reported that tyrosine-to-phenylalanine substitutions of AAV capsid serotypes 8 and 9 also result in higher transduction efficiencies.94  In addition to increasing transduction efficiency of AAV vectors, it 16  has been established that the substitution of multiple capsid tyrosine residues with phenylalanine results in an ability to transduce cell populations deeper into the retina from the site of injection.95  These findings suggest that not only does the substitution of capsid tyrosine residues allow the vector to evade host cell defense mechanisms, it may also augment the extracellular transport kinetics and penetration abilities.  One hypothesized mechanism for this occurrence is an alteration of heparan sulfate binding, where a decreased binding affinity results in less vector sequestration at the retinal inner limiting membrane (ILM) and greater outer retinal transduction rates.96  However, if heparan binding is decreased too much, outer retinal transduction rates suffer, suggesting that a certain amount of binding affinity is necessary for the vector to enter the retina.96  1.3 AAV for Retinal Gene Therapy  1.3.1 Delivery route of AAV vectors for retinal gene therapy Intravitreal and subretinal injections are the two methods most extensively utilized for the delivery of AAV vectors to the retina.97  Choice of method is based primarily on the location of a targeted cell population, as AAV vectors are unable to efficiently traverse the retina of large animal models and humans.  Gene therapy of the photoreceptors and RPE, which are the cell populations most often affected by inherited retinal dystrophies, currently requires subretinal delivery of the AAV vector.  One of the primary benefits to subretinal administration is that the subretinal space is a highly immune-privileged site, with very little risk of a humoral response affecting the transduction efficiency of the AAV vector.98  Even when the dose of a particular AAV serotype would cause a toxic immune reaction following systemic administration, subretinal injection does not result in adverse effects.99, 100   17  Unfortunately, the method of subretinal delivery is invasive, inducing a large retinal detachment by injection of the vector solution into the space between the RPE and photoreceptor outer segments.  The resulting detachment often resolves within 2 days.23, 101  Studies in humans and animals with retinal detachments have shown that photoreceptor cell apoptosis starts to occur within 24 hours, with peak levels at 3 days post-detachment.102, 103  In some photoreceptors that do not undergo apoptosis, there is destruction of the outer segments and reorganization of the inner segments.104  In a study evaluating the long-term safety of subretinal AAV injections in dogs and a non-human primate, the authors reported that there were no deficits in full-field electroretinography and no abnormalities noted on fluorescein angiography for up to 36 months post-injection.105  A recent study by Nork et al. evaluated the effects of subretinal delivery of a balanced salt solution in cynomolgus macaques.101  They described that the regions of retinal detachment showed significant functional deficits (as measured by multifocal electroretinogram) immediately following subretinal injection, but returned to near normal levels by 3 months post-injection.  Although the effect on photoreceptor function was minimal and transient, they also noted disorganization of rod photoreceptor outer segments and redistribution of rhodopsin and L/M-cone opsin pigments.101  In a report from human retinal gene therapy clinical trials, foveal thinning was observed in patients treated with subretinal injections that caused a detachment involving the fovea.26  As the fovea is a pivotal structure for visual acuity in humans, this region is a very important target area for gene therapy.  Regrettably, as the authors of the report discussed, subretinal injection does not appear to be a suitable method for targeting the fovea in humans.26  To date, the distribution of therapeutic effect following subretinal injection has been limited to the region of retinal detachment, which presents the quandary of causing damage to a vital tissue in order to provide therapeutic treatment.14 18  IVT is a more commonly practiced intraocular drug delivery method, and is less invasive than subretinal injection.95  IVT should theoretically distribute the vector across a much larger portion of the retina when compared to the limited region impacted by subretinal injections.  Distribution of transgene expression from the central retina to the periphery was documented in both rodent and non-human primate models following intravitreal AAV injection.94, 106-108  Through methods of rational mutagenesis and directed evolution, transduction efficiency can be optimized for both inner and outer retinal cell populations over the entire retina in rodents; however, efficiency remains limited and regionally variable in large animal models.107-109  The vitreal cavity is also considered an immune-privileged site, with antigen presentation resulting in an immune-deviant systemic response.110  However, the formation of systemic neutralizing antibodies still occurs, which causes an inhibitory effect following IVT of the same AAV capsid serotype into the contralateral eye.98  The use of a different capsid serotype or an AAV vector with carefully selected capsid amino acid substitutions in the partner eye could circumvent this effect.48, 111  Another potential disadvantage to IVT of AAV vectors is an increased risk of off-target transgene expression in both ocular and non-ocular tissues, including the brain.112  AAV8 has the capability of crossing the first synapse in the lateral geniculate nucleus of the brain following subretinal delivery in rats and dogs, while AAV2 does not exhibit this ability after both subretinal injection and IVT.112, 113  This supports the use of target cell-specific promoters, which would significantly limit the risk of off-target transgene expression in the brain.112 A less extensively evaluated method for the delivery of AAV vectors for retinal gene therapy is systemic administration.  This lack of attention is likely due to the higher potential for hypersensitivity reactions to the transgene product, neutralizing antibody interference, and transduction of off-target tissues increasing the opportunity for insertional mutagenesis.46  In 19  2012, Dalkara et al. described widespread transduction of all retinal layers and the brain following intravenous administration of a next-generation tyrosine mutant AAV vector with a ubiquitous promoter to neonatal mice.114  When they incorporated a photoreceptor-specific promoter in place of the ubiquitous promoter, they only observed transduction in the photoreceptors, without any evidence of off-target expression in the retina or brain.114  These results suggest that the use a tissue-specific promoter would evade the risks of transgene hypersensitivity and off-target insertional mutagenesis, however it would not likely negate the inhibitory effects of any systemic neutralizing antibodies to the AAV capsid serotype being used.  Unfortunately, these vectors also require administration to neonates, prior to establishment of the blood-retinal barrier.114  Through the use of AAV constructs with novel capsid structures, safe and effective retinal gene therapy via a systemic route in the presence of neutralizing antibodies may be possible in the future.48 1.3.2 Extracellular barriers to intravitreal retinal gene therapy There are physical barriers present in the inner retina and vitreal cavity which may prevent effective transduction of the outer retinal cells with AAV vectors following IVT.  One of the barriers is the ILM, a basement membrane that is located between the Müller cell end feet and the vitreous body and mediates the adhesion between the neural retina and the vitreous body.115  The ILM is composed of collagen, laminin, and a variable distribution of carbohydrates.115  It is also rich in heparan sulfate proteoglycans, which are the primary receptor bound by AAV2 prior to transport across the cell membrane in cultured cells.116, 117  These along with additional components including chondroitin sulfate are negatively charged, suggesting that the ILM could also pose an electrostatic barrier to transport of cationic or neutral charged substances from the vitreal cavity to the retina.118  The thickness of the ILM varies between species, with rodents and 20  rabbits having a relatively thin membrane compared to the thicker membranes of canines and non-human primates.119  ILM thickness is also topographically variable in non-human primates, being a thin membrane over the foveal pit, retinal vessels, and peripheral retina, and a significantly thicker membrane in the parafoveal and peripapillary regions.120  There is separation of Müller cell endfeet over the retinal vessels resulting in the presence of pores within the ILM, and vitreous humor is reported to extend through these pores.121, 122  The pores of the ILM range in size from 10 to 25 nm, acting as a filtration barrier between the vitreous body and retina.118  The nerve fiber layer, which consists of RGC axons, could also pose a barrier to AAV penetration from the vitreal cavity into the cellular layers of the retina.  This layer also exhibits a topographical distribution similar to the ILM, being thicker over the central retina and thinner peripherally.106  Several studies have evaluated the penetration of various substances across the ILM in animal models.  Dalkara et al. fluorescently labeled AAV capsids of various serotypes and visualized many of the viral particles collecting at the vitreoretinal junction following IVT in rats.119  They then used a non-specific protease to mildly digest the ILM, injected at the same time as the AAV vectors.  The addition of the protease resulted in significantly increased transduction of retinal cell layers, with AAV capsid serotype 5 exhibiting strong expression in the photoreceptors and retinal pigmented epithelium.119  These findings highlight a strong correlation between the integrity of the ILM and the inability of AAV vectors to reach the various retinal cell layers.  Another study described increased retinal penetration of AAV vectors following IVT in neonatal rats compared to adult rats, which also suggested that the viral vectors had difficulty navigating the more developed ILM in adults.123  Recent work using nanoparticles has led to the specific characterization of macromolecule transport across the ILM.  A study in 2003 established that 21  strongly anionic nanoparticles could rapidly traverse the entire retinal thickness following IVT.  The authors described that the particles settled on the ILM by one hour post-injection, some were observed traversing the retina at 6 hours post-injection, and then the particles were preferentially accumulating in the RPE by 18 to 24 hours post-injection.124  In 2009, Kim et al. illustrated co-localization of fluorescent dye conjugated nanoparticles with Müller cells in the ILM and other layers of the retina.118  A follow-up study by Koo et al. used transmission electron microscopy to demonstrate that the nanoparticles penetrated the ILM through a process of clathrin-mediated endocytosis into Müller cells, and then appeared to be transported to the outer plexiform layer via transcytosis.125  Interestingly, both of the studies also described significantly more efficient ILM penetration when the nanoparticles had a strong anionic charge of around -20mV as opposed to cationic or neutral charges.118, 125  AAV2 capsid has been shown to have a weak anionic zeta potential of -8.6mV, so some accumulation of viral particles at the ILM could be expected based on zeta potential alone.125, 126  The information from these studies may be very useful for the development of AAV vectors with greater capabilities of traversing the thicker ILM of humans and large animal models. In addition to the ILM, some have suggested that the viscosity or the electrostatic charge of the vitreous body may inhibit the transport of intravitreally injected substances to the inner retina, which could lead to variable AAV transduction efficiency between patients depending on the placement of the IVT.45, 127  The nanoparticle studies mentioned above described the intravitreal distribution of the particles in addition to the retinal transport.  In those studies, they found that particles with a strong cationic charge tended to aggregate in the vitreous body and were not distributed to the retina, while particles with a strong anionic charge were quickly transported to the retinal surface.118, 125  They also found that glycol chitosan particles, which contain many 22  primary amine groups and a strong cationic charge, were able to distribute through the vitreous body and reach the retina but did not penetrate through the ILM.125  They proposed that the increased vitreal transport was due to the presence of anti-fouling glycol groups on the nanoparticles, which mask the amine groups.  In addition to the physical and chemical properties of the material being injected, simulations of the distribution of intravitreally administered substances have revealed that the placement and volume of the injection and any movement of the eye following IVT will affect the distribution within the vitreous body.128-130  One study demonstrated that injections made adjacent to the retina achieve retinal drug exposure of more than five times an injection into the central vitreous, and over forty times an injection made adjacent to the hyaloid membrane.128 This evidence suggests that IVT of AAV vectors for retinal gene therapy should be placed as close as possible to the retina. 1.3.3 Intravitreal AAV for gene therapy in animal models In 2009, Hellstrom et al. reported the retinal cellular tropism and transduction efficiencies of wild-type AAV capsid serotypes 1-6 and 8 following IVT in adult rats.74  They found that AAV2 was most efficient, and all but AAV6 predominantly targeted RGCs.  Outer retinal transduction was inefficient for all vector serotypes, although AAV3 appeared to exhibit the greatest ability to transduce photoreceptors.74  IVT of wild-type AAV2 was capable of transducing RGCs and providing therapeutic rescue in a mouse model of Leber hereditary optic neuropathy.131  In three different studies, wild-type AAV2 vectors expressing CNTF or BDNF transduced sufficient RGCs and promoted RGC survival after optic nerve crush, intraocular pressure elevation, and optic nerve transection in rats.42, 43, 132  Although wild-type AAV vectors appear to be proficient at widespread RGC transduction following IVT in rodents, outer retinal transduction is significantly less efficient in retinas with normal morphology.74, 131  When administered into 23  rodent models possessing a degenerate retina, the efficiency of the vectors can change substantially.  Therapeutic rescue was achieved following IVT of AAV in rodent models of retinoschisis, while a lower level of transduction occurred when injected into wild-type mice.133  This pattern of increased photoreceptor transduction was also demonstrated using a wild-type AAV5 vector in the Dystrophin Dp71-null mouse model, which has compromised ILM barriers.134   The pattern of retinal transduction by wild-type AAV is less impressive in large animal models.  A study using wild-type AAV2 injected via IVT into normal primates found that transduction occurred only within inner retinal cells around the fovea and in the peripheral retina, and outer retinal transduction was absent.106  Lukason et al. reported as similar pattern of RGC transduction in primates following IVT of wild-type AAV2 driving expression of an anti-VEGF molecule.45  The limited transduction, however, was sufficient to reduce the choroidal neovascularization response following laser injury.  These results validate the need for alteration of AAV capsids to generate vectors capable of producing more efficient retinal transduction in humans and large animal models after IVT.  They also demonstrate that a limited amount of transgene expression may be adequate to treat some retinal diseases. Two methods have primarily been used recently to alter the amino acid makeup of AAV capsids in order to optimize their ability to reach and transduce the target cell while avoiding recognition of the host immune system.  