Structural Characterization of Adeno-Associated Virus Serotypes 1 and 6 Glycan Interactions

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Structural Characterization of Adeno-Associated Virus Serotypes 1 and 6 Glycan Interactions
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english
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Ng, Robert
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University of Florida
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Doctorate ( Ph.D.)
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University of Florida
Degree Disciplines:
Medical Sciences, Biochemistry and Molecular Biology (IDP)
Committee Chair:
Agbandje-Mckenna, Mavis
Committee Members:
Snyder, Richard O
Srivastava, Arun
Flanegan, James B
Muzyczka, Nicholas

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Subjects / Keywords:
aav -- capsid -- cryo-em -- glycan -- interaction -- parvovirus -- receptor -- resolution -- serotype -- structure -- therapy -- virus -- x-ray
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
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Medical Sciences thesis, Ph.D.
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theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
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Abstract:
Adeno-Associated Viruses (AAVs) are small ssDNA viruses with the ability to package non-genomic DNA for therapeutic gene delivery. Due to their non-pathogenicity and ability to transduce non-dividing and dividing cells, these viruses have generated significant interest in their development as therapeutic vectors. These properties dictate the tissue of choice for a particular gene delivery application. Towards understanding the capsid determinant(s) of these functions, we have employed AAV1 and AAV6, which differ by just 6 of 736 VP residues yet exhibit tissue and transduction differences, as models for receptor attachment site characterization.  AAV1 binds sialic acid (SIA), while AAV6 binds both SIA and heparan sulfate (HS). The crystal structures of AAV1 and AAV6 at 2.5Å and 3.0Å resolution showed 5 of 6 differing AAV1/AAV6 amino acids within the ordered VP structure localized proximal to the icosahedral three-fold axis and identified this capsid region as dictating important functions during infection. A series of reciprocal single residue mutations (AAV1 to AAV6 and AAV6 to AAV1) were thus generated to interrogate the role of the interior and exterior residues in dictating transduction efficiency. Quantitative comparisons of virus titers using ELISA, qPCR, and a HEK293 GFP infectivity assay show no significant differences in capsid assembly, genome packaging, and cellular transduction, respectively, among recombinant wild-type AAV1 and AAV6 and their mutations. In addition, to structurally characterizing the glycan receptor interaction of AAV1 with SIA,X-ray crystallography was used to map it’s binding site on the AAV1 capsid to a region  conserved in AAV6, suggesting that these two viruses utilize the same capsid region for this interaction. Site-directed mutagenesis and biochemical studies are underway to confirm this finding. With respect to HS binding, the docking algorithm DOCK6 was used as to localize a potential binding site to a region on the AAV6 capsid that contains an AAV1/AAV6 E531K residue difference consistent with previous mutagenesis and biochemical data. Data arising from these studies will aid the targeting of the AAV capsid to specific tissues or receptor populations for improved targeted gene delivery through recombinant DNA engineering.
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In the series University of Florida Digital Collections.
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Includes vita.
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Statement of Responsibility:
by Robert Ng.
Thesis:
Thesis (Ph.D.)--University of Florida, 2012.
Local:
Adviser: Agbandje-Mckenna, Mavis.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-08-31

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1 STRUCTURAL CHARACTERIZATION OF ADENO ASSOCIATED VIRUS SEROTYPE S 1 AND 6 GLYCAN INTERACTIONS By ROBERT NG 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 2012

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2 2012 Robert Ng

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3 To my parents, family and friends.

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4 ACKNOWLEDGMENTS I would like to begin by thanking my parents; Ng Mok Meng and Tan Moei Moei for the sacrifices they made to provide me with great opportunities and I am forever grateful because they continue to love, support and encourage me To my brothers (Robin Ng and Ebert Ng) and sisters (Jeny Ng Helen Ng, and Helni Ng), and grandparents, especially (in loving memory) Tan A Kie for valuable advice and warm companionships. I feel extremely indebted and honored to be mentored by Dr. Mav is Agbandje McKenna who believes in my abilities and patientl y nurture the scientist in me. I am always and forever grateful for the encouragement and opportunit ies she has provided me and the personal counseling sessions. This dissertation will be impossible to complete without her tremendous assistance. I would also like to thank Dr. Robert McKenna for being a great teacher and fielder of my incessant queries. My committee members, Dr. James B. Flanegan, Dr. Nicholas Muzyczka, Dr. ArunSrivastava and Dr. Richard Snyder have been extremely encouraging and helpful and provided critical inputs. I would also like t o thank the collaborators, Dr Richard Jude Samulski, Dr Jay Chiorini Dr. David F. Smith ; UF Vector core and the beamline scientists at CHESS for the resources and time invested in my work. I greatly appreciate the help offered by the administrative staff of the IDP, BMB and UF International Center. I am grateful to be a family a way from home. Dr. Lakshmanan Govindasamy, Dr. Hyun Joo Nam, Dr. Antonette Bennett, Dr. Brittney L. Gurda Mike DiMattia and Edward B. Miller deserve special thanks for teaching me the techniques used in the lab and also for invaluable advice; Dr. Art Rob bins, Dr. John Domsic, Dr. Katherine Sippel, Dr. Sujata Halder, Dr. Balendu Avvaru, Dr. Bala subramanian Venkat akrisnan Lawrence Tartaglia, Lauren Drouin, Michelle Zak,

