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Expanding the Utility of AAV Gene Therapy for CEP290-Leber Congenital Amaurosis

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Title:
Expanding the Utility of AAV Gene Therapy for CEP290-Leber Congenital Amaurosis
Creator:
Ryals, Renee Christine
Place of Publication:
[Gainesville, Fla.]
Florida
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University of Florida
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english
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1 online resource (196 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Medical Sciences
Molecular Cell Biology (IDP)
Committee Chair:
HAUSWIRTH,WILLIAM W
Committee Co-Chair:
LEWIN,ALFRED S
Committee Members:
SMITH,WESLEY CLAY
BOYE,SHANNON ELIZABETH
SRIVASTAVA,ARUN
KAY,CHRISTINE NICHOLS
Graduation Date:
8/9/2014

Subjects

Subjects / Keywords:
Animal models ( jstor )
Capsid ( jstor )
Clinical trials ( jstor )
Gene therapy ( jstor )
Genetic mutation ( jstor )
Photoreceptors ( jstor )
Retina ( jstor )
Retinal dystrophies ( jstor )
Sand sheets ( jstor )
Transgenes ( jstor )
Molecular Cell Biology (IDP) -- Dissertations, Academic -- UF
dystrophy -- gene -- photoreceptors -- retina -- therapy
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bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Medical Sciences thesis, Ph.D.

Notes

Abstract:
CEP290-Leber congenital amaurosis (LCA) patients are characterized by severe visual dysfunction, early loss of rod photoreceptors and maintained cone photoreceptors within the fovea. CEP290-LCA patients are excellent candidates for AAV-mediated gene therapy because the genetic causation of their disease is understood and their retinas possess retained cone photoreceptors that can be targeted during treatment. The development of clinical trials for CEP290-LCA patients will require outcome measures capable of detecting visual gains post-treatment, AAV-mediated CEP290 expression and an injection technique that facilitates optimal gene expression in cone photoreceptors and minimizes damage to the retina. The National Eye Institute visual functioning questionnaire 25 (NEI-VFQ-25) was explored as a useful subjective outcome measurement for inherited retinal dystrophy patients, including LCA patients. In order to validate the NEI-VFQ-25, subjective assessment of cone-mediated and rod-mediated vision was correlated to objective electroretinography data. Although subjective assessment of vision did not correlate to objective assessment of vision, subjective assessment of vision via the NEI-VFQ-25 showed clear differences in cone-mediated and rod-mediated vision between all inherited retinal dystrophy groups. Simple overlap and hybrid dual AAV vector systems were generated and evaluated for their ability to express CEP290 in vitro and in vivo. The two dual AAV vector systems evaluated for the delivery of CEP290 were unable to mediate full-length transcript and protein expression in vitro. Furthermore, subretinal delivery of the dual AAV vectors did not restore visual function to rd16;Nrl-/- mice. Novel dual AAV vector technologies are required for the development of an AAV-mediated gene therapy for CEP290-LCA patients. In hopes of establishing the intravitreal injection for the treatment of CEP290-LCA patients, novel AAV capsids were generated and examined for their ability to transduce photoreceptors from the vitreous in two different mouse models. Out of nine capsids evaluated, the AAV2 capsid containing four tyrosine to phenylalanine mutations and one threonine to valine mutation showed enhanced transduction (~22%) of the photoreceptors post-intravitreal delivery. In addition, in vitro and in vivo assays were developed to quantify transduction capabilities of novel AAV capsids and photoreceptor-specific promoters. Overall, these studies contribute to developing an AAV gene therapy for CEP290-LCA. ( en )
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In the series University of Florida Digital Collections.
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Includes vita.
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Includes bibliographical references.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2014.
Local:
Adviser: HAUSWIRTH,WILLIAM W.
Local:
Co-adviser: LEWIN,ALFRED S.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2016-08-31
Statement of Responsibility:
by Renee Christine Ryals.

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UFRGP
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Applicable rights reserved.
Embargo Date:
8/31/2016
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LD1780 2014 ( lcc )

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EXPANDING THE UTILITY OF AAV GENE THERAPY FOR CEP290 LEBER CONGENITAL AMAUROSIS By RENEE RYALS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2014

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© 2014 Renee Ryals

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To Elizabeth, my advisor and friend

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4 ACKNOWLEDGMENTS I thank all the members in the Dr. William Hauswirth, Dr. Shannon Boye and Dr. Clay S mith lab s . Frank Dyka , Ph.D. , Vince Chiodo, Sanford Boye, Santhi Mani , Jingfen Sun , M.D. , Se ok Hong Min, Ph.D., Jim Peterson , Ph.D., Astra Dinculescu, Ph.D., hav e all co ntributed technical assistance to the work in this dissertation . I thank Amde Shifera, M.D. and Jing Zhang , M.D. for their contributions to the clinical work in this dissertation. I thank my undergraduates Jessica Kenny , Paige Davis and Oliver Srok a for their willingness to learn and contribute their time to my project . I give special thanks to my committee members Dr. Shannon Boye, Dr. Christi ne Kay, Dr. Arun Srivasta va, Dr. Al Lewin, Dr. Clay Smith and Dr. William Hauswirth for their t ime and dedi cation to my growth as a translational visual scientist. I thank Susan Porterfield for sharing her experience, strength and hope with me on a daily basis. I thank Steve Oden , Ph.D. for editing this dissertation. Finally, I would like to thank my family for their contin uous encouragement and support for all my endeavors.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 15 Classification of Inherited Retinal Dystrophies ................................ ........................ 15 Treatment Strategies for Patients with Severe Retinal Degeneration ..................... 19 Gene Therapy for Inherited Retinal Dystrophies ................................ ..................... 21 Vectors Available for Retinal Gene Therapy ................................ ........................... 22 CEP290 Leber Conge nital Amaurosis ................................ ................................ .... 27 Developing Pre clinical rAAV Gene Therapy Proof of Concept Studies for CEP290 Leber Congenital Amaurosis ................................ ................................ . 29 2 T HE RELATIONSHIP BETWEEN SUBJECTIVE ASSESSMENT OF CONE MEDIATED AND ROD MEDIATED VISION AND ELECTROPHYSIOLOGICAL PERFORMANCE IN RETINAL DYSTROPHIES ................................ ..................... 35 Clinical Characterization of Inherited Ret inal Dystrophies ................................ ...... 35 Methods ................................ ................................ ................................ .................. 38 Study Population ................................ ................................ .............................. 38 Determination of Visual Acuity and ERG b Wave Amplitudes .......................... 38 Subjective Assessment of Vision ................................ ................................ ...... 39 Statistical Analysis ................................ ................................ ............................ 39 Results ................................ ................................ ................................ .................... 39 Demographics of Study Population ................................ ................................ .. 39 Visual Acuity and ERG b Wave Amplitudes ................................ ..................... 40 Responses to Study Questionnaire ................................ ................................ .. 40 Correlation between Visual Acuity or ERG b Wave Amplitudes and Subjective Assessment of Vision ................................ ................................ .. 41 Discussion ................................ ................................ ................................ .............. 43 3 DEVELOPING AN AAV GENE THERAPY FOR CEP290 LCA PATIENTS PART 1: DUAL AAV VECTORS FOR DELIVERING CEP290 ................................ .......... 53 CEP290 Leber Congenital Amaurosis Patients ................................ ...................... 53

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6 Features of CEP290 ................................ ................................ ............................... 54 Dual AAV Vector Technology ................................ ................................ ................. 57 Animal Models for CEP290 Leber Congenital Amaurosis ................................ ....... 60 Methods ................................ ................................ ................................ .................. 64 Dual Vector Production ................................ ................................ .................... 64 Cell Lines ................................ ................................ ................................ .......... 65 Transfections and Infections ................................ ................................ ............. 66 Tyr23 and MG132 Treatment ................................ ................................ ........... 67 Experimental Animals ................................ ................................ ....................... 67 Subretinal Injections ................................ ................................ ......................... 68 Electroretinographic Analysis ................................ ................................ ........... 69 Immunohistochemistry ................................ ................................ ...................... 69 Rosette Coun ts and Outer Nuclear Layer Thickness ................................ ....... 72 Immunoblot Analysis ................................ ................................ ........................ 73 Results ................................ ................................ ................................ .................... 74 Natural History of the rd16;Nrl / Animal Model ................................ ................. 74 Exogenous Expression of Full length CEP290 ................................ ................. 77 Analysis of Dual AAV Vector Systems In Vitro ................................ ................. 77 Analysis of Dual AAV Vector Systems In Vivo ................................ .................. 79 Discussion ................................ ................................ ................................ .............. 83 4 DEVELOPING AN AAV GENE THERAPY FOR CEP290 LCA PATIENTS PART 2: TARGETING PHOTORECEPTORS VIA INTRAVITREAL DELIVERY ............. 108 Subretinal Injection ................................ ................................ ............................... 108 Intravitreal Injection ................................ ................................ ............................... 109 AAV Transduction ................................ ................................ ................................ . 110 Photoreceptor Specific Promoters ................................ ................................ ........ 113 Methods ................................ ................................ ................................ ................ 114 Vector Production ................................ ................................ ........................... 114 Cell Lines ................................ ................................ ................................ ........ 115 Infections and FACS Analysis ................................ ................................ ........ 116 Animals ................................ ................................ ................................ ........... 116 Ethics Statement ................................ ................................ ............................ 116 Intravitreal Injections ................................ ................................ ...................... 117 Subretinal Injections ................................ ................................ ....................... 117 Fundoscopy ................................ ................................ ................................ .... 118 Retinal Dissociation and FACS Analysis ................................ ........................ 118 Immunohistochemistry ................................ ................................ .................... 120 Results ................................ ................................ ................................ .................. 121 Quantification of In Vitro Transduction Efficiency ................................ ........... 121 Quantification of In Vivo Transduction Efficiency ................................ ............ 122 Qualitative Analysis of Photoreceptor Transduction ................................ ....... 125 MicroRNA Mediated Regulation of Transgene Expression ............................ 127 Qualitative Analysis of Serotype Tropism ................................ ....................... 127 In Vitro Transduction Efficiency of Photoreceptor Specific Promoters ........... 128

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7 Developing an In Vivo Transduction Efficiency Protocol for Photoreceptor Specific Promoters ................................ ................................ ...................... 129 Discussion ................................ ................................ ................................ ............ 132 5 CONCLUSIONS AND FUTURE DIRECTIONS ................................ .................... 158 Developing a Clinical Trial for CEP290 LCA Patients ................................ ........... 158 Practical Outcome Measuremen ts for CEP290 LCA Clinical Trails ...................... 160 Developing an AAV Gene Replacement Therapy for CEP290 LCA ..................... 165 Two Injection Techniques Per tinent to Treating CEP290 LCA Patients ............... 170 Summary ................................ ................................ ................................ .............. 174 LIST OF REFERENCES ................................ ................................ ............................. 176 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 196

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8 LIST OF TABLES Table page 2 1 Demographic characteristics of study subjects. ................................ .................. 48 2 2 ................................ ................. 49 2 3 ................................ ................. 50 3 1 Nomeclature and usage of viral constructs. ................................ ........................ 93 4 1 Nomenclature for capsid mutated vectors with description of amino acid location of mutation. ................................ ................................ ......................... 140 4 2 In vivo transduction efficiency of novel AAV capsids. ................................ ....... 14 1 4 3 Preliminary transduction efficiency of photoreceptor specific promoters three weeks p ost injection. ................................ ................................ ........................ 142

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9 LIST OF FIGURES Figure page 2 1 Objective measurements of visual function. ................................ ....................... 51 2 2 Four correlations found between objective visual function and subjective visual function. ................................ ................................ ................................ .... 52 3 1 Simple overlap and hybrid constructs for CEP290.. ................................ ........... 94 3 2 Quantification of central and peripheral rosettes in the rd16;Nrl / retina from P21 to P80.. ................................ ................................ ................................ ........ 95 3 3 Quantification of central and peripheral ONL thickness in the rd16;Nrl / retina from P21 to P80. ................................ ................................ ................................ . 96 3 4 Photoreceptor protein expression is reduced over time in rd16;Nrl / mice. ......... 97 3 5 Full length CEP290myc expression in mIMCD cells. ................................ .......... 98 3 6 CEP290 and myc protein detection via WB post simple overlap and hybrid infection in HEK293 cells. ................................ ................................ ................... 99 3 7 AAV2 CBA GFP transduction is increased with Tyr23 treatment in HEK293 cells. ................................ ................................ ................................ ................. 100 3 8 AAV2 CBA GFP transduction is increased with MG132 treat ment in HEK293 cells. ................................ ................................ ................................ ................. 101 3 9 CEP290 and myc protein detection via WB post simple overlap and hybrid infection in HE K293 cells with Tyr23 treatment . ................................ ............... 102 3 10 CEP290 and myc protein detection via WB post simple overlap and hybrid infection in HEK293 cells with MG132 treatment. ................................ ............. 103 3 11 Myc tagged CEP290 expression in C57BL/6 mice 4 weeks post subretinal delivery of sim ple over la p and hybrid dual AAV vectors. ................................ .. 104 3 12 Electroretinographic analysis of injected rd16;Nrl / mice. . ................................ 105 3 13 Myc tagged CEP290 expression in rd16;Nrl / mice 4 weeks post subretinal delivery of simple overlap and hybrid dual AAV vectors. ................................ .. 106 3 14 Simple overlap and hybrid AAV d ual vectors did not mediate CEP290myc expression in rd16;Nrl / retinas 4 weeks post subretinal delivery. . ................... 107 4 1 Transduction efficiency of unmodified and capsid mutated vectors in vitro . ..... 143

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10 4 2 Qualitative comparison of unmodified and capsid mutated AAV vectors in vivo . ................................ ................................ ................................ .................. 144 4 3 Quantitative comparison of unmodified and capsid mutated AAV vectors in vivo . ................................ ................................ ................................ .................. 145 4 4 Transduction efficiency of scAAV2 quad Y F and scAAV2 quad Y F+T V in photoreceptors of Rho GFP mice 1 week post intravitreal delivery. ................. 146 4 5 Characterizing two green fluorescent photorecepto r populations in the Rho GFP retina. ................................ ................................ ................................ ....... 147 4 6 In vivo analysis of AAV2 based vectors containing the hGRK1 promoter. . ...... 148 4 7 Representative image of a retinal cross section from a C57BL/6 mouse injected with AAV2 quad Y F+T V. ................................ ................................ ... 149 4 8 In vivo analysis of AAV5 based vectors containing the hGRK1 promoter. ....... 150 4 9 In vivo analysis of AAV8 based vectors containing the hGRK1 promoter. ....... 151 4 10 MicroRNA mediated regulation of transgene expression. ................................ 152 4 11 In vivo , qualitative analysis of AAV2 based vectors containing the ubiquitous, CBA promoter. . ................................ ................................ ................................ . 153 4 12 In vitro transduction efficiency of photoreceptor specific promoters. ................ 154 4 13 Fundoscopy of C57BL/6 mice 3 weeks post co delivery of s c mOPs GFP and sc hGRK1 mCherry ................................ ................................ ................... 155 4 14 Fu ndoscopy of C57BL/6 mice 3 weeks post sc hGRK1 mCherry delivery. ...... 156 4 15 FACS plots quantifying GFP and mCherry expression post subretinal delivery of s c mOPs GFP and sc hGRK1 mCherry ................................ .......... 157

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11 LIST OF ABBREVIATIONS LCA Leber congenita l amaurosis AAV Adeno associated virus CEP290 Centrosomal protein, 290kDa molecular weight RPE65 Retinal pigment epithelium specific protein, 65kDa molecular weight NEI VFQ 25 National Eye Institute Visual Functioning Questionnaire, contains 25 questi ons ERG Electroretinography OCT Optical coherence tomography logMAR Logarithm of the minimum angle of resolution Amp Amplitude ITR Inverted terminal repeat NT N terminus or amino terminus CT C terminus or carboxyl terminus AP Alkaline phosphatase SD Splice donor SA Splice acceptor pA Polyadenylation signal b ps Base pairs mIMCD Mouse inner medullar collecting duct cells HEK293 Human embryonic k idney 293 cells GRK1 G protein coupled receptor kinase 1 CBA Chicken beta actin promoter smCBA Sma ll chicken beta actin promoter mOPs Mouse opsin promoter

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12 hOP Human opsin promoter IRBP241 Interphotoreceptor retinoid binding protein promoter, 241 base pairs in length BSS Balance salt solution WB Western blot v g Vector genomes GC L Gan glion cell la yer INL Inner nuclear layer ONL Outer nuclear layer IS Inner segments of photoreceptors OS Outer segments of photoreceptors RPE Retinal pigment epithelium GFP Green fluorescent protein sc Self complimentary

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13 Abstract of Dissertation Present ed to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EXPAN D ING THE UTILITY OF AAV GENE THERAPY FOR CEP290 LEBER CONGENITAL AMAUROSIS By Renee Ryals Augus t 2014 Chair: William Hauswirth Major: Medical Science CEP290 Leber congenital amaurosis (LCA) patients are characterized by severe visual dysfunction, early loss of rod photoreceptors and maintained cone photoreceptors within the fovea. CEP290 LCA pati ents are excellent candidates for AAV mediated gene therapy because the genetic causation of their disease is understood and their retinas possess retained cone photoreceptors that can be targeted during treatment. The development of clinical trials for CE P290 LCA patients will require outcome measures capable of detecting visual gains post treatment, AAV mediated CEP290 expression and an injection technique that facilitates optimal gene expression in cone photoreceptors and minimizes damage to the retina. The National Eye Institute visual functioning questionnaire 25 (NEI VFQ 25) was explored as a useful subjective outcome measurement for inherited retinal dystrophy patients, including LCA patients. In order to validate the NEI VFQ 25, subjective assessment of cone mediated and rod mediated vision was correlated to objective electroretinography data. Although subjective assessment of vision did not correlate to objective assessment of vision, subjective assessment of vision via the NEI VFQ 25 showed clear di fferences in cone mediated and rod mediated vision between all inherited retinal dystrophy groups.

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14 Simple overlap and hybrid dual AAV vector systems were generated and evaluated for their ability to express CEP290 in vitro and in vivo . The two dual AAV vec tor systems evaluated for the delivery of CEP290 were unable to mediate full length transcript and protein expression in vitro . Furthermore, s ubretinal delivery of the dual AAV vectors did not restore visual function to rd16;Nrl / mice. Novel dual AAV vec tor technologies are required for the development of an AAV mediated gene therapy for CEP290 LCA patients. In hopes of establishing the intravitreal injection for the treatment of CEP290 LCA patients, novel AAV capsids were generated and examined for their ability to transduce photoreceptors from the vitreous in two different mouse models. Out of nine capsids evaluated, the AAV2 capsid containing four tyrosine to phenylalanine mutations and one threonine to valine mutation showed enhanced transduction (~22% ) of the photoreceptors post intravitreal delivery. In addition, i n vitro and in vivo assay s were developed to quantify transduction capabilities of nove l AAV capsids and photoreceptor specific promoters. Overall, these studies contribute to developing a n AAV gene therapy for CEP290 LCA .

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15 CHAPTER 1 INTRODUCTION Classification of Inherited Retinal Dystrophies The retina is a multilayer tissue consisting of a ganglion cell layer, an inner nuclear cell layer, a photoreceptor cell layer and a retinal pigment epithelium cell layer. The retinal pigment epithelium is situated on the apical side of the photoreceptors and plays a critical role in photoreceptor cell survival, replenishing visual pigment and phagocytosis (Rodieck, 1998) . The photoreceptors are responsible for c aptur ing light and initiating the p hototransduction cascade. The human retina consists of 60 125 million rod photoreceptors , predominately located in the peripheral parts of the retina, that mediate nighttime vision and peripheral vision as well as 3.2 6.5 million cone photoreceptors , predominately located in the fovea within the macula of the retina, that mediate daytime vision, color vision and central vision (Rodieck, 1998;Nent wich and Rudolph, 2013) . The subsequent retinal layers propagat e an electrical si gnal that is sent to the brain for visual processing (Rodieck, 1998) . Approximately 5% of the vision loss i n the western world is due to inherited retinal dystrophies (Kohl and Biskup, 2013) . Inherited retinal dystrophies are Mendelian genetic conditions that lead to retinal cell death or dysfunction which results in vision loss (Hamel, 2014) . Inheri ted r etinal dystroph ies can present in a stationary or progressive fashion. The dysfunction observed in patients with profound functional defects early in life is often stationary. However, dysfunction that occurs over time due to slow retinal cell death i s progressive. Many inherited retinal dystrophies lead to a progressive appearance of pigmentary deposits in the retina resulting from changes in the retinal pigment epithelium. Depending on the localization of the pigment deposits and observed retinal atr ophy,

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16 clinicians can distinguish peripheral pigmentary retinopathies from macular dystrophies (Hamel, 2014) . Patients with pigmentary retinopathies often have abnormal peripheral visual fields , report difficulties seeing at low light intensities , but maintain their reading c apacities until late in life . On the other hand, pa tients with macular dystrophies often lose their reading capabilities and face recognition capabilities, but have normal peripheral visual fields (Hamel, 2014) . Pigmentary retinopathies can be further categorized based on ph otoreceptor function. If patients present with rod only dysfunction they can be classified as rod dystrophy patients ( i.e. congenital stationary night blindness). If patients present with cone only dysfunction they can be classified as cone dystrophy patie nts ( i.e. achromatopsia). Some patients present with both rod and cone dysfunction, patients can either be classified as rod cone dystrophy patients ( i.e. retinitis pigmentosa) indicating the rods are more affected than the cones or cone rod dystrophy pati ents indicating the cones are more affected than the rods (Kohl and Biskup, 2013;Hamel, 2014) . When severe degeneration is present at birth in both the peripheral and macular areas of the retina the inherited retinal dystrophy is referred to as Leber congenital amaurosis (Kohl and Biskup, 2013;Nentwich and Rudolph, 2013;Hamel, 2014) . Among inherited retinal dystrophies there are also vitreoretinopathies in which ret inal glial cells and vessels are abnormal due to retinal detachment, vitreous hemorrhages and photoreceptor degeneration. When the choroid is affected, these conditions can be referred to as chorioretinopathies. Finally, hereditary optic neuropathies can a lso cause blindness. Even though hereditary optic neuropathies result in degeneration of retinal ganglion cells they are no t classified as

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17 inherited retinal dystrophies because these patients present with a normal retina and isolated atrophy of the optic d isc head (Hamel, 201 4) . M any techniques are used to assess photoreceptor function and overall retinal health d uring the development of a differential diagnosis for inherited retinal dystrophy patients . Electroretinography (ERG) measures the electrophysiological response gen erated from the retina upon exposure to light. This tool is unique in its ability to isolate and assess rod and cone function (Fishman et al., 2001;Marmor et al., 2009) . This reliable measurement of photorecept or function is widely used to diagnose and classify inherited retinal dystrophy patients. In addition to ERG data, c linicians can quanti fy the light sensitivity of isolated photoreceptors in a select region of the retina using microperimetry. Spectral doma in optic coherence tomography is an imaging technique that shows clinicians the morphology of the retina. Most importantly, topographic abnormalities of the photoreceptor cell bodies can be visualized around the fovea. Fundus autofluorescence imaging is a rapid, non invasive technique that . In addition to the extensive testing, clinicians directly observe the retina during a clinical exam and take a thorough personal and family history. Obtaining a thorough family history can help the clinician decipher the inheritance pattern of the disease. The combination of diagnostic testing, a clinical exam and taking a thorough history has proven to be an effective strategy to diagnose and classify inherite d retinal dystrophy patients (Tsui, 2007) . The ex isting diagnostic strategies, while effective, still fail to correctly categorize all inherited retinal dystrophy patients due to the phenotypic overlap and heterogeneous nature of the diseases (Tsui, 2007) . A detailed personal and family history can help in

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18 early diagnostic stages when a practitioner nee ds to decipher between a n inherited retinal dystrophy, an inflammatory disease or an infectious syndrome. Furthermore, diagnosis between a rod or cone dystrophy. For example, patients with rod dystrophies typically experience night blindness (Fishman, 2013) , whereas patients with cone dys trophies usually experience low visual acuity, impaired color vision and photosensitivity (Genead et al., 2011) . In many studies, clinicians use the National Eye Institute Visual Functioning Questionnaire 25 (N EI VFQ subjective quality of vision. The NEI VFQ 25 is a standard visual functioning questionnaire that assesses visual changes after implementation of novel visual therapies or interventions, characterizes vision related quality of life and is used as an outcome measurement in many clinical trials (Mitchell et al., 2013;Lightman et al., 2013;Rakic et al., 2013;Ryan et al., 2013;Renieri et al., 2013;Le et al., 2014) . The NEI VFQ 25 is a r apid, non invasive, cost efficient way to quickly monitor changes in clinical characterization of inherited answers to question s on the NEI VFQ 25 been correlated to their objective electrophysiological data. In Chapter 2 , I investigate if the NEI VFQ 25 could be useful in the diagnosis of inherited retinal dystrophy patients. Early and accurate diagnosis of inherited retinal dyst rophy patients is an important step in developing proper treatments for this patient population. The investigation of an additional diagnostic tool aims to provide clinicians with a rapid, cost efficient, non invasive way to classify inherited retinal dyst rophy patients.

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19 Soon after clinicians started to clinically characterize these inherited retinal diseases, researchers began to study the molecular causation of each disease. Currently, more than 200 distinct genes have been linked to specific gene product malfunctions (Kohl and Biskup, 2013;Daiger et al., 2014;Hamel, 2014) . This new genetic information has shifted the clinical care of inherited retinal dystrophy patients. After obtaining a differential diagnosi s, patients are strongly encouraged to get genetic testing to verify the genetic component of their disease. Genetic testing, as well as counseling, can help provide the patient with a more accurate prognosis, a probable disease progression, a disease inhe eligibility for clinical trials. Due to next generation sequencing technologies, at least 14 diagnostic panels for genetic screening are available which can correctly identify the genetic cause of inherite d retinal dystrophies in 55 80% of cases (Kohl and Biskup, 2013) . However, treatment options remain limited even for patients with a confirmed diagnosis. Treatment Strategies for Patients with Severe Retinal Degeneration Treatment strategies differ depending on the severity of retinal degeneration. Patients with severe retinal degeneration have lost the majority of their photoreceptor s and retinal pigment epithelium. Secondary circuitry cells , including bipolar, amacrine and Müller cells , predominately located in the inner nuclear layer, as well as ganglion cells are usually the only remaining cells in their retina. Scientists have dev eloped three distinct approaches to treat inherited retinal dystrophy patients with severe retinal degeneration. In the first approach, existing retinal cells are modified to become light sensors (Garg and Federman, 2013) . So called optogenetic approaches are still in the pre clinical stages though the data do look promising. One group of investigators from

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20 Japan expressed channelrhodopsin, a light absorbing molecule, i n the retinal ganglion cells of a mouse retina, which restored visual responses to light (Tomita et al., 2009;Tomita et al., 2010;Bi et al., 2006) . In the second approach, a visual prosthesis system is used to electrically stimulate the retina, usually the ganglion cell layer. The Argus II Retinal Prosthesis System has been implanted in blind patients with severe outer retinal degeneration. Results of the study show that patients performed statistically better w ith the system on versus off in object localization tasks and motion discrimination tasks. The results from the phase I/II clinical trial led to the approval of the Argus II Retinal Prosthesis System by the United States Food and Drug Administration (Humayun et al., 2012) . In the third approach, lost photoreceptors and retinal pigment epithelium are regenerated with the use of stem cells. Currently, five clinical trials are using stem cells to treat retinal dise ase, specifically retinitis pigmentosa and Stargardt disease. These stem cell clinical trials are attempting to rescue retinal pigment epithelium function by subretinal injection of human embryonic stem cell derived retinal pigment epithelium or to initiat e retinal repair with intravitreal injection of bone marrow derived hematopoietic stem cells. In the future, stem cell researchers would like to derive retinal pigment epithelium and photoreceptors from induced pluripotent stem cells, which could provide p ersonalized medic in al options (Garg and Federman, 2013) . Overall, studies evaluating the efficacy of these three therapeutic options (optogenetics, visual prosthesis a nd stem cells) whether in rodents or in humans have produced promising results and support continued study and investigation of these therapies for th e treatment of inherited retinal dystrophy patients with severe retinal degeneration.

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21 Gene Therapy for In herited Retinal Dystrophies Patients that present with minimal retinal degeneration have different therapeutic options. These patients usually retain patches of healthy retinal pigment epithelium and photoreceptor cell bodies which can be targeted for trea tment. Over the past decade, gene therapy has become a promising treatment for inherited retinal dystrophy patients with minimal retinal degeneration. Gene therapy involves the delivery of therapeutic DNA to the defective cell. Expression of the delivered gene can provide therapeutic levels of a protein that was previously missing (recessively inherited disorders) (Narfstrom et al., 2003;Boye et al., 2011;Sundaram et al., 2012) . Alternatively, expression of the delivered gene can silence mutant alleles that cause an abnormal gain of function phenotype (dominantly inherited disorders) (Lewin et al., 1998;Narfstrom et al., 2008;Boye et al., 2011;Jiang et al., 2011;Petrs Silva et al., 2012;Sundaram et al., 2012;Mao et al., 2012) . Retinal gene therapy has been particularly successful due to three main features of the eye. First, the eye is a small compartment that is isolated from the rest of the body by the blood retinal barrier. Delivery of the DNA can occur locally instead of systemically, which makes transduction of the desired tissues more feasible and reduces the amount of DNA delivered outside the eye. Second, the eye has a unique immune system that suppresses the na tural immune response to foreign antigens. Due to the suppressed immune response, exogenous DNA and the vehicles used to deliver therapeutic DNA can be tolerated in the eye. Finally, the delivered DNA has the potential for long term expression due to the r etina consisting predominately of post mitotic cells that will carry the infected DNA until the end of their lifetime (Ali, 2012) .