The first is rational mutagenesis, which involves the intentional substitution of surface exposed tyrosine, serine, or threonine residues with phenylalanine, alanine, or valine, respectively.68  Combinations of these surface amino acid substitutions can have an additive effect on these improvements.  Recently, Kay et al. found that the addition of a threonine-to-valine substitution to an AAV2 vector containing four capsid tyrosine-to-24  phenylalanine substitutions resulted in 3.5 times the number of photoreceptors transduced after IVT in mice.108  When the quadruple tyrosine-to-phenylalanine mutant AAV2 vector was delivered via IVT in dogs, it was able to transduce nearly double the amount of photoreceptors than a wild-type self-complimentary AAV2 vector, although photoreceptor transduction by both vectors was mostly limited to regions underneath retinal vessels.109   The second method developed recently to optimize the capsid of AAV vectors for retinal gene therapy is directed evolution.  This process involves the mutation of a wild-type AAV cap gene using error prone PCR, random peptide insertion, or capsid DNA shuffling, to generate a large library of capsid variant vectors.135  These vectors are then subjected to a challenge, either in vitro or in vivo, that may be a barrier to the efficiency of the vector, for example: Neutralizing antibodies (NAb) specific to the AAV serotype, an intact ILM after IVT, or a receptor specific to a target cell population.  The vector particles that are able to overcome the challenge are then isolated and subjected to additional challenges, until eventually the most successful variant is identified and replicated.48, 107, 135  Using this technology, a capsid variant of AAV6 demonstrated significantly increased transduction efficiency of retinal Müller cells in rats following IVT when compared to wild-type AAV2 and AAV6.136  Also the 7m8 vector, generated from a combination of AAV2 mutants and capsid shuffling of AAV serotypes 1, 2, 4, 5, 6, 8, and 9, was capable of producing widespread inner and outer retinal transduction in wild-type mice.107  It was also capable of producing structural and functional rescue in a mouse model of retinoschisis, as well as functional rescue in the rd12 mouse model of LCA, demonstrating the capability to transduce RPE from the vitreous.107  When the 7m8 vector was delivered to normal primates, it produced more efficient inner- and outer-retinal transduction when compared to the quadruple tyrosine-to-phenylalanine mutant AAV2 vector.107 25  1.4 Immune Responses to AAV 1.4.1 Ocular defense mechanisms The eye is considered an immune privileged site, the result of a process termed anterior chamber-associated immune deviation (ACAID).  ACAID is initiated by specialized antigen presenting cells (APCs) that are naturally present within the anterior chamber of the eye, and to a lesser degree in the vitreous humor.137, 138  When a foreign antigen is introduced into the anterior chamber, vitreous cavity, or subretinal space, these APCs collect the antigen and carry it from the eye into the bloodstream through the angular aqueous plexus.139-141  The APCs are preferentially trafficked to the marginal zone of the spleen, where they secrete cell signaling -2 in order to form cell clusters with and present the antigen to natural killer T-lymphocytes and splenic B-lymphocytes.138  These cell clusters then convert naïve CD4+ and CD8+ T-lymphocytes into regulatory T-lymphocytes, which play an anti-inflammatory role in response to antigen stimulation.142  The regulatory CD4+ T-lymphocytes then are trafficked to secondary lymphoid organs to play a systemic antigen-specific immunosuppressive role, while the regulatory CD8+ T-lymphocytes are trafficked to the eye and other peripheral tissues to prevent an inflammatory response to the antigen.143 ACAID is supported by an immunosuppressive intraocular environment that is maintained by the pigmented epithelial cells of the iris, ciliary body, and retina, as well as the corneal endothelium and a specialized set of retinal dendritic cells.142, 144  These cells produce an anti-inflammatory intraocular state through the expression of membrane-bound ligands and soluble factors capable of either suppressing activation of pro-inflammatory T-lymphocytes, converting them into 26  regulatory T-lymphocytes, or inducing apoptotic pathways within the cells.138, 142, 144  These -melanocyte stimulating hormone, thrombospondin-1, and Fas-ligand.138, 142  This environment can be disrupted in cases of chronic uveitis or following exposure to a large enough dose of pro-inflammatory cytokines, leading to inhibition of ACAID and loss of intraocular immunosuppression.145, 146  1.4.2 Humoral response to AAV The presence of circulating NAb can significantly impact transduction efficiency and therapeutic success when AAV treatments are administered.48  Humans and large animal models can possess preexisting NAb to AAV serotypes, with prevalence in humans to the AAV2 serotype reported as high as 72%.55, 147  When dosing into the subretinal space, it is possible to administer multiple intraocular doses without a significant effect on transduction or generation of a significant NAb response.98, 100, 148  In 2008, Li et al. demonstrated that intravitreal AAV injection generates a humoral response resulting in the production of AAV capsid-specific NAb, precluding the effectiveness of additional doses using the same delivery method.98  When IVT was followed by subretinal injection, however, the authors observed no effect on transduction efficiency.98  The reason for the different immune responses between the two delivery routes is unknown, but is thought to be related to an increased systemic exposure of AAV following IVT.98  More recently, Li et al. reported a method for evasion of capsid-specific NAb by substituting amino acids from one AAV capsid serotype into another AAV vector at defined sites, resulting in elevated levels of transduction both in vitro and in vivo.111  This was done through site-directed mutagenesis of an AAV2 vector genome to insert amino acids from AAV1 into a specific capsid region of the final vector, which the authors termed AAV2.5.111  However, it has also been described that non-27  human primates with pre-existing NAb to AAV2 demonstrated a reduction in transgene expression following IVT of both wild-type and capsid mutant AAV2 variants.149   1.4.3 Cell-mediated responses to AAV Systemic administration of AAV risks development of both capsid and transgene-specific cytotoxic T lymphocyte reactions, which can not only reduce the transduction efficiency of the vector, but can also result in destruction of transduced cells, damaging overall health of the host.150, 151  Cell-mediated immune responses are driven by toll-like receptor-9 signaling by CpG motifs in the viral DNA genome, and lack of these motifs prevents both capsid and transgene-specific responses.152  Removal of CpG sequences from the transgene may result in evasion of immune system recognition, however the transcription efficiency of CpG depleted transgenes within host cells may be depleted.153  A heparan binding motif on the AAV2 capsid also contributes to generation of CD8+ cell-mediated immunity to the capsid.154 Humans appear to be able to most readily generate an adaptive immune response to capsid antigen, while animal models are much more resistant, which is hypothesized to be a result of preexisting primed anti-AAV capsid CD8+ T-lymphocytes in humans as a result of previous AAV exposure.155  The result is a gap in translation of results from experimental animals into human clinical trials, as the previously-exposed human scenario is difficult to recreate in animal models.155-157     An additional risk is the development of immune reactions to an expressed transgene, which is dependent upon both the antigenicity of the protein as well as the pattern of inheritance for the disease being treated.158  In general, gene therapy for autosomal recessive diseases carries the highest risk, since a novel transgene is expressed to which the host immune system has never been exposed.159  Rodents appear to be resistant to developing inflammatory responses to many 28  antigenic transgene products, thought to be mediated by induction of regulatory T-lymphocytes or immunological ignorance.160, 161  Inflammation within the target organ unrelated to the AAV vector capsid or transgene can induce a reduction of transgene expression through suppression of transcription by cytotoxic T-lymphocytes.162 Despite the immune-privileged status of the eye, intraocular AAV injections have resulted in the development of immune reactions in some animal models.  Engineered AAV vectors are becoming more efficient, and with that increased efficiency also comes an increased risk of recognition by the immune system. Subretinal injection of AAV9, AAV2, AAV8, and AAV5 vectors produced a retinal inflammatory response and production of NAb titers to both capsid and the GFP transgene in cats.163  AAV2 and AAV8 vectors driving GFP expression are capable of producing a dose-dependent adaptive immune response following subretinal delivery in primates.164  The 7m8 vector developed through the process of directed evolution delivered via IVT in primates generated what was presumed to be an inflammatory response to GFP.107 All three of these studies utilized the ubiquitous cytomegalovirus promoter, allowing GFP expression in both target and off-target cell populations.  This supports the use of tissue-specific promoters, when available, to reduce the risk of transgene-specific cytotoxic T-cell responses.  IVT of the 7m8 vector using the neuronal promoter cx36 resulted in a similar pattern of GFP expression in primate RGCs without generation of an immune reaction.107 1.5 Study Hypotheses and Specific Aims The main focus of the work described in this thesis was to evaluate the ability of intravitreally administered next-generation AAV vectors to abrogate the limitations of the more invasive subretinal delivery route.  As described above, the primary limitations of the subretinal route are 29  a confined region of retinal transduction, a higher potential for retinal damage, an inability to transduce inner retinal cell populations, and the overall intricacy of the injection technique.  While the intravitreal method used in these studies is intended to be a less precarious technique, one drawback would be the potential for vector transduction of a large number of off-target cell populations.  This problem also warranted investigation in our study design.  To commence the process of addressing the above limitations, we performed studies in three phases, with the hypotheses and specific aims of each described below: 1.5.1 Safety and efficacy study The purpose of this phase of the study was to compare the safety and efficacy of two different intravitreally administered vectors at ten-fold different viral titers.  The vectors used for this phase included recombinant AAV serotype 2/2 containing three surface exposed capsid tyrosine residues replaced by phenylalanine (AAV2 (triple Y-F)), and a recombinant AAV serotype 2/2 with three surface exposed capsid tyrosine residues replaced by phenylalanine and one surface exposed capsid threonine residue replaced by valine (AAV2 (triple Y-F + T-V)).  Both vectors -actin (CBA) promoter and were intended to primarily target cells within the ganglion cell layer of the retina following IVT, with the expectation that other cell populations deeper into the retina would be transduced to a lesser degree.  The safety of the vectors would be judged by evaluating the severity of complications that arose during the eight-week period following vector injection.  Previous studies have shown that the effectiveness of AAV gene therapy following inner retinal injury is dependent upon transgene expression within the affected cell populations over a large area of the retina.42, 43  The efficacy of each vector in our study would be determined by an assessment of the distribution of transgene expression and 30  the ability to efficiently transduce cells of the retinal ganglion cell layer.  The specific aims of this study phase were:  1) To demonstrate the retinal area of transgene expression of each vector construct in vivo through the use of imaging techniques, and  2) To demonstrate efficiency of inner retinal cell transduction of each vector by performing immunohistochemistry on retinal cryosections using antibodies to label targeted cell populations. The hypotheses for this phase of the study consisted of:  1) Both vectors will exhibit an acceptable level of safety following IVT, 2) The AAV2 (triple Y-F + T-V) vector will result in a more widespread area of retinal transduction compared to the AAV2 (triple Y-F) vector following IVT, and  3) The AAV2 (triple Y-F + T-V) vector will more efficiently transduce cells within the retinal ganglion cell layer than the AAV2 (triple Y-F) vector following IVT. 1.5.2 Photoreceptor-targeted Vector Study This study was performed to evaluate the ability of two next-generation AAV vectors to penetrate into the outer layers of the retina and specifically transduce the photoreceptor cell populations.  The vectors used for this phase included recombinant AAV serotype 2/2 containing four surface exposed capsid tyrosine residues replaced by phenylalanine (AAV2 (quad Y-F)), and a recombinant AAV serotype 2/2 with four surface exposed capsid tyrosine residues replaced by phenylalanine and one surface exposed capsid threonine residue replaced by valine (AAV2 (quad Y-F + T-V)).  The capsid amino acid substitutions present on these vectors make 31  them leading candidates for overcoming potential barriers to retinal transport including the ILM and nerve fiber layer.  To mediate the risk of off-target protein expression, our packaged reporter transgenes were driven by highly photoreceptor-specific promoters.  Both of the vectors used in this study were packaged into two different constructs carrying either the interphotoreceptor retinoid-binding protein (IRBP) promoter or the guanine nucleotide binding protein alpha transducing activity polypeptide 2 (GNAT2) promoter with an IRBP enhancer.  The specific aims of this study were:  1) To document initial onset and regional distribution of transgene expression using in vivo imaging techniques,  2) To objectively quantify transduction efficiency of each vector construct using immunohistochemistry, and  3) To demonstrate restriction of transgene expression to rod and cone photoreceptors following IVT of the vectors using immunohistochemistry. The hypotheses for this study included:  1) IVT of the AAV2 (quad Y-F + T-V) vector would result in faster onset of transgene expression compared to the AAV2 (quad Y-F) vector,  2) IVT of the AAV2 (quad Y-F + T-V) vector would produce more efficient photoreceptor transduction compared to the AAV2 (quad Y-F) vector, and  3) IRBP and GNAT2 promoter constructs packaged separately within the vectors would restrict reporter transgene expression to outer-retinal photoreceptors and cone photoreceptors, respectively. 32  1.5.3 Vitrectomy Study The purpose of this study phase was to evaluate the effect of vitrectomy on intravitreally administered AAV vectors.  The most effective vector from the safety and efficacy phase, AAV2 (triple Y-F + T-V), was used to compare the transduction efficacy and distribution between vitrectomized and non-vitrectomized eyes.  Given that the vitreous body has been perceived as a significant barrier to the widespread delivery of intravitreally administered substances across the retinal surface, performance of a vitrectomy prior to injection would likely improve the retinal exposure of the AAV vector injection.45, 118, 125, 127  Another possible benefit of performing a vitrectomy prior to intravitreal AAV injection would be the potential for disruption of the inner limiting membrane, another proposed barrier to retinal transduction.119  The specific aims of this study phase were:  1) To demonstrate the retinal transduction speed and area of transgene expression in both vitrectomized and non-vitrectomized eyes in vivo through the use of imaging techniques, and  2) To quantify transduction efficiency of retinal cell populations in both vitrectomized and non-vitrectomized eyes by performing immunohistochemistry on retinal cryosections using antibodies to label targeted cell populations. The hypotheses for this phase of the study included:  1) Performing a vitrectomy prior to AAV injection will result in greater speed of retinal transduction when compared to eyes without vitrectomy,  2) Performing a vitrectomy prior to AAV injection will result in more widespread distribution of retinal transduction when compared to eyes without vitrectomy, and 33   3) Performing a vitrectomy prior to AAV injection will result in the transduction of a greater number of retinal cell populations.   