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5 Mayank Aggarwal, Yu Shan Tseng, Bridget Lins, Harald Messer, Chris Boone, F arzaneh Tond nevis and Lin Ya Huang for their help and t o share great moments with me. Special thanks to Dr. Sujata Halder, who helped me during this last year in the program and the process of writing this dissertation. I would like to thank the high school, MD, and un dergraduate students that I had taught over the years, especially Angela McCall, Yomi Fabunmi, and Jordan Zeldin for being patient with me. For my one year warmness to share and teach me during my first year in US. I also thank my rotation mentors Dr. Sergei Zolotukhin and Dr. Scot t Greishaber who helped and mentored me imm ensely in my first year in IDP. I am grateful to have been blessed with such an amazing group of friends for being present at all the special events in my life including birthdays, farewells, new years and always being there when I needed you. Last but no t least, my heartfelt thanks to my lovely girl friend Yue Liu whose unconditional love, tolerance and support has helped me tremendously in this memorable journey.

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6 TABLE OF CONTENTS page A CKNOWLEDGEMENT S ......... 5 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVIATIONS ................................ ................................ ........................... 12 ABSTRACT ................................ ................................ ................................ ................... 14 CHAPTER 1 BACKGROUND AND INTRODUCTION ................................ ................................ 16 Gene Therapy and Gene Delivery Vectors ................................ ............................. 16 Non viral Delivery Vectors ................................ ................................ ................ 17 Cationic Polymer System ................................ ................................ ........... 17 Inorganic Nanoparticles ................................ ................................ ............. 18 Lipi ds ................................ ................................ ................................ ......... 19 Multifunctional Envelope type Nano Device (MEND) ................................ 20 Current Progress in Non viral Gene therapy ................................ .............. 20 Viruses as natural nucleic acid delivery systems ................................ .............. 21 Gammaretroviral Vectors ................................ ................................ ........... 22 Lentiviral Vectors ................................ ................................ ....................... 24 Adenoviral Vectors ................................ ................................ ..................... 25 Adeno associated Viral (AAV) Vectors ................................ ...................... 26 Examples of Viral Vector Mediated Gene Delivery Therapy ...................... 27 Gene Therapy for Severe Acquired Disease: Cancer ................................ 28 Gene Therapy for Severe Acquired Disease: Acquired Immune Deficiencies Syndrome (AIDS) ................................ ............................... 30 Gene Therapy for Genetic Disorder: Severe Combined Immuno deficiencies ................................ ................................ ............................. 31 Gene Therapy for Genetic Disorder: Lysosomal Storage Disease (LSD) .. 32 Gene Therapy for Genetic Disorder: Cystic Fibrosis ................................ .. 32 (LCA) 2 ................................ ................................ ................................ ... 33 Gene Therapy for Genetic Disorder: Hemophilia B ................................ .... 33 Lessons and Future Perspectives in Viral Vector Gene Therapy ............... 34 Introduction to Parvoviruses ................................ ................................ ................... 37 Viral Genome and Capsid: Architectures and Functions ................................ ......... 38 Parvovirus Capsid Structure ................................ ................................ ................... 40 Dependovirus: Overview of Adeno associated viruses (AAVs) ............................... 42 AAV Capsid Structure and Capsid Cell Surface Receptor Interactions ............... 43