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22 Vectors Available for Retinal Gene Therapy The success of retinal gene therapy has also be en dependent on the many vectors available to deliver therapeutic DNA to the retina. An ideal vector should be able to accommodate the therapeutic gene construct, transduce the target tissue and mediate sustained gene expression without a significant immun e response (Sundaram et al., 2012) . Currently, therapeutic vectors for retinal gene delivery are available in the form of nanoparticles and viral vectors. Nanoparticles consist of a peptide or polymer ba se that condenses or encapsulates the relevant DNA (Jin et al., 2009;Herzog and Zolotukhin, 2010;Sundaram et al., 2012) . The size and charge of the nanoparticles can be modulated to aid in enhanced gene deliver y (Jin et al., 2009) . Nanoparticles have successfully delivered AB CA4 to Abca4 deficient mice, a model of Stargardt disease and Rds to rds +/ m ice , a model of retinitis pigmentosa. In both of these studies, photoreceptor function was restored according to ERG analysis (Cai et al., 2010;Han et al., 2012) . Nanoparticles are relatively easy to prepare, have low immunogenicity and can accommodate therapeutic genes of any size. Despite these advantages and the successful gene delivery with nanoparticles, overall, gene expression is minimal and transient (Jin et al., 2009;Herzog and Zolotukhin, 2010;Sundaram et al., 2012) . Nature has provided us with an alternative method for infecting cells. Viruses have evolved to enter cells efficient ly and deliver their genome into the host nucleus. Thus, three different viruses have been manipulated for gene delivery to the retina. Adenoviruses have an icosahedral capsid that contains a 36kb double stranded, linear genome (Murray and Green, 1973;Burnett et al., 1985;Burnett, 1985) . Segments of early viral genes were deleted to create first and second generation adenoviral vectors. These adenoviral vectors were engineered to deliver a 14kb therapeutic gene of interest

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23 (Engelhardt et al., 1994;Bennett et al., 1996;Rakoczy et al., 1998;Mori et al., 2002;Herzog and Zolotukhin, 2010) . In 1997, Cayouette et al. showed adenoviral vector mediated ciliary neurotrophic fa ctor expression in the rd/rd mouse retina, a model of retinitis pigmentosa. The successful gene delivery and expression led to slower retinal degeneration in the rd/rd mouse (Cayouette and Gravel, 1997) . However, by 2003, multiple studies reported that a denoviral vector s have limited retinal transduction capabilities post intravitreal and subretinal injection due to strong immune response s . After subretinal delivery of adenoviral vector s in wild type mice , reporter protein expression was o nly observed in the retinal pigm ent epithelium. Similarly, reporter protein expression was only observed in Müller cells after intravitreal delivery. T ransgene expression was consistently transient due to a cell mediated immune response (Mori et al., 2002;Bennett, 2003) . Adenoviral vectors have seen limited use for retinal gene therapy due to their inability to transduce photoreceptors and the immunogenicity of the virus. As mentioned previously, most retinal dystrophies involve photoreceptor dysfunction. Therefore, gene expression in the photoreceptors will be most beneficial for treating retinal dystrophies. Investigators have recently been able to achieve moderate levels of photoreceptor transduction with adenovirus serotype five by delet ing the RGD domain in the ad5 penton base ( Ad5DeltaRGD ) . This capsid carrying a reporter protein cassette driven by the photoreceptor specific mouse opsin promoter has shown photoreceptor specific expression after subretinal injection (Sweigard et al., 2010) . In addition, h elper dependent adenoviruses (Hd Ad) have been engineered to lessen the immunogenicity of the virus. Hd Ad contain only the adenovirus inverted terminal repeats that are necessary for DNA replica tion and the adenovirus packaging

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24 signal necessary for encapsidation of the therapeutic gene of interest (Kumar Singh, 2008;Herzog and Zolotukhin, 2010) . Continued development and advancement of adenoviral vect ors is needed for their use in retinal gene therapy clinical trials. The lentivirus is another virus currently being used for retinal gene therapy. Lentiviruses are enveloped HIV 1 RNA viruses (Naldini et al., 1996;Zufferey et al., 1998;Vigna and Naldini, 2000;Herzog and Zolotukhin, 2010) . L entiviral vectors contain up to 10kb of a therapeutic gene of interest and the packaging sequence needed to encapsidate the gene of interest (Bemelmans et al., 2006;Kong et al., 2008;Herzog and Zolotukhin, 2010;Williams and Lopes, 2012;Herzog and Zolotukhin, 2010) . Initial studies demonstrated that lentiviral vectors carrying repo r ter protein cassette s could transduce the retinal pigment epithelium after subretinal or intravitreal delivery. Transduction of adult photoreceptors was observed infrequently. However, transduction of photoreceptors was observed more frequently if transduction occurred during photoreceptor maturation (Bainbridge et al., 2001;Harvey et al., 2002) . In 2006, a l entiviral vector mediated RPE65 gene expression in the R pe 65 / mouse model of Leber congenital amaurosis. Stable gene expression was observed in the retin al pigment epithelium and retinal function was restored according to ERG analysis (Bemelmans et al., 2006) . Furthermore, Kong et al. 2008 reported lentiviral vector mediated ABCA4 expression in Abca4 / mice re tinas for at least two months post subretinal delivery. ABCA4 gene expression reduced amounts of A2E, a toxic product that accumulates in the retinal pigment epithelium in this Stargardt animal model (Kong et al ., 2008) . Lentiviral vectors have also mediated MYO7A expression in the retinal pigment epithelium of an Usher mouse model (Williams and Lopes, 2012) . Taken together, these

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25 successful pre c linical data laid the foundation for lentiviral gene therapy clinical trials for inherited retinal dystrophy patients. Currently, two clinical trials are using lentiviral vectors to deliver ABCA4 to Stargardt patients and MYO7A to Usher 1B patients (Kumar Singh, 2008;Oxford BioMedica, 2013) . Although some reports suggest that lentiviral vectors can transduce post mitotic photoreceptors (Kong et al., 2008;Oxford BioMedica, 2013) , most of the published d ata indicate that lentiviral vectors can only efficiently target the retinal pigment epithelium (Bainbridge et al., 2001;Harvey et al., 2002;Bennett, 2003;Bemelmans et al., 2006;Williams and Lopes, 2012) . Retin al pigment epithelium transduction and restoration are important components when treating inherited retinal dystrophy patients as both the retinal pigment epithelium and photoreceptors are affected in these dystrophies. However, broad application of lentiv iral vectors will depend on their ability to transduce photoreceptors and restore photoreceptor dysfunction. The adeno associated virus (AAV) is the third virus that has been modulated for retinal gene delivery. AAV is a parvovirus. Parvoviruses are small , non enveloped viruses that contain a 5kb linear, single stranded genome (Berns and Rose, 1970;Berns et al., 1986;Cassinotti et al., 1988;Herzog and Zolotukhin, 2010) . Recombinant adeno associated viral vector s (rAAV) contain AAV inverted terminal repeats, needed for replication, which flank the therapeutic gene of interest (Ponnazhagan et al., 1997;Herzog and Zolotukhin, 2010;Sundaram et al., 2012) . By 1997 , two gr oups demonstrated rAAV2 vector mediated gene expression in the photoreceptors and retinal pigment epithelium after subretinal delivery in a wild type mouse. In addition, rAAV2 mediated gene expression was observed in the ganglion cells after intravitreal d elivery.

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26 Results from these studies concluded that rAAV mediated gene expression is stable for many months post injection, no observable immune responses are generated post ocular delivery and efficient photoreceptor transduction is feasible with rAAV (Grant et al., 1997;Flannery et al., 1997a) . Since rAAV has the ability to transduce both cell types relevant to the treatment of retinal dystrophies (photoreceptors and retinal pigment epithelium), is non pathogenic and delivered DNA predominately forms an episome in the nucleus, it has been the most widely used vector for retinal gene t herapy. By 2008 , more than eight different inherited retinal dystrophy animal models had been rescued by stable therapeutic gene expression mediated by rAAV. Th ese studies included specific mouse models of autosomal recessive retinitis pigmentosa, Leber c ongenital amaurosis, achromatopsia and X linked retinoschisis (Bok et al., 2002;Guy et al., 2002;Min et al., 2005;Batten et al., 2005;Pang et al., 2006;Alexander et al., 2007;Pang et al., 2008;Stieger and Lorenz , 2008) . Furthermore, multiple studies demonstrated that delivery of siRNA and ribozymes by rAAV knocked down gene expression in the retina and successfully rescued autosomal dominant retinitis pigmentosa mouse and rat models (LaVail et al., 2000;Teusner et al., 2006;Gorbatyuk et al., 2005;Gorbatyuk et al., 2007a;Gorbatyuk et al., 2007b;Stieger and Lorenz, 2008) . Extensive pre clinical, proof of concept studies were performed in a mouse and dog model of RPE65 L eber congenital amaurosis. Th ese studies reported that rAAV2 mediated RPE65 expression could rescue rod photoreceptor function in both animal models (Acland et al., 2001;Acland et al., 2005;Jacobson et al., 2006 a;Pang et al., 2006) . Furthermore, cone photoreceptor f unction was restored after rAAV mediated expression of guanylate cyclase in the guanylate cyclase knockout mouse, a model of

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27 Leber congenital amaurosis (Ha ire et al., 2006;Boye et al., 2011) . These successful pre clinical studi es laid the foundation for rAAV mediated gene therapy clinical trials. Three separate phase I/II clinical trials have assessed the safety and efficacy of subretinal rAAV mediated ther apy for RPE65 deficient Leber congenital amaurosis patients (Bainbridge et al., 2008;Hauswirth et al., 2008;Maguire et al., 2008) . In all three trials, patients demonstrated improvements in their rod mediated v ision during light sensitivity, pupillometry and mobility tests (Boughton, 2009;Boye et al., 2013) . Furthermore, some patients in the phase I/2 clinical trial for choroideremia had measureable improvements in t heir visual acuity and light sensitivity after rAAV C BA hREP1 (Rab escort protein 1) mediated gene therapy. Choroideremia is an X linked recessive inherited retinal dystrophy that leads to degeneration of the choriocapillaris, retinal pigment epithelium an d photoreceptors (Rubin et al., 1966;Alexander and Fishman, 1985;Francis et al., 2005;MacLaren et al., 2014) . In the near future, two different multicenter clinical trials will be using rAAV to treat more RPE65 Leber congenital amaurosis patients and CNGB3 achromatopsia patients. Overall, rAAV gene therapy has improved visual responses in inherited retinal dystrophy patients (Boye et al., 2013) . Due to this success , clinical trials are expanding to treat more patients. CEP290 Leber Congenital Amaurosis The success of the RPE65 Leber congenital amaurosis clinical trials has encouraged clinicians and scientists to demonstrate that rAAV gene therapy can be a therapeut ic option for multiple types of inherited retinal diseases. With the goal of expanding the utility of rAAV gene therapy for inherited retinal dystrophy patients, my dissertation focuses specifically on developing pre clinical rAAV gene therapy strategies f or the treatment of CEP290 LCA patients. Leber congenital amaurosis (LCA) is the

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28 most severe form of pediatric inherited retinal dystrophy. Patients present with severe visual dysfunction at birth, nystagmus, a sluggish pupillary response and a non detecta ble electroretinogram (Leber, 1869) . LCA has an estimated incidence of 1 in 30,000 newborns and a worldwide prevalence of approximately 200,000 patients (Yzer et al., 2012) . So far, 18 distinct genes have been linked to the reti nal dysfunction seen in 70% of LCA cases (Chang et al., 2006;Cideciyan et al., 2007;Cideciyan et al., 2011a;Baye et al., 2011;Yzer et al., 2012) . Of the eighteen genes identified, the centrosomal protein 290 (C EP290) gene is the most frequently mutated and accounts for approximately 20% of LCA cases. CEP290 spans ove r 52 exons, has an approximate 7.4 kb coding sequence and encodes a protein containing 2,472 amino acids. The CEP290 protein is ubiquitously expresse d in ciliated cells and plays an important role in protein trafficking in photoreceptors (Sedmak and Wolfrum, 2011;Omran, 2010;Craige et al., 2010;Betleja and Cole, 2010) . In addition to LCA, mutations in CEP29 0 can also lead to systemic ciliopathies including nephronophthisis, Joubert, Bardet Biedl and Meckel Gruber syndromes. Patients present with malfunctions in multiple organs including the kidney, brain and testis (Sayer et al., 2006;Tsang et al., 2008;Travaglini et al., 2009;Garcia Gonzalo et al., 2011;Benzing and Schermer, 2011;Sang et al., 2011;Rachel et al., 2012b;Gascue et al., 2012;Shiba and Yokoyama, 2012) . The majority of CEP290 LCA patients carry an int ronic mutation, c.2991+1655A>G, which introduces a cryptic exon into the CEP290 mRNA. This cryptic exon inserts a stop codon (p.Cys998X) in the transcript which terminates transcription and results in a loss of CEP290 protein. However, the cryptic exon is usually observed

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29 in only 50% of the mRNA. Thus, patients have varying amounts of normal CEP290 gene expression which influences the phenotypic outcome in each patient (Chang et al., 2006;Collin et al., 2012;Copp ieters et al., 2010;Cideciyan et al., 2011a;Yzer et al., 2012) . Many clinicians have been struggling to clearly describe the clinical features of CEP290 LCA patients. In most cases, CEP290 LCA is a severe, progressive, rod cone dystrophy (Yzer et al., 2012) . Although these patients present with profound blindness, one typical feature is a sparing or retention of the outer nuclear layer and photoreceptors within the fovea (Cideciyan et al., 2007;Cideciyan et al., 2011a;Yzer et al., 2012) . Studies have also shown structurally sound, melanized retinal pigment epithelium within central retinal regions where the ellipse , the junction between inner and outer segments, rema ins intact (Cideciyan et al., 2007;Cideciyan et al., 2011a;Yzer et al., 2012) . Overall, CEP290 LCA is characterized by severe vision loss, the loss of rod photoreceptors within the first decade of life and the retention of the outer nuclear layer, cone photoreceptors and retinal pigment epithelium within the fovea for multiple decades of life. CEP290 LCA patients are excellent candidates for gene therapy for three main reasons. First the genetic causation of the disease is well understood and identifiable. Secondly, patients have retained cell bodies to target in the fovea . Finally, as for all retinal degeneration patients, CEP290 LCA patients have the most to gain from correction of their central vision. Develo ping Pre c linical rAAV Gene Therapy Proof of Concept Studies for CEP290 L eber C ongenital A maurosis Since the CEP290 LCA patient population is well characterized and has retained photoreceptors available for treatment, there is a need to develop rAAV gene therapy for these patients. However, three distinct components of the pre c linical rAAV gene

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30 therapy proof of concept studies need to be optimized before treating the CEP290 LCA patients. Chapters 3 and 4 of my dissertation will address these three compone nts and illustrate where the pre c linical rAAV gene therapy proof of concept studies for CEP290 LCA stand today. The first necessary component of a pre clinical gene therapy study is a well characterized animal model that appropriately mimics a human dise ase. Currently, there are two mouse models, the rd16 and rd16;Nrl / , that mimic CEP290 LCA patients (Chang et al., 2006;Cideciyan et al., 2011a) . The rd16 mouse model mimics the rapid rod degeneration seen in CEP290 LCA patients and contains a genetic mutation similar to the most common mutation identified in patients. However, the rapid rod degeneration has not allowed for an effective treatment window . Furthermore, the rd16 animal model is missing the cells t hat would be targeted in the patient population. Therefore, in 2011, Cideciyan et al. modified the existing rd16 mouse model and generated the all cone rd16;Nrl / mouse. Knocking out the neural retina leucine zipper ( N rl ) gene prevents rod formation and l eads to an all cone retina (Cideciyan et al., 2011a) . The all cone rd16;Nrl / mouse model overcomes the aforementioned limitations of the rd16 mouse model. The rd16;Nrl / retina degenerates slowly and mimics the preserved cones available to target in many patients. Thus, t he rd16;Nrl / animal model is an appropria te model for pre clinical proof of concept studies for CEP290 LCA patients. However, a complete natural history and understanding of the disease pro gression in this animal model has not been elucidated. In Chapter 3 , I present data contributing to the natural history of the rd16;Nrl / animal model. This natural history

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31 data in turn further the understanding of this animal model and identifies an opti mal treatment window. As mentioned above, CEP290 has a coding sequence of approximately 7.4 kb, almost double the packaging capacity of one rAAV capsid (Chang et al., 2006;Williams, 2008;Mullins et al., 2012) . Therefore, a rAAV system that allows for the delivery of large transgenes needs to be optimized for the delivery of CEP290 . In Chapter 3 , I will further explain the packaging limitations of rAAV and the methodologies being developed to deliver large transg enes. Briefly, Hirsch et al. 2010 have shown successful 9kb rAAV mediated gene expression with the use of novel dual rAAV vector technologies. The process of making dual rAAV vectors includes cloning the therapeutic gene in two different DNA plasmids. Then , the two separate viruses are created and delivered to the cell . After the DNA is delivered to the nucleus of the cells, homologous recombination of the two delivered DNA constructs mediates the form ation the therapeutic gene of the correct size (Hirsch et al., 2010;Dong et al., 2010) . I used novel dual vector technologies to develop two different dual rAAV vector systems for the treatment of CEP290 LCA. This project attempted to overcome the packaging limitati on of rAAV and prove that CEP290 , a large transgene, can be expressed post rAAV delivery. Currently, subretinal injection is the standard injection technique used in the ongoing rAAV gene therapy clinical trials (Bainbridge et al., 2008;Hauswirth et al., 2008;Maguire et al., 2008) . A subretinal injection is an invasive procedure that requires retrobulbar or general anesthesia depending on the age of the patient. First, a standard victrectomy is performed. Then, the vector is introduced into the subretinal space with a 39 gauge injection cannula (Jacobson et al., 2012a) . The bleb of the delivered virus

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32 causes a subretinal detachment between the retinal pigment epithel ium and photoreceptor layer which resolves back to the normal state within 24 hours. The existing clinical trials have suggested that this procedure is effective and safe (Bainbridge et al., 2008;Hauswirth et al ., 2008;Maguire et al., 2008) . However, Jacobson et al. reported that a subretinal injection performed under a cone rich fovea resulted in decreased visual acuity and foveal thinning in some LCA2 patients (Jaco bson et al., 2012a) . These results suggest that subretinal injections may not be beneficial for treatment of all inherited retinal dystrophies, especially CEP290 LCA patients where the fovea w ill be the main therapeutic target. Therefore, an alternative t ype of injection is needed to successfully treat cone rich foveae. An intravitreal injection, where the virus is injected into the vitreous cavity, could be a safer and more effective alternative to subretinal injections. Intravitreal injections are less i nvasive and do not require anesthesia or a victrectomy. They are performed as an outpatient procedure and are well established for the treatment of age related macular degeneration (Dikmetas et al., 2013;Wolf an d Kampik, 2014) . However, intravitreal injections have yet to be validated for the treatment of inherited retinal dystrophy patients. When the therapeutic rAAV vector is delivered to the vitreous it must penetrate through the entire retina to reach the p hotoreceptors for gene delivery. As of 2003, rAAV2 vector could mediate gene expression only in ganglion cells and not photoreceptors post intravitreal delivery (Mori et al., 2002) . Th ese data suggest there is a need for viral vectors capable of transducing photoreceptors after intravitreal delivery. Creation of these viral vectors would help validate the intravitreal injection as an

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33 additional delivery method for rAAV gene therapy studies. Although AAV2 was wel l established and mediated gene expression in HIV 1, cystic fibrosis and hemoglobinopathy studies in the 1990s (Chatterjee et al., 1992;Walsh et al., 1992;Flotte et al., 1993;Flotte, 1993;Bartlett et al., 1998) , it was not until the 2000s that multiple AAV serotypes (AAV1 9) were characterized in a wild type mouse retina (Rabinowitz et al., 2002a;Lebherz et al., 2008) . Studies that compared the tropism of these nine serotypes reported that rAAV2, rAAV1, rAAV5, rAAV8, rAAV9 transduced photoreceptors and retinal pigment epithelium in the mouse retina post subretinal injection. Subsequent studies indicated that rAAV5 and rAAV8 transduced the photoreceptors most efficient ly (Auricchio, 2011) . To enhance transduction efficiency of these wild type capsids, surface exposed tyrosine (Y) residues on the AAV capsid can be changed to phenylalanine (F) residues by site directed mutagenes is. These Y F modifications help the capsid avoid proteosomal degradation, subsequently increasing viral trafficking to the nucleus. Increasing the amount of DNA delivered to the nucleus leads to the observed increase in transduction efficiency (Zhong et al., 2008a;Zhong et al., 2008b;Petrs Silva et al., 2011;Aslanidi et al., 2013;Gabriel et al., 2013;Zhong et al., 2007) . These novel capsids containing the Y F modifications have led to robust increases in transd uction efficiency post subretinal delivery. More importantly, four Y F the retina post intravitreal delivery. This increase in retinal penetration suggests that these cap sid modifications may advance rAAV intravitreal gene delivery (Petrs Silva et al., 2009;Petrs Silva et al., 2011) .

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34 In Chapter 4 , I characterize transduction capabilities of novel, modified AAV2, AAV5 and AAV8 capsids in vitro and in vivo post intravitreal delivery. This project aims to validate the intravitreal injection as an alternat ive delivery method, which will be necessary for the treatment of CEP290 LCA patients. Furthermore, the methodologies used to qu antify transduction efficiency of the novel rAAV capsids can help researchers cho o se an optimal capsid to deliver their therapeutic transgene in pre clinical gene therapy studies. The overarching goal of the studies presented in this dissertation is to lay the foundation for an AAV gene therapy clinical trial for non syndromic CEP290 LCA patients. In Chapter 5, I propose three important components required for a gene therapy clinical trial for CEP290 LCA patients and demonstrate how each one of my studies c ontributed to these components.

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35 CHAPTER 2 THE RELATIONSHIP BETWEEN SUBJECTIVE ASSESSMENT OF CONE MEDIATED AND ROD MEDIATED VISION AND ELECTROPHYSIOLOGICAL PERFORMANCE IN RETINAL DYSTROPHIES Clinical Characterization of Inherited Retinal Dystrophies As new therapeutic strategies and phase I/II/III clinical trials become available for patients, early and accurate diagnosis of retinal dystrophies becomes increasingly important (Jacobson et al., 2012b;Salvatore e t al., 2013;Birch et al., 2013;Salvatore et al., 2013) . Currently, clinicians use a variety of tools including a patient history, clinical exam, electrophysiological testing, imaging and perimetry to clinically characterize inherited retinal dystrophy pat ients. These tools help clinicians narrow a diagnostic differential, which pinpoints the relevant sequence panel to use for genetic testing. However, inherited retinal dystrophies remain difficult to characterize. A single class of inherited retinal dystro ph ies can be caused by mutations on several distinct genes. For example, 18 distinct genes have been linked to the retinal dysfunction seen in 70% of Leber congenital amaurosis (LCA) patients (Yzer et al., 2012) . Furthermore, multiple mutations within a specific gene can lead to different phenotypic presentations. For example, when evaluating a subpopulation of LCA patients, CEP290 LCA patients, multiple mutations within CEP290 can lead to retinal dys function (Cideciyan et al., 2007) . The large variation in genetic mutations leads to variable phenotypic presentations of the disease. On the other hand, some patients might present with a very distinct phenotype, but due to the limited number of sequence panels available for genetic testing , the precise genetic mutation these patients carry may not be elucidated. For these reasons, diagnosing and identifying specific genetic mutations in all inherited retinal dystrophy pa tients remains challenging (Tsui, 2007) .

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36 As menti oned previously, p igmentary retinopathies can be further categorized based on photoreceptor function. B road disease classifications for inherited retinal dystrophies can include rod dystrophy, cone dystrophy, cone rod dystrophy and rod cone dystrophy (Tsui, 2007) . Electroretinography (ERG) is a critical t ool used to classify patients into one of these inherited retinal dystrophy categories. A full field ERG measures the electrical signal generated from the retina during phototransduction (Fishman et al., 2001) . ERGs ar e administered according to the International Society for the Clinical Electrophysiology of Vision (ISCEV) standards and test five specific responses (1) scotopic rod response; (2) scotopic combined rod cone response; (3) scotopic inner circuitry response; (4) photopic cone response and (5) photopic 30 Hz flicker (Fishman et al., 2001;Marmor et al., 2009) . This standard ERG protocol has been able to effectively characterize many inherited retinal dystrophies (Fishman et al., 2001) . Furthermore, ERG has been a criti cal tool in pre clinical, proof of concept animal studies evaluating the efficacy of gene therapy treatments for many retinal dystrophies (Acland et al., 2005;Alexander et al., 2007;Boye et al., 2010) . Multiple studies have established that ERG is an effective component in the diagnosis and management of inherited retinal dystrophy patients , as well as being an accepted o utcome measure in retinal gene therapy clinical trials (Pennesi et al., 2011;Fishman et al., 2001;Maguire et al., 2009;Simonelli et al., 2010;Birch et al., 2013;Testa et al., 2013;McAnany et al., 2013a) . In ad dition to electrophysiological data, a clinical exam and an extensive history remain important parts of the diagnostic process. A detailed personal history, family history and clinical exam will allow a practitioner to suspect a n inherited retinal

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37 dystroph y rather than an inflammatory or infectious syndrome. Several key questions in a history can be asked to narrow a differential between a rod or cone dystrophy. For example, patients with rod dystrophies typically experience night blindness (Fishman, 2013) , whereas patients with cone dystrophies usually experience low visual acuity, impaired color vision and photosensit ivity (Genead et al., 2011) . In many studies, clinicians use the National Eye Institute Visual Functioning Questionnaire 25 (NEI VFQ VFQ 25 is a st andard visual functioning questionnaire used to assess visual changes after implementing novel visual aids, to characterize vision related quality of life and as an outcome measurement in many clinical trials (M itchell et al., 2013;Lightman et al., 2013;Rakic et al., 2013;Ryan et al., 2013;Le et al., 2014;Renieri et al., 2013) . The NEI VFQ 25 is a rapid, non invasive, cost efficient way to quickly monitor changes in VFQ 25 has n ot been used for the clinical characterization of inherited retinal dystrophy patients or correlated to ERG performance. Screening inherited retinal dystrophy patients with the NEI VFQ 25 could help physicians narrow their differential and elucidate clini cal features typical of a particular inherited retinal dystrophy prior to ERG testing. Questionnaire answers found to be associated with certain ERG patterns could be used to characterize disease progression and could potentially be used as an outcome meas urement in inherited retinal d ystrophy clinical trials. Therefore, a modified version of the NEI VFQ 25 was evaluated for the use of diagnosing inherited retinal dystrophy patients. In order to validate the NEI VFQ 25, the relationship between inherited r

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38 clinically confirmed disease by ERG performance and their subjective quality of cone mediated vision and rod mediated vision was investigated. Methods Study Population A cross sectional study was carried out on a total of 75 pat ients diagnosed with cone dystrophy, cone rod dystrophy, rod cone dystrophy (including r etinitis p igmentosa patients) or Leber congenital amaurosis (LCA). These diagnoses were ascertained from both clinical exam and electrophysiological performance. These patients were chosen from 120 consecutive patients who received an ERG evaluation at a single practice between March 2012 and July 2013. All patients diagnosed with one of the above retinal dystrophies were included in the study. Patients with inflammatory diseases, toxic disorders or optic neuropathies were excluded from the study. Clinical diagnoses and demographic information were gathered by reviewing the medical records of the patients. The study was approved by the Institutional Review Board of the Un iversity of Florida and all procedures adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained from all the participants. For patients under eighteen years of age, the parents provided informed consent while the children gave as sent. Determination of Visual Acuity and ERG b Wave Amplitudes corrected Snellen visual acuity. Right and left eye visual acuities were averaged and converted to logMAR acuity for analysis. Full field ERG examination was carried out under ISCEV standards using Dawson Trick Litzkow electrodes (LKC UTAS Visual Electrodiagnostic Test System, LKC Technologies, Inc, Gainthersburg, MD) (Fishman et al., 2001;Marmor et al., 2009) . Right and left eye ERG b wave amplitudes were averaged and used in statistical

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39 analysis. Right and left eye visual acuities and b wave amplitudes were not significantly different for each of the four patient groups (data not shown). Subje ctive Assessment of Vision Patients answered questions from a modified version of the NEI VFQ 25. Our modified NEI VFQ 25 consisted of fourteen questions taken directly from the NEI VFQ 25. Those fourteen questions addressed overall vision, eyesight, brigh t light vision, nystagmus, color vision, night vision, light sensitivity, central vision, peripheral vision, reading difficulties and dark adaptation capabilities. However, only responses to six questions most specifically addressing cone mediated and rod mediated vision are reported here and used in statistical analysis. Statistical Analysis The photopic and scotopic ERG b wave amplitudes were normalized to age matched normal values and presented as a percent of normal. Comparison of two sample means for quantitative parametric data was conducted using the two tailed between visual acuity or ERG b wave amplitude and subjective assessment of vision. A correlation coeffi Results Demographics of Study Population A total of 75 patients were enrolled in the study: cone dystr ophy (n=8), cone rod dystrophy (n=17), rod cone dystrophy (n=42) and LCA (n=8). The age and gender distribution of the study population is presented in Table 2 1. The age of the patients ranged from 4 to 88 years old. There were 33 males and 42 females. LC A patients were

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40 younger than cone dystrophy, cone rod dystrophy and rod cone dystrophy patients ( p 1). Visual Acuity and ERG b Wave Amplitudes The mean logMAR was 0.89±0.27 for cone dystrophy, 1.01±0.64 for cone rod dystrophy, 0.82±0.86 for rod cone dystrophy and 1.15±0.60 for LCA patients (Figure 2 1A). When comparing mean visual acuities among the four groups , no significant differences were found between any two pairs of the patient groups. The normalized photopic b wave amplitudes ranged between 0% to 44% of normal for cone dystrophy, 0% to 91% of normal for cone rod dystrophy and 0% to 158% of normal for rod cone dystrophy patients (Figure 2 1B). All LCA patients had no recordable photopic b wave amplitudes which are presented as 0% of normal (Figure 2 1B). Cone rod dys trophy patients had significantly higher photopic b wave amplitudes compared to cone dystrophy and LCA patients ( p <0.05). Furthermore, rod cone dystrophy patients had significantly higher photopic b wave amplitudes compared to LCA patients ( p <0.01) (Figure 2 1B). The normalized scotopic b wave amplitudes ranged from 93% to 127% of normal for cone dystrophy, 0% to 121% of normal for cone rod dystrophy and 0% to 121% of normal for rod cone dystrophy patients (Figure 2 1C). All LCA patients had no recordable s cotopic b wave amplitudes which are presented as 0% of normal (Figure 2 1C). Comparisons between any two groups showed statistically significant differences in the normalized scotopic b wave amplitudes (Figure 2 1C). Responses to Study Question naire cone mediated and rod mediated vision are reported in Table 2 2. Seventy five percent