34  CHAPTER 2 SAFETY AND EFFICACY STUDY 2.1 Materials and Methods 2.1.1 Animals All procedures were performed in accordance with regulations of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and under approval of the Michigan State University Institutional Animal Care and Use Committee.  Four normal juvenile purpose-bred Curly-coated Retriever dogs maintained at a Michigan State University animal housing facility were used in this study.  The animals were housed on a 12-hour light:dark cycle. 2.1.2 Ophthalmic examinations and retinal imaging Slit-lamp biomicroscopy (SL, Kowa, Japan), indirect ophthalmoscopy(Dualite, Keeler, Windsor, UK), and high-resolution digital fundus photography (RetCam II, Clarity Medical Systems, Pleasanton, CA) were performed pre-injection, three times weekly for four weeks post-injection, and twice weekly thereafter to monitor for any post-operative complications.  Fundus photographs were collected using white-light as well as 471 nm wavelength-light to excite green fluorescent protein (GFP) with a 510 nm emission filter to record fluorescence.  The dogs were sedated as needed for imaging and examinations using intravenous administration of a combination of either 0.03 mg/kg acepromazine (Vedco, St. Joseph, MO) and 0.1 mg/kg butorphanol (Zoetis, Kalamazoo, MI), or 3 mg/kg ketamine (Fort Dodge Animal Health, Overland Park, KS) and 0.6 mg/kg xylazine (Akorn, Inc, Decatur, IL).  During the sixth week post-injection, high-resolution confocal scanning laser autofluorescence imaging was performed 35  using the 488 nm laser on the Spectralis HRA/OCT (cSLO, Spectralis, Heidelberg Engineering, Carlsbad, CA) to capture GFP fluorescence. 2.1.3 Vectors and vector preparation Recombinant AAV vectors were produced and titered by the Center for Vision Research group of Drs. William Hauswirth and Shannon Boye at the University of Florida.  The vectors used were recombinant AAV serotype 2/2 containing three surface exposed capsid tyrosine residues replaced by phenylalanine (AAV2 (triple Y-F)), and a recombinant AAV serotype 2/2 with three surface exposed capsid tyrosine residues replaced by phenylalanine and one surface exposed capsid threonine residue replaced by valine (AAV2 (triple Y-F + T-V)).  Both vectors expressed humanized GFP driven by the chicken -actin (CBA) promoter.  Stock vector preparations were diluted with sterile balanced salt solution to the working titers of 1.3 x 1012 vg/ml and 1.3 x 1011 vg/ml. 2.1.4 Intravitreal injections Twenty four hours pre-injection, dogs were started on a regimen of 1 mg/kg prednisone (West-Ward Pharmaceutical Corp., Eatontown, NJ) (rounded to the nearest 5 mg) orally twice daily.  Twenty minutes prior to injection, both eyes were dilated with 1% tropicamide (Alcon Laboratories, Fort Worth, TX) and 10% phenylephrine (Akorn, Inc, Lake Forest, IL).  Following premedication with 0.2 mg/kg acepromazine intramuscularly, dogs were induced with intravenous ketamine and valium (Hospira, Inc., Lake Forest, IL) to effect and maintained on isoflurane (Abbott Laboratories, North Chicago, IL) delivered in 100% oxygen through an endotracheal tube.  The dogs were positioned in dorsal recumbency with neck ventroflexion to align the palpebral fissure within the viewing field of the operating microscope (Zeiss OpM1 36  Operating Microscope; Carl Zeiss, Inc, Thornwood NY).  The eye was prepared with 1:50 povidone-iodine solution, an eyelid speculum was fitted, and the globe was positioned using conjunctival stay sutures of 4-0 silk.  A commercial injector (RetinaJect; SurModics Inc, Irvine, California) was inserted through the sclera 5-6mm posterior to the limbus in the region of the pars plana and advanced toward the retina under direct visualization through a vitreoretinal surgery contact lens (Acrivet Vit. Lens, Acrivet, Hennigsdorf, Germany).  The 39-gauge extendable cannula of the injector was advanced until immediately adjacent the retinal surface.  A volume of 200µl of vector was injected as close as possible to the retinal surface in the region of the visual streak nasally and temporally, and directly over the optic nerve head.  The four dogs were randomized into two treatment groups.  Both dogs in treatment group 1 received AAV2 (triple Y-F), contralateral eyes were randomly selected for administration of low titer (1.3 x 1011 vg/ml) or high titer (1.3 x 1012 vg/ml) vector preparation (Table 4). Both dogs in treatment group 2 received AAV2 (triple Y-F + T-V), with contralateral eyes similarly randomized for low/high titer vector preparation (Table 4).   Aqueous paracentesis withdrawing 200µl was performed on all eyes immediately post-injection to relieve elevation of intraocular pressure resulting from the fluid volume added during the intravitreal injection. 4 mg of triamcinolone (Bristol-Myers Squibb, New York, NY) was administered subconjunctivally prior to anesthesia recovery to reduce expected post-operative uveitis.  Post-operatively, the dogs received a preventative ten day course of oral 14-22 mg/kg amoxicillin-clavulanic acid antibiotic (Zoetis, Kalamazoo, MI) treatment, a tapered three week course of oral prednisone, and topical treatments of atropine and neomycin-polymixin B-dexamethasone ointments (Bausch & Lomb, Rochester, NY) applied to both eyes twice daily for two weeks and once daily thereafter.  37  Table 4. Vector dosing arrangement with vector titer dose and timing of euthanasia for safety and efficacy study.  Dog 1  AAV2 (triple Y-F) 4 weeks Dog 2   AAV2 (triple Y-F) 8 weeks Dog 3   AAV2  (triple Y-F + T-V) 8 weeks Dog 4   AAV2 (triple Y-F + T-V) 4 weeks Left Eye 1.3x10^11 vg/ml 1.3x10^12 vg/ml 1.3x10^11 vg/ml 1.3x10^12 vg/ml Right Eye 1.3x10^12 vg/ml 1.3x10^11 vg/ml 1.3x10^12 vg/ml 1.3x10^11 vg/ml  2.1.5 Globe collection Dogs 1 and 4 were euthanized via overdose of sodium pentobarbital (Vortech Pharmaceuticals, Dearborn, MI) four weeks post-injection.  Following enucleation, both eyes were immersed in 4% paraformaldehyde (PFA) chilled on ice (PFA powder 95% (Sigma-Aldrich, St Louis, MO, USA) in phosphate buffered saline (PBS) pH 7.4) for four hours.   The anterior segment was removed via dissection along the pars plana, and the eyecups were immersed in 2% PFA for another 20 hours at 4°C.  The eyecups were desiccated by immersion in 15% sucrose for 24 hours, followed by immersion in 30% sucrose solution for an additional 24 hours at 4°C.  They were then cut into 9 segments as described in Figure 1 and embedded in optimal cutting temperature medium (ThermoFisher Scientific, Pittsburgh PA) and stored in a -80°C freezer.  Dogs 2 and 3 were euthanized eight weeks post-injection.  Following administration of the sodium pentobarbital overdose, the chest cavity was opened and 4% PFA was infused intravenously through a trocar placed into the left ventricle.  The globes were enucleated and processed for embedding as described above.  In addition, the optic nerves, optic chiasm, lateral geniculate nuclei (LGN), and superior colliculus were collected and embedded whole in the same manner as described above for retinal cryosections.  38   Figure 1. Diagram depicting the nine eyecup sections embedded from each eye in the safety and efficacy study.  Each section was labeled with Neuronal nuclei (NeuN) antibody and the proportion of GFP positive cells within the ganglion cell layer was quantified. N, nasal; T, temporal.  2.1.6 Immunohistochemistry 20µm thickness frozen retinal sections were collected using a cryomicrotome (Leica CM3050-S cryostat, Leica Microsystems, Buffalo Grove, IL) onto poly-lysine coated slides, and stored in a -80°C freezer.  Following defrosting, non-specific antibody binding sites were blocked with 10% normal goat serum and 0.1% triton x-100 in 0.01M PBS applied to the slides for one hour, followed by a PBS wash.  Slides were incubated with primary antibodies (Table 5) overnight at            39  4°C.  Following a 20 minute application of 10% normal goat serum and washing with PBS, secondary antibodies included goat anti-mouse and goat anti-rabbit covalently bound to the Texas Red fluorophore were applied and incubated for one hour at room temperature.  Antifade reagent with 4',6-diamidino-2-phenylindole (DAPI) was then applied prior to affixing glass cover slips (ProLong Gold, Life Technologies, Grand Island NY).  Sections of optic nerve, optic chiasm, LGN, and superior colliculus were labeled with the GFP antibody and DAPI only and mounted on slides as described above.  Following excitation at wavelength appropriate for the specific fluorophores, representative images were captured (Nikon Eclipse 80i, Nikon Instruments Inc., Melville, NY, USA).  Table 5. Primary and secondary antibodies used for retinal immunohistochemistry on safety and efficacy study. Target antigen Primary antibody details and working dilution Manufacturer Targeted cell population Secondary antibody and concentration (all from Invitrogen Corp, Carlsbad, CA) Green fluorescent protein Rabbit FITC conjugated, 1:1000 Invitrogen Corp, Carlsbad, CA GFP transduced cells109 N/A Neuronal Nuclei (NeuN) Mouse monoclonal 1:2000 Millipore, Billerica, MA Ganglion and Amacrine cells165 Alexafluor 546 goat anti-mouse 1:500 Protein kinase C alpha Mouse monoclonal 1:500 BD Biosciences, San Diego, CA Rod bipolar cells109 Alexafluor 546 goat anti-mouse 1:500 L/M-opsin Rabbit polyclonal 1:500 Millipore, Billerica, MA L/M-cone outer segments166 Alexafluor 546 goat anti-rabbit 1:500 S-opsin Rabbit polyclonal 1:500 Millipore, Billerica, MA S-cone outer segments109 Alexafluor 546 goat anti-rabbit 1:500 Human Cone arrestin (LUMif)167 Rabbit polyclonal 1:5000 Kind gift of Cheryl M. Craft, University of Southern California Cone photoreceptors109 Alexafluor 546 goat anti-rabbit 1:500    40  2.1.7 Speed of onset evaluation The rate of onset of GFP expression for each injected eye was evaluated using analysis of the fluorescent fundic images.  All images were equally enhanced using Microsoft Office Picture Manager (Microsoft Corporation, Redmond WA).   2.1.8 Cellular transduction efficiency Neuronal Nuclei antibody labeled retinal cryosections were examined from each of the nine segments of each eye after the identifying information was masked on each slide.  For each vector titer at each time point the ratio of GFP-positive cells within the RGC layer compared to the overall number of RGC layer cells was determined. 2.1.9 Statistical evaluation Transduction efficiency was compared between high and low dose titers for each vector at both 4 and 8 weeks post-IVT using a one-tailed paired t-test.  The high dose transduction efficiencies of each vector was compared using a two-tailed -test.  Results were considered significant if p<0.05. 2.2 Results and Discussion 2.2.1 Ophthalmic examinations Aside from expected mild post-operative intraocular inflammation, characterized by grade 1 out of 4 aqueous humor flare, (grade 1 = Tyndall effect just barely visible with slit-lamp magnification in a darkened exam room) in all eyes, and focal retinal hemorrhages present on one eye each of two dogs, no adverse effects following IVT were noted in any of the four dogs. These changes were present at examination 24 hours following injection.  The hemorrhages 41  occurred in the left eye of Dog 2 and the right eye of Dog 3.  Aqueous flare had resolved in all eyes by 48 hours post-IVT, retinal hemorrhages had decreased in size by seven days post-IVT.  One dog developed a 3mm diameter superficial corneal ulceration one week post-IVT, suspected to be the result of fundoscopic imaging.  The ulceration resolved without complications within three days.   There was a lack of clinically-appreciable adverse reactions in all four dogs following recovery from the IVT procedure.  This suggested that both the high and low dose of both vectors used in this study were safe for use in future studies using the same or similar capsid-mutant vectors with planned termination dates as long as 8 weeks post-IVT. 2.2.2 Speed of GFP expression onset The right eye of Dog 1 was the first to exhibit GFP fluorescence, localized to the optic nerve head, at day 7 post-IVT (Figure 2).  This eye had received the higher titer of AAV2 (triple Y-F).  The remaining high titer eyes all exhibited GFP fluorescence over the optic nerve head by day 10 post-IVT.  The first to exhibit GFP fluorescence of the eyes receiving low vector titers was the left eye of Dog 3 and the right eye of Dog 2, both at day 23 post-IVT.  These eyes had been injected with AAV2 (triple Y-F + T-V) and AAV2 (triple Y-F), respectively. 42   Figure 2. Enhanced in vivo 471 nm-fluorescent light fundoscopic images demonstrating retinal GFP expression. Dog 1 exhibited the most rapid onset of GFP expression, presenting in the optic nerve of the eye receiving high dose AAV2 (triple Y-F) at day 7 post-IVT (a, open arrow). At four weeks post-IVT, fluorescence was much stronger but still limited to the optic nerve (b, solid arrow).  Dog 4 first showed appreciable fluorescence within the optic nerve of the AAV2 (triple Y-F + T-V) high dose eye at day 10 post-IVT (c, open arrow). By 8-weeks post-IVT, strong fluorescence extended from the optic nerve into the superior retina (d, solid arrow). Some background tapetal autofluorescence can be seen in the enhanced fluorescent images (arrowheads).  Images enhanced using Microsoft Picture Manager (Microsoft Corporation, Redmond WA). IVT, intravitreal injection; GFP, green fluorescent protein. These data suggest that time of GFP expression onset was similar between vectors, but that the high dose of both vectors was able to induce GFP expression in a more rapid manner than the low dose.  43  2.2.3 RGC layer transduction efficiency The transduction efficiency for all regions of each eye is reported in Table 6.  Both AAV2 (triple Y-F + T-V) and AAV2 (triple Y-F) vectors showed an increase in transduction efficiency from 4 to 8 weeks post-IVT at both low and high titers.  The AAV2 (triple Y-F + T-V) vector at a titer of 1.3 x 1012 transduced a higher proportion of RGC layer cells compared to the AAV2 (triple Y-F) vector at the same titer, although the observed increase was not significant (p=0.06, unpaired t-test).  Both vectors performed significantly better at the high titer (1012) compared to the low titer (1011, p<0.02, paired t-test).  Peak regional efficiency was 31.0% for the AAV2 (triple Y-F + T-V) vector and 22.2% for the AAV2 (triple Y-F) vector (Table 6).  Both vectors exhibited a similar pattern of retinal GFP expression, with regional variations trending toward greater transduction efficiency within nasal and central compared to temporal retinal regions (Figure 3). Table 6. RGC layer transduction efficiency (%) by retinal region (refer to Figure 1) for safety and efficacy study. Retinal Section Dog 1  OD (4wk) Dog 1  OS (4wk) Dog 2  OD (8wk) Dog 2  OS (8wk) Dog 3  OD (8wk) Dog 3  OS (8wk) Dog 4  OD (4wk) Dog 4  OS (4wk) 1 16.7 0 18.8 24.0 31.0 24.2 0 17.9 2 22.2 0 7.7 14.3 22.2 14.8 0 7.1 3 5.5 0 0 8.3 30.7 15.4 0 18.8 4 9.1 0 0 15.4 31.3 13.3 0 23.1 5 6.3 0 0 21.4 21.4 10.9 0 10.7 6 0 0 0 11.1 22.3 8.4 0 0 7 0 0 5.8 0 14.2 0 0 0 8 0 0 0 0 10.5 0 0 0 9 0 0 0 0 9.3 0 0 0  44   Figure 3. Immunohistochemical labeling of retinal cryosections with the antibody NeuN demonstrating regional variation in transduction efficiency observed with both vectors.  The AAV2 (triple Y-F) high dose eye from Dog 2 and AAV2 (triple Y-F + T-V) high dose eye from Dog 3 both exhibited an ability to efficiently transduce RGC layer cells in the nasal retinal sections at 8-weeks post-IVT (a and c, respectively), as shown by colabeling of cell nuclei with NeuN and GFP antibodies. A lower level of transduction efficiency was present in the temporal sections from the same eyes (b and d, respectively). Images captured using a fluorescent microscope (Nikon Eclipse 80i, Nikon Instruments Inc., Melville, NY, USA) with a 20x objective lens. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; IVT, intravitreal injection; GFP, green fluorescent protein; NeuN, neuronal nuclei.    The efficiency results, combined with the ophthalmic examination results, suggest that the AAV2 (triple Y-F + T-V) vector at the high dose should be utilized for future studies, as it is more efficient than the AAV2 (triple Y-F) vector and carries a good safety margin through 8 weeks post-IVT.  The variation observed in transduction efficiency between retinal regions warrants further investigation in future studies.  45  2.2.4 Extraocular GFP expression Sections from the optic nerves and optic chiasm of both dogs that were euthanized at 8 weeks post-IVT exhibited regional GFP fluorescence within nerve axons (Figure 4).  Sections of the LGN and superior colliculus also contained GFP fluorescence within nerve axons exclusively, aside from the left LGN of Dog 3, which contained some GFP positive nuclei surrounding a large blood vessel (Figure 5).  Interestingly, the right LGN of Dog 2 had stronger GFP expression than the left LGN, corresponding to the observed nasally-oriented GFP expression within the left eye. The extraocular expression of GFP observed following IVT of both vectors is concerning when considering the risk of insertional mutagenesis, or of off-target transgene expression inducing immune reactions in central nervous tissues. Although the GFP expression was limited to the axons of RGC neurons that originate within the eye, there were some cell bodies within the LGN of Dog 3 that expressed GFP, suggesting transduction occurred within the off-target nervous tissue.  This observation greatly supports the use of cell-specific promoter sequences that would inhibit the expression of an AAV transgene product within off-target cell populations, which should minimize the risk of unintentional adverse effects on non-target tissues.  46   Figure 4. GFP expression within the optic nerves of dogs euthanized at 8 weeks post-IVT.  Regional GFP expression was strong in the optic nerve axons of the AAV2 (triple Y-F) high dose eye of Dog 2 (a) and in both AAV2 (triple Y-F + T-V) high dose (c) and low dose (d) eyes of Dog 3.  The AAV2 (triple Y-F) low dose eye of Dog 2 had limited GFP expression (b). Images captured using a fluorescent microscope (Nikon Eclipse 80i, Nikon Instruments Inc., Melville, NY, USA) with a 4x objective lens.  IVT, intravitreal injection; GFP, green fluorescent protein.  47   Figure 5. GFP expression within the LGN and superior colliculus of dogs euthanized at 8 weeks post-IVT.  GFP expression was strongest in the right LGN of Dog 2 (a), which would receive the nasal optic nerve axons from the left eye injected with high dose AAV2 (triple Y-F).  GFP expression was weaker in the left LGN of Dog 2 (b).  GFP expression was present within nerve axons bilaterally in the LGN of Dog 3 (c,d) and in the superior colliculi of both dogs (e, Dog 2 pictured).  Cell nuclei surrounding a larger vessel within the left LGN of Dog 3 also expressed GFP (f).  Images captured using a fluorescent microscope (Nikon Eclipse 80i, Nikon Instruments Inc., Melville, NY, USA) with a 4x objective lens for images a-d, a 40x objective lens for e, and a 20x objective lens for f. LGN, lateral geniculate nucleus; IVT, intravitreal injection; GFP, green fluorescent protein.  48  CHAPTER 3 PHOTORECEPTOR-TARGETED VECTOR STUDY 3.1 Introduction Adeno-associated virus (AAV) vectors have numerous advantageous properties that have been exploited for successful gene therapy including the ability to transduce post-mitotic cell populations, induction of long-term transgene expression, and low immunogenicity within human patients and animal models.49, 168 Subretinal injection is the most commonly utilized delivery route for targeting of AAV vectors to retinal photoreceptors.97 Major disadvantages of subretinal delivery include the induced retinal detachment may worsen the underlying disease process the therapy is intended to treat, and the necessity of a trained surgeon with specialized equipment for routine injection success.26, 97, 101 Interest in circumventing these disadvantages has driven the development of AAV vector variants which readily penetrate the retina and transduce photoreceptors following intravitreal injection (IVT). Unfortunately, IVT results in increased exposure of AAV particles to the immune system98, and is more likely to produce off-target transgene expression in non-ocular tissues, including the brain112. To circumvent these adverse effects, photoreceptor-specific promoters may be used, which have been shown to inhibit AAV-mediated transgene expression in off-target cell populations following subretinal, intravitreal, and intravascular administration.107, 114, 169   Recent advances in AAV vector development, including rational mutagenesis and the technique directed evolution, have made widespread outer retinal transduction possible in rodent models following IVT.107, 108 Alteration of the capsid amino acid constituents of AAV vectors can increase their overall efficiency. Substitution of specific capsid tyrosine residues for 49  phenylalanine (Y-F) promotes evasion of intracellular proteasomal degradation pathways67, and decreases the affinity of vectors for heparan sulfate proteoglycan receptors on the inner limiting membrane (ILM) surface96. Serine and threonine residue substitutions are thought to further diminish intracellular phosphorylation and ubiquitination leading to enhanced transport of AAV vector particles into the nucleus.68 An additional threonine-to-valine (T-V) substitution in an AAV2 vector containing four Y-F substitutions was recently shown to increase photoreceptor transduction in mice 3.5-fold over a vector containing the Y-F substitutions alone.108 Fundamental differences in retinal structure and physiology make translation of results from rodent models to human subjects challenging, as various studies have demonstrated identical AAV vector constructs can have significantly attenuated responses when used in large animal models.106, 107, 109 Dogs are an important preclinical model for the development of retinal gene therapy, as they exhibit inherited retinal diseases that mimic those observed in humans, often the result of a mutation in an analogous gene.37 They also possess similar retinal anatomy to humans, including a recently described fovea-like region of high cone photoreceptor density.170 Efficacy studies performed in canine models have provided the foundational justification for clinical trials to be performed in multiple human retinal dystrophies.18, 40, 148, 169, 171 The primary aim of our study was to evaluate the photoreceptor transduction efficiency of two novel AAV2 vectors containing capsid amino acid substitutions following IVT in dogs. A green fluorescent protein (GFP) reporter transgene was driven by either the interphotoreceptor binding protein (IRBP) promoter or guanine nucleotide binding protein alpha transducing activity polypeptide 2 promoter with an IRBP enhancer (GNAT2/IRBP) to restrict expression to targeted rod and/or cone photoreceptors. Both promoters produce robust photoreceptor transduction following subretinal injection in dogs when packaged with AAV5 or AAV8 capsids.169, 172 As a 50  secondary aim, we investigated the correlation of regional transduction efficiency variation with ILM thickness measurements.   3.2 Results and Discussion 3.2.1 GFP expression and safety Two novel capsid substituted vectors, AAV2 (quad Y-F + T-V) and AAV2 (quad Y-F), with GFP transgene expression driven by either the IRBP or GNAT/IRBP promoter, were delivered separately via IVT into the right or left eye of six dogs (Table 7) immediately adjacent to the retinal surface along the visual streak (Figure 6a). A total of 4 x 1011 vector genomes were injected in a volume of 200µL. At six and eight weeks post-IVT, confocal scanning laser ophthalmoscopy (cSLO) showed the six eyes injected with IRBP vectors [AAV2 (quad Y-F + T-V) IRBP or AAV2 (quad Y-F) IRBP] had increased GFP fluorescence above background tapetal autofluorescence in regions surrounding the retinal vasculature (Figure 7a-d). Of the six eyes injected with GNAT2/IRBP vectors [AAV2 (quad Y-F + T-V) GNAT2/IRBP or AAV2 (quad Y-F) GNAT2/IRBP], none showed increased fluorescence on cSLO at either time point post-IVT. Increased fluorescence was not observed in either eye of the six dogs using fluorescent fundus camera imaging at any time point. Aside from expected mild post-operative intraocular inflammation, characterized by grade 1 out of 4 aqueous humor flare, (grade 1 = Tyndall effect just barely visible with slit-lamp magnification in a darkened exam room) in all eyes and mild settled hyphema in two of eight eyes, no adverse effects following IVT were noted in any of the six dogs. These changes were present at examination 24 hours following injection, but had resolved by 48 hours post-IVT. This degree of inflammation is typical as a response to IVT and 51  subretinal dosing procedures in our laboratory, and does not appear to be a vector-directed reaction due to rapid resolution of clinical signs. Table 7. Intravitreal dosing arrangement and AAV vector descriptions for photoreceptor-targeted vector study.  Beagle 1 Beagle 2 Beagle 3 Mixed Breed Dog 4 Mixed Breed Dog 5 Mixed Breed Dog 6 Left Eye AAV2(quad Y-F) IRBP AAV2(quad Y-F + T-V)  IRBP AAV2(quad Y-F) IRBP AAV2(quad Y-F + T-V)  GNAT2/IRBP AAV2(quad Y-F) GNAT2/IRBP AAV2(quad Y-F + T-V)  GNAT2/IRBP Right Eye AAV2(quad Y-F + T-V)  IRBP AAV2(quad Y-F) IRBP AAV2(quad Y-F + T-V)  IRBP AAV2(quad Y-F) GNAT2/IRBP AAV2(quad Y-F + T-V)  GNAT2/IRBP AAV2(quad Y-F) GNAT2/IRBP Y- tyrosine, F- phenylalanine, T- threonine, V- valine, IRBP- interphotoreceptor binding protein, GNAT2- guanine nucleotide binding protein alpha transducing activity polypeptide 2  52   Figure 6. Photoreceptor transduction efficiency following IVT of AAV2 (quad Y-F + T-V) IRBP and AAV2 (quad Y-F) IRBP. 53   Figure 7.  Localization of maximal photoreceptor transduction following IVT of AAV2 (quad Y-F + T-V) IRBP and AAV2 (quad Y-F) IRBP. 54  3.2.2 AAV vector retinal transduction efficiency At eight weeks post-IVT, sagittal eyecup cryosections were taken from all eyes through the optic nerve head (ONH) as well as 2-4mm nasal and temporal of the ONH, and used for quantification of rod and cone photoreceptors expressing GFP. Overall, AAV2 (quad Y-F) IRBP and AAV2 (quad Y-F + T-V) IRBP vectors transduced 4.3 ±0.8% and 4.6 ±1.4% of cones, and 2.2 ±0.3% and 2.0 ±1.2% of rods, respectively (Figure 6b). These rates were not significantly different (cones: p=0.43 and rods: p=0.42). Up to 11-fold increases in transduction rates of photoreceptors were noted beneath retinal blood vessels, with up to 31% of cones and 25% of rods expressing GFP (Figure 7e,f).  These regions were excluded from statistical analysis to avoid bias when comparing regions with limited retinal vasculature (such as the cone rich area centralis and visual streak) with other retinal regions.  The GNAT2/IRBP vectors did not transduce a sufficient number of cone photoreceptors to allow quantification or statistical comparison.  The major aim of our study was to evaluate the ability of novel AAV vectors to transduce photoreceptors in dogs following IVT. We found both IRBP vectors were capable of photoreceptor-specific transgene delivery following IVT, an original finding in a large animal model. Previous studies injecting wild-type AAV2 intravitreally in dogs and primates have reported absent or limited photoreceptor transduction.106, 173 Recently, capsid-mutant AAV2 vectors with ubiquitous promoters were reported to produce high levels of photoreceptor transduction beneath retinal vessels following IVT in dogs and primates.107, 109 It is promising that the IRBP vectors in our study also produced strong transduction in these regions, at similar efficiency to the AAV2 (quad Y-F) vector capsid paired with the strong ubiquitous smCBA promoter used for IVT by Mowat et al.109 Greater than 90% of photoreceptors were transduced by this vector following subretinal injection.109 It must be noted that the vector dose used for IVT 55  in our study was roughly threefold higher. Although transduction efficiency was not quantified in a study reporting transgene expression in primate photoreceptors following IVT of a novel mutant AAV2 paired with the ubiquitous CMV promoter, confocal images of regions beneath retinal vessels appear to demonstrate comparable results to those found in our study.107 That vector was injected at a dose more than tenfold higher than our study.  Transduction efficiency in our study was lower than previously reported in mice using the same vector capsids paired with the strong smCBA promoter; however, the transgenes were packaged in a self-complimentary manner for the mouse study which increases transgene expression levels, while the vectors in our study had single-stranded genomes.108 Single-stranded genomes allow larger transgenes to be delivered, making them more applicable for replacement of defective genes causing human retinal dystrophies.174 Kay et al. showed a significant increase in the number of photoreceptors transduced when AAV2 (quad Y-F + T-V) was compared to the AAV2 (quad Y-F) following IVT in mice.108 A similar effect was not observed in our canine model, as the vector transduction efficiencies were not statistically different in any region. The reason for the lack of difference between the efficiencies of the vectors in the canine is unclear; and could be attributed to differences in cell surface receptors or intracellular trafficking at any level between the ILM and the outer nuclear layer. The primary receptor recognized by AAV2 is heparan sulfate proteoglycan, although additional co-receptors exist.49 A recent study demonstrated that alterations to the capsid amino acid composition of AAV2 affects the vector affinity for these cell surface receptors, thereby impacting IVT mediated transduction.96 Species variation in the composition and density of these receptors on the ILM or other retinal cell membranes would theoretically produce differences in the ability of AAV vectors to transduce 56  the photoreceptors following IVT. Differences in endosomal transport and capsid processing67 of the vectors between species could also account for the differences we observed.  Our findings are preliminary for large animal models and further optimization of vector efficiency is required for future therapeutic use. A minimum photoreceptor transduction threshold likely exists that must be exceeded to obtain a clinically significant therapeutic response, but is likely to vary between different disease states and even individual animals, as has been shown previously.40 Photoreceptor-specific AAV vectors capable of producing therapeutic rescue following IVT in rodent models of retinoschisis showed a lower level of transduction when injected into wild-type mice.133 Although our initial transduction efficiency was limited in normal dogs, we are very interested to assess vector efficiency in a number of our canine models of retinal dystrophy. If required, further optimization techniques might include additional vector capsid amino acid substitutions or manipulation of the host ILM to potentiate retinal penetration. 3.2.3 AAV vector retinal tropism As capsid engineering has advanced to improve transduction efficiency of next-generation AAV vectors, off-target expression of transgenes has emerged as a significant safety concern.58, 88 The IRBP vectors both drove GFP expression in LM-cones, S-cones, and rods exclusively, without any off-target retinal transduction observed (Figure 8).  