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41 whereas only 52.9% of cone rod dystrophy, 50% of rod cone dystrophy and 37.5% of cone rod dystrophy, 38.1% of rod cone dystrophy and 12.5% of LCA patients reported rod dystrophy, 19.0% of rod cone whereas only 58.8% of cone rod dystrophy, 54.7% of rod cone dystrophy or 62.5% of of patients with cone dystrophies had worse overall eyesight, bright light vision, color vision and light sensitivity compared to patients with cone rod dystroph y, rod cone dystrophy or LCA. Subjective assessment of rod mediated vision showed that patients with rod cone dystrophies or LCA reported worse night vision compared to patients with cone rod or cone dystrophies. For instance, 76.2% of rod cone dystrophy a nd 62.5% of rod Unexpectedly, the majority of patients in our study reported difficulties adaptin g to the dark; cone dystrophy (62.5%), cone rod dystrophy (82.4%), rod cone dystrophy (76.2%) and LCA (87.5%). Correlation between Visual Acuity or ERG b Wave Amplitudes and Subjective Assessment of Vision Spearman's correlation test was carried out to ex amine the relationship between visual acuity or ERG b wave amplitudes and patient responses to the study

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42 questionnaire. The results of these analyses are presented in Table 2 3.Out of the total 14 strong correlations, seven involved the correlation between visual acuity and questionnaire responses. From those seven cas es, statistically significant positive correlations were found in six associations with decreased visual acuity being associated with questionnaire responses indicating low subjective assessment of vision. Questionnaire responses assessing eyesight correla ted strongly with visual acuities in cone p <0.05), rod p <0.01) and LCA p <0.05) patients, but such a correlation was not found in cone dystrophy p >0.05) (Figure 2 2A C). In addition, questionnaire responses assessing bright light vision correlated strongly with visual acuities in rod cone dystrophy patients, but not in cone or cone rod dystrophy patients; although such a strong correlation was found in LCA patients the correlation was not statistically significant. Furthermore, questionnaire responses assessing color vision showed a strong correlation with visual acuities only in rod cone dystrophy patients. Interestingly, a strong correlation between questionnaire responses assessing n ight vision and visual acuities was found only in cone rod dystrophy patients. The other seven strong correlations involved correlations between ERG performance and questionnaire responses (Table 2 3). In six of those cases there were statistically signifi cant negative correlations, indicating the association of decreased ERG b wave amplitudes with poor subjective assessment of vision. There was a strong correlation between night vision and photopic ERG in cone 0.54, p <0.05). There was also a strong positive correlation between night vision and

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43 photopic ERG performance for cone dystrophy patients, but the correlation was not statistically significant. Questionnaire responses assessing overall eyesight, bright light vision and light sensitivity correlated strongly with scotopic ERG performance in cone dystrophy patients. Furtherm ore, questionnaire responses assessing overall eyesight correlated strongly with scotopic ERG performance in cone rod dystrophy patients. Questionnaire responses assessing night vision correlated strongly with scotopic ERG performance in cone rod dystrophy p <0.01) (also shown in Figure 2 2D). No strong correlations were found between any of the questionnaire responses and photopic or scotopic ERG performance for either rod cone dystrophy or LCA patients. Discussion This study demonstrates the limitations of using subjective visual performance analysis in categorizing inherited retinal dystrophy patients and emphasizes the necessity of ERG as an objective measure. Out of 40 associations assessed comparing ERG performance and subjective resp onses, only six associations showed statistically significant correlations. Only one strong correlation was found between rod mediated vision (night vision) and scotopic ERG performance. Cone subjective assessment of their night vis ion did correlate to scotopic ERG performance. Th ese data suggest that the NEI VFQ 25 has only limited utility in characterizing dystrophy patients as it was only able to correlate cone vision with their scotopic ERG performan ce and not able to correlate co ne rod mediated vision (eyesight, bright light, color vision and light sensitivity) to their photopic ERG performance. Furthermore, cone mediated and rod mediated vision did not correlate strongly wit h photopic and scotopic ERG performance

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44 for cone dystrophy, rod cone dystrophy or LCA patients. Overall, th ese data suggest that the NEI VFQ 25 may not be useful in characterizing subjective visual performance of inherited retinal dystrophy patients for di agnostic purposes, as this subjective analysis was not found to correlate consistently to objective ERG data. Overall, q uestionnaire responses showed expected differences in cone mediated vision and rod mediated vision between inherited retinal dystrophy g roups. Subjective assessment of vision showed that 75% percent of cone dystrophy patients reported st, only 33.3% of rod cone inherited retinal dystrophy patients reported difficulties adapting to the dark, sugg esting that this question was not helpful in differentiating dystrophy categories. Furthermore, four statistically significant strong correlations were found between cone mediated vision (eyesight, bright light, color vision and light sensitivity) and scot opic ERG performance and one statistically significant strong correlation between rod mediated vision (night vision) and photopic ERG performance. These were unexpected results, as it was hypothesized that cone mediated vision questions would correlate to photopic performance and rod mediated vision questions would correlate to scotopic performance. ERG is an accepted, standard measure in the clinical characterization of inherited retinal dystrophy patients (Fish man et al., 2001;Marmor et al., 2009) . The goal was to validate the modified NEI VFQ 25 by showing correlation to this accepted standard. However, the data presented

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45 in C hapter 2 does not support that the NEI VFQ 25 can be reliably correlated to ERG measu rements. Many studies have characterized inherited retinal dystrophy patients in relation to clinical signs or symptoms (Wilkie et al., 2000;den Hollander et al., 2008;Genead et al., 2011;Fishman, 2013) . Patie nts with achromatopsia (a common cone dystrophy) consistently report lack of color discrimination, poor visual acuity and photophobia (Genead et al., 2011) . Cone rod dystrophy patients report an early loss of v isual acuity and color discrimination. Depending on the disease progression, cone rod dystrophy patients may also report a decline in their nighttime vision and peripheral vision (Wilkie et al., 2000) . Retiniti s pigmentosa , the most frequently inherited form of night blindness, can be classified as a rod cone dystrophy. Patients have reported severe loss of peripheral vision followed by an eventual loss in central vision (Fishman, 2013) . L eber congenital amaurosis patients commonly report loss of their nighttime vision and daytime vision as they suffer from severe photorecep tor dysfunction in both peripheral and central areas of the retina (den Hollander et al., 2008) . Because inherited retinal dystrophy patients are so well characterized by their subjective visual function and their electrophysiological performance (Wilkie et al., 200 0;Fishman et al., 2001;den Hollander et al., 2008;Genead et al., 2011;Fishman, 2013) , it was thought that the subjective questionnaire answers would correlate to ERG performance. It was hypothesized that the cone dystrophy group would have the worst cone dysfunction compared to cone rod and rod cone dystrophy groups. Interestingly, visual acuity and photopic b wave amplitudes compared to cone rod dystrophy and rod -

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46 c one dystrophy patients. Furthermore, it was thought that more of the cone dystrophy to the dark. These findings suggest that additional objective measurements may b e needed to reliably assess cone function and that the NEI VFQ 25 questions addressing cone mediated vision may be insufficiently elucidating discrepancies between cone dystrophy, cone rod dystrophy and rod cone dystrophy patients. There were many limitat ions to this study . Firstly, subjective analysis differently. Secondly, a limited number of inherited retinal dystrophy patients participated in this study. The groups v ary in size due to the prevalence of each inherited retinal dystrophy. Retinitis pigmentosa is much more prevalent than cone rod dystrophy and L eber congenital amaurosis (Fishman et al., 2001;Tsui, 2007) . T he study population was limited and could benefit from a longer enrollment period to increase cone dy strophy, rod dystrophy and L eber congenital amaurosis patient populations. Thirdly, multifocal ERG would have been a better objective measurement to analyze cone function. However, our institution did not have age matched normative data for the multifocal ERG system. Lastly, isolated photoreceptor function is measured by the full field ERG a wave. In this study we used b wave amplitudes as an assessment of photoreceptor function in order to utilize the age matched normative data we had . Collection of age ma tched normative data on both the multifocal ERG and full field ERG systems would have allowed us to provide more relevant objective measurements of rod and cone function.

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47 In conclusion, ERG performance and subjective analysis of cone mediated and rod med iated vision did not correlate consistently . O bjective measurements of vision may not reflect visual function assessed by patients. Instead of validat ing the NEI VFQ 2 5 by comparing it to objective measures , results from longitudinal studies assessing rod mediate and cone mediated vision of inherited retinal dystro phies with the NEI VFQ 25 would indicate if the questionnaire responses change overtime and correlate within a specific patient population. In addition, a new, targeted questionnaire could be desi gned to more specifically address visual function in inherited retinal dystrophy patients. Inherited r etinal dystrophy patients may have difficulties in accurately categorizing their visual function due to having abnormal vision from early ages in life, pr ogressive changes over time or variations in day to day visual function depending on lighting conditions. These factors may account for some of the discrepancies between the subjective and objective data. Continued long term studies that monitor subjective visual performance in inherited retinal dystrophy patients would be beneficial since clinicians and researchers continue to rely upon the VFQ as a relevant outcome measure in clinical trials.

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48 Table 2 1. Demographic characteristics of study subjects. Type of dystrophy Sample size Age in years: mean SD (range) Gender Males: Number (%) Females: Number (%) Cone 8 38.1±19.2 (6 75) 4 (50%) 4 (50%) Cone rod 17 42.9±16. 6 (11 68) 8 (47%) 9 (53%) Rod cone 42 45.2 ±20.3 (7 88) 17 (40%) 25 (60%) LCA 8 14. 9 ±7.2* (4 27) 4 (50%) 4 (50%) *LCA vs. cone, cone rod and rod LCA, Leber congenital amaurosis; SD, standard deviation

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49 Table 2 . Question Cone (n=8): Number (%) Cone rod (n=17): Number (%) Rod cone (n=42): Number (%) LCA (n=8): Number (%) Excellent 0 (0.0) 0 (0.0) 2 (4.8) 1 (12.5) Good 1 (12.5) 3 (17.6) 11 (26.2) 2 (25.0) Fair 1 (12.5) 5 (29.4) 8 (19.0) 2 (25.0) Poor 6 (75.0) 5 (29.4 ) 8 (19.0) 1 (12.5) Very poor 0 (0.0) 4 (23.5) 11 (26.2) 1 (12.5) Completely blind 0 (0.0) 0 (0.0) 2 (4.8) 1 (12.5) 2. In bright light, how good is your vision? Excellent 1 (12.5) 0 (0.0) 3 (7.1) 1 (12.5) Good 0 (0.0) 4 (23.5) 14 (33.3) 4 (50.0) Fair 3 (37.5) 6 (35.3) 9 (21.4) 2 (25.0) Poor 2 (25.0) 5 (29.4) 11 (26.2) 0 (0.0) Very poor 2 (25.0) 2 (11.8) 5 (11.9) 1 (12.5) Completely blind 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 3. At the pr Excellent 1 (12.5) 1 (5.9) 9 (21.4) 1 (12.5) Good 1 (12.5) 6 (35.3) 10 (23.8) 3 (37.5) Fair 3 (37.5) 4 (23.5) 10 (23.8) 2 (25.0) Poor 0 (0.0) 4 (23.5) 5 (11.9) 1 (12.5) Very poor 3 (37.5) 2 (11.8) 8 (19.0) 1 (12.5) 4. At the present time, how would you describe your sensitivity to light using both eyes? Not sensitive 1 (12.5) 2 (11.8) 10 (23.8) 1 (12.5) Mildly sensitive 0 (0.0) 2 (11.8) 4 (9.5) 1 (12.5) Moderately sensitive 0 (0.0) 3 (17.6) 5 (11.9) 1 (12.5) Sensitive 5 (62.5) 6 (35.3) 14 (33.3) 3 (37.5) Very sensitive 2 (25.0) 4 (23.5) 9 (21.4) 2 (25.0) 5. At the present time, would you say your night vision using Excellent 0 (0.0) 0 (0.0) 1 (2.4) 0 (0.0) Good 3 (37.5) 2 (11.8) 6 (14.3) 0 (0.0) Fair 3 (37.5) 5 (29.4) 3 (7.1) 3 (37.5) Poor 1 (12.5) 4 (23.5) 12 (28.6) 0 (0.0) Very poor 1 (12.5) 6 (35.3) 20 (47.6) 5 (62.5) 6. Does it take you longer than the average person to adapt to the dark when you come indoors from a bright sunny day? Takes me less time 0 (0.0) 0 (0.0) 0 (0.0) 1 (12.5) Takes me the average time 3 (37.5) 3 (17.6) 10 (23. 8) 0 (0.0) Takes me much longer 5 (62.5) 14 (82.4) 32 (76.2) 7 (87.5) LCA, Leber congenital amaurosis

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50 Table 2 Visual Acuity Photopic ERG b wave amplitude Scotopic ERG b wave amplitude Cone Eyesight 0.17 0.24 0.76* Bright Light 0.12 0.22 0.69* Color Vision 0.20 0.31 0.30 Light Sensitivity 0.03 0.23 0.87 # Night Vision 0.13 0.61 0.15 Cone rod Eyesight 0.56 # 0.24 0.55* Bright Light 0.17 0.06 0.09 Color V ision 0.39 0.22 0.34 Light Sensitivity 0.11 0.08 0.28 Night Vision 0.57 # 0.54* 0.76 # Rod cone Eyesight 0.63 # 0.36 0.19 Bright Light 0.63 # 0.15 0.08 Color Vision 0.53 # 0.20 0.03 Light Sensitivity 0.34 0.07 0.12 Night Vision 0.28 0.38 0.28 LCA Eyesight 0.79 # 0.00 0.00 Bright Light 0.58 0.00 0.00 Color Vision 0.04 0.00 0.00 Light Sensitivity 0.20 0.00 0.00 Night Vision 0.06 0.00 0.00 * p <0.05 (one tail), # p < 0.01 (one tail) A indicates a strong correlation (bolded). LCA, Leber congenital amaurosis; ERG, electroretinography

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51 Figure 2 1. Objective measurements of visual function. (A) Average visual acuities and st andard deviations of all cone dystrophy (n=8), cone rod dystrophy (n=17), rod cone dystrophy (n=42) and LCA (n=8) patients. (B,C) Photopic and scotopic b wave amplitudes were recorded from ERG waveforms. Amplitudes were compared to age matched normal value s and calculated as percent of normal. Percentages were averaged and graphed for cone dystrophy, cone rod dystrophy, rod cone dystrophy and LCA groups. P values were calculated using a two t test. LCA, Leber congenital amaurosis; ERG, elec troretinography

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52 Figure 2 2. Four correlations found between objective visual function and subjective excellent, 2 good, 3 fair, 4 poor, 5 very poor or 6 compl axis) against visual acuities (y axis) for cone rod dystrophy (A), rod cone dystrophy (B) and LCA (C) patients. excellent, 2 good, 3 fair, 4 poor or 5 were plotted (x axis) against normalized scotopic b wave amplitudes (y axis) for cone p values. LCA, Leber congenital amaurosis; Amp, amplitude; ERG, electroretinography

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53 CHAPTER 3 DEVELOPING AN AAV GENE THERAPY FOR CEP290 LCA PATIENTS PART 1: DUAL AAV VECTORS FOR DELIVERING CEP290 CEP290 L eber C ongenital A maurosis Patients Retina specialists have found three distinct features tha t delineate CEP290 LCA patients. The first feature, which usually occurs early in life, is profound blindness (Cideciyan et al., 2007;Coppieters et al., 2010;Cideciyan et al., 2011a;Yzer et al., 2012;McAnany et al., 2013b) . In a recent study, 64% of the 43 CEP290 LCA patients evaluated presented with a visual acuity of count fingers , hand motion, light perception or no light perception in their better seeing eye (McAn any et al., 2013b) . Visual acuity can decline over time, but after these patients reach a non measurable chart visual acuity not much additional loss in visual function occurs (McAnany et al., 2013b) . The seco nd characteristic of CEP290 LCA patients is the rapid degeneration of rod photoreceptors (Cideciyan et al., 2007;Cideciyan et al., 2011a;Yzer et al., 2012) . In some patients, rod degeneration has occurred so qu ickly and entirely that physicians questioned whether rod photoreceptors existed and participated in phototransduction. Studies were conducted to verify rod photoreceptor existence by extensively studying the retinal pigment epithelium in CEP290 LCA patien ts (Cideciyan et al., 2011a) . Physicians can observe lipofuscin accumulation in the retinal pigment epithelium by short wavelength autofluorescence. Lipofuscin is a pigment that accumulates in the retinal pigme nt epithelium when p hotoreceptor outer segments that contain photoisomerization retinoid products generated during phototransduction are phagocytized . Lipofuscin was observed in regions of the retina devoid of photoreceptors indicating that these regions d id contain rod photoreceptors at one time (Cideciyan et al., 2011a) . To explain these observations, it is hypothesized that the rod

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54 photoreceptors in CEP290 LCA patients differentiate normally during developmen t, but the outer segments do not elaborate normally and become structurally unstable after maturation causing the rapid rod photoreceptor degeneration (Cideciyan et al., 2011a) . The third feature observed in CE P290 LCA patients is retained cone photoreceptor nuclei and healthy patches of retinal pigment epithelium within the fovea (Cideciyan et al., 2007;Cideciyan et al., 2011a;Yzer et al., 2012) . This characteristic seems to contradict the severe vision loss these patients experience, but upon further investigation the cone photoreceptors have structural abnormalities in the inner and outer segments that inhibit proper functioning and contribute to the minimal visual function of these patients (Cideciyan et al., 2011a) . However, the retained cone nuclei and healthy patches of retinal pigment epithelium in the fovea represent potential targets for therapy. Restoring functio n to the remaining cone photoreceptors could CEP290 LCA patients are good candidates for g ene therapy for several reasons ; most importantly their retina s retain cells to target. In addition, there is only one m alfunctioning gene in these cells causing the loss of function. Theoretically the CEP290 gene could be delivered to these cone nuclei, restor ing CEP290 function to these photoreceptors and restor ing cone mediated vision to these patients. Features of CEP2 90 The CEP290 gene spans over 85kb, 52 exons and encodes a protein of 2,472 amino acids. This large gene has a coding sequence of ~7.5kb (Chang et al., 2006;Coppieters et al., 2010;Drivas et al., 2013a) . Due to the large size of this gene and protein, some researchers hypothesize that replacing half of the gene or replacing functional segments of the gene would elicit therapy (Baye et al., 2011;Drivas et al.,

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55 2013a) . However, there are two main reasons why the delivery and replacement of the entire gene would be the most optimal therapy option for CEP290 LCA patients. Firstly, over 112 distinct mutations have so far been identified in the CEP290 gene (Coppieters et al., 2010) . Approximately 43 of these genetic mutations can cause LCA alone and approximately 17 mutations can cause LCA in combination with Meckel Grüber syndrome, Senior Loken syndrome, cerebello oculo renal syndrome and Joubert syndrome. These 60 genetic mutations occur in locations spanning the entire CEP290 protein. The most prevalent mutation is an intronic c.2991+1655A>G mutation which occurs in 26% of all LCA cases in Northwestern Europe. This mutation introduces a cryptic exon into 50% of the CEP290 mRNA . The cryptic exon contains a stop codon (p.Cys998X) which terminates transcription and results in a loss of CEP290 protein. However, this mutation has a significantly lower prevalence in Southern Europe, Korea, Sout hern India and Saudi Arabia (Coppieters et al., 2010) . Therefore, to ensure that most CEP290 LCA patients can be treated, regardless of the causative mutation, it would be most beneficial to design a strategy to replace the entire gene. The second rationale for replacing full length CEP290 to maximize the therapeutic benefit, considers the location and function of the protein. Photoreceptors contain modified primary cilia (S ung and Chuang, 2010;Murga Zamalloa et al., 2010;Sedmak and Wolfrum, 2011;Rachel et al., 2012a;Drivas et al., 2013a) . Within the transition zone of the photoreceptor there are four distinct compartments comprising its primary cilia. The four compartments are a distal cilium or axoneme, the connecting cilium/transition zone, the basal body and periciliary complex/ciliary pocket (Rachel et al., 2012a) . Elegant structural work in Chlamydomonas indicated that CEP290 localizes

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56 to the ciliary Y linkers within the connecting cilium/transition zone that link the ciliary membrane to the microtubule doublets. The Y links are thought to form a barrier to passi ve diffusion between the cilium and the cytosol and possibly act as a scaffold for the cellular machinery responsible for intraflagellar transport (Craige et al., 2010;Benzing and Schermer, 2011;Shiba and Yokoya ma, 2012;Garcia Gonzalo and Reiter, 2012) . CEP290 likely plays an important role in the organization of this diffusion barrier through the recruitment of a number of interacting partners (Chang et al., 2006;Cra ige et al., 2010;Drivas et al., 2013a) . In the photoreceptor, this diffusion barrier is critical for proper transportation of phototransduction proteins from the inner segments to the outer segments and outer segment maintenance (Chang et al., 2006;Cideciyan et al., 2011a;Drivas et al., 2013b) . The CEP290 protein has 13 coiled coiled domains, a region with homology to structural maintenance of chromosomes, chromosomal segregation ATPases, a bipartite nuclear lo calization sequence, six RepA/Rep+ protein KID motifs, three tropomyosin homology domains and an ATP/GTP binding site motif A (Chang et al., 2006;Coppieters et al., 2010;Drivas et al., 2013a) . Upon further inve stigation of these protein domains, amino acids 1 362 seem to play a crucial role in CEP290 binding to membranes, whereas amino acids 1695 1966, corresponding to the myosin tail homology domain, play a crucial role in microtubule binding (Chang et al., 2006;Drivas et al., 2013a) . Both the N terminal portion of the protein and the C terminal portion of the protein are important for linking the ciliary membrane to the microtubule doublets. Furthermore, the CEP290 protein recruits and binds many proteins. CP110 binds to the N terminal portion of CEP290 and inhibits CEP290 protein activity (Rachel et al., 2012a;Drivas et al., 2013a) . Rab8 binds to the

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57 central region of CE P290 and facilitates the elongation of the cilium (Rachel et al., 2012a) . CEP290 immunoprecipitation experiments using retinal extracts from WT mi ce suggests that CEP290 can interact with retinitis pigmentosa proteins (RPGRIP1), dynactin and dynein subunits (p50 and p150 Glued ), kinesin subunits (KIF3 and KAP3), tubulin), centrosomes (PCM1, centrin and pericentrin) and cohesion complexes (SMC1 and SMC3) (Chang et al., 2006) . The exact binding locations of the aforeme ntioned proteins have yet to be elucidated. Overall, every domain within the CEP290 protein functions to interact with either membranes, microtubules, ciliary proteins or phototransduction proteins. These data taken together suggest that to restore CEP290 function completely the entire protein needs to be re established. Dual AAV Vector Technology Research in this dissertation explores the ability of adeno associated viral (AAV) vectors to mediate full length CEP290 expression. AAV vectors have been the m ost widely used delivery system for retinal gene therapy due to their ability to transduce both of the cell types relevant to the treatment of retinal dystrophies (photoreceptors and retinal pigment epithelium), their safety profile and their established e fficacy in treating inherited retinal dystrophy patients in multiple clinical trials (Bainbridge et al., 2008;Hauswirth et al., 2008;Maguire et al., 2008;MacLaren et al., 2014) . One AAV capsid can package a max imum of ~4.7kb of DNA and as previously mentioned the coding sequence of CEP290 is ~7.5kb. Therefore, b efore AAV gene therapy can be applied in a clinical setting, the packaging limitation of the AAV capsid must be expanded for AAV mediated CEP290 expressi on to be possible . Four distinct AAV technologies have been explored and characterized for the delivery of large transgenes (Lai et al., 2005;Ghosh et al., 2008;Grose et al., 2012;Zhang and Duan, 2012;Dyka et

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58 al ., 2014a;Colella et al., 2014) . One approach enables large transgene expression through the use of heterogeneous AAV vectors. During the packaging of a large transgene (>5.3kb) a heterogeneous population of partially packaged transgene exists in the final AAV prep (Hirsch et al., 2010) sequence and continues sequentially until the capsid reaches its capacity of ~5kb. transgene fragments of opposite polarity. Although only fragments of the large transgene are delivered to the cell, full length protein has been detected from these heterogeneous length DNA AAV vectors (Wu et al., 2010;Grose et al., 2012;Dyka et al., 2014a) . It is hypothesized that upon delivery of the partially packaged cDNAs , large gene reconstruction can occ ur through recombination at overlapping homologous sequences or direct annealing of opposite polarity transgene fragments followed by OH ends (Wu et al., 2010;Hirsch et al., 2010) . Heterogeneous AAV vectors were not explored in this dissertation for the delivery of full length CEP290 due to their decreased packaging efficiency, decreased transduction efficiency and the potential complications in re ceiving regulatory approval for therapeutic application (Hirsch et al., 2010;Dong et al., 2010;Dyka et al., 2014a) . An alternative techno logy being investigated for AAV mediated expression of large transgenes is trans splicing vectors. To create t rans splicing AAV vectors a large transgene is split into two separate constructs. One construct contains the promoter the and an engineer ed splice donor si te . T portion of the gene , an engineered splice acceptor site and a polyadenylation signal.

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59 Upon co infection, the AAV genomes undergo head to tail recombination through the viral inverted terminal repeats (ITRs). The engineered splice signals remove the ITR junction du ring RNA splicing and restore full length transgene expression. An optimal gene splitting site must be present in order to efficiently express a gene with t rans splicing AAV vectors (Yan et al., 2005;Lai et al., 2005;Ghosh et al., 2008) . Simple overlap vectors, a second alternative dual AAV vector technology, can also mediate large transgene expression. In this case the two separate constructs lack the engineered splice signals, but instead the two constructs sh are a common central sequence approximately 1kb in size. Upon co infection of the simple overlap vectors, homologous recombination occurs between the overlapping sequences to create a full length transgene. Transduction efficiency is largely dependent on t he recombination efficiency of the overlapping sequence (Ghosh et al., 2008) . Both trans splicing and simple overlap vectors have mediated full length expression of large cDNAs in vivo (Lai et al., 2005;Zhang and Duan, 2012;Colella et al., 2014;Dyka et al., 2014a) . Dual AAV technology was further advanced with the development of hybrid vectors. Hybrid vectors were deve loped to generate a generic dual vector strategy that would be applicable to any large transgene. The first hybrid vectors contained an 872bp alkaline phosphatase (AP) gene fragment isolated from the middle one third of the human placental AP complementary DNA. Ghosh et al. 2008 have previously demonstrated that this fragment of DNA tends to be highly recombinogenic in the context of overlapping vectors. Hybrid dual vectors contain strong splice donor and acceptor signals as well as the highly recombinogeni c AP gene fragment ultimately combining trans splicing and simple overlap technology. However, the insertion of the AP DNA fragment allows for

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60 transgene independent reconstitution. Recently, two groups of researchers independently showed successful dual ve ctor mediated ABCA4 and MYO7A and Usher1B patients (Lopes et al., 2013;Dyka et al., 2014a;Colella et al., 2014) . The resu lts from both research groups suggested that the hybrid dual vectors mediate large transgene expression most efficiently in vivo when compared to trans splicing or simple overlap vectors. Although published data suggest that the hybrid vectors will likely be the most optimal dual vector technology for large transgene delivery it is important to note that dual vector systems may have unique requirements for each large transgene. For example, Zhang et al. 2012 created three hybrid vector systems and one simpl e overlap vector system for the expression of a second generation mini dystrophin gene. Due to the highly recombinogenic nature of the mini dystrophin gene, the simple overlap vector system had the highest transduction efficiency in vivo compared to the hy brid vector systems (Zhang and Duan, 2012) . In this dissertation, simple overlap and hybrid dual AAV vectors were investigated for their ability to mediate full length CEP290 expression. Simple overlap and hybrid dual AAV vector technologies were chosen to explore the recombinogenic properties of the CEP290 gene itself and to explore the utility of the transgene independent reconstitution. Animal Models for CEP290 Leber Congenital Amaurosis Relevant pre clinical animal models are essential for verifying CEP290 dual AAV vectors in vivo . Two naturally occurring animal models are known with mutations in CEP290 , they are a pedigree of Abyssinian cats and the rd16 mouse (Coppieters et al., 2010) . Both models display an autosomal recessive inheritance pattern and possess retinas that progressively degenerate. The genetic defect causing retinal degeneration

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61 in the Abyssinian cat consists of a nucleotide substitution in intron 50. This nucleotide substitution creates a strong splice donor site resulting in an elongation of e xon 50 and a frameshift with a premature termination codon. This results in the production of a truncated CEP290 protein, shortened by 159 amino acids (Coppieters et al., 2010) . Retinal degeneration is usually observable within 12 18 months, but the onset is highly variable among affected cats. At eight months of age and onward retinal dysfunction can be measured by full field ERG. Retinal dysfunction can also be monitored by pupillary light responses, which become sluggi sh with disease progression. It is hypothesized that defects in protein transport at the connecting cilia lead to degeneration of membranes in the basal part of the rod outer segments in the early stage of the disease (Co ppieters et al., 2010) . The rd16 mouse model displays an early onset retinal dystrophy caused by a homozygous in frame deletion of 897bps. The deletion results in a reduced amount of a truncated protein lacking amino acids 1599 1897 which correspond t o the myosin tail homology domain (Chang et al., 2006;Coppieters et al., 2010) . The minimal expression of the truncated CEP290 protein results in many distinguishable phenotypes. Firstly, fundus examination sho ws retinal degeneration in the rd16 animal model as early as one to two months of age. Secondly, at 18 days of age, rd16 mice have severely reduced scotopic and photopic ERG b wave amplitudes. By four weeks of age, ERG waveforms are non recordable (Chang et al., 2006) . Thirdly, histology of rd16 retinas shows severe reduction in outer nuclear layer thickness at three weeks of age. At 30 days of age, only two rows of nuclei are remaining in the outer nuclear laye r (Chang et al., 2006;Cideciyan et al., 2011a) . Fourthly, co immunoprecipitation experiments show

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62 that the truncated CEP290 protein isolated from rd16 retinal extracts recruit s 50 times more retinitis pigmento sa G protein receptors (RPGR ORF15) than CEP290 protein isolated from WT retinal extracts (Chang et al., 2006) . This enhanced binding property of truncated CEP290 protein has also been reported in a more recent study that used truncated CEP290 constructs to investigate the microtubule and membrane binding domains of the CEP290 protein (Drivas et al., 2013a) . Finally, immuno electron microscopy (EM) experiments were performed to assess the structur al features of rd16 photoreceptors. Immuno EM experiments showed intact, properly positioned basal bodies and connecting cilium in the remaining photoreceptors of rd16 retinas. However, the axonemes were reduced or sometimes absent leading to shortened and malformed outer segments. From these results it was concluded that cilia biogenesis was initiated, but outer segments remained rudimentary and failed to extend in the rd16 retina (Chang et al., 2006;Cideciyan e t al., 2011a) . Realizing that this structural abnormality in rd16 photoreceptors could have an effect on the localization of phototransduction proteins , immuno EM as well as immunocytochemistry experiments were performed to assess the localization of RPGR ORF15, arrestin and rhodopsin. All three of these proteins were improperly localized to the inner segments of P12 rd16 retinas. In WT retinas, the aforementioned proteins were properly distributed in the outer segments which were structurally sound. Overa ll, it is hypothesized that the misrouting of phototransduction proteins is the underlying cause of photoreceptor degeneration in the rd16 retina (Chang et al., 2006;Cideciyan et al., 2011a) . The rd16 mouse mod el mimics the rapid rod degeneration seen in CEP290 LCA patients. However, the rapid photoreceptor degeneration has not allowed for an effective treatment window . Furthermore, due to

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63 this rapid degeneration of the entire retina the rd16 ess the retained cone nuclei that would be targeted in the patient population. Therefore, in 2011, the existing rd16 mice were bred with Nrl / mice to generate an all cone retina rd16;Nrl / mouse. Knocking out the neural retina leucine zipper gene ( NRL ) prevents rod formation and leads to an all cone retina (Cideciyan et al., 2011a) . The rd16;Nrl / mouse model overcomes the aforementioned limitations of the rd16 mouse model. Firstly, retinal degeneration occu rs much slower. Histology of rd16;Nrl / retinas showed significant survival of cone photoreceptors at three to four months of age (Cideciyan et al., 2011a) . Secondly, the all cone retina mimics the preserved c ells available to target in patients. Distinct phenotypes of this animal model include reduced ERG waveforms, structural abnormalities of the photoreceptors and improper localization of phototransduction proteins. Cideciyan et al. showed that rd16;Nrl / mice have reduced S cone and M cone ERG signals at ages two to four months compared to Nrl / mice. Structural analysis of rd16;Nrl / photoreceptors showed proper inner segment, basal body and co nnecting cilium morphology. However, these photoreceptors co ntained a reduced number of outer segments most of which were in correctly formed (Cideciyan et al., 2011a) . Similar to the rd16 retina, a predominate phototransduction protein, S opsin, was incorrectly loca lize d to the inner segments in three month old rd16;Nrl / mice. Overall, Cideciyan et al. 2011 reported that cone cell loss resulting from the CEP290 mutation was slow even though cone dysfunction was profound and this models the cone cells retained in patien ts (Cideciyan et al., 2011a) . These studies therefore suggest that the rd16;Nrl / animal model is an appropria te model for pre clinical proof of concept studies for CEP290 LCA patients.