This finding is in line with the report of Beltran et al., in which use of the IRBP promoter limited transgene expression to canine rods and cones following subretinal injection.169 By limiting transgene expression in off-target tissues through the use of a cell-specific promoter, risk of developing a transgene-directed immune response is minimized.46 In five of six eyes that received the GNAT2/IRBP vectors, limited GFP 57  expression was present only within cone photoreceptors beneath the retinal blood vessels (Figure 9a,b). In one of six eyes (dog 4, Table 7), off-target transduction occurred in the superior nasal retina following IVT of AAV2 (quad Y-F + T-V) GNAT2/IRBP; GFP expression was present within all retinal cellular layers at this site (Figure 9c-f). When paired with an AAV5 vector, the GNAT2/IRBP promoter/enhancer combination produced transgene expression limited to cones and a small number of rods with no inner retinal expression in mice175, and in all cone subclasses exclusively in dogs172, following subretinal injection. It is unclear why the off-target expression within the inner retinal layers was only seen in one region of one eye in our study; one possibility is the affected region was exposed to a higher dose of vector as a result of the IVT procedure.   58    Figure 8.  Photoreceptor-specific transduction following IVT of AAV2 (quad Y-F) IRBP and AAV2 (quad Y-F + T-V) IRBP.  Representative photomicrographs demonstrate GFP expression was limited to rod and cone photoreceptors 8 weeks post-IVT (a,d).  Transduction of L/M- and S-cone photoreceptor subtypes is demonstrated (white arrows) in panels (b,e), and (c,f), respectively.  Co-labeling with the L/M-opsin and S-opsin antibodies is implied by approximation of a GFP positive inner segment with an opsin-labeled outer segment in this confocal microscopy section. hCAR, human cone arrestin; RPE, retinal pigmented epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 50m. 59   Figure 9. Retinal transduction following intravitreal injection of AAV2 (quad Y-F) GNAT2/IRBP and AAV2 (quad Y-F + T-V) GNAT2/IRBP.  In 3 of 3 eyes receiving AAV2 (quad Y-F) GNAT2/IRBP (a) and 2 of 3 eyes receiving AAV2 (quad Y-F + T-V) GNAT2/IRBP (b) GFP expression was limited to a very small number of cone photoreceptors underlying retinal vessels.  In 1 of 3 eyes receiving AAV2 (quad Y-F + T-V) GNAT2/IRBP off-target GFP expression was observed by cells of the inner nuclear and ganglion cell layers in the superior nasal retinal region. There was no GFP co-labeling with the bipolar cell-antibody (c), but co-labeling was present with the ganglion cell and amacrine cell-specific neuronal nuclei (NeuN) antibody (d).  A large number of cone photoreceptors were transduced in this region, including both L/M cones (e) and S cones (f) (solid arrows).  hCAR, human cone arrestin; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 50m. 60   3.2.4 Distribution of photoreceptor transduction Variation in transduction rates produced by the IRBP vectors was observed between retinal regions (Figure 6c, Table 8), and a consistent pattern in the geographic distribution emerged during data analysis. The central and nasal visual streak regions (Figure 6a, regions 2 and 5) showed significantly higher (p<0.05) transduction rates than temporal retinal regions (Figure 6a, regions 7-9). Notably, 15.6% of cones were transduced in the central region of the cone rich visual streak. However, transduction efficiency was greatly attenuated in the temporal region containing the area centralis (Figure 6, region 8), a structure in dogs similar to the human macula.170 This variation in transduction efficiency could not be easily explained by procedural variables such as patient positioning or the dosing location, as the dogs were placed in dorsal recumbency. With the injections performed by a right handed surgeon, this resulted in a temporal approach in the right eye and a nasal approach in the left eye. The injection device was intentionally moved across the region of the visual streak during vector administration to expose both the nasal and temporal retina.  It has been suggested that variation in ILM thickness may account for differences in transduction efficiency of AAV vectors delivered by IVT in canines and primates.106, 109 The ILM acts as a barrier to transport of not only AAV119, 176, 177, but also stem cells178 and nanoparticles118, 125. To investigate this hypothesis, retinal sections labeled with anti-laminin antibody were used to measure ILM thickness. Overall, there were no significant differences in ILM thickness detected between the retinal regions (Figure 10, p=0.24). Linear correlation analysis showed there was no correlation between the ILM thickness and average photoreceptor transduction rates (cones: r2=0.17, rods: r2=0.21) (Figure 10d,e). This result suggests ILM thickness alone does not account 61  for regional variation in retinal transgene expression in the dog, and some other property of the temporal retina or vitreous humor affects vector transduction efficiency. Both heparan sulfate proteoglycan and laminin receptors are reported to be present within the ILM of rodents.119, 177 It is possible a variation in the density of these receptors exists between retinal regions in the dog, which differentially sequesters AAV2 particles as they are transiting into the retina.  This will be a future direction of investigation in our laboratory. Table 8. Mean photoreceptor transduction and statistical comparison of regions for photoreceptor-targeted vector study. Region  (Illustrated in Figure 6a) Percent cones transduced  (± SD) Regions p<0.05 Percent rods transduced (± SD) Regions p<0.05 1  Nasal Superior 4.1 (± 5.4)  2.5 (± 2.0) 6 2  Nasal Visual Streak 7.8 (± 5.3) 6,7,8 4.3 (± 3.4) 3,6,7,8 3  Nasal Inferior 3.8 (± 3.4) 5 1.2 (± 0.6) 2,5,6 4  Central Superior 5.9 (± 6.5)  2.7 (± 3.2) 6,7 5  Central Visual Streak 15.6 (± 5.9) 3,6,7,8 6.6 (± 6.9) 3,6,7,8,9 6  Central Inferior 0.7 (± 1.6) 2,5 0.3 (± 0.3) 1,2,3,4,5 7  Temporal Superior 0.5 (± 1.1) 2,5,8 0.6 (± 0.7) 2,4,5 8  Temporal Visual Streak 1.5 (±2.3) 2,5,7 0.5 (± 0.5) 2,5 9  Temporal Inferior 0 (± 0) * 0.2 (± 0.4) 5 *Region 9 cones could not be included in linear regression analysis due to value of 0.   62    Figure 10. Assessment of regional retinal inner limiting membrane thickness.  Like previous studies, our results demonstrate increased retinal AAV transduction in regions adjacent to and beneath the retinal vasculature.106, 107, 109, 179 There is a stronger attachment of 63  vitreous cortex to the retina in regions where the retinal vasculature exists.120, 180 The ILM is thinner over these regions in primates, and there is separation of Muller cell endfeet over the vessels resulting in the presence of pores within the ILM.121 Vitreous humor is reported to extend through these pores, which may account for vector accumulation at these sites and increased transduction within these regions.122 These reported findings, in conjunction with our inability to link ILM thickness with outer retinal transduction efficiency, suggest that differences in composition of the ILM may alter AAV permeability to a greater degree than ILM thickness.  In conclusion, based on our primary aim, we demonstrated both AAV2 (quad Y-F) IRBP and AAV2 (quad Y-F + T-V) IRBP were able to produce photoreceptor-specific transgene expression following IVT in dogs. The addition of a threonine-to-valine capsid substitution did not significantly increase transduction efficiency of the vector as observed previously in mice.  AAV2 (quad Y-F + T-V) GNAT2/IRBP and AAV2 (quad Y-F) GNAT2/IRBP were considerably less efficient, and some off-target retinal GFP expression was observed. Following evaluation of our secondary aim, it appears ILM thickness is not significantly variable across the retina in dogs. ILM thickness alone, therefore, is unlikely to account for regional variations in vector transduction efficiency. Identification of a specific property of the ILM and/or vitreous humor that contributes to regional variation in AAV transduction efficiency would contribute to advancement in design of therapeutic vectors meant for IVT in human patients. 3.3 Materials and Methods 3.3.1 Animals Three purpose-bred ten-month-old Beagle dogs (Marshall BioResources, North Rose, NY) were used for injection of the vectors containing the IRBP promoter; and three mixed-breed purpose-64  bred dogs (ages 9, 16, and 58 months) were used for injection of vectors containing the GNAT2/IRBP promoter. All dogs were socially housed with a 12-hour light:dark cycle. Animal care was in compliance with the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research, and all procedures were performed following approval by the Institutional Animal Care and Use Committee. 3.3.2 AAV vectors Four recombinant AAV vector constructs containing capsid amino acid substitutions were manufactured and purified at the Retinal Gene Therapy Vector Lab, University of Florida College of Medicine using previously described methods.108, 181 One AAV2 vector was mutated for substitution of four surface-exposed capsid tyrosine residues with phenylalanine [Y272F, Y444F, Y500F, and Y730F; referred to as AAV2 (quad Y-F)]. A second AAV2 vector was mutated to include the same four tyrosine to phenylalanine substitutions, plus an additional substitution of a surface-exposed threonine to valine [T491V; referred to as AAV2 (quad Y-F + T-V)]. Two separate promoters were used: 1) the 1.3kb human interphotoreceptor binding protein promoter (IRBP), reported to specifically target rod and cone photoreceptors following subretinal injection169; and 2) the 277-bp 5'-flanking sequence of the human guanine nucleotide binding protein alpha transducing activity polypeptide 2 promoter coupled with the 214-bp IRBP enhancer (GNAT2/IRBP)182, which targets cone photoreceptors following subretinal injection in mice, with a small number of rod photoreceptors transduced.175 Both promoters drove expression of GFP. Each capsid type was used to separately package each of the two promoter/reporter gene constructs, yielding a total of four vectors (Table 7).  65  3.3.3 Intravitreal injections Vectors were prepared for injection by diluting stock supplies to a titer of 2 x 1012 vg ml-1 using sterile balanced salt solution (Alcon Laboratories, Fort Worth, TX). 200µl of vector solution was injected into the vitreous humor immediately anterior to the retinal surface in a transverse plane along the visual streak (Figure 6) using a RetinaJect injector (SurModics, Inc., Eden Prairie, MN) as previously described.109, 127 Direct visualization of the injector tip in close proximity to its shadow on the tapetal retina allows solution to be injected immediately adjacent to the retinal surface. Previous work in our laboratory using retinoid therapy showed that injection adjacent to the retinal surface produced more consistent results; therefore, we adopted this method for IVT of AAV vectors to potentially reduce sequestration of vector particles within the vitreous humor.127 Postoperative treatment included antibacterial and anti-inflammatory medications as previously described for IVT and subretinal injections in our laboratory.109 The vector dose was selected based on results from a preliminary safety study utilizing similar vectors containing the ubiquitous CBA promoter (unpublished data). Briefly: four eyes were administered 1.3 x 1012 vg ml-1 by IVT and showed increased GFP expression compared to four eyes administered 1.3 x 1011 vg ml-1. No adverse effects were observed at either vector titer, so the higher titer from this safety study was selected.  3.3.4 Ophthalmic examinations and imaging Post-IVT all dogs received regular ophthalmic examinations and fluorescence fundus imaging as previously described.109 Confocal scanning laser ophthalmoscopy utilizing 488 nm laser-induced fluorescence (cSLO, Spectralis, Heidelberg Engineering, Carlsbad, CA) was performed at 6 and 8 weeks post-injection under general anesthesia. 66  3.3.5 Eyecup collection and sectioning All dogs were euthanized 8 weeks post-injection with an intravenous injection of sodium pentobarbital (Fatal Plus, Vortech Pharmaceuticals, Dearborn, MI). Eyes were immediately enucleated and processed as previously described.166 14µm thick sagittal cryosections, three sections per slide, were collected (Leica CM3050-S cryostat, Leica Microsystems, Buffalo Grove, IL) from the central, nasal, and temporal eyecups (Figure 6), and stored at -20°C. 3.3.6 Immunohistochemistry and cell quantification Details of primary and secondary antibodies used for immunohistochemical labeling can be found in Table 9. Representative images from the superior, central, and inferior aspect of sagittal retinal cryosections collected from the central, nasal, and temporal regions of each eyecup (Figure 6) were captured using a confocal laser scanning microscope (Olympus FluoView fv1000 Confocal, Center Valley, PA) at 40x magnification. Slides labeled with cone arrestin (hCAR) and GFP antibodies were used for counting purposes with regions overlying retinal vasculature avoided. Identifying information was masked, images randomized, and retinal cells counted by a single observer (RFB). For each image, separate counts were produced for all DAPI stained nuclei within in the outer nuclear layer (ONL), GFP-labeled ONL nuclei, hCAR-labeled ONL nuclei, and GFP-labeled ONL nuclei co-labeled with hCAR.    67  Table 9. Primary and secondary antibodies used for retinal immunohistochemistry for photoreceptor-targeted vector study. Target antigen Primary antibody details and working dilution Manufacturer Targeted cell population Secondary antibody and concentration (all from Invitrogen Corp, Carlsbad, CA) Cone arrestin (LUMif)167 Rabbit polyclonal 1:5000 Kind gift of Cheryl M. Craft, University of Southern California Cone photoreceptors109 Alexafluor 546 goat anti-rabbit 1:500 Green fluorescent protein Rabbit FITC conjugated, 1:1000 Invitrogen Corp, Carlsbad, CA GFP transduced cells109 N/A L/M-opsin Rabbit polyclonal 1:500 Millipore, Billerica, MA L/M-cone outer segments166 Alexafluor 546 goat anti-rabbit 1:500 S-opsin Rabbit polyclonal 1:500 Millipore, Billerica, MA S-cone outer segments109 Alexafluor 546 goat anti-rabbit 1:500 Protein kinase C alpha Mouse monoclonal 1:500 BD Biosciences, San Diego, CA Rod bipolar cells109 Alexafluor 546 goat anti-mouse 1:500 Neuronal Nuclei Mouse monoclonal 1:2000 Millipore, Billerica, MA Ganglion and Amacrine cells165 Alexafluor 546 goat anti-mouse 1:500 Laminin183 Rabbit polyclonal Dako, Carpinteria, CA Inner limiting membrane119 N/A  3.3.7 Evaluation of ILM thickness Six globes collected from recently euthanized normal three-month-old mixed-breed dogs and fixed in 2% paraformaldehyde were submitted to the Michigan State University Diagnostic Center for Population and Animal Health for labeling with an anti-laminin antibody. The globes were post-fixed for 24 hours in 10% formaldehyde, and then paraffin embedded. Sagittal retinal sections were obtained from each globe to match the sections evaluated for vector transduction 68  efficiency. The labeled slides were imaged using white light microscopy (Nikon Eclipse 80i, Nikon Instruments Inc., Melville, NY, USA) to obtain representative 40x images from the nine retinal regions detailed in Figure 6. The thickness of the ILM from each region was measured using computer software (Adobe Photoshop CS4; San Jose, CA, USA). 3.3.8 Statistical analysis Differences in overall retinal rod and cone photoreceptor transduction efficiency for each vector, as well as transduction efficiency between regions of the retina, were compared using generalized linear regression analysis with a Tweedie distribution (SPSS, IBM, Armonk, NY). To clarify effects of significant categorical predictors, we applied a Bonferroni adjustment. Differences in ILM thickness between regions of the retina were compared using ANOVA (Excel, Microsoft, Redmond, WA). Results were considered significant if p<0.05. Linear correlation was used to compare the ILM thickness with the photoreceptor transduction rates by region (Excel, Microsoft, Redmond, WA). Data are displayed as mean ± standard deviation.   69  CHAPTER 4 VITRECTOMY STUDY 4.1 Introduction Neuroprotective strategies are a critical focus in the development of novel therapeutic interventions to prevent retinal ganglion cell (RGC) loss secondary to glaucoma.184 Direct supplementation of exogenous neurotrophic factors via intravitreal injection (IVT) results in RGC preservation in rodent glaucoma models. Unfortunately, these effects are short-lived, typically persisting less than 2 weeks.42, 185-187 Although significant advancement has been made in  sustained-release device technology, the steady-state delivery of neurotrophic support remains limited to between 6 months and 3 years.188 As an alternative to sustained-release devices, adeno-associated virus (AAV)-mediated gene therapy appears promising. Since AAV vectors can stably transduce post-mitotic inner retinal cells, long-term transgene expression resulting in a life-time therapeutic effect becomes a theoretical possibility.189 Evaluation of IVT-delivered AAV vectors in small animal models has demonstrated rescue of RGCs following axonal insult; however, this effect appears to be variable by retinal region.190 AAV delivered via IVT in large animal models also produces variable RGC transduction between retinal regions.106, 107, 109 We recently reported a similar finding when using photoreceptor-targeted AAV vectors in dogs, where retinal tissue penetrating ability was significantly inhibited over the temporal retina.191 Development of a method that generates consistent, widespread retinal transduction is pivotal to the advancement of AAV gene therapy in management of glaucoma as well as outer retinal diseases. 70  Immune reactions following the administration of viral vectors reduce therapeutic benefits while simultaneously inflicting damage on transduced cells and harming the patient.151 The eye has long been recognized as an immune-privileged site, limiting the development of immune reactions following both subretinal and IVT delivery.192 However, reports of immune reactions in animal models following intraocular delivery have become more frequent as dosing strategies are modified to employ more efficient, engineered AAV vectors with escalation of administration titers.56, 107, 163 When an unexpected immune reaction occurs, it is paramount to determine whether it is directed against the vector capsid or the expressed transgene. Animal models appear to be resistant to the development of cell-mediated capsid reactions; however, transgene reactions have been described.151, 193 Risk of these reactions is significantly increased with gene therapy for diseases caused by recessive null mutations, since a novel transgene is expressed to which the host immune system has never been exposed.159 Core vitrectomy is routinely used in human patients prior to subretinal injection of AAV vectors, which provides space for the advancing subretinal bleb and limits elevation of intraocular pressure immediately post-injection.26 It is also commonly used in management of retinal detachments and proliferative diabetic retinopathy. A primary aim of our study was to evaluate the effect of vitrectomy on the regional distribution of transduction following IVT of AAV in dogs, as well as a qualitative assessment of its effect on retinal penetration ability. A secondary aim of our study was to characterize the efficiency of AAV capsid subtype 2 with three capsid tyrosine-to-phenylalanine substitutions and one threonine-to-valine substitution [AAV2 (triple Y-F + T-V)], carrying the green fluorescent protein (GFP) reporter transgene driven by the constitutive chicken -actin (CBA) promoter, for transduction of RGCs in the dog model. AAV2 (triple Y-F) has been previously shown to increase transduction efficiency in RGCs >30-fold 71  when compared to wild-type AAV2 in mice.95 A preliminary dose-range finding and safety study in our laboratory suggested that AAV2 (triple Y-F + T-V) is more efficient than AAV2 (triple Y-F) following IVT in dogs, and we hypothesized that core vitrectomy with posterior hyaloid membrane peeling may further potentiate this transduction efficiency. When a marked intraocular, presumed immune reaction occurred part way through the study, focus was redirected toward characterization of the reaction, including procedural and/or vector-induced variables responsible for the immune response. 4.2 Results and Discussion 4.2.1 Preliminary safety and dose-range finding study The vector dose used in our study was selected based on results from a preliminary dose-range finding and safety pilot study utilizing the same vector (unpublished data). In the pilot study eight normal dog eyes were administered either AAV2 (triple Y-F + T-V) or AAV2 (triple Y-F), both utilizing CBA promoter to drive GFP expression. Four eyes received IVT of 2.6 x 1011 vector genomes and had higher transduction rates compared to four eyes administered a total of 2.6 x 1010 vector genomes. AAV2 (triple Y-F + T-V) transduced cells within the GCL, our intended cellular target, more efficiently than AAV2 (triple Y-F) (Table 10). Aside from expected mild post-operative intraocular inflammation, characterized by grade 1 out of 4 aqueous humor flare (Table 11) in all eyes, which cleared within 48 hours post-IVT, no adverse effects were noted in any of the eight eyes. Based on these findings, AAV2 (triple Y-F + T-V) at a dose of 2.6 x 1011 vector genomes was selected for the study described below.  72  Table 10. Average (peak) retinal ganglion cell layer transduction percentage from pilot safety and efficacy study. Weeks post-IVT AAV2 (triple Y-F) 2.6 x 1011 vg AAV2 (triple Y-F) 2.6 x 1011 vg AAV2 (triple Y-F + T-V) 2.6 x 1010 vg AAV2 (triple Y-F + T-V) 2.6 x 1010 vg 4 weeks 0 (0) 6.6 (22.2) 0 (0) 8.6 (23.1) 8 weeks 3.6 (18.8) 10.5 (24.0) 9.7 (24.2) 21.4 (31.3)  Table 11. Definition of grading scheme for anterior uveitis.1 Grade Description 1 Tyndall effect just barely visible with slit-lamp magnification in a darkened exam room 2 Moderate Tyndall effect with slit-lamp magnification in a darkened exam room 3 Marked Tyndall effect with slit-lamp magnification in a darkened exam room 4 Intense Tyndall effect with slit-lamp magnification in a darkened exam room and presence of fibrin, hypopyon, or hyphema  4.2.2 Ophthalmic examinations and GFP fluorescence Core vitrectomy with posterior hyaloid membrane peeling was performed in the right eye of three dogs, four weeks prior to vector injection.  Twelve days after vitrectomy Dog 1 developed endophthalmitis of the right eye (grade 2/4 aqueous humor flare [Table 11], miosis, and vitreal cloudiness) and was treated with systemic corticosteroids and antibiotics. The endophthalmitis resolved within one week. Regional retinal thinning, evidenced by patchy tapetal hyperreflectivity, was the only abnormality present on ophthalmic examination at the time of vector injection. AAV2 (triple Y-F + T-V) was delivered via IVT into both vitrectomized and non-vitrectomized eyes of all three dogs immediately adjacent to the retinal surface along the visual streak. A total of 2.6 x 1011 vector genomes were injected in a volume of 200 µl. During the first four weeks post-IVT, all eyes were normal aside from expected post-procedural mild 73  anterior uveitis (grade 1/4 aqueous humor flare) at examination 24 hours following injection, with resolution by 48 hours.  At two weeks post-IVT, confocal scanning laser ophthalmoscopy (cSLO) showed widespread increased GFP fluorescence above background tapetal autofluorescence in the non-vitrectomized eye of all three dogs (Figure 11a). The multifocal pattern with curvilinear streaks coursing toward the optic nerve head suggested the fluorescence was mostly resultant from RGC and nerve fiber layer expression of GFP. Subjectively, the intensity and distribution of retinal fluorescence increased in the non-vitrectomized eye of all three dogs as the study progressed. The vitrectomized eyes of Dogs 2 and 3 were grossly devoid of retinal GFP fluorescence apart from focal fluorescence involving the optic nerve head beginning at two weeks post-IVT (Figure 11b). Dog 1 showed weak multifocal GFP fluorescence in the inferotemporal fundus region and adjacent to the retinal vasculature in the vitrectomized eye, which had previously been affected by endophthalmitis following vitrectomy. 74   Figure 11. Comparison of ganglion cell layer transduction efficiency of non-vitrectomized and vitrectomized eyes following IVT of AAV2 (triple Y-F + T-V). Representative confocal scanning laser ophthalmoscopy images (a,b) obtained at 5 weeks post-injection demonstrate widespread fluorescence in non-vitrectomized eye (a) versus fluorescence limited to the optic nerve head of the vitrectomized eye (b). The low level of background autofluorescence in the superior retina of image (b) is the expected appearance of the canine tapetum. Immunohistochemical labeling of retinal cryosections with Neuronal Nuclei antibody (NeuN, c,d) demonstrating a higher number 75  Figure 11 (cont of cells colabeling with GFP in non-vitrectomized eye (c, white arrows) compared to absent transduction in vitrectomized eye (d). e: Overall ganglion cell layer transduction efficiency for non-vitrectomized eyes and vitrectomized eyes 6 weeks post-IVT. Non-vitrectomized eyes had significantly higher levels of ganglion cell layer transduction (p=0.04; unpaired t-test). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; IVT, intravitreal injection; GFP, green fluorescent protein; DAPI, 4',6-diamidino-2-phenylindole.. Scale bar = 50 m. * There is no transduction efficiency calculation for the vitrectomized eye of Dog 1 due to loss of sample for histopathological analysis.  At four weeks post-IVT, Dog 3 developed acute bilateral endophthalmitis (grade 3/4 aqueous flare, miosis, and vitreal cloudiness) and was treated with topical and systemic corticosteroids and systemic antibiotics. The inflammation improved within one week but did not fully resolve, with vitreal cloudiness persisting. It was unclear what caused this inflammation; the acute and bilateral nature of the inflammation was suggestive of an immune reaction to the AAV capsid or transgene product, however a lack of inflammatory signs in the other two dogs precluded the omission of inflammation or infection secondary to the IVT surgical procedure as a possible etiology, so the study was continued. At six weeks post-IVT, Dog 1 developed acute endophthalmitis in the vitrectomized eye (grade 3/4 aqueous flare, miosis, and vitreal cloudiness), and retinitis (diffuse tapetal hyporeflectivity and increased vessel tortuosity) in the non-vitrectomized eye (Figure 12b). Timing of the inflammatory reactions observed at four and six weeks post-IVT is similar to that reported for both capsid and transgene-specific cytotoxic T-lymphocyte reactions in humans and large animal models.150, 151 This led us to hypothesize the inflammation was vector-induced, and a decision was made to terminate the study to collect samples critical for definitive determination of the etiology and to preserve retinal tissues necessary for evaluation of vector transduction distribution and efficiency. Dog 1 was euthanized immediately due to the rapidly progressive inflammation, and Dogs 2 and 3 were euthanized the 76  following day. Unfortunately, the abrupt decision to euthanize Dog 1 for humane reasons prior to planning assays for characterization of the immune reaction and vector biodistribution precluded collection of some samples from this animal. Aside from the described intraocular inflammation, all three dogs were systemically healthy throughout the course of the study. Gonioscopic imaging (488 nm blue light) was performed on Dog 2 at six weeks post-IVT. Fluorescence was observed within the ciliary cleft of the vitrectomized eye, while the non-vitrectomized eye did not have appreciable fluorescence (Figure 13a,b). At the time of eyecup collection six weeks post-IVT, the cornea, iris, and ciliary body from dogs 2 and 3 were also collected and cryosections were prepared. Both vitrectomized and non-vitrectomized eyes expressed GFP within the ciliary body (Figure 13e,f). Vitrectomized eyes had subjectively increased GFP expression within the iris and trabecular meshwork (Figure 13c,d).  77   Figure 12. Demonstration of retinitis following IVT of AAV2 (triple Y-F + T-V). White light fundoscopic imaging showing the normal fundus of the non-vitrectomized eye of Dog 1 at 4 weeks post-IVT (a), and acute retinitis that developed during week 6 (b).  Fluorescent microscopic images of retinas from non-vitrectomized eyes labeled with GFAP shown in c and d to demonstrate glial activation of Müller cells as a result of inflammation. c: Retina from Dog 2 without gross evidence of inflammation, showing expected strong GFAP labeling limited predominantly to the nerve fiber layer. d: Retina from Dog 3 with chronic inflammation, showing GFAP labeling extending from the nerve fiber layer into the inner and outer retinal layers. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; IVT, intravitreal injection; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; DAPI, 4',6-diamidino-2-phenylindole.. Scale bar = 50 m.  78   Figure 13. Comparison of GFP fluorescence in the anterior segments of vitrectomized and non-vitrectomized eyes. Fluorescent gonioscopic imaging showing fluorescence within the ciliary cleft of vitrectomized eye of Dog 2 at 6 weeks post-IVT (a, arrow). Note the pectinate ligament fibrils demonstrated by the dark bands crossing the region of fluorescence. Non-vitrectomized eye of Dog 2 shows no appreciable fluorescence within the cliary cleft (b, arrow). Immunohistochemical labeling of cryosections of the anterior segment of vitrectomized eye (c,e) showed GFP expression within the trabecular meshwork outflow pathway (c, arrow), and expression within the ciliary body epithelium (e, arrow). The anterior segment sections from non-vitrectomized eye (d,f) showed less prominent GFP expression within the trabecular meshwork outflow pathway (d, arrow), but comparable expression within the ciliary body epithelium (f, arrow).  IVT, intravitreal injection; GFP, green fluorescent protein.  Scale bar = 100 µm. 79  The lack of appreciable retinal GFP expression in vitrectomized eyes was unexpected. We originally hypothesized that vector solution injected within an intact vitreous humor could be sequestered by the gel-like collagen matrix, preventing widespread exposure of the retina. Our results suggest that the intact vitreous humor is actually beneficial for retinal exposure to the vector solution. Flow of aqueous humor within the posterior segment of an eye with intact vitreous humor is in a posterior direction (from the hyaloid membrane toward the retina).129 We now postulate that loss of the posterior vitreous humor disrupts this flow and creates increased reflux of vector solution across the anterior hyaloid membrane into the anterior chamber. This is supported by the increased level of GFP expression within anterior segment structures in vitrectomized eyes (Figure 13). 4.2.3 AAV vector retinal transduction efficiency and tropism At six weeks post-IVT, sagittal eyecup cryosections were taken from all eyes through the optic nerve head as well as 2-4 mm nasal and temporal of the optic nerve head. Quantification of cells expressing GFP within the retinal ganglion cell layer (GCL) revealed vitrectomized eyes had either very limited or absent GFP expression (Figure 11d). Non-vitrectomized eyes showed an overall mean transduction rate of 15.7 ±5.9% of cells within the GCL (Figure 11c). The transduction rate difference between vitrectomized eyes and non-vitrectomized eyes was significant (p=0.04). Variation in GCL transduction rates was observed between sagittal retinal regions, with a mean of 20.8 ±4.5% and 21.8 ±14.7% cells transduced in the nasal and central sagittal planes, respectively, versus 4.6 ±4.6% cells transduced in the temporal sagittal plane (Figure 14). This difference was not statistically significant (p=0.13). Peak transduction rates were as high as 25.8% in the nasal plane and 37.3% in the central plane. We recently reported a similar pattern following IVT of photoreceptor-specific AAV vectors in dogs.191 Transduction 80  occurred within all other retinal cellular layers predominantly in regions underneath the retinal vasculature in non-vitrectomized eyes, with both rod and cone photoreceptors as well as rod bipolar cells expressing GFP (Figure 15). This pattern of increased AAV penetration at the site of retinal vessels has been described previously following IVT.109, 191 These retinal cell populations are important targets for retinal gene therapy. 81   Figure 14. Regional variation observed in non-vitrectomized eyes of all three dogs.  Confocal scanning laser ophthalmoscopy showed more prominent GFP fluorescence in the nasal and central sagittal planes than the temporal plane (a).  