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64 With the existing dual AAV vector technology and an reasonable pre clinical animal model, treatment strategies can be evaluated for CEP290 LCA. I first investigated morphological changes and phototransduction protein content in the rd16;Nrl / retina from P21 to P80 to specify a n optimal treatment window for vector delivery . Secondly, I created novel CEP290 dual AAV vector systems that could potentially restore CEP290 function to the rd16;Nrl / animal model. Thirdly, I investigated the utility of the CEP290 dual vector systems i n vitro and in vivo . The purpose of these experiments is to develop a pre clinical AAV gene therapy for the treatment of CEP290 LCA. Methods Dual Vector Production Two separate plasmids were constructed to produce the simple overlap vectors (Figure 3 1A) . The vector plasmid corresponding to the N terminal sequence was erted terminal repeat , a small hCEP290 c omplementary DNA ending at position 3,622. T he C terminal sequence vector plasmid was cloned to contain bases 2,948 to 3,622 of hCEP290 c o mplementary DNA hCEP290 c omplementary DNA, a 30 base pair sequence coding for a myc tag, a polyadenylation inv erted terminal repeat. The internal over lapping CEP290 coding sequence was 675 base pairs long. Two separate plasmids were also constructed to produce the hybrid vectors (Figure 3 1B). The N terminal to inverted terminal repeat , the sm all chicken beta actin portion of hCEP290 c omplementary DNA, a strong splice donor signal and an alkaline

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65 phosphatase exon sequence. The h ybrid C terminal vector plasmid was cloned to conta in an alkaline phosphatase exon sequence identical to that in the N terminal vector plasmid, a strong hCEP290, a 30 base pair sequence coding for a myc tag , inverted terminal repeat. The hCEP290 c omplementary DNA was divided in half at a natural splice junction at position 3,309 (end of exon 29) and position 3,310 (start of exon 30) (according to CEP290 genomic NG 008417). Simple overlap and hybrid N terminal and C terminal con structs were packaged separately and titered according to standard AAV production methods (Zolotukhin et al., 2002) . Simple overlap viral titers were analyzed with polymerase chain reaction primers designed to anneal to the CEP290 overlapping region. Simple overlap and hybrid N terminal and C terminal constructs were packaged in AAV2 for in vitro experiments and AAV5 for in vivo experiments (Table 3 1). Myc tags were added to the C terminus vectors that do not c ontain promoters and after the CEP290 coding sequence for two reasons. First, all of the systems that were used to val idate these dual vectors contain endogenous CEP290. The myc tag enabled us to distinguish endogenous CEP290 from viral mediated CEP290. Se condly, due to the placement of the myc tag we hypothesized that myc expression would only occur if the full length gene product was formed. Cell Lines The HEK293 and mIMCD cells (purchased from ATCC) were routinely passaged by dissociation in 0.05% (w/v) trypsin and 0.02% (w/v) EDTA, followed by re plating at a split ratio ranging from 1:3 to 1:6 and 1:10 to 1:20, respectively in T75 flasks. HEK293 cells were used between passage number 40 and 60 and mIMCD cells were used between passage number 2 and 11. Cell morphology and growth rates were closely

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66 monitored at all steps to ensure cell line identity. The HEK293 cells were maintained in Glucose containing 5% fetal bovine serum (FB S), 400mM L glutamine, 4500mg/L glucose and 50mg/ml gentamicin (Hyclone, Logan, Utah, Cat#SH30022.02). The mIMCD cells were maintained in ATCC formulated DMEM: F 12 medium with HEPES containing L glutamine, 10% fetal bovine serum , 1% penicillin/streptomyci n (ATCC, Manassas, Virginia, Cat#30 2006). Cultures were incubated at 37°C in 7% CO 2 . Transfections and Infections mIMCD cells were seeded at a cell density of 5.5E4 cells in each well of an eight chamber slide (Lab Tek II Chamber Slide w/Cover, LAB TEK, R ochester, New York, length smCBA hCEP290myc plasmid using polyethylenimine (PEI) (Polysciences, Warrington, rately in serum free DMEM: F 12 media. DNA and PEI mixtures were combined and incubated 12 media was added to each we ll. Transfected slides were then incubated at 37°C for three days. HEK293 cells were seeded in six well plates. The next morning, old media was discarded and 3mls of 10% DMEM was added to each well. In the afternoon, when cells reached 80% confluency (~1. 0E6 cells/well), cells were transfected with 10ug of the full length smCBA hCEP290myc plasmid using a CaCl 2 method. Briefly, 10ug of DNA was mixed with 1M CaCl 2 and H 2 O. Then, Hepes buffered saline was added to the mixture. After an 80 second incubation, t he mixture was added to the appropriate well. Transfected ce lls were incubated at 37°C for three days. In addition to transfections,

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67 HEK293 cells were infected with 10,000 DNA containing vector particles per cell with a full length virus as well as simple overlap and hybrid viruses (Table 3 1). Briefly, viruses were diluted in balanced salt solution two viruses for dual vector infections) was added to the cells as well as ~ 1ml of serum free DMEM. Cells were incubated for one hour at 37°C. After the incubation, 1ml of 10% DMEM was added to the wells. Finally, cells were incubated at 37°C for three d ays. Tyr23 and MG132 Treatment HEK293 cells were seeded in six well plates. The next morning, old media was discarded and 1ml of 5% DMEM was added to the cells. For the Tyrphostin 23 dose Cat#T7165 5MG) was added to the 1ml of 5% DMEM. Four hours post incubation at 37°C media was discarded and cells were infected with AAV2 CBA GFP at 10,000 (SIGMA, Saint Louis, Missouri, Cat#M7449 1ML) was added to 1ml of 5% DMEM. Four hours post incubation at 37° C cells were rinsed once with phosphate buffered saline then infected with AAV2 CBA GFP at 10,000 particles per cell. Three days post infection green fluorescence was imaged using an Olympus IX70 Inverted Fluorescent Microscope equipped with a QImaging Retiga 4000R Camera with RGB HM 5 color filter and QImaging QCapture Pro 6.0 software (QImaging Surrey, BC, Canada). Once an o ptimal dose was established HE Experimental Animals C57BL/6 mice, purchased from the Jackson Laboratory (Bar Harbor, Maine) . Nrl / mice and rd16;Nrl / mice, generously provide d by Anand Swaroop (University of

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68 Michigan), were bred and maintained at the University of Florida Health Science Center Animal Care Services Facility under 12 hour/12 hour light/dark cycle. All experiments itutional Animal Care and Use Committee and were conducted in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and with National Institutes of Health regulations. Subretinal Injections Prior to subretinal inject ions, mice were topically administered 1% atropine eye drops followed by 2.5% phenylephrine hydrochloride eye drops. When mouse eyes were fully dilated, mice were anesthetized with ketamine (72mg/kg)/ xylazine (4 mg/kg). All subretinal injections were perfo rmed as previously described (Timmers et al., 2001;Pang et al., 2006) .One to two month old C57BL/6 mice were injected subretinally controls and remained un injected. P21 rd16;Nrl / mice were injected subretinally with were used as controls and remained un injected. P35 rd16;Nrl / mice were injected .0E12vg/ml or 1.0E11vg/ml. The 1.0E12 vg/ml or remained un injected. All NT an d CT simple overlap and hybrid viruses were diluted in BSS to the desired titer and then mixed at a 1:1 ratio prior to injection.

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69 Electroretinographic Analysis Prior to ERG analysis, mouse eyes were dilated with 1% atropine eye drops and 2.5% phenylephrine hydrochloride eye drops. After mouse eyes were fully dilated, mice were anesthetized with ketamine (72 mg/kg)/ xylazine (4 mg/kg). Cone function was evaluated in un injected Nrl / mice and treated rd16;Nrl / mice with photopic and 15 Hz flicker electroret inograms (ERG) using a Diagnosys LLC system with Espion V6.0.52 software (Diagnosys, Lowell, Massachusetts). Briefly, after an overnight dark adaptation mi ce were light adapted for five minut es. Then, mice were exposed to five flashes of xenon light at 600 cd.s/m 2 to obtain a photopic response. During the 15 Hz flicker test, mice were exposed to 50 flashes of a white 6500K light at 100cd.s/m 2 . These tests were performed four weeks post injection on the following groups: Nrl / un injected eyes (n=12), rd16;N rl / un injected eyes (n=19), rd16;Nrl / BSS injected eyes (n=4), rd16;Nrl / hybrid CT (1.0E11vg/ml) injected eyes (n=3), rd16;Nrl / hybrid CT (1.0E12vg/ml) injected eyes (n=3), rd16;Nrl / simple overlap (1.0E11vg/ml) injected eyes (n =4), rd16;Nrl / simple overlap (1.0E12 vg/ml) injected eyes (n=5), rd16;Nrl / hybrid (1.0E11vg/ml) injected eyes (n=10) and rd16;Nrl / hybrid (1.0E12vg/ml) injected eyes (n=10). Photopic and flicker b wave amplitudes were recorded, averaged and reported as mean ± stand ard deviation. A standard t test was used to calculate p values between groups. Significant difference was defined as a p Immunohistochemistry Multiple different samples were prepared for IHC. First, un injected rd16;Nrl / eyes were harvested on P21 , P40, P60 and P80 for the natural history study. Secondly, injected C57BL/6 and rd16;Nrl / eyes were harvested four weeks post injection (after ERG a nalysis) and assessed for viral mediated myc expression. Eyes were enucleated

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70 and the retinal tissue was prepared for cryoprotection and sectioning as previously described (Boye et al., 2011) . Sections were first rinsed three times with phosphate buffered saline . Then, sections were incuba ted with 0.5% Triton X 1 00 for one hour followed by a 30 minute incubation with a blocking solution of 1% bovine serum albumin (BSA). P21, P40, P60 and P80 rd16;Nrl / retinal sections were then incubated overnight at 4°C in a lectin PNA from Arachis hypogaea (peanut), Alexa fluo r 594 conjugate (Invitrogen, Eugene, Oregon, Cat#L 32459) and a rabbit polyclonal antibody specific to cone transducin al t2 , Santa Cruz Biotechnology, Dallas, Texas, Cat# sc 390) or a rabbit polyclonal antibody specific to cone S opsin (Rb X opsin, blue, Millipore, Billerica, Massachusetts, Cat#AB5407) diluted in 0.3% Triton X 100/1% BSA at 1:400, 1:500 or 1:3 00, respectively. Injected C57BL/6 and rd16;Nrl / retinal sections were incubated overnight at 4°C in a mouse monoclonal antibody specific to acetylated alpha tubulin (1:2,000, abcam, Cambridge, Massachusetts, Cat#ab24610) and a rabbit polyclonal antibody specific to myc (1:5,000, abcam, Cambridge, Massachusetts, Cat#ab9106) diluted in 0.3% Triton X 100/1% BSA. The following day, P21, P40, P60 and P80 rd16;Nrl / sections were rinsed with phosphate buffered saline and incubated for one hour at room tempera ture in goat anti rabbit IgG secondary antibody Alexa fluor 488 (Invitrogen, Eugene, Oregon, Cat#A11008) diluted in phosphate buffered saline at 1:500. Injected C57BL/6 and rd16;Nrl / sections were rinsed with phosphate buffered saline and incubated for o ne hour at room temperature with goat anti mouse IgG secondary antibody Alexa fluor 594 (Invitrogen, Eugene, Oregon, Cat#11032) and a goat anti rabbit IgG secondary antibody Alexa fluor 488 (Invitrogen, Eugene, Oregon, Cat#A11008) diluted in phosphate buff ered saline at 1:500. Finally, all sections were

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71 diaminio 2 phenylindole (DAPI) for five minutes at room temperature. Transfected mouse Inner Medullar Collecting Duct (mIMCD) cells were stained in a similar manner. First, the cell s were incubated in 4% paraformaldehyde for 20 minutes at 4°C. Then, cells were rinsed with 50mM NH 4 Cl in phosphate buffered saline for ten minutes at room temperature. Cells were incubated in 0.5% Triton X 100 for 15 minutes followed by a 30 minute incuba tion with a blocking solution of 1% BSA. Cells were then incubated for 45 minutes in a mouse monoclonal antibody specific to acetylated alpha tubulin (abcam, Cambridge, Massachusetts, Cat#ab24610) and a rabbit polyclonal antibody specific to myc (abcam, Ca mbridge, Massachusetts, Cat#ab9106) diluted in 0.3% Triton X 100/1% BSA at 1:1,000 and 1:500, respectively. After the primary antibody incubation, cells were rinsed twice with phosphate buffered saline and then incubated in goat anti mouse IgG secondary an tibody Alexa fluor 594 (Invitrogen, Eugene, Oregon, Cat#11032) and a goat anti rabbit IgG secondary antibody Alexa fluor 488 (Invitrogen, Eugene, Oregon, Cat#A11008) diluted in phosphate buffered saline at 1:500. Finally, slides containing the cells were c over slipped with SlowFade Gold antifade reagent with diamidino 2 phenylindole ( DAPI ) (Life Technologies, Grand Island, New York, Cat#S36939). In order to count the number of rosettes in the rd16;Nrl / retinal sections, P21, P40, P60 and P80 rd16;Nr l / retinal sections were imaged using a fluorescent Axiophot microscope (Zeiss, Thornwood, NY) as previously described (Boye et al., 2011) . Images were captured at 5X only and exposure settings were consisten t across images. All other images were captured using the Ultra View Vox Confocal Imaging System

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72 containing the Volocity 6.3.1 software and a Nikon Eclipse TE 2000 E microscope (Perkin Elmer, Waltham, Massachusetts). A variety of magnifications were used, but exposure settings were consistent across images at each magnification. Rosette Counts and Outer Nuclear Layer Thickness 5X, DAPI images of P21, P40, P60 and P80 rd16;Nrl / retinal sections were used to obtain rosette counts. Peripheral rosette counts were made by counting the total number of rosettes in the two most peripheral retinal sections on one slide. Photoreceptor r osette counts were performed on three d ifferent eyes corresponding to three different animals for each age group (n=6 for each age g roup). Central rosette counts were made by counting the total number of rosettes in the central most retinal section on one slide (n=3 for each age group). Central retinal sections were identified by visualization of the optic nerve head bulb (data not sho wn). 20X, DAPI images of P21, P40, P60 and P80 rd16;Nrl / retinal sections were used to obtain outer nuclear layer (ONL) thickness measurements. Volocity 6.3.1 software was used to analyze the 20X image and calculate an ONL thickness. Briefly, one point was chosen to represent the bottom of the ONL and another point was chosen to represent the top of the ONL. Then, the software measured and reported the were located. Periph eral ONL thickness was measured in four different locations in the two most peripheral retinal sections on one slide. ONL thickness measurements were performed on three different eyes corresponding to three different animals for each age group (n=24 for ea ch age group). Similarly, central ONL thickness measurements were made in four different locations in the central most retinal section on on e slide. This was performed on three different eyes for each age group (n=12 for each age group).

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73 Peripheral and cen tral rosette counts and ONL thickness measurements were averaged t test was performed to identify differences between the age groups. A p considered significant. Immunoblot Analysis At four weeks post injection (after ERG analysis) or three days post infection/transfection, retinas from rd16;Nrl / mice and treated HEK293 cells were harvested. Retinas and 293 cells were immediately placed in a sucrose buffer conta ining a 100X protease inhibitor (Protease Inhibitor Cocktail Kit, Thermo Scientific, Rockford, Illinois, Cat #78410) and a 100X phosphatase inhibitor (Halt Phosphatase Inhibitor Cocktail, Thermo Scientific, Rockford, Illinois, Cat #78420). The samples were then sonicated and centrifuged at 14,000rpm for 15 minutes. The protein concentration of the supernatant was determined with a Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, Illinois, Cat#23227). Samples were separated on a gradient 4 15% poly acrylamide gel (Mini PROTEAN TGX Gel, Bio Rad, Hercules, California, Cat#456 1084) (100µg for retina samples and 50µg for cell samples) and transferred onto a transfer membrane (Immobilon FL, Millipore, Billerica, Massachusetts, Cat#IPFL00010) for one hour in transfer buffer (25mM Tris, 192mM glycine) containing 5% methanol. The next day, blots were incubated in blocking buffer (ODYSSEY Infra red Imaging System Blocking Buffer, Li Cor, Lincoln , Nebraska, Cat#927 40000) for one hour. Blots were then labeled w ith a mouse monoclonal antibody recognizing myc (1:100, Santa Cruz Biotechnology, Dallas, Texas, Cat#sc actin (1:5000, abcam, Cambridge, Massachusetts, Cat#ab34731) or a rabbit polyclonal antibody raised against CEP 290 (1:2000, generously provided by Anand Swaroop,

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74 actin (1:5000, SIGMA, Saint Louis, Missouri, Cat#A2228) diluted in blocking buffer and 10% NaN 3 for one hour. After the hour incubation, secondar y antibodies including goat anti mouse immunoglobulin conjugated to CW800 (ODYSSEY Infradred Imaging System Blocking Buffer, Li Cor, Lincoln, Nebraska, Cat#926 32210) and goat anti rabbit conjugated with IR680 (ODYSSEY Infradred Imaging System Blocking Buf fer, Li Cor, Lincoln, Nebraska, Cat#926 32221) and were diluted in phosphate buffered saline , phosphate buffered saline tween and blocking bluffer and applied to the blots. The secondary incubation lasted for one hour. Finally, blots were rinsed with phosp hate buffered saline and then imaged with an infrared imaging system (Odyssey, Odyssey 2.1 software, Li Cor, Lincoln, Nebraska). Results Natural History of the rd16;Nrl / Animal Model With the goal of finding an effective treatment window for the rd16;Nrl / animal model, retinal morphology and phototransduction protein content was monitored closely in rd16;Nrl / retinas from P21 to P80. Cones in the Nrl / retina form rosettes due to excess amount of cones in the cone only retina. Th is large amount of co nes leads to the formation of an overpopulated layer or surface that buckles inwardly, similar to the infoldings of the cerebral cortex. These rosettes resolve over time due to the deterioration of the cells within the rosettes (Daniele et al., 2005) . Rosette formation and deterioration is an important morphological component of cone only retinas which has not been well characterized in the rd16;Nrl / retina. Therefore, to characterize rosette formation and det erioration, rosettes were counted in rd16;Nrl / retinal cross sections at P21, P40, P60 and P80 (Figure 3 2). The average number of rosettes in P21 rd16;Nrl /

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75 mice was 29.7±1.2 in the central retina and 31.0±7.5 in the peripheral retina. At P40, the aver age number of rosettes was 23.7±5.5 in the central retina and 22.2±7.0 in the peripheral retina. P60 r d16;Nrl / mice had an average of 14.5±2.1 rosettes in their central retina and 16.0±1.5 rosettes in their peripheral retina. Finally, at P80, the average number of rosettes was 15.0±1.0 in the central retina and 13.8±4.6 in the peripheral retina. Overall, the number of rosettes declined over time. However, the number of rosettes in P21 retinal sections and P40 retinal sections was not significantly differe nt. In addition, the number of rosettes in P60 and P80 retinal sections was not significantly different. Therefore, the significant decline in the number of rosettes occurred between P40 and P60. P21 retinal sections had a significantly different number of rosettes compared to P60 and P80 retinal sections in both the central and peripheral retina ( p when comparing P40 and P80 retinal sections in the peripheral retina ( p 3 2A). Figure 3 2B I shows representative 5X images of DAPI stained rd16;Nrl / central and peripheral retinal sections at P21, P40, P60 and P80 that were used to quantify rosettes. ONL thickness was measured in rd16;Nrl / mice at P21, P40, P60 and P80 to moni tor early retinal degeneration (Figure 3 3). Figure 3 3A H contains representative 20X images of DAPI stained central and peripheral retinal sections that were used to obtain ONL thickness measurements. At P21 the average ONL thickness was 3I).

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76 retina and 41.0±4.1 Overall, ONL thickness declined from P21 to P80 in both the central and peripheral retina. However, the ONL thickness at P60 was not significantly different from the ONL thickness at P80. The ONL thickness at P21 was significantly different than the ONL thickness at P40, P60 and P80 in both the central and peripheral retina ( p addition, the ONL thickn ess at P40 was significantly different than the ONL thickness at P80 in the central and peripheral retina and P60 in the peripheral retina ( p 3 3I). Previous studies have shown that S opsin is incorrectly localized to the cell body and out er plexiform layer region s in three month old rd16;Nrl / photoreceptors (Cideciyan et al., 2011a) . However, S opsin and other phototransduction proteins have not been monitored over time or at early ages in th is animal model. Therefore, to observe phototransduction protein content over time, rd16;Nrl / retinas were harvested at P21, P40, P60 and P80, prepared for IHC and stained for S opsin and the alpha subunit of cone transducin (GNAT2) (Figure 3 4). Represe ntative 40X images showing S opsin and GNAT2 abundance in the central and peripheral retina of rd16;Nrl / mice are shown in Figure 3 4A P. Overall, S opsin and GNAT2 levels decrease in the central retina from P21 to P80 (Figure 3 4A H). The most noticeabl e reduction occurs at P60 (Figure3 4C&G). Furthermore, GNAT2 levels decrease from P21 to P80 in the peripheral retina with the most dramatic decrease occurring at P60 (Figure 3 4I L). In contrast, S opsin levels decrease more rapidly in the peripheral reti na. By P40 there is an appreciable difference in the S opsin level (Figure 3 4M P). Overall, rosette

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77 abundance, ONL thickness and phototransduction protein content decreases from P21 to P80. The most significant decrease occurs between P40 and P60. Therefo re, therapeutic interventions should occur prior to the significant morphological changes and phototransduction protein decline. Exogenous Expression of Full length CEP290 Immunocytochemistry analysis in cultured cells has shown that CEP290 expressi on co localizes with centrosomal and pericentriolar markers (Chang et al., 2006;Sang et al., 2011) . To first verify that our full length CEP290 plasmid expresses CEP290 in biologically relevant locations, mIMCD cells were transfected with a smCBA hCEP290myc plasmid. Figure 3 5 shows representative images of un transfected and transfected mIMCD cells stained with an antibody specific for myc and acetylated alpha tubulin. Primary cilia are present in un transfecte d cells; however myc expression was undetectable (Figure 3 5A). Myc expression, representing CEP290 expression, was localized to the base of the cilia (Figure 3 5B,D,F,G), the centrosome (Figure 3 5C) and the cytoplasm (Figure 3 5B F) of these cells. These experiments show that exogenously expressed CEP290 can localize to biologically relevant locations. Analysis of Dual AAV Vector Systems In Vitro Simple overlap and hybrid constructs were first tested in an in vitro system. HEK293 cells were transfected with a full length plasmid and infected with a full length virus, simple overlap viruses and hy brid viruses (Table 3 1). Viral mediated CEP290 and myc expression was analyzed via Western Blot (WB) 3 days post transfection/infection (Figure 3 6). Endogenou s levels of CEP290 in HEK293 cells are shown in control samples (lanes 1 3). There was an increase in CEP290 expression after transfection with the full length plasmid visible in lane 4. However, there was no

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78 increase in CEP290 expression pos t infections ( lanes 5 7). Viral mediated CEP290 expression was also assessed by detecting myc expression. Myc expression was present post transfection (lane 4), but not present in control samples (lanes 1 3) or post infections (lanes 5 7) (Figure 3 6). Simple overlap a nd hybrid dual AAV vectors failed to mediate detectable levels of pr otein. However, dual AAV vector mediated expression could be occurring at very low levels not detectable by WB. To enhance transduction efficiency of the dual AAV vector systems HEK293 cel ls were treated with a specific epidermal growth factor receptor protein tyrosine kinase (EGFR PTK) inhibitor Tyrphostin 23 (Tyr23) or a proteasome inhibitor MG132 prior to simple overlap and hybrid infections. Viral second strand synthesis and viral intra cellular trafficking to the nucleus are two rate limiting steps of viral transduction. Studies have shown that EGFR PTK phosphorylates a cellular protein that inhibits viral second strand DNA synthesis and surfaced exposed tyrosines on AAV capsids which ta rget the capsid for ubiquitination and subsequently degradation by the proteasome (Zhong et al., 2007;Zhong et al., 2008a;Zhong et al., 2008b;Cheng et al., 2012) . Zhong et al. 2007 reported that EGFR PTK inhibi tors increased viral second strand synthesis and increased viral trafficking to the nucleus which significantly increased viral transduction efficiency (Zhong et al., 2007;Zhong et al., 2008c) . First, differen t concentrations of Tyr23 and MG132 were tested on HEK293 cells (Figure 3 7&3 8). Figure 3 7 contains representative 10X images of GFP expression in Tyr23 treated HEK293 cells three days post infection with AAV2 CBA GFP. GFP expression was undetectable in untreated HEK293 cells (Figure 3 7A). GFP expression was detectable in HEK293 cells infected with AAV2 CBA GFP (Figure 3 7B) and

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79 7D), 7E). GFP express ion was comparable between cells 7E&F). Figure 3 8 contains representative 10X images of GFP expression in MG132 treated HEK293 cells three days post infection with AAV2 CBA GFP. GFP expression was not det ectable in untreated HEK293 cells (Figure 3 8A). However, GFP expression was detectable in HEK293 cells infected with AAV2 CBA GFP (Figure 3 8B) and increased with the (Figure 3 8E) a 8F). Maximum GFP expression was observed infection with simple overlap an d hybrid infections. WB analysis of protein lysates from cells treated with Tyr23 and infected with simple overlap and hybrid viruses showed no increase in CEP290 expression (from endogenous levels) and no viral mediated myc expression (Figure 3 9). WB ana lysis of protein lysates from cells treated with MG132 and infected with simple overlap and hybrid viruses did show an increase in CEP290 expression (Figure 3 1 0 lanes 5&7). However, no viral mediated myc expressio n was detected to confirm viral mediated f ull length transgene expression (Figure 3 10). Increasing viral transduction efficiency did not elicit detectable viral mediated full length CEP290 expression via WB. Analysis of Dual AAV Vector Systems In Vivo Simple overlap and hybrid viruses were first tested in WT ( C57BL/6 ) mice. C57BL/6 mice were subretinally injected with simple overlap and hybrid viruses. Four weeks post injection, eyes were harvested, prepared for IHC and stained for myc and

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80 acetylated alpha tubulin. Representative 20X and 60X imag es of stained C57BL/6 retinal cross sections are shown in Figure 3 11. Acetylated alpha tubulin was visualized in the connecting cilium and outer segments of photoreceptors (Figure 3 11A F). Myc expression was not detectable in un injected or hybrid inject ed C57BL/6 retinas (Figure 3 11A,B,E,F). However, detectable levels of myc expression were observed in the inner segments of C57BL/6 retinas post simple overlap delivery (Figure 3 11C&D). Simple overlap and hybrid viruses were then tested in rd16;Nrl / m ice. Four weeks post injection with simple overlap and hybrid viruses electroretinographic analysis was performed (Figure 3 12). The average photopic b wave amplitude was injected Nrl / injected rd16;Nrl / eyes. Rd16;Nrl / mice have significantly reduced cone function measured by photopic ERG ( p Rd16;Nrl / mice injected with balanced saline solution had an average un injected rd16,Nrl / mice (p=0.08). Rd16;Nrl / mice injected with the hybrid CT virus had Rd16;Nrl / mice injected with the h ybrid CT virus at 1.0E12vg/ml had significantly decreased photopic ERG responses compared to un injected rd16;Nrl / mice ( p Rd16;Nrl / mice injected with simple overlap viruses at 1.0E11vg/ml and 1.0E12vg/ml had an average photopic b wave amplitud of mice injected with simple overlap viruses at 1.0E11vg/ml were not significantly different than photopic b wave amplitudes of un injected rd16;Nrl / mice. However, when mice were injected with simple overlap viruses at 1.0E12vg/ml photopic b wave