b: Quantification of ganglion cell layer transduction within sagittal retinal cryosections labeled with the Neuronal Nuclei antibody showed higher transduction rates in the nasal and central sections and lower rates in the temporal section.  This difference was not statistically significant (p=0.13, ANOVA). GFP, green fluorescent protein. 82   Figure 15. Transduction of all retinal layers following IVT of AAV2 (triple Y-F + T-V), driven by the constitutive chicken -actin promoter in non-vitrectomized eyes (a).  Representative photomicrographs demonstrate GFP expression in cells co-labeled with PKC (b, white arrows) and LM-opsin (c, white arrows), showing transduction of rod bipolar cells and LM-cone photoreceptors, respectively. Solid box inset images (b,c) provide 2.5x magnification of region demarcated by dashed boxes to demonstrate co-labeling. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; PKC, protein kinase-C alpha; LM-opsin, long/medium-wavelength cone opsin; IVT, intravitreal injection; GFP, green fluorescent protein; DAPI, 4',6-diamidino-2-phenylindole. Scale bar = 50 m. 83   Direct comparison of our GCL transduction efficiency results to other reports utilizing IVT of AAV vectors in large animal models is challenging, as quantification of GCL transduction rate has not been previously reported. By subjectively comparing our in vivo imaging to images published from other studies, our results appear to suggest AAV2 (triple Y-F + T-V) generates higher GCL transduction compared to both wild-type AAV2 and AAV2 (quad Y-F) vectors previously used in dogs and primates.106, 107, 109 Optimization of capsid amino acid substitutions has been shown to decrease intracellular proteasomal degradation of AAV vectors.67, 68 cSLO images from non-vitrectomized eyes in our study seem to demonstrate comparable levels of in vivo GFP expression to the novel 7m8 vector used in primates as reported by Dalkara et al. although cross-species comparisons should be interpreted with caution.107 4.2.4 Microscopic evaluation of retinitis solution and submitted for histopathological evaluation. Hematoxylin and eosin-stained retinal sections revealed that the clinically acute retinitis was characterized by mononuclear inflammatory cells, suggestive of an immune-mediated reaction as opposed to an infectious etiology. Retinal sections were labeled with cluster of differentiation (CD) 3 and 20 antibodies to identify T- and B-lymphocytes, respectively. Mononuclear inflammatory cells within the retina labeled positive with both CD3 and CD20 antibodies, indicating a heterogeneous lymphocytic inflammatory response (Figure 16a,b). Paraformaldehyde-fixed retinal cryosections from the non-vitrectomized eye of Dog 1 and both eyes of Dogs 2 and 3 were labeled with CD4 and CD8 antibodies to differentiate between T-helper and cytotoxic T-lymphocytes, respectively. Dog 2 did not have any labeled 84  cells. Positive labeling for both antibodies was observed in both eyes of Dog 3 (Figure 16c,d), as well as the non-vitrectomized eye of Dog 1. Subjectively, CD-positive inflammatory cells were more abundant within the retina of the non-vitrectomized eye of Dog 3 compared to the vitrectomized eye. The vitrectomized eye of Dog 1 could not be labeled with CD4 and CD8 due osections with glial fibrillary acidic protein antibody showed increased expression deep to the nerve fiber layer in Dog 3 compared to Dog 2, indicating glial transformation of retinal Müller cells secondary to chronic retinal inflammation (Figure 12c,d).  Collectively, these results are consistent with a delayed-type hypersensitivity immune reaction, similar to that reported following systemic and intraocular AAV delivery in large animal models.151, 164 Presence of both CD4+ and CD8+ T-lymphocytes as well as B-lymphocytes within the inflamed retinas is reminiscent of the hepatic inflammatory cell profile described by Gao et al. following intraportal injection of AAV7 vectors driving GFP expression in primates.151 AAV2 and AAV8 vectors driving GFP expression are capable of producing a dose-dependent adaptive immune response following subretinal delivery in primates.164 This not only leads to suppression of transgene expression, but also destruction of the affected retinal tissues.107, 164 The timing of the reaction in our study is slightly later than described following intraportal injection of AAV, but much earlier than that reported following subretinal injection or IVT in non-vitrectomized eyes.107, 151, 164   85   Figure 16. Immunohistochemical characterization of the mononuclear cell populations in eyes with retinitis. Photomicrographs characterizing the cellular component of the retinal inflammatory response from vitrectomized eye of Dog 1 (a,b) and non-vitrectomized eye of Dog 3 (c,d) through the use of cluster of differentiation (CD) labeling. Positive immunolabeling of mononuclear inflammatory cells with CD20 (a) and CD3 (b) demonstrates a mixed response involving both B-cells and T-cells, respectively.  Positive immunolabeling of inflammatory cells with CD4 (c) and CD8 (d) demonstrates that both T-helper and cytotoxic T-cells, respectively, were present.  ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 100 m.  4.2.5 Immune response assays A neutralizing antibody (NAb) assay was performed four weeks post-IVT to detect humoral responses directed against AAV2 vector. A robust NAb response occurred in all dogs. Reciprocal serum dilutions required to achieve less than 50% neutralization of AAV transduction measured between 20,480 and 81,920 in Dogs 2 and 3, and >81,920 (the highest dilution tested) 86  in Dog 1. Serum from a naïve animal housed in the same vivarium tested <5 (the lowest dilution tested). The generation of a humoral immune response is expected following IVT. A study in mice demonstrated IVT generates capsid-specific NAb capable of suppressing transgene expression when the same vector was injected via IVT into the contralateral eye.98 Peripheral blood mononuclear cells (PBMCs) were isolated from Dogs 2 and 3 at six weeks post-IVT and challenged against vector-specific antigens. PBMCs from Dog 2 exhibited a robust stimulatory response to GFP antigen, but not to AAV2 (triple Y-F) capsid antigen (Table 12). The reactivity of PBMCs from Dog 2 to GFP is consistent with previous studies where subretinal, intraportal, and intravenous AAV injection in primates and dogs generated an adaptive immune response to GFP.151, 158, 164 Capsid-directed T-lymphocyte responses were not detected in any of these studies. The presence of GFP-primed PBMCs in Dog 2 suggests that a T-lymphocyte inflammatory reaction would have eventually developed in this animal.151 Dog 3 did not generate a significant stimulatory response to either test antigen or the control antigen Candida albicans. We suspect this is an effect of immunosuppressive corticosteroid treatment (oral prednisone 1 mg/kg/day) it received for bilateral endophthalmitis for two weeks prior to PBMC collection. The PBMC results from Dog 2 are highly suggestive of a GFP transgene-specific T-lymphocyte response, however a direct link cannot be definitively made between this response and the immune reactions seen in Dog 1 and Dog 3 due to absence of a PBMC sample from Dog 1. Future experiments in our laboratory will include PBMC collections in all animals prior to and serially following AAV injection, in order to definitively characterize any immune responses that may occur.  87   Table 12. Peripheral blood mononuclear cell (PBMC) stimulation index following challenge with vector-derived antigens. (Positive result >2) Subject Positive Control Antigens Vector Capsid Transgene  Phytohemagglutinin (PHA) Candida albicans AAV2 GFP Dog 2 80.55 19.67 0.61 4.60 Dog 3* 21.36 1.81 1.35 0.84 *Dog 3 had received two weeks of immunosuppressive prednisone therapy prior to PBMC collection.  The eye is considered an immune privileged site, capable of generating an immune deviant response to intraocular antigens, although this process can be overcome by inflammation or a strong antigen.137, 138, 145, 194 We suspect the vitrectomy procedure greatly increased the amount of AAV vector transiting into the anterior chamber and aqueous humor outflow pathways, resulting in significant transduction of off-target cell populations and exposure of GFP antigen to the systemic immune system. AAV is unable to efficiently transduce antigen presenting cells (APCs), therefore GFP antigens must be exogenously taken up by APCs in order to be presented for T-193 The increased level of immunogenic GFP antigen within the anterior segment of vitrectomized eyes likely overwhelmed existing immune-deviation mechanisms, resulting in a delayed-type hypersensitivity response instead of immune tolerance.137 A lack of response to capsid antigen may have resulted from evasion of proteasomal degradation due to the presence of Y-F and T-V substitutions, decreasing the amount of capsid antigen available for cross presentation.  88  4.2.6 Biodistribution of AAV vector Ocular and non-ocular tissue samples were collected from Dogs 2 and 3 for quantitative PCR (qPCR) biodistribution analysis of AAV vector genomes (Table 13). AAV vector genomes were present in the aqueous humor and optic nerves of vitrectomized and non-vitrectomized eyes. A much greater quantity of vector genomes was present in the aqueous humor of the non-vitrectomized eye of Dog 2 compared to the vitrectomized eye. We suspect this indicates a slow in a much slower clearance of vector across the anterior hyaloid membrane. The vitrectomized eye, however, may have had more rapid clearance of vector into the aqueous humor post-IVT, and the lower number of genomes in the fluid aqueous humor present 6-weeks later represents a steady state level within the eye, with most of vector already within anterior uveal tissues or filtered out of aqueous drainage pathways. This theory is supported by the increased level of GFP expression within the anterior uveal tissues and trabecular meshwork of the vitrectomized eyes (Figure 13). To explore this theory, future studies in our laboratory will include serial aqueous humor samples following IVT of AAV vectors, as well as the collection of the ciliary body and trabecular meshwork tissues, for qPCR analysis.   89  Table 13. Biodistribution of AAV genomes determined by qPCR  (Number of vector genomes/µg DNA, 00) Tissue Dog 2 Dog 3 Aqueous Humor OD (vitrectomized) 54,275 2,505 Aqueous Humor OS (non- vitrectomized) 6,220,225 1,525 Optic Nerve OD (vitrectomized) 90 145 Optic Nerve OS (non- vitrectomized) 710 10 Optic Chiasm 31 52 Cerebrum 0 0 Cerebellum 0 14 Parotid Gland 0 0 Submandibular Lymph Node 0 0 Lung 0 0 Heart 10 0 Liver 53 3 Pancreas 0 0 Spleen 611 581 Kidney 6 19  qPCR also detected vector DNA in splenic tissue, but not in any other sampled non-ocular tissues. This trafficking of vector genomes to the spleen has been described after intravenous or intraportal injection of AAV.151, 158 Following exposure to an intraocular antigen, specialized APCs responsible for immune deviation preferentially migrate to the spleen, where they generate regulatory T-lymphocytes to promote antigen tolerance.195 We suspect this established mechanism is responsible for vector genomes collected within splenic tissue but not in other organs. 90  Our study had a number of limitations. The unexpected and acute nature of the immune reactions resulted in expedited changes in the study protocol. The immediate euthanasia of Dog 1 due to welfare concerns over the rapidly developing severe intraocular inflammation resulted in a lack of collecting valuable immune assay samples for that animafixation of the vitrectomized eye of Dog 1 to achieve better histopathological characterization of the inflammatory response led to an inability to label sections from that eye with CD4 and CD8 antibodies as well as exclusion of the eye from transduction efficiency assessments, which in turn reduced statistical power. The immunosuppressive corticosteroid treatment of Dog 3 likely inhibited the expected PBMC reactivity to GFP antigen. Therefore, our conclusions regarding the specificity of the immune responses are based on results from a very limited number of animals. In addition, the GFP-specific PBMC result from Dog 2 could have been further characterized using methods such as intracellular cytokine staining and flow cytometry to verify the effector functions of the T-lymphocytes.159 In conclusion, our study demonstrates that core vitrectomy with posterior hyaloid membrane peeling occurring prior to IVT delivery of AAV vectors does not enhance retinal transduction in the dog, but instead significantly reduces transduction. We also demonstrated that the dog is capable of generating a delayed-type hypersensitivity response to the GFP transgene following IVT of an AAV vector. Based on comparison to previous studies in our laboratory in which similar doses of the same vector were used without adverse complications, development of this immune reaction appears to be potentiated by the vitrectomy procedure. Therefore, we urge caution when considering IVT delivery of AAV vectors for retinal gene therapy in patients that have had a prior vitrectomy. Additional studies are warranted to evaluate whether a similar pattern would occur in eyes affected by advanced vitreal syneresis. There was no evidence of a 91  capsid-directed T-lymphocyte response, a finding that is promising for future studies utilizing AAV2 (triple Y-F + T-V), although the absence of a collected PBMC sample from Dog 1 compels us to make this statement with some reservation. The vector was able to transduce inner retinal neurons, including RGCs and bipolar cells, and may be considered for use in future efficacy studies in animal models of inner retinal disease. 4.3 Materials and Methods 4.3.1 Animals Three purpose-bred 10-month-old Beagle dogs (Marshall BioResources, North Rose, NY) were used in the study. Power analysis using data from a preliminary pilot study revealed that an N of three dogs would be required to achieve a power of >0.8 when using a paired one-tailed t-test. All dogs were socially housed with a 12-hour light:dark cycle. Animal care was in compliance with the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research, and all procedures were performed following approval by the Michigan State University Institutional Animal Care and Use Committee. 4.3.2 Vitrectomy Core vitrectomy with posterior hyaloid membrane peeling was performed in the right eye via three-port pars plana approach in all three dogs one month prior to vector injection. Briefly, three 23 gauge scleral ports (Alcon, Fort Worth, TX) were placed to facilitate infusion, illumination, and vitrectomy probe insertion into the vitreous humor. A 23-gauge vitrectomy probe (Alcon Accurus, Fort Worth, TX) was used to remove the posterior half of the vitreous humor and replace it with balanced salt solution (BSS, Alcon, Fort Worth, TX) under direct visualization through an operating microscope (Zeiss S8, Thornwood, NY) with noncontact, indirect 92  visualization system (Oculus BIOM, Port St. Lucie, FL) installed. Microfiltered triamcinolone crystals (Bristol-Myers Squibb, New York, NY) were injected to facilitate visualization and removal of posterior vitreous cortical fibers, followed by placement of the vitrector over the optic nerve head to induce detachment and peeling of the posterior hyaloid membrane.  4.3.3 AAV vector The recombinant AAV vector construct containing capsid amino acid substitutions was manufactured and purified at the University of Florida College of Medicine using previously described methods.108, 181 All AAV vectors underwent testing for endotoxin and were determined to contain less than 5 EU/ml. The AAV2 based capsid was mutated by substitution of three surface-exposed capsid tyrosine residues with phenylalanine and one threonine residue with valine (Y444F, Y500F, Y730F, and T491V; referred to as AAV2 (triple Y-F + T-V)). This capsid variant has previously been shown to maximally avoid proteasomal degradation with up to 90% of vector genomes reaching the nucleus of infected cells.69 The full length, ubiquitous chicken -actin (CBA) promoter was used to drive expression of the reporter, GFP. 4.3.4 Intravitreal injections Vectors were prepared for injection by diluting stock supplies to a titer of 1.3 x 1012 vg ml-1 vector solution was injected into the vitreous humor of both eyes immediately anterior to the retinal surface in a transverse plane along the visual streak using a RetinaJect injector (SurModics, Inc., Eden Prairie, MN) as previously described.109, 127 Postoperative treatment included antibacterial and anti-inflammatory medications as previously described.109  93  4.3.5 Ophthalmic examinations and imaging Post-IVT all dogs received regular ophthalmic examinations and fluorescence fundus imaging as previously described.109 Confocal scanning laser ophthalmoscopy utilizing 488 nm laser-induced fluorescence (cSLO, Spectralis, Heidelberg Engineering, Carlsbad, CA) was performed weekly post-injection under general anesthesia. 