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81 amplitudes were significantly decreased compared to un injected rd16;Nrl / mice ( p Rd16;Nrl / mice injected with hybrid viruses at 1.0E11vg/ml and 1.0E12vg/ml had average photopi respectively. Photopic b wave amplitudes of rd16;Nrl / mice injected with hybrid viruses at both titers were significantly decreased when compared to un injected rd16;Nrl / mice ( p 12). Cone function was also measured in Nrl / and treated rd16;Nrl / mice with a 15 Hz flicker ERG (Figure 3 for un injected Nrl / injected rd16;Nrl / mice. Rd16;Nrl / m ice have significantly reduced cone function compared to Nrl / mice measured by flicker ERG ( p Rd16;Nrl / mice injected with balance saline solution had an from un injected rd16;Nrl / flicker responses ( p =0.15). Rd16;Nrl / mice injected with the were not significantly different between un injected rd16;Nrl / mice and rd16;Nrl / mice injected with the hybrid CT virus at 1.0E11vg/ml. However, rd16;Nrl / mice injected with the hybrid CT virus at 1.0E12vg/ml had a significantly decreased flicker b wave amplitude compared to un injected animals ( p Rd16;Nrl / mice injected with simple overlap viruses at 1.0E11vg/ml and 1.0E12vg/ml had an average flicker b wave mice injected with simple overlap viruses at 1.0E11 vg/ml and 1.0E12vg/ml were significantly reduced compared to flicker b wave amplitudes of un injected rd16;Nrl /

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82 mice ( p Rd16;Nrl / mice injected with the hybrid viruses at 1.0E11vg/ml and 1.0E12vg/ml had average flicker b wave amplitudes of 19.4± respectively. Flicker b wave amplitudes of rd16;Nrl / mice injected with the hybrid viruses at both titers were not significantly different compared to un injected rd16;Nrl / mice (Figure 3 12). Overall, cone function, measure by ER G, remained stable or significantly decreased after subretinal injections of dual vectors in rd16;Nrl / mice. After ERG analysis some rd16;Nrl / eyes were harvested and prepared for IHC (Figure 3 13) and some retinas were harvested for WB (Figure 3 14) . Retinal sections were stained for acetylated alpha tubulin and myc to assess viral mediated protein. Figure 3 13 contains representative 60X images of retinal cross sections from control eyes, simple overlap injected eyes (1.0E11vg/ml and 1.0E12vg/ml) an d hybrid injected eyes (1.0E11vg/ml and 1.0E12vg/ml). Acetylated alpha tubulin was observed in the connecting cilium and outer segments of the photoreceptors (Figure 3 13A F). Minimal m yc expression was det ected post simple overlap delivery at 1E11vg/ml ( Figure 3 13C). Myc expression was not detected in un injected or hybrid injected retinas (Figure 3 13A ,B,E,F ). Viral mediated myc expression was also analyzed in rd16;Nrl / retinas post ERG analysis using WB (Figure 3 14). Figure 3 14A shows the lack of m yc expression detected in un injected eyes (lanes 2&4) and rd16;Nrl / retinas injected with the simple overlap viruses at 1.0E11vg/ml (lane 1) and 1.0E12vg/ml (lane 3). Figure 3 14B shows the lack of myc expression detected in un injected eyes (lane1), BS S injected eyes (lane 2), hybrid CT at 1.0E11vg/ml injected eyes (lane 7), hybrid CT at 1.0E12vg/ml injected eyes (lane 8), hybrid at 1.0E11vg/ml injected eyes (lanes 3&4) and hybrid at

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83 1.0E12vg/ml inje cted eyes (lanes 5&6). No viral mediated myc expressio n was detected. However, the myc antibody did recognize the epitope tag protein (lane 5 in Figure 3 14A and lane 9 in Figure 3 14B). Discussion The advancement of dual AAV vector technology is required for the creation of an AAV mediated therapy for CEP 290 LCA patients. Engineered dual AAV vectors must deliver the CEP290 gene to photoreceptors and mediate CEP290 expression at therapeutic levels. CEP290 dual AAV vectors will be validated by their ability to restore visual function to the rd16;Nrl / mouse model. In C hapter 3 , a gene dependent (simple overlap) dual vector system and a gene independent (hybrid) dual vector system were generated and examined for their ability to express CEP290. Although the results indicate that exogenously expressed full len gth CEP290 protein can localize to biologically relevant locations, for example the base of the primary cilia and the centrosomes, the dual AAV vectors were unable to mediate full length CEP290 transcript (data not shown) and protein in vitro . Minimal amou nts of v iral mediated myc expression was detected in the inner segments of C57BL/6 and rd16;Nrl / retinas mice post simple overlap delivery . However, subretinal delivery of the dual AAV vectors did not restore visual function to rd16; Nrl / mice. The rd1 6; Nrl / mouse is a suitable model for evaluating gene replacement therapies for CEP290 LCA. However, previous studies have only characterized the phenotype of this mouse model at three to four months of age, long after therapeutic interventions would tak e place (Cideciyan et al., 2011b) . In this study, disease progression in rd16; Nrl / retinas was characterized at early ages (P21 to P80) in order to designate an optimal treatment window for this animal model . The outer nuclear layer thickness, the quantity of rosettes and the abundance of S -

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84 opsin and GNAT2, two critical phototransduction proteins, was significantly decreased by P60 in both central and peripheral parts of the retina. Gene therapy interventions work when a sufficient number of cell bodies are available to target and ample amounts of phototransduction proteins are present to initiate the phototransduction cascade in response to light. Data from our studies show that there were many cone photorece ptor cell bodies and outer segments present at P80 suggesting that a targetable cell population is present in the rd16; Nrl / retina. S opsin and GNAT2 levels were maintained through P40, but at P60 appreciable decreases were observed. In order to obtain maximum therapeutic gains, AAV mediated gene expression must occur prior to the phototransduction protein decline. To ensure that gene therapy success is not limited by phototransduction content, exogenous CEP290 expression must occur prior to P60 in the r d16; Nrl / retina. Gene therapies should be delivered to the rd16; Nrl / retina on P35 to allow for maximum protein expression prior to phototransduction protein decline. Since simple overlap and hybrid dual AAV vectors have successfully mediated large t ransgene expression of photoreceptors in two different animal models (Lopes et al., 2013;Colella et al., 2014;Dyka et al., 2014a) , it was hypothesized that similar simple overlap and hybrid dual AAV vector syst ems would successfully mediate CEP290 expression. Although the dual vector technology described in this study was consistent to the technology described in previously published studies, our results suggest the same simple overlap and hybrid dual vector tec hnology will not be applicable for CEP290 (Lopes et al., 2013;Dyka et al., 2014a) . All constructs used in this study were sequenced verified. Maximum viral infectivity and transduction was achieved during in

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85 vi tro experiments by packaging constructs in an AAV2 capsid known to efficiently infect HEK293 cells and by treating cells with Tyrphostin 23 or MG132 known to increase viral trafficking to the nucleus. After controlling for these well k nown limitations to v iral mediated expression, we hypothesize that specific features in the CEP290 coding sequence are responsible for the malfunctioning dual AAV vectors and new cloning strategies are required for the creation of an AAV mediated therapy for CEP290 LCA patient s. A highly recombinogenic overlap region is necessary for producing efficient simple overlap vectors. However, key features that facilitate homologous recombination are unknown. One hypothesis suggests that secondary structure and GC content of the overl ap sequence may contribute to the recombinogenic properties of the NT and CT constructs. To investigate this hypothesis, the overlap sequence used in our simple overlap vectors was analyzed for palindromic sequences which are known to create hairpin like s econdary structures within DNA. Results from an online palindromic sequence finder (Bioinfoproject from Genome Analysis Tools for Biotechnology) revealed 34 palindromic sequences within the 675bp overlap sequence. Twenty eight of these sequences were 4bps long and consisted mainly of restriction sites. The remaining sequences were 6bps, 8bps or 9bps long. These results suggest the overlap sequence has a high potential for forming secondary structure, but it remains unclear what affect these potentials have on the recombination properties of the NT and CT constructs. In addition , the GC content of the overlap sequence was analyzed by Vector NTI. The content contributed to a reduced number of recombination events in the overlapping

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86 region, but it does suggest that the GC content can be increased to test the hypothesis. In summary, low G C content and potential secondary structures in our overlap sequence could have inhibited recombination events between the NT and CT constructs. Reducing potential secondary structure as well as increasing GC content of the overlap region should be explore d in future CEP290 simple overlap designs. In order to postulate to what extent these features should be modulated, the GC content and secondary structure in the CEP290 overlap sequence should be compared to overlapping sequences with proven utility in dua l vector system s ( alkaline phosphatase sequence, MYO7A complementary DNA overlap , ABCA4 complementary DNA overlap ). Once key features enhancing homologous recombination events of dual vectors are identified, modification of the CEP290 coding sequence wil l be necessary to incorporate these features. Sequence optimization offers one possibility to manipulate the CEP290 coding sequence without altering the amino acid sequence. One example of sequence optimization, c odon optimization , has been used to remove cryptic splice sites, increase GC content and increase the codon adaptation index of the Factor VIII coding sequence (Ward et al., 2011) . These modifications to the coding sequence greatly enhanced translation of the Factor VIII protein, which plays an important role in blood coagulation (Ward et al., 2011) . Many online programs and companies specialize in sequence optimization and should be sought out for optimizing a novel overlap sequence for future CEP290 dual vectors (Ward et al., 2011;Sack et al., 2012;Chin et al., 2014) . Homologous recombination occurs at a high rate during the S phase of the cell cycle when a large amount of DNA replication occurs. Constant close interactions between the DNA segments facilitate homologous recombination. Exploring ways to

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87 concentrate simple overlap NT and CT DNA replication in order to maximize interactions between the constructs may also facilitate homologous recombination. Since it is unknown whether any region of the CEP290 coding sequence is able to facilitate homologous recombination of the NT and CT constructs, a gene independent system (hybrid) was generated to overcome this p otential gene specific limitation. However, the hybrid system was also unable to produce full length CEP290 transcript and protein. The hybrid dual vectors were created by dividing the coding sequence at a natural exon junction, cloning strong splice donor and strong splice acceptor sites at the end and beginning of the junction and cloning a highly recombinogenic alkaline phosphatase (AP) sequence outside of the exon junction. Full length DNA constructs are created in the nucleus when inverted terminal rep eats (ITRs) contained in the NT construct bind to the ITRs contained in the CT construct or when homologous recombination at the AP sequence occurs. After the DNA constructs have annealed, full length message production is dependent on proper splicing at t he exon junction. Since no full length transcript was observed post infection with our hybrid vectors, either the NT and CT constructs never annealed to create a full length DNA or aberrant splicing occurred. Since the AP sequence and engineered splice sig nals have proven to be effective in multiple hybrid systems (Zhang and Duan, 2012;Colella et al., 2014;Dyka et al., 2014a) , it is unlikely that hybrid dual vectors malfunctioned due to the se elements. However, the chosen CEP290 exon junction may be incapable of facilitating proper splicing. W e assumed that dividing the CEP290 coding sequence at a natural exon would

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88 underst ood. Given that the CEP290 coding sequence consists of 52 exons, transcription of this large transgene may have unique features. Basic studies investigating mRNA transcript assembly of endogenous CEP290 isolated from HEK293 cells and mouse photoreceptors would advance our understanding of CEP290 splicing patterns. Results from these studies could reveal alternat ive splicing patterns, novel usage of the exons and superior exon junctions for dual vector designs. For creation of our hybrid dual vectors, the C EP290 coding sequence was divided at exon junction 29 30. Keeping in mind the size constraints of the AAV capsid, it is possible to explore the utility of exon junction 30 31 and 31 32. Overall, further investigation is needed to determine why our hybrid d ual vectors did not mediate full length transcript production . Assuming that optimizing dual AAV vector technologies w ill lead to expression of viral mediated CEP290, optimizing therapeutic expression levels will be the next critical step. Based on the si ze of the transition zone in the connecting cilium of the photoreceptor it is assumed that relative low levels of CEP290 expression are required for restoration of function. In addition, a recent study discovered that high levels of CEP290 expression can b e toxic in a human cell line (Burnight et al., 2014) suggesting that viruses mediating low levels of CEP290 expression will be imperative for successful therapy . Weak er photoreceptor specific promoters and the endogenous CEP290 promoter should be explored for their ability to express CEP290 at levels that provide a therapeutic benefit without toxic side effects. On the other hand, it is possible that dual AAV vector systems will mediate CEP290 expression below therapeutic levels. When comparing protein leve ls post delivery with a single AAV vector, dual AAV vectors are 88% less efficient (Carvalho et al., 2014) . To overcome a potential exp ression limitation

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89 of dual vectors, enhanced recombination between overlap sequences is required for simple overlap dual AAV vector systems and enhance recombination of inverted terminal repeats (ITRs) is required for trans splicing and hybrid dual AAV vec tor systems. As mentioned previously , key features that facilitate homologous recombination remain unknown . However, hybrid inverted terminal repeats can be incorporated to facilitate enhanced recombination of ITRs . Hybrid ITRs utilize two non homologous I TRs, AAV5 and AAV2, to manipulate the orientation of NT and CT ITR annealing. As mentioned previously, NT and CT ITR interactions facilitate the creation of full length DNA constructs in the nucleus prior to transcription. The strength of the ITR interacti on is dependent on ITR homology. When both NT and CT constructs 25% chance that the ITR binding/circularization will occur in an orientation that allows for full length t ranscription (Yan et al., 2005). However, with the addition of AAV5/AAV2 hybrid ITRs, only specific pairs chance that the ITR binding/circularization will occur in an orientation that allows for full l ength transcription (Yan et al., 2005) . The addition of hybrid ITRs to standard trans splicing dual AAV vectors led to a 6 10 fold increase in protein expression (Yan et al., 2005) . Trans splicing and hybrid d ual AAV vectors containing hybrid ITRs should be investigated if a more efficient dual vector system is required for therapeutic CEP290 expression. For now, future studies should focus on producing dual AAV vectors that mediate full length CEP290 transcript and protein in vitro . Results from our transfection experiments suggest that HEK293 cells are a sufficient in vitro model for verification of

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90 dual AAV vectors. Once dual AAV vectors are verified in vitro , proof of concept stu dies can be attempted in the rd16;Nrl / animal model. If transcript and protein expression are not evident in relevant mouse models, but are confirmed in vitro , a mouse C ep290 coding sequence may be required for expression in the mouse models. Although th e human CEP290 coding sequence and protein are 86% and 89% identical to the mouse coding sequence and protein, it is uncertain if the human CEP290 coding sequence and protein are functional in mouse tissue. In addition, if transcript and protein expression are verified in vitro and in relevant mouse models, but fail to restore the rd16;Nrl / phenotype an additional factor may be required. R d16;Nrl / photoreceptors contain basal bodies, inner segments and connecting cilium , but the photoreceptors have eith er incorrectly formed outer segments or no outer segments at all (Cideciyan et al., 2011a). It is reasonable to hypothesize that exogenous CEP290 expression will be able to incorporate into malfunctioning transition zone s and facilitate protein trafficking , but it remains unclear if CEP290 expression will facilitate the formation or restoration of the outer segments or if outer segment re storation will be required for visual restoration . If outer segment re storation is necessary, administration of ciliary n eurotrophic factor (CNTF), which promotes cone outer segment regeneration (Wen et al., 2012) as well as exogenous CEP290 expression may be required to fully restore visual function to the rd16; Nrl / animal model. Dual AAV vector designs will inher ently be limited by the size of the CEP290 coding sequence. At this point, the size constraints of the AAV capsid will limit the length of the overlap sequence used in the simple overlap systems and limit the number of natural exon junctions available for hybrid designs. Sequence optimization and

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91 transcript analysis of endogenous CEP290 may lead to optimized cloning strategies. Natural history studies of the rd16; Nrl / mouse suggested that gene therapy interventions should occur prior to the observed phot otransduction protein decline. However, phototransduction protein decline was assessed by S opsin and GNAT2 levels . Although these are the two most abundant phototransduction proteins in Nrl / cone photoreceptors, several other proteins play critical role s in the phototransduction cascade and their content should be analyzed as well . Furthermore, phototransduction protein content should be quantified and compared to a measure of visual function. Results from these studies could reveal the relationship betw een the loss of phototransduction proteins and visual function decline. In order to verify the dual AAV vectors in vitro , viral mediated protein and transcript was analyzed. Since neither dual vector system mediate d transcript or protein production, one c ould postulate that viral DNA did not reach the nucleus in sufficient amounts . Although attempts were made to maximize AAV infectivity and transduction, episomal DNA was not extracted from cells and viral genome content was not quantified. Q uantifying the viral payload in nuclear fractions of cells could be used to exclude viral transduction efficiency as a limitation and could led to alternat ive hypothesizes for why the dual AAV vectors are not functional. In future studies, after dual AAV vectors are conf irmed in vitro , dual AAV mediated CEP290 expression should be confirmed in the Nrl / retina prior to proof of concept studies in rd16; Nrl / mice. Although C57BL/6 mice are commonly used as controls for degenerative mouse models, the all cone Nrl / mous e is the preferred control animal model for the degenerative rd16;Nrl / mouse due to their shared all cone retina and morphology.

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92 Since both simple overlap and hybrid dual vector systems failed to mediate full length CEP290 mRNA and protein, I currently p refer the h ypothesi s that specific features of CEP290 are hindering the success of our dual AAV vectors.

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93 Table 3 1. Nomeclature and usage of viral constructs.

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94 Figure 3 1. Simple overlap and hybrid constructs for CEP290. (A) Two separate plasmids were constructed to produce the simple overlap vectors. The NT The CT wa s cloned to contain bases 2,948 to 3,622 of hCEP290 cds, the rest Two separate plasmids were constructed to produce the hybrid vectors. The NT vector plasmid was cloned to contain: a AP exon sequence. Hybrid vector plasmid CT was cloned to contain an AP portion of was divided roughly in half at a natural splice junction at position 3309 of the hCEP290 cds (end of exon 29) and position 3310 (start of exon 30).

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95 Figure 3 2. Quantification of central and p eripheral rosettes in the rd16;Nrl / retina from P21 to P80. (A) Bar graph indicating the average number of rosettes counted in central (n=3) and peripheral (n=6) retina slices from rd16;Nrl / mice at ages P21, P40, P60 and P80. P values were calculated using a two tailed t test. (B I) Representative 5X images of central (B E) and peripheral (F I) retina slices from rd16;Nrl / mice at P21, P40, P60 and P80. Nuclei were counterstained with DAPI (grayscale).

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96 Figure 3 3. Quantification of cent ral and peripheral ONL thickness in the rd16;Nrl / retina from P21 to P80. (A H) Representative 20X images of central (A D) and peripheral (E H) retinal cross sections highlighting ONL thickness of rd16;Nrl / retinas at P21, P40, P60 and P80. Nuclei were counterstained with DAPI (blue). (I) Bar graph indicating the average ONL thickness measured in central (n=12) and peripheral (n=24) retinal cross sections from rd16;Nrl / mice at ages P21, P40, P60 and P80. P values were calculated using a two tailed St t test. RPE retinal pigment epithelium, ONL outer nuclear layer, INL inner nuclear layer, GCL ganglion cell layer.

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97 Figure 3 4. Photoreceptor protein expression is reduced over time in rd16;Nrl / mice. Representative images of retinal cross se ctions (40X) from central (A H) and peripheral (I P) retina of P21, P40, P60 and P80 rd16;Nrl / mice. Retinal cross sections were immunostained for the presence of the alpha subunit of cone transducin (GNAT2) (A D&I L) or S cone opsin (E H&M P) and PNA (A P). Despite maintenance of cone outer segment sheaths (PNA), both GNAT2 and S opsin expression are markedly reduced by P60 in both central and peripheral retina.

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98 Figure 3 5. Full length CEP290myc expression in mIMCD cells. Mouse inner medullar collec ting duct cells were transfected with a smCBA hCEP290myc plasmid. Three days post transfection cells were stained for myc (green) and acetylated alpha tubulin (red). (A) Representative 60X image of un transfected mIMCD cells. Primary cilia were present in un transfected cells. However, myc expression was undetectable. (B G) Representative 60X images of transfected mIMCDs. Myc expression, representing CEP290 expression, was localized to the base of the cilia (B,D,F,G), the centrosome (C) and the cytoplasm (B ,D,E,F) post transfection.

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99 Figure 3 6. CEP290 and myc protein detection via WB post simple overlap and hybrid infection in HEK293 cells. Samples 1 7 were probed for CEP290 (~300kDa red) and actin (~45kDa green) expression (top). The same samples we re also probed for myc (~300kDa green) and actin (~45kDa red) expression (bottom). Samples consisted of protein lysate (50 g) harvested from HEK293 cells that were untreated (control lane 1), transfected with no DNA (control lane 2), infected with no vir us (control lane 3), transfected with the full length smCBA hCEP290myc plasmid (transfection, full length lane 4), infected at 10,000p/cell with AAV5 smCBA hCEP290myc (infection, full length lane 5), infected at 10,000p/cell with AAV2 smCBA hCEP290NT + AAV 2 hCEP290mycCT (infection, simple overlap lane 6) or infected at 10,000p/cell with AAV2 smCBA hCEP290NT APSD + AAV2 hCEP290mycCT APSA (infection, hybrid lane 7). An increase in CEP290 expression and detectable myc expression was observed post transfection with the full length plasmid.

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100 Figure 3 7. AAV2 CBA GFP transduction is increased with Tyr23 treatment in HEK293 cells. (A) Representative 10X image of untreated HEK293 cells. (B) Representative 10X image of GFP expression in HEK293 cells 3 days post i nfection with AAV2 CBA GFP. (C F) Representative 10X images of GFP expression in HEK293 cells 3 days post treatment with M (C), 150 M CBA GFP. Exposure settings were consistent between images.

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101 Figure 3 8. AAV2 CBA GFP transduction is increased with MG132 treatment in HEK293 cells. (A) Repr esentative 10X image of untreated HEK293 cells. (B) Representative 10X image of GFP expression in HEK293 cells 3 days post infection with AAV2 CBA GFP. (C F) Representative 10X images of GFP expression in HEK293 cells 3 days post treatment with 1 M (C), 2 M (D), 3 M (E) or 5 M (F) of MG132 prior to infection with AAV2 CBA GFP. Exposure settings were consistent between images.

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102 Figure 3 9. CEP290 and myc protein detection via WB post simple overlap and hybrid infection in HEK293 cells with Tyr23 treatmen t. Samples 1 7 were probed for CEP290 (~300kDa red) and actin (~45kDa green) expression (top). The same samples were also probed for myc (~300kDa green) and actin (~45kDa red) expression (bottom). Samples consisted of protein lysate (50 g) harvested fr om HEK293 cells that were untreated (control lane 1), transfected with smCBA hCEP290myc (transfection, full length lane 2), treated with 250 M Tyr23 (Tyrphostin 23 treatment lane 3), infected at 10,000p/cell with AAV2 smCBA hCEP290NT + AAV2 hCEP290mycCT (i nfection, simple overlap lane 4), treated with M Tyr23 then infected at 10,000p/cell with AAV2 smCBA hCEP290NT + AAV2 hCEP290mycCT (infection, simple overlap plus Tyrphostin 23 treatment lane 5), infected at 10,000p/cell with AAV2 smCBA hCEP290NT APSD + AAV2 hCEP290mycCT APSA (infection, hybrid 10,000p/cell with AAV2 smCBA hCEP290NT APSD + AAV2 hCEP290mycCT APSA (infection, hybrid plus Tyrphostin 23 treatment lane 7). Integrated intensities were calcu lated by obtaining the intensity of the ~300kDa CEP290 band from each sample and the intensity of the ~45kDa actin band from each sample. The CEP290 band intensity was divided by the actin band intensity. An increase in CEP290 expression and detectable myc expression was observed post transfection with the full length plasmid.

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103 Figure 3 10. CEP290 and myc protein detection via WB post simple overlap and hybrid infection in HEK293 cells with MG132 treatment. Samples 1 7 were probed for CEP290 (~30 0kDa red) and actin (~45kDa green) expression (top). The same samples were also probed for myc (~300kDa green) and actin (~45kDa red) expression (bottom). Samples consisted of protein lysate (50 g) harvested from HEK293 cells that were untreated (control lane 1), tr ansfected with smCBA hCEP290myc (transfection, full length lane 2), treated with 5 M MG132 (MG132 treatment lane 3), infected at 10,000p/cell with AAV2 smCBA hCEP290NT + AAV2 hCEP290mycCT (infection, simple overlap lane 4), treated with 5 M MG132 then infe cted at 10,000p/cell with AAV2 smCBA hCEP290NT + AAV2 hCEP290mycCT (infection, simple overlap plus MG132 treatment lane 5), infected at 10,000p/cell with AAV2 smCBA hCEP290NT APSD + AAV2 hCEP290mycCT APSA (infection, hybrid lane 6) or treated with 5 M MG13 2 then infected at 10,000p/cell with AAV2 smCBA hCEP290NT APSD + AAV2 hCEP290mycCT APSA (infection, hybrid plus MG132 treatment lane 7). Integrated intensities were calculated by obtaining the intensity of the ~300kDa CEP290 band from each sample and the i ntensity of the ~45kDa actin band from each sample. The CEP290 band intensity was divided by the actin band intensity. An increase in CEP290 expression was observed post transfection with the full length plasmid, simple overlap infection with MG132 treatment and hybrid infec tion w ith MG132 treatment. Detectable myc expression was observed post transfection with the full length plasmid.

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1 04 Figure 3 11. Myc tagged CEP290 expression in C57BL/6 mice 4 weeks post subretinal delivery of simple overlap and hybrid dual AAV vectors. Representative 20X and 60X images of un injected WT retina cross sections (A&B), simple overlap injected WT retina cross sections (C&D) and hybrid injected WT retina cross sections (E&F). Retinal cross sections were stained with antibodies specific for myc (green) and acetylated alpha tubulin (red). Nuclei were counterstained wit h DAPI (blue). Detectible viral mediated myc expression was seen in WT retinas post simple overlap delivery. OS outer segments, IS inner segments, ONL outer nuclear layer, INL inner nuclear layer, GCL ganglion cell layer.

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105 Figure 3 12. Electroretinographic analysis of injected rd16; Nrl / mice. Average photopic and flicker b wave amplitudes and standard deviations of un injected Nrl / eyes (n=12), un injected rd16; Nrl / eyes (n =19), rd16;Nrl / mouse eyes injected with BSS (n=4), AAV5 hybrid CT vector at 1.0E11vg/ml (n=3), AAV5 hybrid CT vector (n=3), AAV5 simple overlap vectors at 1.0E11vg/ml (n=4), AAV5 simple overlap vectors at 1.0E12vg/ml (n=5), AAV5 hybrid vectors at 1.0E11 vg/ml (n=10) or AAV5 hybrid vectors at 1.0E12vg/ml (n=10). P values were calculated using a two t test. Simple overlap and hybrid dual AAV vectors did not improve cone function measured by ERG.

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106 Figure 3 13. Myc tagged CEP290 expressio n in rd16;Nrl / mice 4 weeks post subretinal delivery of simple overlap and hybrid dual AAV vectors. Representative 60X images of an un injected rd16;Nrl / retina cross section (A), a AAV5 hybrid CT injected rd16;Nrl / retina cross section (B), simple ov erlap injected rd16:Nrl / retina cross sections (C&D) and hybrid injected rd16;Nrl / retina cross sections (E&F). Viral titers were diluted to either 1.0E11vg/ml (C&E) or 1.0E12vg/ml (B,D,F). Retinal cross sections were stained with antibodies specific f or myc (green) and acetylated alpha tubulin (red). Nuclei were counterstained wit h DAPI (blue). Detectible viral mediated myc expression was seen in WT retinas post simple overlap delivery. IS inner segment, ONL outer nuclear layer.

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107 Figure 3 14. Simple overlap and hybrid AAV dual vectors did not mediate CEP290myc expression in rd16;Nrl / retinas 4 weeks post subretinal delivery. 100 g of protein harvested un injected and injected rd16;Nrl / retinas were probed for myc (~300kDa green) and beta actin (~45kDa red) expression (A&B). (A) No myc expression was detected in protein harvested from 2 retinas injected with AAV5 simple overlap vectors at 1.0E11vg/ml (lane 1), 2 un injected retinas (lane 2), 2 retinas injected with AAV5 simple overlap vectors at 1.0E12vg/ml (lane 3) and 2 un injected retinas (lane 4). The myc antibody was confirmed with an epitope control (lane 5). (B) No myc exp ression was detected in protein harvested from 2 un injected retinas (lane 1), 2 retinas injected with BSS (lane 2), 2 retinas injected with AAV5 hybrid vectors at 1.0E11vg/ml (lanes 3&4), 2 retinas injected with AAV5 hybrid vectors at 1.0E12vg/ml injected eyes (lanes 5&6), 2 retinas injected with AAV5 hybrid CT vector at 1.0E11vg/ml (lane 7), 2 retinas injected with AAV5 hybrid CT vector at 1.0E12vg/ml (lane 8). The myc antibody was confirmed with an epitope control (lane 9).

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108 CHAPTER 4 DEVELOPING AN AAV G ENE THERAPY FOR CEP290 LCA PATIENTS PART 2: TARGETING PHOTORECEPTORS VIA INTRAVITREAL DELIVERY * Subretinal Injection While an AAV system is being developed to express CEP290 protein an optimal delivery method for the virus needs to be established specifica lly for CEP290 LCA patients. The retained cone photoreceptors within CEP290 been identified as the target cell population for the therapeutic virus (Cideciyan et al., 2007;Cideciyan et a l., 2011a;Yzer et al., 2012) . The current method available to deliver therapeutic AAV to the fovea i nvolves a subretinal injection. Preliminary RPE65 LCA clinical trials demonstrated the ability to deliver therapeutic transgene to the retinal pigment epit helium by subretinal injection, which restored retinal function and visually evoked behavior to patients (Maguire et al., 2008;Bainbridge et al., 2008;Cideciyan et al., 2009) . However, a subretinal injection is an invasive procedure which requires anesthesia, a standard vitrectomy and leads to a subretinal detachment between the retinal pigment epithelium and photoreceptor layer which resolves back to the normal attached state within 24 hours , at least as determ ined by fundus examination . Thus, t he RPE65 LCA clinical trials have validated the subretinal injection and proven its safety and efficacy for treating RPE65 LCA patients (Bainbridge et al., 2008;Hauswirth et al ., 2008;Maguire et al., 2008) . However, surgical requirements are different for CEP290 * Reprinted with permission from Kay CN, Ryals RC, Aslanidi GV, Min SH, Ruan Q, Sun J, Dyka FM, Kasuga D, Ayala AE, Van VK, Agbandje McKenna M, Hauswirth WW, Boye SL, Boye SE (2013) Targeting photoreceptors via intravitreal delivery using novel, capsid mutated AAV vectors. PLoS One 8:e62097.

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109 LCA patients. Firstly, the retinal pigment epithelium was the main target for RPE65 LCA patients whereas cone photoreceptors will be the main target for CEP290 LCA pati ents. Secondly, RPE65 LCA patients have retained cell bodies outside of the fovea that in theory can respond to treatment. In the combined preliminary RPE65 LCA clinical trials, sixteen of thirty subretinal injections detached the macula including the fove a (Bainbridge et al., 2008;Maguire et al., 2009;Jacobson et al., 2012a) . All but two of these patients did have restoration in visual function as defined by the outcome measures in the trials (improvement in fu ll field sensitivity and pupillary light reflex). 2012a report foveal detachments resulted in a decrease in visual acuity (in two out of five treated LCA2 patients) as well as foveal thinning (in both LCA2 patients and non human prim ates) (Jacobson et al., 2006b;Jacobson et al., 2012a) . More importantly, those two patients with foveal thinning had the most foveal photoreceptors at baseline measurements , making the loss more substantial (Jacobson et al., 2012a) . If subfoveal detachment during a subretinal injection is more harmful than beneficial for patients then an alternative injection method needs to be developed for patients requiring treatm ent primarily within their fovea. Intravitreal Injection Another well established i njection method widely used in o phthalmology clinics for the treatment of age related macular degeneration is the intravitreal injection (Dikmetas et al., 2013;Wolf and Kampik, 2014) . Intravitreal injections are less invasive, require only surface anesthesia and a vi trectomy is not needed. Vitreal injections are routinely performed as an outpatient procedure. An intravitreal injec tion delivers the virus to the vitreous humor of the eye . This would require the vector to penetrate through part of the vitreous and then through the neural retinal cell layers to reach the

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110 photoreceptors . If feasible, an intravitreal approach could lead to transduction of foveal cones without disrupting the retinal pigment epithelium and photoreceptors giving CEP290 LCA patients the best chance to recover foveal cone function . Overall, treating inherited retinal dystrophy patients with intravitreal inject ions instead of subretinal injections would be more cost efficient, faster and safer. AAV Tran s duction However, AAV viruses that can penetrate the retina post intravitreal delivery need to be developed and validated. Identifying vectors capable of transdu cing photoreceptors via the vitreous will rely partially on identifying which serotypes have native tropism for this cell type following local delivery. Several serotypes have been used to successfully target transgenes to photoreceptors following subretin al injection, including AAV2, AAV5 and AAV8 with all three demonstrating efficacy in pr oof of concept experiments across multiple species (mouse, rat, dog, pig and non human primate) (Ali et al., 1996;Bennett et al., 1999;Auricchio et al., 2001;Acland et al., 2001;Yang et al., 2002;Lotery et al., 2003;Weber et al., 2003;Allocca et al., 2007a;Stieger and Lorenz, 2008;Petersen Jones et al., 2009;Vandenberghe et al., 2011;Mussolino et al., 201 1;Vandenberghe et al., 2011;Boye et al., 2012) . Studies comparing the relative transduction efficiency of several AAV serotypes following subretinal delivery in the rodent show that both AAV5 and AAV8 transduce photoreceptors more efficiently than AAV2, w ith AAV8 being the most efficient (Rabinowitz et al., 2002b;Yang et al., 2002;Allocca et al., 2007b;Boye et al., 2011;Pang et al., 2011) . Previous studies showed that AAV2 and AAV8 vectors containing point muta tions of surface exposed tyrosine residues (tyrosine to phenylalanine,Y F) displayed increased transgene expression in a variety of retinal cell types relative to

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111 unmodified vectors following both subretinal and intravitreal delivery (Petrs Silva et al., 2009;Petrs Silva et al., 2011) . Of the vectors tested, an AAV2 triple mutant (triple Y F) exhibited the highest transduction efficiency following intravitreal injection whereas the quadruple mutant (quad Y F) ex hibited the novel property of enhanced transduction in the outer retina (Petrs Silva et al., 2011) . Further improvements in transduction efficiency may be achieved via directed mutagenesis of surface exposed th reonine (T) residues to either valine (V) or alanine (A) residues. Both Y F and T V/T A mutations increase efficiency by decreasing phosphorylation of capsid and subsequent ubiquitination as part of the proteosomal degradation pathway (Zhong et al., 2008a;Aslanidi et al., 2013;Gabriel et al., 2013) . In C hapter 4 , AAV2, AAV5 and AAV8 based vectors containing a combination of Y F and T V mutations are compared for their ability to transduce photoreceptors followin g intravitreal injection. The transduction profile of intravitreally delivered AAV is heavily dependent upon the injection procedure itself. Due to the small size of the mouse eye, trans scleral, intravitreal injections can result in damage to the retina that might allow delivery of some vector directly to the subretinal space. Therefore, an optimized intravitreal injection method was used to reduce this damage . This in turn reduced injection variability and allowed for more accurate comparisons to be made among vectors. Many novel capsids are being engineered by either rational mutagenesis or directed evolution to enhance cell specificity. Rational mutagenesis includes making site specific modifications to the surface of the AAV capsid to evade proteosoma l degradation as mentioned above, to modulate cellular receptor binding and to evade the immune system recognition (Klimczak et al., 2009;Petrs Silva et al., 2011;Pulicherla and

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112 Asokan, 2011;Li et al., 2012;Day et al., 2014) . Directed evolution involves creating diverse viral genetic capsid libraries based on wild type AAVs. Libraries are generated by error prone polymerase chain reaction, random peptide insertion or capsid DNA shuffling. Each novel capsid parti cle in the resulting vector library encapsidates the genome encoding that capsid, which can be harnessed later for identification. The viral libraries are then packaged and injected into the vitreous. The capsid gene sequences that are able to reach the ph otoreceptors are recovered, packaged and subjected to additional rounds of selection, which may further evolve the capsid toward the selected trait of transducing photoreceptors . The final recovered variants are individually screened and the most successfu lly variants are determined (Dalkara et al., 2013;Day et al., 2014) . These two methodologies have led to a large increase in the number of capsids that could be useful for targeting photoreceptors via an intrav itreal approach. Developing a methodology to compare the in vivo photoreceptors transduction efficiency of these novel capsids would be beneficial in order to select an optimal AAV capsid for use in subsequent preclinical proof of concept studies. Therefor e, we designed a novel method for quantifying transduction efficiency in vivo using knock in mice bearing a human rhodopsin enhanced green fluorescent protein (EGFP) fusion gene (Rho GFP mice), AAV vectors driving mCherry expression and subsequent fluoresc photoreceptor (GFP negative, mCherry positive cell population) (Wensel et al., 2005) . This method for scoring intravitreally delivered, AAV mediated photoreceptor transduction can be

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113 applied tow ard development of additional vectors intended for the treatment of inherited retinal disease. Photoreceptor Specific Promoters Specific targeting of the photoreceptors post intravitreal delivery does not rely solely on the capsid. In order to isolate tra nsgene expression in a specific cell type, the promoter driving transgene expression is equally as important. Since cone photoreceptors w ill be the main target cell population for CEP290 LCA patients it is vital to examine photoreceptor specific promoters. Intravitreal injections of the AAV2, AAV5 and AAV8 based vectors containing a combination of Y F and T V mutations were performed with constructs containing a photoreceptor specific promoter (the human rhodopsin kinase (hGRK1) promoter) in hopes of valida ting a capsid and promoter combination that would generate robust transduction in the photoreceptor s post intravitreal injection (Khani et al., 2007;Boye et al., 2012) . Photoreceptor specific promoters are cont inuously being generated and enhanced to elicit robust transgene expression to the rods, S cones, M cone and L cones (Glushakova et al., 2006;Khani et al., 2007;Li et al., 2008;Komaromy et al., 2008;Beltran et a l., 2010;Komaromy et al., 2010;Dyka et al., 2014b) . Furthermore, researchers attempt to shorten large promoters (~1kb or larger) to allow for the insertion of a larger therapeutic transgene in the AAV cassette. For example, the ubiquitous chicken beta act in (CBA) promoter originally ~1600bps has been modified to ~900bps (smCBA) and still elicits similar transduction patterns (Haire et al., 2006) . With the growing number of photoreceptor specific promoters a qua ntitative method to compare the transduction efficiency between promoters would be useful for choosing an optimal promoter for preclinical proof of concept studies. Therefore, in C hapter 4 , a high -

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114 throughput transduction efficiency protocol, previously est ablished for quantifying AAV capsid transduction efficiency, was modified for the use of novel photoreceptor specific promoters (Ryals et al., 2011) . Finally, an in vivo quantification o f transduction efficiency of photoreceptor specific promoters was attempted to verify in vitro findings. Methods Vector Production For analysis of novel capsids , the following vector plasmid constructs were cloned and packag ed in both unmodified AAV serotypes two , five , and eight and capsid mutant derivatives of these serotypes: self complementary small chicken actin driving mCherry expression (sc smCBA mCherry), standard (non self complimentary) human rhodopsin kinase driving green fluorescent protein expression (hGRK1 GFP) and standard full length chicken actin driving green fluorescent protein expression ( CBA GFP). Promoter constructs were identical to those previously described (Burger et al., 2004;Haire et al., 2006;Khani et al., 2007) . A hGRK1 GFP miR181c construct was also generated and packaged in AAV2 quad Y F+T V by inserting four tandem copies of complementary sequence for mature miR of miRNA distribution: http://mirneye.tigem.it/) immediately downstream of GFP, similar to Karali et al. (Karali et al., 2011) . AAV2, AAV5 and AAV8 capsid mutants were generated by site directed mutagenesis of surface exposed tyrosine and threonine residues with the QuickChange Multi Site Directed Mutagenesis Kit (Agilent Technologies, CA, Cat# 200514). Selected tyrosine residues were mutated to phenylalanine (Y F) whereas threonine residues were mutated to valine residues (T V) (Zhong et al., 2008b) . Table 4 1 describes the location of the amino acid mutations for the experimental mutant vectors. All vectors

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115 were packaged, purified, and titered according to previously described methods (Zolotukhin et al., 2002;Jacobson et al., 2006b) . For in vitro transdu c tion efficiency analysis of photoreceptor specific promoters , the following promoters were tested: a cone and rod specific hGRK1 promoter, a rod specific mou se opsin promoter (mOPs), a rod specific human opsin promoter (hOP), a cone specific interphotorec eptor retinoid binding protein promoter (IRBP241) and the ubiquitous smCBA promoter. The following constructs were generated and packaged in AAV2 quad Y F+T V: sc hGRK1 mCherry, sc mOPs mCherry, sc hOP mCherry, sc IRBP241 mCherry and sc smCBA mCherry. The hGRK1, mOPs and smCBA promoters were identical to those previously described (Flannery et al., 1997b;Haire et al., 2006;Khani et al., 2007;Boye et al., 2012) . The IRBP241 promoter consists of bps 216 to +19 of the original IRBP promoter characterized in 1990 (Liou et al., 1990) . The hOP promoter is 99% identical to 537 base pairs on the human chromosome 3 (129210157 129210693). For in vivo transduction efficiency analysis of photoreceptor specific promoters the following constructs were packaged in AAV5 double Y F: sc mOPs GFP and sc hGRK1 mCherry. All vectors were packaged, purified, and titered according to previously described methods (Zolotukhin et al., 2002) . Cell Lines 661W cone cells (generously provided by Dr. Muayyad R. Al Ubaidi, University of Oklahoma Health Sciences Center, Oklahoma City, OK) were passaged by dissociation in 0.05% (w/v) trypsin and 0.02% (w/v ) EDTA, followed by replating at a split ratio ranging from 1:3 to 1:5 in T75 flasks (Tan et al., 2004;Ryals et al., 2011) . Cells were maintained in DMEM containing 10% fetal bovine serum , 300mg/l glutamine, 23 mg/l

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116 putrescine, 40ml of mercaptoethanol, and 40mg of hydrocortisone 21 hemisuccinate and progesterone. The media also contained penicillin (90units/ml) and streptomycin (0.09mg/ml). Cultures were incubated at 37°C (al Ubaidi et al., 2008) . Infections and FACS Analysis 661W cells were plated in 96 well plates at a concentration of 2.0E4 cells/well. The following day, cells were infected at 10,000p/cell with sc smCBA mCherry packaged in unmodified and modified AAV2, AAV5 or AAV8 vectors for novel capsid analysis and sc hGRK1 mCherry, sc mOPs mCherry, sc hOP mCherry, sc IRBP241 mCherry and sc smCBA mCherry packa ged in AAV2 quad Y F+T V for photoreceptor specific promoter analysis. Three days post infection, fluorescent microscopy at a fixed exposure was performed, cells were detached and FACS analysis was used to quantify reporter protein (mCherry) fluorescence. Transduction efficiency (mCherry expression) of each AAV vector and promoter was calculated by multiplying the percentage of positive cells by the mean flu orescence intensity in each sample (Ryals et al., 2011) . Animals Vectors were injected in one two month old C57BL /6 mice (Jackson Laboratory, Bar Harbor, ME) and in one month old heterozygote Rho GFP mice, knock in mice bearing human rhodopsin GFP fusion gene (generously provided by Dr. Alecia Gross , University of Alabama at Birmingham). Ethics Statement animal care facilities and were handled in accordance with the ARVO statement for Use of Animals in Ophthalmic and Vision Research and the guidelines of the Institutional

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117 Animal Care and Use Committee of the University of Florida. Animal work performed in Intravitreal Injections Prior to vector administration, mice were anesthetized wit h ketamine (72mg/kg)/xylazine (4mg/kg) by intraperitoneal injection. Eyes were dilated with 1% delivered to the intravitreal cavity of adult mice. An aperture was made 0.5mm posterior to the limbus with a 32 gauge ½ inch needle on a tuberculin syringe (BD, Franklin Lakes, NJ) followed by introduction of a blunt 33 gauge needle on a Hamilton syringe. GenTeal gel, 0.3% (Novartis) was applied to the corneal surface and a glass co verslip was laid onto this interface for visualization through the microscope to guide the needle into the vitreous cavity without retinal or lenticular perforation. Extreme care was taken with this visualization technique to confirm that no retinal perfor ation occurred. For experiments evaluating activity of the hGRK1 promoter in C57BL/6 mice, 7.5E9vg of AAV2 based vectors, 8.5E10vg of AAV5 single Y F, 5.3E9vg of AAV5 double Y F, 1.3E11vg of AAV8 double Y F and 6.0E10vg of AAV8 double Y F+T V were delivere d. For experiments evaluating the CBA promoter in C57BL/6 mice, 1.5E10vg of all vectors were delivered. To evaluate transduction of vectors containing microRNA target sequence in C57BL/6 mice, 1.5E10vg w ere delivered. All Rho GFP mice were injected intravi treally with 1.5E9vg. Subretinal Injections Prior to subretinal injections, mice were topically administered 1% atropine eye drops followed by 2.5% phenylephrine hydrochloride eye drops. When mouse eyes were fully dilated, mice were anesthetized with ketam ine (72mg/kg)/ xylazine (4mg/kg).

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118 All subretinal injections were performed as previously described (Timmers et al., 2001;Pang et al., 2006) . One to two month old C57BL /6 mice were injected subretinally with eit F mOPs scAAV5 double Y F hGRK1 Y F mOPs GFP/ hGRK1 mCherry mixed at a 1:1 ratio (n=3). Three mice were injected subretinally with 1µl o f scAAV5 double Y F mOPs GFP (1.0E9vg delivered) one week later they were injected in the same eye with scAAV5 double Y F hGRK1 mCherry (1.0E9vg delivered). Contralateral eyes were used as controls and remained un injected. Fundoscopy At four weeks post in jection for novel capsid analysis and three weeks post injection for photoreceptor specific promoter analysis, fundoscopy was performed using a Micron III camera (Phoenix Research Laboratories, Pleasanton, CA). Bright field, green fluorescent and red fluor escent images were taken to visualize retinal health, GFP expression and mCherry expression, respectively. Exposure settings were constant between experiments. Retinal Dissociation and FACS Analysis Four weeks post injection and one week post injection for select capsids, Rho GFP retinas were harvested and dissociated with the papain dissociation system (Worthington Biochemical Corporation, NJ, Cat #3150). FACS analysis was used to quantify the percentage of cells that were non fluorescent (negative, box B Figure 4 3A ), GFP positive ( photoreceptors , box A Figure 4 3A ), mCherry positive (any retinal cells transduced with vector, box D Figure 4 3A ) and both GFP and mCherry positive ( photoreceptors transduced by vector, box C Figure 4 3A ). The percentage of tot al

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119 retinal cells transduced was calculated by dividing of total percentage of mCherry positive cells by the total percentage of cells [(C+D/A+B+C+D)X100]. The percentage of mCherry positive photoreceptor s was calculated as the ratio of cells both GFP and m Cherry positive relative to total GFP positive photoreceptor s [(C/A +C )X100]. The PR) transduction was calculated by dividing the percentage of mCherry positive cells by the total percentage of cells [(D/A+B+C+D)X100]. For ph otoreceptor specific promote r analysis, three weeks post injection C57BL/6 retinas were harvested and dissociated with the papain dissociation system (Worthington Biochemical Corporation, NJ, Cat #3150). FACS analysis was used to quantify the percentage of cells that were non fluorescent (negative, box B Figure 4 15A ), GFP positive ( box A Figure 4 15A ), mCherry positive ( box D Figure 4 15A ) and GFP and mCherry positive ( box C Figure 4 15A ). Total sc mOPs GFP transduction, predominately rods, was calculated by dividing the total percentage of GFP positive cells by the total percentage of cells [(A+C/A+B+C+D)X100]. Total sc hGRK1 mCherry transduction was calculated by dividing the total percentage of mCherry positive cells by the total percentage of cells [(C +D/A+B+C+D)X100]. On target sc mOPs GFP transduction was calculated by dividing the percentage of GFP positive cells by the total percentage of cells [(A/A+B+C+D)X100]. On target sc hGRK1 mCherry transduction was calculated by dividing percentage of mCherr y positive cells by total percentage of cells [(D/A+B+C+D)X100]. Total sc mOPs GFP /sc hGRK1 mCherry transduction was calculated by diving the total percentage of GFP and mCherry positive cells by the total percentage of cells [(A+C+D/A+B+C+D)X100]. Since sc mOPs GFP should

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120 predominately target the rods the percentage of rod transduction was calculated as the ratio of cells both GFP and mCherry positive relative to total GFP positive cells [(C/C+A)X100]. To characterize the two GFP positive cell population s seen in Rho GFP retinas, one single retina was harvested from a one month old heterozygote Rho GFP mouse and dissociated with the papain dissociation system (Worthington Biochemical Corporation, NJ, Cat #3150). Cells were re suspended in 1.5ml of 2% feta l bovine serum phosphate buffered saline and sorted using a BD FACS Aria TM llu equipped with FACS Diva 6.1.2 software (BD Biosciences, San Jose, CA). During the cell sorting, GFP was excited using a 50mW, 488nm "Sapphire" laser (Coherent, Inc, Santa Clara, CA). Fluorescence was collected using a 530/30nm bandpass filter. In addition, mCherry was excited using a 150mW, 532nm "Compass" laser (Coherent, Inc, Santa Clara, CA). Fluorescence was collected using a 610/20 nm bandpass filter. 10,000 cells from popu lation one and two shown in Figure 4 5 were directly sorted into a flat bottom 96 well plate. The plate was spun at 1,000r pm for five minutes. Cell populations one and two were imaged using an EVOS fl Digital Inverted Microscope (Advanced Microscopy Group, Life Technologies, Grand Island, NY). Bright field and green fluorescent images were captured, overlaid and saved for analysis. Exposure settings were kept consistent when imaging the two populations. Immunohistochemistry Immediately after fundoscopy, eyes were enucleated and tissue was prepared for cryoprotection and sectioning as previously described (Boye et al., 2011) . Briefly, after rinsing with phosphate buffered saline , sections were incuba ted with 0. 5% Triton X 100 for one hour followed by a 30 minute incubation with a blocking solution of 1% bovine

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121 serum albumin (BSA). Retinal sections were then incubated overnight at 4°C in a rabbit polyclonal antibody raised against GFP (generously provided by Dr. Clay Smith, University of Florida, Gainesville, Florida) diluted in 0.3% Triton X 100/1% BSA at 1:1,000. The following day, sections were rinsed with phosphate buffered saline and incubated for one hour at room temperature in anti rabbit IgG secondary anti body Alexa fluor 488 (Invitrogen, Eugene, Oregon, Cat#A11008) diluted in phosphate buffered saline diami d i n o 2 phenylindole (DAPI) for five minutes at room temperature. Retinal sections were imaged using a fluorescent Axiophot microscope (Zeiss, Thornwood, NY) as previously described (Boye et al., 2011) . Images were captured at 5X, 20X and 40X. Exposure settings were consistent across images at each magni fication. Results Quantification of In Vitro Transduction Efficiency 661W mouse cone photoreceptor cells were infected with unmodified or capsid mutated, self complimentary AAV vectors containing the smCBA promoter driving mCherry expression in order to q uantify the relative transduction efficiencies of all vectors. FACS analysis provided a measure of the relative transduction efficiency (mCherry expression) across samples. Figure 4 1 shows mCherry expression, in arbitrary units, for each capsid tested: sc AAV2, scAAV2 quad Y F, scAAV2 quad Y F+T V, scAAV5, scAAV5 single Y F, scAAV5 double Y F, scAAV8, scAAV8 double Y F, scAAV8 double Y F+T V. The following modified capsids scAAV2 quad Y F, scAAV5 single Y F, scAAV5 d ouble Y F and scAAV8 double Y F mediated significantly increased levels of mCherry expression compared to their unmodified parent serotype scAAV2, scAAV5 or scAAV8 ( p F+T V and

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122 scAAV8 double Y F+T V mediated significantly increased levels of mCherry expressio n compared to their parent unmodified serotype and their corresponding Y F only modified capsid ( p 1). This preliminary screen revealed that capsid modifications did increase transduction efficiencies compared to unmodified capsids. Overall , scAAV2 quad Y F+T V transduced cone cells most efficiently and scAAV8 transduced cone cells least efficiently. Increases in scAAV2 quad Y F+T V mediated mCherry expression were ~10 fold above the scAAV2 baseline (Figure 4 1). Quantification of In Vivo Tr ansduction Efficiency Following in vitro screening, identical vectors were evaluated for their relative ability to transduce photoreceptors in vivo following intravitreal injection in 1 month old, heterozygote Rho GFP mice (1.5E9vg delivered). Fundoscopy a t 4 weeks post injection showed qualitatively that mCherry expression was enhanced with the addition of capsid mutations to each serotype (Figure 4 2). Rho GFP mouse retinas injected intravitreally with scAAV2 quad Y F+T V smCBA mCherry exhibited the highe st qualitative levels of mCherry expression (Figure 4 2C). Levels of transgene expression achieved following intravitreal injection of scAAV2 quad Y F, scAAV5 double Y F and scAAV8 double Y F+T V were approximately equivalent. To quantify the relative abi lity of each vector to transduce photoreceptor s, intravitreally injected Rho GFP retinas were dissociated and FACS analysis was performed. Cells were sorted into four populations: 1) non fluorescent: indicating un transduced, non photoreceptor retinal cell indicating un transduced photoreceptor indicating transduced photoreceptor indicating transduced non photoreceptor reti 3). As

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123 shown in Figure 4 3A and 4 3 B, an un injected Rho GFP retina contains two photoreceptor non photoreceptor s) whereas a Rho GFP retina inject ed with scAAV2 quad Y F+T V contains all four populations of cells. The relative percentage of mCherry positive photoreceptor s following intravitreal injection of all vectors is shown in Figure 4 3C. Addition of quad Y F and quad Y F+T V mutations to the A AV2 capsid surface resulted in ~3.5 fold and ~13 fold increases in the percentage of mCherry positive photoreceptor s, respectively. Unmodified scAAV2 transduced an average of 1.7% of photoreceptor s from the vitreous whereas scAAV2 quad Y F and scAAV2 quad Y F+T V transduced an average of 6.1% and 21.8%, respectively. scAAV2 quad Y F+T V transduced the highest number of photoreceptor s of all vectors tested. Retinas injected with unmodified and modified AAV5 and AAV8 based vectors exhibited lower efficiencies of photoreceptor transduction. Consistent with fundoscopic observations, appreciable photoreceptor transduction was seen following intravitreal injection of scAAV2 quad Y F, scAAV5 double Y F and scAAV8 double Y F+T V. The average percent of mCherry posit ive photoreceptor s in retinas injected with scAAV5, scAAV5 single Y F and scAAV5 double Y F was 2.0%, 1.7% and 5.9%, respectively. The average percent of mCherry positive photoreceptors in retinas injected with scAAV8, scAAV8 double Y F and scAAV8 double Y F+T V was 1.9%, 1.4% and 2.9%, respectively (Figure 4 3C). We also found that quantitative comparisons could be made using this methodology at just 1 week post intravitreal injection with scAAV2 based vectors (Figure 4 4). While fewer total photoreceptor s expressed detectable levels of

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124 mCherry at this early time point, the pattern remained the same, with scAAV2 quad Y F+T V mediating the highest levels of transgene expression in photoreceptor s. In addition to calculating the average percentage of mCherry p ositive photoreceptor s, average percentage of total retinal cells transduced and average photoreceptor ) cells transduced for non mutated and mutated capsids was calculated at four weeks post injection and one week post inje ction for select mutated capsids (Table 4 3). Average percentage of total retinal cells addition of the capsid mutations except when comparing AAV8 to AAV8 double Y F. Of f target transduction was expected with the use of a smCBA promoter. However, the best performer AAV2 quad Y F+T V only had an average off target transduction percentage of 13.4 (Table 4 2). The sc smCBA mCherry construct packaged in AAV2 quad Y F+T V was intravitreally injected twice (at a 3 day interval) to see if the percentage of total retinal cells transduced and percentage of mCherry positive photoreceptor s would increase. An average total retinal cell transduction of 25.5% was achieved with one intr avitreal AAV2 quad Y F+T V administration whereas an average total retinal cell transduction of 32.0% was achieved after two intravitreal administrations. Furthermore, the average percentage of mCherry positive photoreceptor s increased from 21.8% to 32.1% after double injection with AAV2 quad Y F+T V (Table 4 2). AAV2 quad Y F and AAV2 quad Y F+T V were analyzed one week post injection. One week post injection an average total retinal cell transduction of 12.2%, an average photoreceptor transduction of 11% and an average off target cell transduction of 7.9% was achieved with AAV2 quad Y F+T V (Table 4 2). At one week post injection, total retinal cell

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125 transduction and photoreceptor transduction was approximately 1/3 of the transduction observed at 4 weeks po st injection for AAV2 quad Y F and 1/2 of the transduction observed 4 weeks post injection with and AAV2 quad Y F+T V (Table 4 2). Upon FACS analysis of Rho GFP retinas, two distinct GFP positive cell populations were visible. Cell population one had mea surable amounts of red fluorescence (10 2 10 3 Texas Red intensity) and a reduced GFP fluorescence (10 2 10 3 FITC inte nsity) whereas cell population two had minimal red fluorescence (0 10 2 Texas Red intensity) and a strong GFP fluorescence (10 3 10 5 FTIC inten sity) (Figure 4 5A). During the sorting and capturing of these two individual cell populations the forward scatter and side scatter analysis indicated that the rod cells in population one was larger in size than those in population two (Figure 4 5B). Micro scopy of the individual cell populations showed an appreciable difference in size, but no appreciable difference in GFP fluorescence. Population one cells did appear larger than population two cells (Figure 4 5C). Qualitative Analysis of Photoreceptor Tr ansduction With the intention to restrict transgene expression to photoreceptor s following intra vitreal delivery of AAV, the photoreceptor specific hGRK1 promoter was incorporated into unmodified and capsid mutated vectors. All vectors in this set of exper iments were single stranded and not self complimentary due to the need to accommodate larger promoters and transgenes, which are more relevant for treatment of inherited retinal diseases. Representative fundus images of C57BL/6 mice and their immunostained retinal sections taken 4 weeks post intravitreal injection with AAV2, AAV2 quad Y F and AAV2 quad Y F+T V are shown in Figure 4 6 (7.5E9vg delivered for all vectors). Consistent with the quantification results shown in Figure 4 3, very few

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126 photoreceptor s expressed GFP following intravitreal injection of AAV2 or AAV2 quad Y F (Figure 4 6B&D). However, robust GFP expression was seen in the photoreceptors following injection of AAV2 quad Y F+T V (Figure 4 6F). AAV2 quad Y F+T V mediated transgene expression w as evident in photoreceptor s throughout the retina rather than in one specific location (Figure 4 7). This representative section, in conjunction with surgical observations and fundoscopy, support the premise that the injection procedure did not involve re tinal perforation and was, in fact, intravitreal (Figure 4 7). Although reports have shown that the hGRK1 promoter has exclusive activity in rods and cones of mouse and non human primate when delivered subretinally, hGRK1 mediated transgene expression was observed in ganglion cells of injected mice (Figure 4 6D&F and Figure 4 7) (Khani et al., 2007;Boye et al., 2012) . In vivo quantification data in Rho GFP mice revealed relatively low levels of photoreceptor tr ansduction following intravitreal delivery of 1.5E9vg of AAV5 and AAV8 based vectors (Figure 4 3). Therefore, in order to maximize expression and qualitatively analyze general transduction patterns, higher titers of AAV5 and AAV8 based vectors were used fo r the following experiments. For analysis of AAV5 single Y F and AAV5 double Y F vectors 8.5E10vg and 5.3E9vg were delivered, respectively. Fundus images paired with fluorescent images of retinal cross sections showed minimal photoreceptor transduction fol lowing intravitreal injection of AAV5 single Y F and AAV5 double Y F (Fig. 4 8). A pattern of peripapillary tropism was evident, with photoreceptors around the optic nerve exhibiting the most prominent transgene expression (Figure 4 8A,B,D,E). photorecepto r transduction was found in scattered peripheral retinal sections of AAV5 single Y F injected eyes (Figure 4 8C), with expression typically found near the retinal