4.3.6 Immune reaction assays Prior to euthanasia, serum was collected from all dogs for analysis of NAb titers and whole blood samples were collected from dogs 2 and 3 for PBMC isolation. A control serum sample was also obtained from an age-matched AAV-naïve, colony dog. NAb titers were determined via Nab assay utilizing ARPE-19 cells and an AAV2 (triple Y-F + T-V)-mCherry vector at 5000 particles per cell, as previously described.196 PBMCs were isolated as previously described197, then immediately frozen at -80°C to maintain cell viability prior to antigen challenge analysis. Anti-AAV2 (triple Y-F + T-V) and GFP antigen-specific lymphocyte proliferation responses were assessed as previously described.198 Briefly, lymphocytes were cultured and separated into four groups with three (controls) and six (unknowns) cultures per group: unstimulated (as negative control), stimulated with AAV2 (triple Y-F + T-V) (5000, 500, and 5After 5 days of incubation the stimulation index (SI) was defined as (mean counts per minute of [3H]thymidine from stimulated cells)/(mean counts per minute of [3H]thymidine from unstimulated cells). On the basis of antigen-specific lymphocyte proliferation response (ASR) results at baseline in other nonocular studies, SI values greater than 2.0199 or 3.0198 have been considered significant. The viability of each lymphocyte culture was confirmed by positive 94  controls with mitogen-induced proliferation in response to phytohemagglutinin (PHA) (10, 1.0, Candida albicans (10, 1.0, and 0.1 . 4.3.7 Eyecup collection and sectioning Following the development of bilateral endophthalmitis in two of the three dogs, all dogs were euthanized 6 weeks post-IVT with an intravenous injection of sodium pentobarbital (Fatal Plus, Vortech Pharmaceuticals, Dearborn, MI). Eyes were immediately enucleated and processed as previously described166solution for histopathologic processing to characterize the inflammatory response. Twenty micron-thick sagittal cryosections, three sections per slide, were collected (Leica CM3050-S cryostat, Leica Microsystems, Buffalo Grove, IL) and stored at -20°C. 4.3.8 Immunohistochemistry and cell quantification Details of primary and secondary antibodies used for immunohistochemical labeling can be found in Table 14-fixed retinal sections from the vitrectomized eye of Dog 1 were used for labeling with CD 3 and CD 20 antibodies, all other antibodies were used to label paraformaldehyde-fixed sections. Unfortunately, this meant the vitrectomized eye of Dog 1 was excluded from analysis of vector transduction efficiency. Sagittal sections labeled with Neuronal Nuclei (NeuN) antibody were used for counting purposes. Identifying information was masked, and NeuN positive cells in the GCL counted at 20x magnification by a single observer (RFB). Cells colabeled with NeuN and GFP were also quantified for each section.  Representative images were captured using a confocal laser scanning microscope (Olympus FluoView fv1000 Confocal, Center Valley, PA) at 20x magnification. 95   Table 14. Primary and secondary antibodies used for retinal immunohistochemistry on vitrectomy study. Target antigen Primary antibody details and working dilution Manufacturer Targeted cell population Secondary antibody and concentration (all from Invitrogen Corp, Carlsbad, CA) Green fluorescent protein Rabbit FITC conjugated, 1:1000 Invitrogen Corp, Carlsbad, CA GFP transduced cells109 N/A Neuronal Nuclei Mouse monoclonal 1:2000 Millipore, Billerica, MA Ganglion and Amacrine cells165 Alexafluor 546 goat anti-mouse 1:500 Protein kinase C alpha Mouse monoclonal 1:500 BD Biosciences, San Diego, CA Rod bipolar cells109 Alexafluor 546 goat anti-mouse 1:500 L/M-opsin Rabbit polyclonal 1:500 Millipore, Billerica, MA L/M-cone outer segments166 Alexafluor 546 goat anti-rabbit 1:500 S-opsin Rabbit polyclonal 1:500 Millipore, Billerica, MA S-cone outer segments109 Alexafluor 546 goat anti-rabbit 1:500 Human Cone arrestin (LUMif)167 Rabbit polyclonal 1:5000 Kind gift of Cheryl M. Craft, University of Southern California Cone photoreceptors109 Alexafluor 546 goat anti-rabbit 1:500 Glial fibrillary acidic protein (GFAP) Mouse monoclonal 1:300 Cell Signaling Technology, Danvers, MA Glial activation of Müller cells Alexafluor 546 goat anti-mouse 1:500 CD3 Rabbit polyclonal   DakoCytomation, Inc., Carpenteria, CA T-lymphocytes200 N/A CD4 Rat Monoclonal AbDSerotec, Raleigh, North Carolina T-helper lymphocytes201 N/A CD8 Rat Monoclonal AbDSerotec, Raleigh, North Carolina Cytotoxic T-lymphocytes201 N/A CD20 Mouse monoclonal DakoCytomation, Inc., Carpenteria, CA B-lymphocytes200 N/A   96  4.3.9 AAV Biodistribution analysis Following euthanasia, ocular and non-ocular tissue samples (see Table 13) were collected from dogs 2 and 3 and frozen at -80°C. Ocular and non-ocular tissue biodistribution of CBA-GFP vector genomes was determined via quantitative PCR (qPCR) analysis as previously described.202 4.3.10 Statistical analysis Differences in overall GCL transduction efficiency between vitrectomized (Dogs 2 and 3) and non--test. Differences in GCL transduction efficiency between sagittal regions within non-vitrectomized eyes of all three dogs were compared using ANOVA (Excel, Microsoft, Redmond, WA). Results were considered significant if p<0.05. Data are displayed as mean ± standard deviation.   97  CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS 5.1 Photoreceptor-Targeted Vector Study This study demonstrated photoreceptor-specific transgene expression was possible following IVT of AAV2 (quad Y-F) IRBP and AAV2 (quad Y-F + T-V) IRBP, with both vectors transducing rod and cone photoreceptors at similar rates.  The efficiency of the vectors was not ideal, although higher levels of transduction were observed at the site of larger retinal vessels.  One theory that could explain this phenomenon is ILM disruption by the large vessels, resulting in greater permeability at these sites.  If the disruption of ILM architecture is in fact responsible for the apparent increased AAV penetration at these locations, it would support the use of ILM manipulation prior to IVT of AAV vectors targeting the outer retina.  This could be achieved using digestive substances such as proteases, or through direct surgical manipulation.  Proteases have been shown to increase retinal permeability to AAV, however use of these substances presents a risk of damage to the underlying retinal structures in addition to the ILM.119  Surgical shown would likely be detrimental to transduction efficiency of the AAV vector administered into the vitreous.203  As an alternative to manipulation of the host ILM, focus instead should be directed at further manipulation of AAV vectors in order to increase retinal penetration ability.  This will likely require a comprehensive understanding of properties inherent to the ILM that inhibit AAV transport.   Our study suggests that overall ILM thickness does not play as prominent a role as previously thought.  The fact that we observed such a difference in transduction rates between the temporal 98  retinal regions compared to nasal and central regions, while ILM thickness did not significantly change between these regions, advocates the exploration of other ILM factors that may pose a barrier to AAV.  One barrier that warrants additional investigation is the composition of heparan sulfate receptors on the ILM surface.  These receptors are a primary target for AAV2 capsids, and binding to these receptors at the ILM could result in sequestration of the AAV particle at the surface or internalization of the vector into the Müller cell to result in either proteasomal degradation or transcellular transport to the outer limiting membrane of the retina.  Therefore, a higher number of heparan sulfate receptors could either be beneficial or detrimental to overall outer retinal efficiency of AAV vectors.  The manipulation of AAV capsids to affect heparan sulfate binding has been shown to affect retinal penetrating ability, however there appears to be a limit to the benefit induced by these alterations.96  This may be due to an imbalance between the optimal level of heparan sulfate binding ability to promote ILM passage and for target cell membrane binding.  An alternative to capsid manipulation may be use of pharmacologic substances to either competitively bind or upregulate the ILM heparan sulfate receptors prior to IVT of AAV. Another potential focus for refinement of AAV penetration ability to the outer retina is the zeta potential of the capsid.  Strongly anionic nanoparticles are more capable of reaching the outer retina following IVT when compared to more neutral or cationic particles.118, 125  Substitution of cationic amino acids (lysine, arginine, and histidine) in the capsid of AAV particles with either neutral or anionic (aspartate, glutamate, and cysteine) residues could theoretically increase the ability of the vectors to move through the vitreous and avoid sequestration at the ILM.  The effect of these capsid substitutions on target cell binding and capsid processing would need to be evaluated. 99  The GNAT2/IRBP promoter used in our study lacked the ability to efficiently transduce cone photoreceptors following IVT of both AAV2 (quad Y-F) GNAT2/IRBP and AAV2 (quad Y-F + T-V) GNAT2/IRBP.  This promoter had previously performed much more desirably after subretinal injection.172, 175  It is possible that the decreased ability of the vectors to penetrate the retina and reach the outer retinal layers may have accounted for most of this apparent difference in performance.  Another potential contributing factor is alteration of the intracellular trafficking and nuclear processing of the AAV vectors when delivered via IVT instead of the subretinal route.  Our more favorable results when using the same AAV capsids with the IRBP promoter seem to suggest that binding to cone photoreceptors and transport from the cell membrane to the nucleus should not be significant limiting factors.  Nuclear processing of the genomes containing the GNAT2/IRBP promoter should hypothetically be just as robust as that reported in the subretinal injection studies.  Hence, it is unclear why this capsid/promoter combination did not perform as expected. The off-target GFP expression in the superior nasal retina of one of the eyes injected with AAV2 (quad Y-F + T-V) GNAT2/IRBP is also perplexing.  Given the poor performance of this vector in all other retinal regions, it is unclear what occurred at the site of the off-target expression.  One theory is that a much higher concentration of vector was present at that location due to the dosing procedure.  The injections were all carried out by a right-handed surgeon, and injection cannula insertion through the sclera into the vitreous of the left eye in this case would occur in the superior nasal quadrant.  This may have resulted in an unintended increase of vector dose over the region of off-target expression.  100  5.2 Vitrectomy Study Our study demonstrates that AAV2 (triple Y-F + T-V) is capable of inducing widespread and efficient transduction of RGCs following IVT.  This efficiency is attenuated when posterior vitrectomy is performed prior to IVT of the vector, and vitrectomy potentiates the recognition of the GFP transgene by the immune system.  These findings have significant implications for the future use of AAV2 (triple Y-F + T-V) for retinal gene therapy.  Although the vector appears to be efficient enough for therapeutic application in many inner retinal disease models, careful consideration of patient selection and restriction of non-target cell population transduction both need to be emphasized. Prior vitrectomy appears to increase the release of vector particles into the anterior chamber of the eye following IVT.  This is in agreement with the study by Friedrich et al.128, where injections close to the retina in eyes with an intact vitreous had significantly greater retinal exposure and vitreal concentrations of drug compared to injections adjacent to the hyaloid membrane.  Vitrectomy likely results in rapid diffusion of the injected vector away from the site of injection near the retina and to the hyaloid membrane, where it is then filtered out of the eye through the aqueous humor drainage pathways.  Not only does this significantly reduce the amount of retinal transduction that occurs, it also results in a much larger amount of vector exposure to the systemic immune system.  AAV vector particles theoretically leave the eye through the iridocorneal angle, enter the conventional and unconventional aqueous outflow pathways, and end up in the bloodstream.  There, the AAV capsid antigen is capable of being recognized by the immune system to generate a cytotoxic T-cell response as well as neutralizing antibodies to the capsid.  Transduction of off-target tissues is also highly possible.  Although capsid-directed cytotoxic T-cell reactions were not observed in our study in dogs, a reaction in 101  humans is still highly possible.  In addition to the exposure of capsid antigen, transduction of cells within the aqueous outflow pathways introduces the potential for exposure of the transgene product to the immune system through cross presentation.  This could result in the production of both cytotoxic T-cell and neutralizing antibody responses directed at the transgene.  These immune responses to AAV-associated antigens would not only reduce transduction efficiency, but could also result in the destruction of transduced cells and damage to vital host tissues.  Therefore, it is advisable to avoid IVT of AAV vectors for retinal gene therapy in any patient with altered vitreal integrity, including prior vitrectomy or advanced vitreal syneresis.  Unfortunately, these vitreous states are commonly encountered in patients with retinal diseases, either as part of the disease process or a result of previous treatment attempts. Use of cell-specific promotor sequences or miRNA to restrict transgene expression to only the cell population of interest has been previously shown to limit immune reactions to AAV vector transgene products.107, 108  With both capsid and transgene exposure limited to the vitreous body and inner retina, the eye is more likely to generate an immune-deviant response to the AAV-associated antigens, resulting in ignorance of the systemic immune system and lack of any deleterious inflammatory responses.  In addition, restricting use of IVT for the delivery of AAV vectors to non-recessively inherited disease conditions would also help to mitigate the risk of transgene-directed immune reactions, as the transgene product is not recognized by the immune system as a foreign antigen and therefore does not result in an inflammatory response.  Through the employment of the described methods to limit off-target transgene expression and careful patient selection, it is possible that AAV2 (triple Y-F + T-V) may be an effective vector for retinal gene therapy of inner retinal diseases.  102  5.3 Conclusion In conclusion, the studies described above provide valuable information that will contribute to the advancement of AAV vector use in retinal gene therapy.  They also revealed some additional questions that warrant further investigation.  Our hope is that the knowledge gained through these investigations will provide some of the additional groundwork needed to eventually produce versatile AAV vectors that can be delivered intravitreally to treat a wide range of inherited and acquired retinal diseases.    103             BIBLIOGRAPHY   104  BIBLIOGRAPHY  1. Berger W, Kloeckener-Gruissem B, Neidhardt J. The molecular basis of human retinal and vitreoretinal diseases. Prog Retin Eye Res 2010; 29(5): 335-375.  2. 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