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127 vasculature. For analysis of AAV8 double Y F and AAV8 double Y F+T V 1.3E11vg and 6.0E10vg we re delivered, respectively. Fundus images (at 4 weeks post injection) paired with fluorescent images of retinal cross sections show minimal photoreceptor transduction following intravitreal injection of either vector (Figure 4 9). Similar to AAV5 based vec tors, a pattern of peripapillary tropism was seen following injection of modified AAV8 vectors (Figure 4 9A,B,D,E). MicroRNA Mediated Regulation of Transgene Expression In order to mitigate the observed off target transgene expression in ganglion cells fol lowing intravitreal delivery of hGRK1 containing AAV vectors, a target sequence for miR181, an miRNA shown to be expressed exclusively in ganglion cells and inner retina, was incorporated into the AAV vectors (Atlas of miRNA distribution: http://mirneye.ti gem.it/) immediately downstream of GFP, similar to Karali et al. (Karali et al., 2011) . The intended effect was to degrade vector derived transcripts and inhibit synthesis of viral mediated protein in all cells of the retina except photoreceptor s. Both hGRK1 GFP and hGRK1 GFP miR181c were packaged in AAV2 quad Y F+T V and delivered intravitreally to C57BL/6 mice (1.5E10vg). At 4 weeks post intravitreal injection, fundoscopy and IHC on frozen retina cross section s revealed that addition of miR181c to the vector construct did eliminate off target expression (Figure 4 10). Although hGRK1 GFPmiR181c mediated GFP expression was exclusive to photoreceptor s, it was also appreciably decreased (Figure 4 10). Qualitative A nalysis of Serotype Tropism Because mutations in genes expressed in retinal cell types other than photoreceptor s can also cause or result in retinal degeneration, the ubiquitous CBA promoter was incorporated into vectors to ascertain what other retinal cel ls types were

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128 targeted following intravitreal injection of our strongest capsid mutated vectors (Figure 4 11). All vectors were delivered intravitreally at a dose of 1.5E10vg. AAV2 quad Y F and AAV2 quad Y F+T V vectors were chosen for testing based on the ir performance in the photoreceptor targeting experiments described above. AAV2 triple Y F+T V was chosen based on the documented efficiency of AAV2 triple Y F in multiple in vitro and in vivo settings (Petrs Si lva et al., 2011;Ryals et al., 2011) . All AAV2 based vectors mediated robust, pan retinal GFP expression (Figure 4 11A,B,D,E,G,H), with GFP found throughout the inner and middle retina (Figure 4 11B,C,E,F,H,I). AAV2 quad Y F+T V and AAV2 triple Y F+T V me diated GFP expression was also seen in photoreceptor cells bodies (Figure 4 11C&I). In Vitro Transduction Efficiency of Photoreceptor Specific P romoters In order to quantify transduction efficiency of photoreceptor specific promoters in vitro one rod and cone specific promoter (hGRK1), two rod specific promoters (mOPs, hOP), one cone specific promoter (IRBP241) and a ubiquitous promoter (smCBA) were evaluated in the 661W mouse cone photoreceptor cell line. Self complimentary constructs containing the afore mentioned promoters driving mCherry expression were created and packaged in AAV2 quad Y F+T V. Three days post infection FACS analysis provided a measure of relative transduction efficiency (mCherry expression) across samples. Figure 4 12 shows mCherry exp ression, in arbitrary units, for each promoter tested: hGRK1, mOPs, hOP, IRBP241 and smCBA. Non infected 661W cells had a mCherry expression value of 4.7E3 which was significantly lower than mCherry expression measured post infections (data not shown). The hGRK1, mOPs, hOP and IRBP241 promoters had transduction efficiencies of 1.5E4, 1.3E4, 1.8E4 and 1.8E4, respectively (Figure 4 12). No significant di fferences were found between

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129 photoreceptor specific promoters. The ubiquitous smCBA promoter had a transduc tion efficiency of 2.9E4, which was significant ly increased compared to all photoreceptor specific promoters ( p 12). This prelimina ry screen indicates that all photoreceptor specific promoters tested thus far can mediate expression in 661W cells. This transduction efficiency protocol will continue to be a useful screen for promoters prior to in vivo testing. Developing an In Vivo Transduc tion Efficiency Protocol for Photoreceptor Specific Promoters Quantifyin g transduction efficiency of photoreceptor specific promoters was originally tested in Rho GFP mice post subretinal delivery. However, severe retinal degeneration was observed after all subretinal injections regardless of the promoter strength and titer of the virus (data not shown). Most photoreceptor specific promoters are first characterized in C57BL/6 retinas post subretinal delivery . Thus, quantifying in vivo transduction eff iciency of photoreceptor specific promoters post subretinal delivery in C57BL/6 mice is most comparable to previously published qual itative characterizations of photoreceptor specific promoters. Subretinal quanti fication of photoreceptor specific promoters could also indicate which promoter works most efficiently and would be a good candidate for intravitreal approaches. The protocol design includes labeling C57BL/6 rods with scAAV5 double Y F mOPs GFP. At the sa me time an experimental promoter driving mCherry expression packaged in AAV5 double Y F can be delivered. Three weeks post injection, single injected retinas can be dissociated and the percentage of cells that are fluorescing red and green, which would ind icate the rod transduction of the experimental promoter . This can then be quantified via FACS. Figure 4 13 shows representative fundus images of

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130 C57BL/6 mice three weeks post subretinal injection with either scAAV5 double Y F mOPs GFP alone (Figure 4 13A&B ), scAAV5 double Y F hGRK1 mCherry alone (Figure 4 13C&D) or scAAV5 double Y F mOPs GFP/ hGRK1 mCherry (Figure 4 13E&F). Robust photoreceptor transduction was observed post subretinal administration of sc mOPs GFP (Figure 4 13A) and sc hGRK1 mCherry (Figur e 4 13D). When both viruses were administered together, photoreceptor transduction appeared the same in both the red and green channel (Figure 4 13E&F). Unfortunately, the visible black areas within the transduced retina indicate retinal degeneration, whic h was evident in most injected retinas. Because two viruses are being administered at the same time with the same capsid, AAV5 double Y F, it is possible that the viruses may be competing for the same cellular receptors and not entering the cell at the sam e ratio. In order to test this hypothesis sc mOPs GFP was subretinally injected first to label the rods. One week later, after the first virus has cleared, sc hGRK1 mCherry was administered subretinally. Figure 4 14 shows representative fundus images three weeks post sc hGRK1 mCherry injection. Again, robust photoreceptor transduction was observed post sc mOPs GFP administration (Figure 4 14A) and sc hGRK1 mCherry administration (Figure 4 14D). However, this time the mCherry expression pattern differed from the GFP expression pattern (Figure 4 14E&F). Retinal degeneration was less evident following this injection procedure. The day after fundoscopy individual retinas were dissociated and prepared for FACS. The percentages of cells that were non fluorescent, green fluorescent, red fluorescent and red and green fluorescent were quantified. Figure 4 15 shows

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131 representative FACS plots showing cell populations for each group. Un injected C57BL/6 retinas had one large population of non fluorescent cells (negative) (Figure 4 15A). C57BL/6 retinas injected with sc mOPs GFP had two populations of cells. One population of cells was non fluorescent (negative) and one was GFP positive (predominately rods) (Figure 4 15B). The average percentage of cells that were GFP posi tive was 38.7 (n=5). Dissociated Rho GFP mice had an average percentage of GFP positive cells of 56.9 (n=5) (data not shown). Subretinal AAV mediated labeling of rods was significantly less efficient than the transgenic labeling of rods ( p Furthermo re, the average percentage of cells that were mCherry positive post subretinal delivery of sc hGRK1 mCherry was only 5.8% (n=8) (Figure 4 15C). Th ese quantitative data do not correlate with the robust expression seen in the fundus images (Figure 4 13&4 14) . This discrepancy could be due to the unknown amount of retinal degeneration occurring in these retinas. Subretinal delivery of both sc mOPs GFP and sc GRK1 mCherry concurrently resulted in an average of 3.2% mCherry and GFP positive cells (Figure 4 15D). Finally, delivering sc hGRK1 mCherry one week after sc mOPs GFP administration resulted in an average of 5.8% mCherry and GFP positive cells (Figure 4 15E). Even though, the percentage of cells that were mCherry and GFP positive increased when waiting one week to deliver the sc hGRK1 mCherry, expression levels were not significantly different between the groups. The average percentage of total retinal cells transduced, the average percentage of rods transduced by the experimental promoter and the average transduction of sc mOPs GFP alone or sc hGRK1 mCherry alone was calculated. Table 4 3 indicates that the hGRK1 promoter has a rod transduction efficiency of 14.2% when

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132 administered with sc mOPs GFP and a rod transduction efficiency of 18.2% whe n administered one week after sc mOPs GFP administration. However, sc hGRK1 mCherry total retinal cell transduction was only 6.1% (Table 4 3), which does not correlate to qualitative findings (Figure 4 13 and 4 14). Furthermore, AAV mediated rod labeling w as significantly reduced compared to the transgenic Rho GFP model. Further advances are needed to validate an in vivo quantitative transduc tion efficiency protocol for photoreceptor specific promoters. Discussion The development of viral vectors capable o f efficiently transducing photoreceptor s via a less invasive delivery method than the previously utilized subretinal injection route would be a critical advance in retinal gene therapy. In recent years, rational mutagenesis and directed evolution have been utilized to identify novel AAV capsid variants with increased transduction efficiency, altered tropism and the ability to evade recognition by the immune system (Petrs Silva et al., 2009;Klimczak et al., 2009;P etrs Silva et al., 2011;Pulicherla and Asokan, 2011;Li et al., 2012) based approach to manipulating the viral capsid to develop customized vectors with distinctive features. Rational mutagenesis of surface e xposed tyrosine, threonine and lysine residues results in increased transduction by inhibiting phosphorylation of the surface exposed residues subsequently reducing ubiquitination and proteosomal degradation of the AAV capsid (Zhong et al., 2008a;Aslanidi et al., 2013;Gabriel et al., 2013) . Previous studies showed that Y F mutations on the AAV2, AAV8 and AAV9 capsid surface led to increased transduction and altered transduction profiles relative to unmodified ve ctors following both subretinal and intravitreal delivery (Petrs Silva et al., 2009;Petrs Silva et al., 2011) . Proof of -

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133 concept studies later showed that incorporation of these mutations led to more pronounced rescue in animal models of inherited retinal disease and in one case, conferred therapy in a particularly aggressive mouse model that was refractory to treatment using an unmodified parent serotype (Boye et al., 2011;Pang et al., 2011;Pang et al., 2012) . Directed evolution can select for desired characteristics without a priori knowledge of the physical determinants, allowing identification of novel vectors that exhibit desired, specific tropisms (Bartel et al., 2012) . Directed evolution has been applied to select AAV variants from combinatorial libraries that demonstrate a diverse range of cellular tropisms in vivo relative to their parent serotypes (Bartel et al., 2012) . In the retina, this technology was used to identify a variant capable of specifically transducing Müller cells via the vitreou s (Klimczak et al., 2009) . With the goal to develop vectors capable of transducing photoreceptor s via intravitreal delivery, structural features of the mammalian retina need to be consider ed. The internal limiting membrane (ILM) which defines the border between the retina and vitreous acts as a physical and biological barrier to AAV transduction following intravitreal injection in the rodent and non human primate retina (Dalkara et al., 2009;Yin et al., 2011) . It has been shown that AAV2 and AAV8 attach to the ILM and accumulate at the vitreoretinal junction, with AAV2 exhibiting the most robust attachment (Dalkara et al., 2009) . However, only AAV2 has mediated detectable transgene expression in the inner retina (Dalkara et al., 2009) . AAV2 binds heparin sulfate proteoglycan (HSPG) which is abundant in the ILM, while AAV8 binding involves the laminin receptor which may mediate a weaker interaction with this structure (Chai and Morris, 1994;Summerford and Samulski, 1998;Akache et al., 2006) . Data in

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134 Chapter 4 ha ve shown that the addition of Y F and T V mutations to the AAV8 capsid modestly improves its ability to transduce inner/middle/outer retina following intravitreal injection demonstrating the importance of both extracellular and intracellular barriers to tran sduction. Standard AAV5 fails to attach or accumulate at the ILM, likely because it relies on sialic acid for initial binding, a monosaccharide absent from the ILM (Kaludov et al., 2001;Cho et al., 2002;Dalkara et al., 2009) . Removal of this physical barrier with protease, however, led to robust gene expression in various cells of the retina, including photoreceptors and retinal pigment epithelium (Dalkara et al., 200 9) . Similar to AAV8, the addition of Y F mutations to the AAV5 capsid surface only modestly improves its ability to transduce the outer retina following intravitreal delivery. Taken together, it is clear that the cellular receptors of the parent AAV serot ype play a key role in influencing vector interaction with this vitreoretinal interface. These results are consistent with findings that AAV2 based vectors have the highest affinity for the ILM suggesting that, as of now, capsid mutants based on this serot ype have the highest potential for targeting a transgene to photoreceptor s via the vitreous. As the capsid biology of AAV8, a strong transducer of photoreceptor s in situ , becomes known, an approach that capitalizes on respective receptor biology of AAV2 a nd AAV8 may yield improved variants (Raupp et al., 2012) . An ideal approach would be to identify variants with the ability to reach/target the tissue of interest through manipulation of capsid receptor biology. This variant would then be further modified to account for intracellular trafficking. A method that utilizes directed evolution to find variants with increased affinity for photoreceptor s that can subsequently be enhanced by incorporation of the appropria te combination of Y F and or T V mutations may

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135 ultimately be the most successful strategy, particularly if powerful quantitative assays can be used to rapidly and accurately assess in vivo vector properties. Methods for quantifying vector transduction eff iciency in a biologically relevant, photoreceptor cell line have been described previously (Ryals et al., 2011) . This method has now been extended to a reliable in vivo assay for quantifying transduction efficiencies of intravitreally delivered AAV vectors in mouse photoreceptors . The quantitative r esults correlated well with qualitative fundoscopic observations. It was demonstrated that quantitative findings could be obtained as early as one week post injection and that, although fewer total cells appear transduced at this early time point relative to 4 weeks post injection, the pattern and relative efficiencies of vectors remained the same. Of all vectors tested, the most robust in vivo expression of photoreceptor s was noted following intravitreal delivery of scAAV2 quad Y F+T V smCBA GFP. Approxima tely 22% of photoreceptor s expressed detectable levels of transgene following intravitreal injection with this capsid mutant. To what extent transduction of 22% of photoreceptor s is capable of preserving retinal structure and/or restoring visual function t o an animal model of IRD is yet to be determined. Likewise, whether further improvements in transduction efficiency of the AAV2 quad Y F+T V can be achieved by additional mutagenesis requires further investigation. Evidence suggests that directed mutagenes is of additional threonine, lysine and serine residues, all of which are more abundant on the AAV2 capsid surface than tyrosine, will similarly reduce phosphorylation/proteosomal degradation of capsids, may further augment AAV mediated transgene expression (Gabriel et al., 2013) . It is expected that this approach has a finite maximum. However, it is important to note that the transduction efficiency of

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136 capsid mutant vectors varies with the target tissue as well as the profile and activity levels of kinases involved in AAV capsid phosphorylation (Aslanidi et al., 2012) . Additionally, it has yet to be d etermined whether initially non surface exposed residues that become available for phosphorylation in later steps of cellular processing (during conformational changes of th e capsid) may also be mutated to improve transduction efficiency. When considering intravitreal delivery of AAV vector intended to transduce distal photoreceptors , emphasis must also be placed on avoiding off target transgene expression. Consistent with p revious reports, the data presented here showed that the hGRK1 promoter drove strong transgene expression in photoreceptors (Khani et al., 2007;Boye et al., 2012) . Unexpectedly, off target expression was also n oted in retinal ganglion cells. Previous studies evaluating GRK1 promoter activity in the retina have utilized AAV serotypes with poor tropism for retinal ganglion cells, namely AAV5 and AAV8 (Khani et al., 2007 ;Boye et al., 2010;Beltran et al., 2010;Boye et al., 2011;Boye et al., 2012) . Therefore, even when such vectors were delivered to the vitreous, transduction of retinal ganglion cells was unlikely. When a parent serotype with strong affinity for retinal ga nglion cells (AAV2) was delivered with a high titer to the vitreous, GRK1 promoter activity in retinal ganglion cells was apparent. Because GRK1 has been shown to promote strong gene expression in both rods and cones of primate retina with no expression in middle retina or retinal pigment epithelium , a ganglion cell/middle retina miRNA target sequence was used to mitigate the observed expression in retinal ganglion cells. Four tandem sequences complimentary to an inner/middle retina specific miRNA were inco rporated into the AAV vectors. A microRNA expression atlas of the

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137 mouse eye indicates that miR 181c is highly expressed in retinal ganglion cells and middle retina and absent in photoreceptors in P60 mouse (http://mirneye.tigem.it/view_state. php?state = P 60&mirna = mmu miR 181c) (Karali et al., 2010) . Incorporation of miR2181c repeat sequences resulted in ablation of expression in retinal ganglion cells; however it also appreciably reduced expression of transgene in photoreceptor s. Attempts are underway to characterize vectors containing fewer miRNA target sequences and/or in a different spatial arrangement with the goal to prevent off target transduction while preserving photoreceptor expression. An important limitation of any AAV transduction study performed in lower order ma mmals is its translatability to the clinic. The ILM is relatively thin and homogenous in rodents. In primate, the ILM is significantly thicker, except for an area in and around the fovea pit and immediately above large blood vessels (Matsumoto et al., 1984 ) . These enhanced vectors will need to be thoroughly tested to determine if the gains in transduction from the mouse vitreous translate to similar improvements in the primate retina, particularly in the relatively exposed cone rich fovea. Thus far the hGR K1 and smCBA promoter s have been tested in non human primates (Yin et al., 2011;Boye et al., 2012) . An AAV5 hGRK1 GFP virus was delivered subretinally, 2.5mm superior temporally to the fovea. However, the bleb did extend under the fovea during delivery of th hGRK1 GFP mediated robust and exclusive expression in both rods and cones in retinal locations that received the virus (Boye et al., 2012) . An AAV2 smCBA GFP virus was administered intravitreally into a non human primate retina. 15 months post injection GFP expression could not be visualized. However, when injected to an eye pretreated with microplasmin, which

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138 induces enzymatic vitreolysis, GF P expression was evident in foveal ganglion cells and Müller cells (Yin et al., 2011) . The hGRK1 promoter seems very promising for transducing photoreceptors in a primate retina. However, this promoter has yet to be validated when delivered intravitreally. Overall, a limited number of studies have analyzed and compared efficiencies of photoreceptor specific promoters in non human primat es. Further investigation of photoreceptor specific promoters delivered both subretinally and intravitreally in non human primates will be necessary to develop treatments for patients with a photoreceptor mediated inherited retinal disease. In order to help researchers chose optimal promoters for non human primate studies, i n C hapt er 4 , transduction efficiencies o f two novel promoters, the cone sp ecific IRPB241 promoter and rod specific hOP promoter as well as previously studied hGRK1, mOPs and smCBA promoters were qua ntified in parallel . In 661W cells, all photoreceptor specific pr omoters mediated protein expression at similar levels. Not surprisingly, the ubiquitous promoter, smCBA, had a significantly higher transduction efficiency. This in vitro assay will continue to be a useful screen to validate viral constructs and compare tr ansduction capabilities. To improve upon the in vitro analysis, an in vivo transduction efficiency quantification was attempted. 38.5% of rod photoreceptor s were successfully labeled in a C57BL/6 mouse by subretinal delivery of scAAV5 double Y F mOPs GFP. However this was significantly reduced from the rod photoreceptor s obtained from the transgenic Rho GFP mice. Furthermore, double labeling of photoreceptor s with scAAV5 double Y F hGRK1 mCherry, whether delivered at the same time or one week later, led to a very low total retinal transduction of 6.1%. This did not agree with mCherry expression seen

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139 in fundus images. Therefore, continued strategies need to be employed to optimize an in vivo transduction efficiency quantification of photoreceptor specific pr omoters. Transgenic Nrl GFP mice show rod exclusive eGFP expression driven by the neural retina leucine zipper promoter (Akimoto et al., 2006) . This transgenic model may be able to withstand subretinal injectio ns without degeneration and the isolated GFP+ rod population may be comparable to the rod population isolated from Rho GFP mice. Studies are currently underway to investigate their usage in this assay. Overall, the work described in C hapter 4 supports co ntinued developm ent of AAV based vectors and photoreceptor specific promoters for the treatment of CEP290 LCA patients and various forms of photoreceptor mediated inherited retinal disease with a surgically less invasive intravitreal injection technique. A s additional capsids and promoters become available, researchers can use the novel in vivo approaches developed here to quantify photoreceptor transduction capabilities p rior to their preclinical proof of concept studies.

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140 Table 4 1. Nomenclature for capsid mutated vectors with description of amino acid location of mutation.

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141 Table 4 2. In vivo transduction efficiency of novel AAV capsids. PR, photoreceptor

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142 Table 4 3. Preliminary transduction efficiency of photoreceptor specific promoters three weeks post injecti on.

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143 Figure 4 1. Transduction efficiency of unmodifi ed and capsid mutated vectors in vitro . 661W cells were infected with scAAV2, scAAV2 quad Y F, scAAV2 quad Y F+T V, scAAV5, scAAV5 single Y F, scAAV5 double Y F, scAAV8, scAAV8 double Y F and scAAV8 double Y F+T V at a multiplicity of infection (MOI) of 10 calculated by multiplying the percentage of positive cells by the mean fluorescence intensity in each sample. P values were calculated using a two t test.

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144 F igure 4 2. Qualitative comparison of unmodified and capsid mutated AAV vectors in vivo . Fundoscopy (red channel only) of Rho GFP mice 4 weeks post intravitreal injection with unmodified and capsid mutated scAAV smCBA mCherry vectors (1.5E9vg delivered). Exposure and gain settings were the same across all images.

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145 Figure 4 3. Quantitative comparison of unmodified and capsid mutated AAV vectors in vivo . Transduction efficiency of unmodified and capsid mutated scAAV2, scAAV5 and scAAV8 vectors in Rho GFP mice. FAC S analysis was used to quantify the percentage of cells that were GFP positive ( photoreceptors ), mCherry positive (any retinal cells transduced with AAV) and both GFP and mCherry positive ( photoreceptor s transduced by AAV). Representative plots for a negat ive control (un injected retina) and 2 pooled retinas injected with scAAV2 quad Y F+T V are shown in (A) and (B), respectively. Cells that were both GFP and mCherry positive are shown in the top right of (A) and (B) and represent the percent of transduced photoreceptor s. The bottom right of (A) and (B) show cells that were mCherry positive, but GFP negative, representing off target transduction. The percentage of mCherry positive photoreceptors (a measure of in vivo photoreceptor transduction efficiency for each vector) in retinas injected with unmodified or capsid mutated scAAV vectors is shown in (C).

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146 Figure 4 4. Transduction efficiency of scAAV2 quad Y F and scAAV2 quad Y F+T V in photoreceptors of Rho GFP mice 1 week post intravitreal delivery. Tran sduction efficiency of scAAV2 quad Y F and scAAV2 quad Y F+T V in Rho GFP mice 1 week post intravitreal injection. Representative FACS plots for 2 pooled retinas injected with scAAV2 quad Y F and 2 pooled retinas injected with scAAV2 quad Y F+T V are shown in (A) and (B), respectively. Cells that were both GFP and mCherry positive are shown in the top right of (A) and (B) and represent the percent of transduced photoreceptor s. The bottom right of (A) and (B) show cells that were mCherry positive, but GFP ne gative, representing off target transduction. The percentage of mCherry positive photoreceptor s (a measure of in vivo photoreceptor transduction efficiency for each vector) in retinas injected with scAAV2 quad Y F and scAAV2 quad Y F+T V are shown in (C).

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147 Figure 4 5. Characterizing two green fluorescent photoreceptor populations in the Rho GFP retina. A single Rho GFP retina was analyzed by FACS. Two green fluorescent populations were observed, sorted and imaged. (A) FACS plot indicating that cells in po pulation 1 have a measurable red fluorescence and a reduced green fluorescence (purple) whereas cells in population 2 have minimal red fluorescence and are highly green florescent (green). (B) A forward and side scatter plot of the two populations indicati ng that cells in population 1 are larger than cells in population 2. (C) The top panel contains four images of cells in population 1 and the bottom panel contains four images of cells from population 2. Bright field and green fluorescent images were overla id. Exposure settings were consistent across images.

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148 Figure 4 6. In vivo analysis of AAV2 based vectors containing the hGRK1 promoter. In vivo analysis of AAV2 based vectors containing the hGRK1 promoter. Fundus images paired with immunohistochemistry of frozen retinal cross sections from C57BL/6 mice taken 4 weeks post injection with AAV2, AAV2 quad Y F and AAV2 quad Y F+T V vectors containing hGRK1 GFP (7.5E9vg delivered). Identical gain and exposures were used for fundoscopy. All tissue sections were imaged at 20X, with identical gain and exposure settings. GFP expression is shown in green. Nuclei were counterstained with DAPI (blue). RPE retinal pigment epithelium, IS/OS inner segments/outer segments, ONL outer nuclear layer, INL inner nuclear layer, GCL ganglion cell layer.

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149 Figure 4 7. Representative image of a retinal cross section from a C57BL/6 mouse injected with AAV2 quad Y F+T V. Representative image of a retinal tissue section from a C57BL/6 mouse injected with AAV2 quad Y F+T V (5.0E9vg de livered), stained for GFP (green) and counterstained with DAPI (blue). Merged images are presented at 10X to visualize the full retina.

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150 Figure 4 8. In vivo analysis of AAV5 based vectors containing the hGRK1 promoter. In vivo analysis of AAV5 based vec tors containing the hGRK1 promoter. Fundus images paired with IHC of frozen retinal cross sections from C57BL/6 mice taken 4 weeks post injection with capsid mutated AAV5 vectors containing hGRK1 GFP. For analysis of AAV5 single Y F and AAV5 double Y F vec tors 8.5E10vg and 5.3E9vg were delivered, respectively. Central retinal tissue sections containing the optic nerve head (B&E) and peripheral retinal cross sections (C&F) are shown. White arrows demarcate the optic nerve head. Identical gain and exposures w ere used for fundoscopy. All cross sections were imaged at 20X, with identical gain and exposure settings. GFP expression is shown in green. Nuclei were counterstained with DAPI (blue). RPE retinal pigment epithelium, IS/OS inner segments/outer segments, O NL outer nuclear layer, INL inner nuclear layer, GCL ganglion cell layer.

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151 Figure 4 9. In vivo analysis of AAV8 based vectors containing the hGRK1 promoter. In vivo analysis of AAV8 based vectors containing the hGRK1 promoter. Fundus images paired wit h immunohistochemistry of frozen retinal cross sections from C57BL/6 mice taken 4 weeks post injection with capsid mutated AAV8 vectors containing hGRK1 GFP. For analysis of AAV8 double Y F and AAV8 double Y F+T V vectors, 1.3E11vg and 6.0E10vg were delive red, respectively. Central retinal cross sections containing the optic nerve head (B&E) and peripheral retinal cross sections (C&F) are shown. White arrows demarcate the optic nerve head. Identical gain and exposures were used for fundoscopy. All cross sec tions were imaged at 20X, with identical gain and exposure settings. GFP expression is shown in green. Nuclei were counterstained with DAPI (blue). RPE retinal pigment epithelium, IS/OS inner segments/outer segments, ONL outer nuclear layer, INL inner nucl ear layer, GCL ganglion cell layer.

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152 Figure 4 10. MicroRNA mediated regulation of transgene expression. Both hGRK1 GFP and hGRK1 GFP miR181c were packaged in AAV2 quad Y F+T V and delivered intravitreally to C57BL/6 mice (1.5E10vg delivered). Fundoscopy at 4 weeks post injection is shown adjacent to immunohistochemistry of frozen retinal cross sections. Identical gain and exposures were used for fundoscopy. All cross sections were imaged at 20X, with identical gain and exposure settings. GFP expression i s shown in green. Nuclei were counterstained with DAPI (blue). RPE retinal pigment epithelium, IS/OS inner segments/outer segments, ONL outer nuclear layer, INL inner nuclear layer, GCL ganglion cell layer.

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153 Figure 4 11. In vivo , qualitative analysis of AAV2 based vectors containing the ubiquitous, CBA promoter. Fundus images paired with immunohistochemistry of frozen retinal cross sections from C57BL/6 mice taken 4 weeks post injection with AAV2 triple Y F+T V, AAV2 quad Y F, and AAV2 quad Y F+T V vector s containing ubiquitous promoter CBA driving GFP (1.5E10vg delivered.) Identical gain and exposures were used for fundoscopy. Retinal sections were imaged at 5X for visualization of the entire retina from periphery to periphery (B,E,H), at 20X for detailed analysis of each retinal cell type (C,F,I) and at 40X for better resolution of outer the retina (insets of C,F,I). All sections were imaged with identical gain and exposure settings. GFP expression is shown in green. Nuclei were counterstained with DAPI ( blue).

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154 Figure 4 12. In vitro transduction e fficiency of photoreceptor specific promoters. 661W cells were infected with sc hGRK1 mCherry, sc mOPs mCherry, sc hOP mCherry, sc IRBP241 mCherry and sc smCBA mCherry all packaged in AAV2 quad Y F+T V at a mu ltiplicity of infection (MOI) of 10,000. mCherry the percentage of positive cells by the mean fluorescence intensity in each sample. P values were calculated using a two tail t test.

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155 Figure 4 13. Fundoscopy of C57BL/6 mice 3 weeks post co delivery of sc mOPs GFP and sc hGRK1 mCherry. Fundoscopy (green channel top, red channel bottom) of C57BL/6 mice 3 weeks post injection with scAAV5 double Y F mOPS GFP (A&B), scAAV5 double Y F hGRK1 mCherry (C&D) or scAAV5 double Y F mOPs GFP and scAAV5 double Y F hGRK1 mCherry at a 1:1 ratio (E&F). 1.0E9vg of each virus was delivered subretinally. Exposure and gain settings were the same across all images.

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156 Figure 4 14. Fu ndoscopy of C57BL/6 mice 3 weeks post sc hGRK1 mCherry delivery. Fundoscopy (green channel top, red channel bottom) of C57BL/6 mice 4 weeks post injection with scAAV5 double Y F mOPS GFP (A&B) or 3 weeks post injection with scAAV5 double Y F hGRK1 mCherry (C&D). The C57BL/6 retina pictured in (E&F) was injected with scAAV5 double Y F mOPs GFP then one week later scAAV5 double Y F hGRK1 mCherry was delivered. 1.0E9vg of each virus was delivered subretinally. Exposure and gain settings were the same across al l images.

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157 Figure 4 15. FACS plots quantifying GFP and mCherry expression post subretinal delivery of sc mOPs GFP and sc hGRK1 mCherry. Each injected C57BL/6 retina was harvested, dissociated and prepared for FACS analysis. (A) Representative FACS plot o f an un injected C57BL/6 retina showing one large population of non fluorescent cells (negative). (B) Representative FACS plot of a C57BL/6 retina injected with scAAV5 double Y F mOPS GFP. One GFP+ cell population and one non fluorescent cell population wa s evident. An average of 38.7% of cells were GFP+ (n=5). (C) Representative FACS plot of a C57BL/6 retina injected with scAAV5 double Y F hGRK1 mCherry. One mCherry+ cell population and one non fluorescent cell population was evident. An average of 5.8% of cells were mCherry+ (n=8). (D) Representative FACS plot of a C57BL/6 retina co injected with scAAV5 double Y F mOPs GFP and scAAV5 double Y F hGRK1 mCherry. Cells were non fluorescent, GFP+, mCherry+ and both GFP+ and mCherry+. The average percentage of c ells that were both GFP+ and mCherry+ was 3.2 (n=3). (E) Representative FACS plot of a C57BL/6 retina injected with scAAV5 double Y F mOPs GFP and then one week later scAAV5 double Y F hGRK1 mCherry was delivered. Cells were non fluorescent, GFP+, mCherry+ and both GFP+ and mCherry+. The average percentage of cells that were both GFP+ and mCherry+ was 5.8 (n=3).

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158 CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS Developing a Clinical Trial for CEP290 LCA Patients The overarching goal of the studies presented in t his dissertation was to lay the foundation for an AAV gene therapy clinical trial for non syndromic CEP290 LCA patients. In general , the treatment strategies described in the AAV mediated RPE65 LCA clinical trials should be adaptable for CEP290 LCA patient s (Maguire et al., 2008;Hauswirth et al., 2008;Cideciyan et al., 2009;Maguire et al., 2009;Simonelli et al., 2010;Jacobson et al., 2012a;Testa et al., 2013;Bainbridge et al., 2008) . However, the treatment strat egy will not be identical due to differing genotypic and phenotypic presentations of CEP290 LCA patients. When designing a clinical trial for CEP290 LCA patients three important components need to be considered. First, retinal dystrophy clinicians must est ablish common procedures that accurately assess visual function, both objectively and subjectively in this severely blind population of patients. Once these common procedures are defined the ones capable of evaluating therapeutic gains after treatment shou ld be chosen as outcome measurements for CEP290 LCA clinical trials. Secondly, a therapeutic virus capable of expressing CEP290 protein in photoreceptors at biologically relevant levels needs to be developed. The ability of dual AAV vectors to mediate lar ge , photoreceptor specific transgene expression is no longer in question. Two groups have proven that simple overlap, trans splicing and hybrid dual vectors can deliver large transgene s , similar in size to CEP290 , to the photoreceptors of a mouse and pig r etina and mediate detectable levels of protein (Dyka et al., 2014a;Colella et al., 2014). It is reasonable to assume that these general dual AAV

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159 technologies can be modified to mediate CEP290 expression. Although this dissertation focuses on developing an AAV gene therapy, it is important to note that a lentiviral strategy, which does not suffer from the packaging limitations found in AAV strategies, is also being investigated (Burnight et al., 2014) . Positive r esults from the lentiviral clinical trials treating patients with Usher and Stargardt disease could provide the inherited retinal dystrophy community with another efficacious option for large transgenes. However, results from the RPE65 LCA and p hase 1/2 choroideremia clinical trials indicate that an AAV mediated therapeutic is the most reliable and efficacious treatment option for inherited retinal dystroph y patient s (Maguire et al., 2008;Hauswirth et al., 2008;Cideciyan et al., 2009;Maguire et al., 2009;Simonelli et al., 2010;Jacobson et al., 2012a;Testa et al., 2013;MacLaren et al., 2014;Bainbridge et al., 2008) . Although researchers are optimizing both AAV mediated and lentiviral mediated strategies for the treatment of CEP290 LCA patients, it is currently unknown which of the two approaches will prove most efficacious. Lastly, the injection technique that will be used to deliver the therapeutic virus is a third important component to consider when optimizing treatment strategies. The subretinal injection has been established as a safe and effective delivery method for AAV mediated gene therapy (Maguire et al., 2008;Hauswirth et al., 2008;Cideciyan et al., 2009;Maguire et al., 2009;Simonelli et al., 2010;Jacobson et al., 2012a;Testa et al., 2013;Bainbridge et al., 2008) . A subfoveal detachment will be required during subretinal administration of the virus in CEP290 LCA patients because foveal cones are the only remaining/targetable cell bodies in their retinas. This potentially poses a problem due to poor outcomes observed in preliminary RPE65 LCA studies (Jacobson et al., 2012a) .

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160 Investigators are now attemptin g to validate the widely used intravitreal injection for treating inherited retinal dystrophy patients (Petrs Silva et al., 2011;Yin et al., 2011;Kay et al., 2013;Dalkara et al., 2013;Day et al., 2014) . More st udies characterizing photoreceptor transduction of AAV capsids and photorec eptor specific promoters in a non human primate are required to fully validate the intravitreal approach, but in the future, CEP290 LCA patients may be a ble to choose between a subf oveal, subretinal injection and an intravitreal injection. The work presented in this dissertation has contributed to all three of these components required for the development of a clinical trial for CEP290 LCA patients. Practical Outcome Measurements fo r CEP290 LCA Clinical Trails Clinical features of non syndromic CEP290 LCA patients include: severe vision loss, roving eye movements, nystagmus, sluggish pupillary response, a non recordable electroretinogram, a foveal outer nuclear layer thickness within the normal li mits up to 5 and beyond, abnormal near infrared autofluorescence topography (a larger and more abrupt boundary), severe rod degeneration within the first decade of life a nd intact visual brain pathway anatomy (Cideciyan et al., 2007;Cideciyan et al., 2011a;Yzer et al., 2012;McAnany et al., 2013b;Boye et al., 2014) . These features need to be taken into consideration when determi ning outcome measurements for a CEP290 LCA clinical trial. Because these patients lose their rod photoreceptors very rapidly, treatments at this time should focus solely on restoring function to foveal cones and preventing the observed slow degeneration of the fovea. Taking these clinical features into consideration as well as the most useful outcome measurements in the RPE65 LCA clinical trial pupillometry, nystagmus testing and cone optimized dark adapted full field

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161 sensitivity tests would be most useful for CEP290 LCA patients (Maguire et al., 2008;Hauswirth et al., 2008;Cideciyan et al., 2009;Maguire et al., 2009;Simonelli et al., 2010;Jacobson et al., 2012a;Testa et al., 2013;Bainbridge et al., 2008) . At th e Chicago Lighthouse, CEP290 LCA patients have been undergoing standard pupil light reflex and dark adapted full field sensitivity testing. Patients show no measurable rod mediated pupil reflex, a slightly abnormal cone mediated reflex and a measurable, bu t reduced melanopsin mediated pupil reflex (Co llison et al., 2014) . To obtain dark adapted FST thresholds both red and blue lights were used to isolate cone and rod function, respectively. CEP290 LCA patients have elevated cone FST thresholds between 7 and 25db relative to normal cone thresholds (Collison et al., 2014) . Investigators at the Chi cago Lighthouse have also developed a light discomfort threshold test for CEP290 eyes are illuminated with a blue light (444nm), green light (52 4nm) or red light (632nm) for 2 seconds at increasing intensity. Patients report when they find the stimulus to be uncomfortable. CEP290 LCA patients had lower light discomfort thresholds for shorter wavelength stimuli versus longer wavelength stimuli (Collison et al., 2014) . All three of these tests would be valid outcome measurements for CEP290 LCA p atients. RPE65 monitored with optical coherence tomography (OCT) (Jacobson et al., 2012a;Cideciyan et al., 2013) . O CT should also be uti lized in CEP290 LCA clinical trials. Monitoring the fovea with OCT will be important for discerning injection d amage and evaluating treatment e ffects on the natural history of foveal degeneration. M onitor ing inner nuclear layer thickness pre and post trea tment with OCT may also provide insights to treatment

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162 efficacy. Morphological changes may occur in treated areas of the retina once function is restored. In a three year follow up of treated RPE65 LCA patients, Testa et al. 2013 found differences in fundus autofluorescence between untreated and treated eyes. Fundus autofluorescence is not routinely used in the clinical trials, but was used in this study to further validate the reconstitution of the retinoid cycle after RPE65 delivery (Testa et al., 2013) . Fundus autofluorescence could also be employed to m onitor treatment e ffects in CEP290 LCA patients because their retinas have an abnormal fundus autofluo resence phenotype that could change after treatment. More than likely, ONL thickness of the fovea, INL thickness of the retina outside the fovea and autofluorescence will not change drastically after treatments. However, these objective measurements will be useful to evaluate all therapeutic gains provided by the treatment. Full field electroretinography (ffERG) analysis was used to characterize inherited retinal dystrophy patients in the study described in C hapter 2 of thi s dissertation. In particular, eight LCA patients were identified by their non recordable f fERGs, which continues to be a unique electrophysiological finding common to all LCA patients. Full field ERG analysis was used in preliminary RPE65 LCA studies (Maguire et al., 2009;Simonelli et al., 2010) . Ho wever, improvements after treatment were not observed with ffERG because the total area of the treatment zones was too small to generate a gross retina response (Maguire et al., 2009) . Although ffERG has not be en useful yet in AAV mediated clinical trials , multi focal ERG (mfERG), which can measure electrical signals generated from an isolated area of the retina (the macula), could be useful to asses cone function within the fovea after a subretinal injection th at detaches the macula including the fovea . However, good fixation is required when obtaining a mfERG

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163 and LCA patients lack normal fixation due to their nystagmus. Maguire et al. reported that investigators were able to test three subjects with mfERG after injection due to their reduced levels of nystagmus (Maguire et al., 2009) . A signal was seen in the electroretinographs, but amplitudes were not reported (Maguire e t al., 2009) . Th ese data suggest that mfERG may be a useful objective tool to asses cone function post order to establish mfERG as a useful outcome measurement, ongoing characterizations of inherited ret inal dystrophy major limitations of the study discussed in Chapter 2 was the omission of mfERG analysis, especially since objective ffERG measurements were being compar ed to subjective assessments of rod and cone mediated vision. Multi focal ERG instead of ffERG would have been a better objective measurement to compare to subjective assessment of cone mediated vision. Overall, ffERGs remain a critical component to the c haracterization of LCA patients (and all inherited retinal dystrophy patients) and mfERG should be investigated thoroughly as an outcome measurement for cone function especially if future AAV mediated clinical trials continue to deliver the virus to the fo vea subretinally . The goal of the study described in Chapter 2 was to validate the well established NEI VFQ 25 as a useful subjective outcome measurement for inherited retinal dystrophy clinical trials (Mitche ll et al., 2013;Lightman et al., 2013;Rakic et al., 2013;Ryan et al., 2013;Le et al., 2014;Renieri et al., 2013) . Although, objective ffERG data did not correlate to subjective analysis, the NEI VFQ 25 may be useful to provide investigators with a documen ted and measurable subjective assessment of rod -

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164 mediated and cone mediated vision. Subjective reporting of vision has been a vital component of the RPE65 LCA clinical trials and should be considered as a useful measurement for future trials. For example, R PE65 LCA patients reported noticeably increased brightness in their treated eye when they awoke from sleep in a darkened environment. After this reporting, dark adaption times were extended for dark adaptation full field sensitivity tests and a greater mag nitude of visual gain was observed (Cideciyan et al., 2009) . First hand accounts from subjects describing their visual improvements along with their desire to have their second eye injected have contributed to moving the RPE65 LCA trials forward. Results from the study described in Chapter 2 indicate that differences in subjective assessment of rod mediated and cone mediated vision can be observed and compared between inherited retinal dystrophy patients. For ex ample, LCA patients had well defined subjective characteristics of their rod and cone mediated vision when analysis was conducted using the modified NEI VFQ 25. When assessing cone mediated vision, 62.5% of LCA mediated vision assessment, 75% of LCA p mediated vision

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165 adapting to the dark when transi tioning to indoor lighting conditions from bright light conditions. In summary, most LCA patients reported better cone mediated vision than rod mediated vision. Furthermore, most LCA patients reported being sensitive to light (a cone mediated response) and hav ing difficult ies adapting to the dark (a rod mediated response). It would be interesting to see if these findings change post treatment . A ny patient reported changes in light sensitivity and dark adaptation would suggest that the treatment is providing quality of life improvements for the patient. Overall, data presented in Chapter 2 suggests that the subjective analysis of cone mediated and rod mediated vision by the NEI VFQ 25 needs continued investigation with a large r number of LCA patients carrying different genetic mutations so it can become clear if it is a useful subjective outcome measurement. Developing a n AAV Gene Replacement Therapy for CEP290 LCA The goal of the study described in Chapter 3 was to create a therapeutic AAV vector for CEP290 LCA patients. Two dual AAV vectors systems, one a gene dependent (simple overlap) system and the other a gene independent (hybrid) system, were developed to express CEP290 in the photoreceptors of rd16;Nrl / mice. The dual AAV vectors were unable to medi ate full length CEP290 transcript and protein in vitro . Furthermore, subretinal delivery of the dual AAV vectors did not restore visual function to rd16; Nrl / mice. I hypothesize that specific features of CEP290 are hindering the success of our dual AAV vectors. Future experiments are needed to clarify why the dual vectors are malfunctioning and to further our understanding of CEP290 gene regulation so that novel dual AAV cloning strateg y can be utilized for effective AAV mediated CEP290 expression.

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166 Fut ure CEP290 simple overlap AAV vectors need to include a highly recombinogenic overlap region . However, key features that facilitate homologous recombination are not well understood . One hypothesis suggests that secondary structure and GC content of the ove rlap sequence may contribute to the recombinogenic properties of the NT and CT constructs. The overlap region utilized in our simple overlap vectors possessed a low GC content and potential secondary structures which may have limited recombination events b etween NT and CT constructs. Reducing potential secondary structure as well as increasing GC content of the overlap sequence should be explored in future CEP290 simple overlap designs. In order to postulate to what extent these features should be modulated , the GC content and secondary structure in the CEP290 overlap sequence should be compared to overlapping sequences with proven utility in dual vector systems (alkaline phosphatase sequence, MYO7A complementary DNA overlap, ABCA4 complementary DNA overlap) . Once key features enhancing homologous recombination events of dual vectors are confirmed , modification of the CEP290 coding sequence will be necessary to incorporate these features. Sequence optimization offers one possibility to manipulate the CEP29 0 coding sequence without altering the amino acid sequence. Many online progra ms and companies specialize in sequence optimization algorithms and should be helpful for optimizing future CEP290 dual vectors (Ward et al., 2011;Sack et al., 2012;Chin et al., 2014) . Homologous recombination occurs at a high rate during the S phase of the cell cycle when a large amount of DNA replication occurs. Post mitotic cells of the retina are therefore relatively inefficient re combi nogenic cells. Thus, e xploring ways to concentrate simple overlap NT and CT DNA replication in order to

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167 maximize interactions between the two constructs may be necessary to facilitate homologous recombination. Future CEP290 trans splicing and hybrid d ual AAV vectors n eed explore the use of alternative natural exon junctions. For now, it is difficult to hypothesize why one natural exon junction would be superior to another because our understanding of native CEP290 gene regulation and transcription effi ciency is very limited. Given that the CEP290 coding sequence consists of 52 exons, transcription of this large transgene may have unique features. Basic studies investigating mRNA transcript assembly of endogenous CEP290 isolated from HEK293 cells and mo use photoreceptors would advance our understanding of CEP290 splicing patterns. Results from these studies could reveal alternat ive splicing patterns, novel usage of the exons and superior exon /intron junctions for dual vector designs . Assuming that optimi z ed dual AAV vector technologies will lead to expression of CEP290, optimizing therapeutic expression levels will be the next critical step. Based on the small size of the transition zone in the connecting cilium of the photoreceptor it is assumed that low levels of CEP290 expression would be required for restoration of function. In addition, a recent study discovered that high levels of CEP290 expression can be toxic in a human cell line (Burnight et al., 2014) suggesting that viruses mediating low levels of CEP290 expression may be imperative for successful therapy . Weak photoreceptor specific promoters and the endogenous CEP290 promoter should be explored for their ability to express CEP290 at levels that provide a therapeutic benefit without toxic side e ffects. On the other hand, it is possible that dual AAV vector systems will mediate CEP290 expression below therapeutic levels. When comparing

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168 protein levels post delivery with a single AAV vector, dual AAV vectors are 88% less efficient (Carvalho et al., 2014) . To overcome a potential expression limitation of dual vectors, enhanced recombination between overlap sequences is required for simple overlap dual AAV vector systems and enhance recombination of inverted terminal repeats (ITRs) is required for trans splicing and hybrid dual AAV vector systems. As mentioned previously, key features that facilitate homologous recombination remain un known. However, hybrid inverted terminal repeats can be incorporated to facilitate enhanced recombination of ITRs. Hybrid ITRs utilize two non homologous ITRs, AAV5 and AAV2, to manipulate the orientation of NT and CT ITR annealing since it occurs at least in part through ITR/ITR interaction . As mentioned previously, NT and CT ITR interactions facilitate the creation of full length DNA constructs in the nucleus prior to transcription. The strength of the ITR interaction is dependent on the homology between left and right hand ITRs . When both NT and CT constructs contain the same ITRs, i.e. only a 25% chance that the ITR binding/circularization will occur in an orientation that allows for full length transc ription (Yan et al., 2005) . However, with the addition of AAV5/AAV2 hybrid ITRs, i.e. only correct ITR recombination will occur in an orien tation that allows for full length transcription (Yan et al., 2005) . The addition of hybrid ITRs to standard trans splicing dual AAV vectors led to a 6 10 fold increase in protein ex pression (Yan et al., 2005) . Trans splicing and hybrid d ual AAV vectors containing such hybrid ITRs should be investigated if a more efficient dual vector system is required for therapeutic CEP290 expression.

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169 For now, future studies should focus on produ cing dual AAV vectors that mediate full length CEP290 transcript and protein in vitro . Results from our transfection experiments suggest that HEK293 cells are a sufficient in vitro model for verification of dual AAV vectors. Once accomplished , proof of con cept studies can be attempted in the rd16;Nrl / animal model. If transcript and protein expression are not evident in relevant mouse models, but are confirmed in vitro , a mouse C ep 290 coding sequence may be required for expression in the mouse models. Alt hough the human CEP290 coding sequence and protein are 86% and 89% identical to the mouse coding sequence and protein, it is uncertain if the human CEP290 coding sequence and protein have functional potential in mouse tissue. In addition, if transcript and protein expression are verified in vitro and in relevant mouse models, but fail to restore the rd16;Nrl / phenotype an additional factor may be required. Rd16;Nrl / photoreceptors contain basal bodies, inner segments and connecting cilium, but the photo receptors have either incorrectly formed outer segments or no outer segments at all (Cideciyan et al., 2011a). It is reasonable to hypothesize that exogenous CEP290 expression will be able to incorporate into malfunctioning transition zones and facilitate protein trafficking, but it remains unclear if CEP290 expression will facilitate the formation or restoration of the outer segments or if outer segment restoration will be required for visual restoration. If outer segment restoration is necessary, administ ration of ciliary neurotrophic factor (CNTF), which promotes cone outer segment regeneration (Wen et al., 2012) as well as exogenous CEP290 expression may be required to fully restore visual function to the rd16; Nrl / animal model.

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170 R esult s from this st udy suggest that novel dual AAV vectors are required for AAV mediated CEP290 expression . Fortunately, cloning strategies have not been exhausted for CEP290 dual AAV vector development. A s more is understood about native CEP290 gene regulation and protein f unction better strategies will be developed. Multiple groups have proven that simple overlap, trans splicing and hybrid dual vectors can deliver large transgenes, similar in size to CEP290, to the photoreceptors of a mouse and pig retina and mediate detect able levels of protein (Lopes et al., 2013;Colella et al., 2014;Dyka et al., 2014a) . Th ese recent dual AAV success es support the continued development of dual AAV vector for CEP290. Two Injection Techniques P ertinent to Treating CEP290 LCA Patients Since the results of the RPE65 LCA clinical trials have been published there has been much controversy over the safety of subretinal injections that detach the fovea. This is a major concern for CEP290 LCA patients who only have foveal cones remaining in their retina, meaning transduction of foveal cones is necessary for treatment. In the combined RPE65 LCA clinical trials, 16 of 30 subretinal injections detached the macula including the fovea (Maguire et al., 2008;Hauswirth et al., 2008;Cideciyan et al., 2009;Maguire et al., 2009;Simonelli et al., 2010;Jacobson et al., 2012a;Testa et al., 2013;Bainbridge et al., 2008) . All but two of these patients did have restoration in visual function as defined by the outcome measures in the trials (improvement in full field light sensitivity, pupillary light reflex and others). However, in a careful study by Jacobson et al. 2012a foveal detachments resulted in a decrease in visual acu ity (in two out of five treated LCA2 patients) as well as foveal thinning . This occurred in both LCA2 patients and non human primates (Jacobson et al., 2006b;Jacobson et al., 2012a) . I m portantly, those two pati ents with foveal thinning had

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171 the most foveal photoreceptors at baseline measurements making the vision loss even more substantial (Jacobson et al., 2012a) . R ecently, MacLaren et al. 2014 reported re sults from six patients that received AAV mediated therapy for choroideremia via subretinal injections, which resulted in subfoveal detachments. No detrimental effects from detachment and treatment of the fovea were reported. Overall, some patients had visual acuity improvements, light sensitivity improvements and no change in their retinal thickness (MacLaren et al., 2014) . In summary, a total of 22 patients have been treated with AAV in a subfoveal subretinal fashion. Ac cordi ng to the published data, only two patients have suffered a loss in visual acuity and a loss in foveal photoreceptors due to a subretinal detachment under the fovea . However, it is unclear whether all studies examined foveal OCT data at a resolution s ufficiently high to detect foveal cone thinning (Maguire et al., 2008;Hauswirth et al., 2008;Cideciyan et al., 2009;Maguire et al., 2009;Simonelli et al., 2010;Jacobson et al., 2012a;Testa et al., 2013;Bainbridg e et al., 2008) . It is thought by some that the potential gain in cone function from treating the fovea outweighs the chance of decreased foveal function (Testa et al., 2013;MacLaren et al., 2014) . CEP290 LCA patients with severe vision loss (HM to NLP) are perfect candidates for subfoveal treatment. However, CEP290 LCA patients with preserved central vision have more to lose if the subfoveal detachment results in loss of visual acuity and foveal thinning. Ove rall, it is very difficult to clearly interpret the RPE65 LCA clinical trial results due to the small number of patients treated and the differing outcome methodologies used between the clinical trials. As more patients are treated in the phase 3 RPE65 LCA clinical trial it may become clear whether sub r etinal injections resulting in subfoveal detachments are safe for CEP290 LCA patients.

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172 As mentioned above , this controversy as well as the desire to optimize treatments for other retinal degenerative disea ses that will require transduction of inner retinal cells (bipolar and ganglion cells) has led to the development of an intravitreal approach for treating inherited retinal dystrophy patients. If CEP290 LCA patients are going to be treated with an intravit real approach then an AAV capsid that can penetrate f oveal cones and mediate therapeutic levels of protein will need to be developed . Since primates are the sole species containing a fovea, studies validating nove l AAV capsids and photoreceptor specific pr omoters need to be performed in non human primates. The in vitro assay and in vivo assay described in Chapter 4 are useful tools researchers can use to quantify the transduction efficiency of their nove l AAV capsids and photoreceptor specific promoters pri or to characterization in such expensive non human primate studies. Thus far, an AAV variant (7m8), derived from directed evolution experiments, has shown efficacy in restoring visual function to a mouse model of retinoschisis and LCA2 post intravitreal d elivery (Dalkara et al., 2013) . The ability of this variant to transduce the retinal pigment epithelium from the vitreous and elicit therapy via RPE65 expression suggests this variant is capable of penetrating all the retinal layers from the vitreous. It was further confirmed that 7m8 CMV GFP could transduce photor eceptors in a non human primate post intravitreal delivery. High levels of GFP expression at 12 weeks post injection caused an observable immune response, which led to the termination of the experiment (Dalkara et al., 2013) . These results suggest that due to the transduction ability of the capsid, reduced doses an d weaker promoter choices may be required to avoid immune responses . Another capsid, AAV2 quad Y F, that has previously been

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173 shown to penetrate the entire retina of a C57BL/6 mouse post intravitreal delivery has been concurrently characterized in the non h uman primate (Petrs Silva et al., 2011;Dalkara et al., 2013) . AAV2 quad Y F CMV GFP did mediate GFP expression in foveal cones, but at much lower levels than the evolved 7m8 variant (Dalkara et al., 2013) . In Chapter 4, a novel AAV mutant capsid AAV2 quad Y F +T V, engi neered by rational mutagenesis, was shown to transduce ~22% of photoreceptors post intravitreal delivery in a Rho GFP retina. More importantly, this resulted in a 3.5 fold increase in photoreceptor transduction compared to AAV2 quad Y F. F urthermore, AAV2 quad Y F + T V mediated abundant pan retinal GFP expression in a C57BL/6 mouse 4 weeks post intravitreal delivery. These results suggest that the 7m8 and the AAV2 quad Y F+T V may be the two best capsid options currently for intravitreal delivery. For now , the optimal photoreceptor specific promoter, which has been characterized in non human primates, is the human rhodopsin kinase promoter (hGRK1) (Boye et al., 2012) . GRK1 has mediated robust and exclusive GFP expression in both rods and cones post subretinal delivery in a non human primate retina (Boye et al., 2012) . However, this promoter needs to be validated in the non human primate post intravitreal delivery to ensure that it does not have leaky expression in the ganglion cells. Results from Chapter 4 and from other studies have indicated that the hGRK1 and the mOPs promoter have at least some activity in ganglion cells when delivered from the vitreous (Petrs Silva et al., 2011) . Future studies should compare 7m8 hGRK1 GFP and AAV2 quad Y F+T V hGRK1 GFP side by side in the non human primate to decide which capsid can transduce foveal photoreceptors the best post intr avitreal delivery. GFP expression should be quantified in foveal cross sections via confocal microscopy.

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174 Th ese data would allow researchers to compare the foveal transduction efficiencies of these two capsids and validate the specificity of the hGRK1 promo ter in a reliable way . Th is would bring researchers one step closer to using an intravitreal approach in the clinic for inherited retinal dystrophy patients. Studies in this dissertation characterized the AAV2 quad Y F+T V in Rho GFP and C57BL/6 mice. Fut ure studies also need to validate the AAV2 quad Y F +T V in a retinal degenerative mouse model. Thus far, AAV2 quad Y F+T V has been able to drive nyctalopin (Nyx) expression in the ON bipolar cells of Nyx nob mice, a model of congenital stationa ry night bl indness. It remains unclear whether the AAV2 quad Y F+ T V mediated nyctalopin expression restores visual function (White et al., 2014) . Summary It is evident that retinal dystrophy clinicians, surgeons and researchers are working diligently to develop therapies for well characterized CEP290 LCA patients. Future clinical trials evaluating therapies for CEP290 LCA patients seem feasible for the following reasons. Firstly, there are a variety of outcome measurements that can be used to monitor CEP290 al function, disease progression and to evaluate therapeutic gains provided by the treatments being investigated. Secondly, it is reasonable to believe that successful strategies will be developed for constructing more novel CEP290 dual AAV vectors as our structure and gene regulation expands. In addition, lentiviral mediated CEP290 expression was verified for the first time (Burnight et al., 2014) . These results provid e promise that gene replacement will be a treatment option for CEP290 LCA patients. Thirdly, two injection methods may be available for CEP290 LCA patients. Perhaps CEP290 LCA patients with very poor vis ion (HM to NLP) will be able to elect a

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175 subretinal in jection while CEP290 LCA patients with preserved central vision will be able to elect an intravitreal injection. As results are published from the lentiviral Usher IB and Stargardt clinical trials, phase 3 RPE65 LCA clinical trials and upcoming CNGB3 Achro matopsia clinical trials, it will become clear which virus and injection method will be the most effective for treating CEP290 LCA patients. Work in this dissertation has directly advanced the development for CEP290 LCA treatment . Firstly, a new method to subjectively assess rod mediate d and cone mediated vision in LCA patients has been investigated that indicated clear, reportable trends for different inherited retinal dystrophy patient populations. Secondly, two dual AAV vector systems were developed for the delivery of CEP290 . Although, they were unable to mediate protein expression, alternative strategies have been suggested . Thirdly, in vitro and in vivo assay s were developed to quantify transduction capabilities of nove l AAV capsids and photoreceptor specific promoters. These novel capsids and promoters are being created to mediate transgene expression to specific re tinal cells when delivered either subretinally or intravitreally. The transduction efficiency assays will help researchers identify the be st capsid and promoter prior to characterization in non human primates. Fourthly, a novel capsid , AAV2 quad Y F+T V, generated by rational design, was characterized and showed an increase in photoreceptor transduction efficiency compared to existing unmodi fied and modified capsids. This capsid may aid in validating an intravitreal approach for treating retinal dystrophies. Overall, the work in this dissertation has advanced the field towards developing a gene therapy for CEP290 LCA.

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196 BIOGRAPHICAL SKETCH R enee Ryals was born and rai sed in Ormond Beach, Florida. In the summer of 2005, she graduated from the International Baccalaureate program at Spruce Creek High School . Soon after graduation from high school, she matriculated at the University of Florida for her undergraduate studies . In May of 2009, Renee received a Bachelor of Science degr ee for her thorough studies in c hemistry. In August of 2010 , Renee was accepted in the interdisciplinary graduate program for biomedical science. She received her Ph.D. from the University of Flori da in the summer of 2014. After graduation , Rene e will move to Portland , Oregon to start her post doctoral fellowship with Dr. Mark Pennesi at the Casey Eye Institute affiliated with the Oregon Health and Science University .