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7 AAV Capsid Internalization and Post Entry Events ................................ ................. 48 Significance ................................ ................................ ................................ ............ 53 2 MATERIAL AND M ETHODS ................................ ................................ .................. 61 Production and Purification of AAV Virus Like Particles (VLPs) ............................. 61 VLP and Vector Concentration ................................ ................................ ............... 62 Negative Stain Electron Microscopy ................................ ................................ ....... 63 AAV6 VLP Structure Determination by Cryo Reconstruction ................................ .. 63 Structure Determination of AAV6 VLP using X ray Crystallography ....................... 64 Structure Determination of AAV1 VLPs with Sialic Acid (SIA) using X ray Crystallography ................................ ................................ ................................ .... 67 Site directed Mutagenesis of A AV1 and AAV6 ................................ ....................... 68 E.Coli competent cells ................................ ..................... 70 Cesium Chloride Plasmid Purification ................................ ................................ ..... 70 Production of Mammalian Expressed Recombinant Virions ................................ ... 72 Purification of rAAV using Ion Exchange Chromatography ................................ ..... 72 Biochemical Characterization of Recombinant Virions ................................ ............ 73 In vitro GFP Infectivity Assay ................................ ................................ .................. 74 In silico modeling and calculation of ligand binding to the AAV capsid ................... 74 Structural Comparison among AAV Serotype Structures ................................ ....... 75 3 STRUCTURE DETERMINATION OF ADENO ASSOCIATED VIRUS SEROTYPE 6 ................................ ................................ ................................ ......... 81 Introduction ................................ ................................ ................................ ............. 81 Results and Disc ussions ................................ ................................ ......................... 83 Structure of AAV6 VLP ................................ ................................ ..................... 83 Comparison of AAV6 Structure to Those of Other AAVs Pinpoints Capsid Regions That Control Differential Tissue Transduction Property ................... 88 4 STRUCTURALLY ANNOTATING AAV1 AND AAV6 GLYCAN BINDING INTERACTIONS ................................ ................................ ................................ ... 109 Introduction ................................ ................................ ................................ ........... 109 Results and Discussions ................................ ................................ ....................... 110 Crystal Structure of AAV1 ................................ ............... 110 Structural Comparison of AAV Serotypes at AAV1 SIA Interacting Residues 112 In silico Docking Mode l of AAV6 Heparan Sulfate ................................ ....... 114 Structural Comparison of AAV Serotypes at AAV6 HS Interacting Residues 116 5 CHARACTERIZING THE TISSUE TRANSDUCTION DETERMINANTS IN AAV1 AND AAV6 ................................ ................................ ................................ .. 128 Introduction ................................ ................................ ................................ ........... 128 Results and Discussion ................................ ................................ ......................... 128 6 SU MMARY AND FUTURE DIRECTIONS ................................ ............................ 134

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8 LIST OF REFERENCES ................................ ................................ ............................. 1 40 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 169

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9 LIST OF TABLES Table page 1 1 Current Ongoing Clinical Trials using Adeno associated Virus Vectors ............. 55 1 2 Adeno associated virus: Cellular Receptors, and Host Range ........................... 56 2 1 Nucleotide sequence of primers used in this study. ................................ ............ 77 3 1 Am ino acid differences between AAV1 and AAV6 and their reported mutants ... 96 3 2 Data Collection, Reduction and Refinement Statistics a ................................ ..... 97 3 3 structures overall and for VRI and VRIV ................................ ............................. 98 3 4 Comparison of residues reported to be involved in heparan sulfate and sialic acid binding for AAV6, AAV1, AAV2, AAV5, andAAV VR942 and the amino acids at equivalent positions in each virus ................................ .......................... 99 4 1 Data Collection, Reduction and Refinement Statistics a ................................ .... 119 4 2 structures overall and for SIA interacting regions (SIAIR) ................................ 120 4 3 structures overall and for HS interacting regions ................................ .............. 121 4 4 Structure alignment of AAV residues involve in SIA and HS interaction. .......... 122 5 1 Biochemical characterization of AAV1 and AAV6 reciprocal mutants a ............. 130

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10 LIST OF FIGURES Figure page 1 1 Genome architecture of gammaretrovirus, lentivirus, Adenovirus, and Adeno associated virus (AAV). ................................ ................................ ...................... 57 1 2 Schematic of the life cycle of Adeno associated Viruses. ................................ ... 58 1 3 Conserved secondary structure superposition of VP for one member from every genus in Parvovirinae subfamily. ................................ .............................. 59 1 4 Surface representation of AAV2. ................................ ................................ ........ 60 2 1 Schematic flow chart of of AAV1 and AAV6 VLP expressions, purifications and structural determinations. ................................ ................................ ............ 78 2 2 Schematic flow chart of mutagenesis and transduction phenotype studies of AAV1 and AAV6 SIA interaction residues mutants. ................................ ............ 79 2 3 Stick representations of sialic acid (SIA) and heparan sulfate (HS). ................... 80 3 1 AAV6 structure.. ................................ ................................ ............................... 101 3 2 Cr ystal structure of AAV6. ................................ ................................ ................ 102 3 3 Comparison of available AAV crystal structures.. ................................ ............. 103 3 4 AAV6 DNA binding site.. ................................ ................................ ................... 104 3 5 Locations of amino acid differences in AAV1 / AAV6 capsids. ......................... 105 3 6 Comparison of AAV surface residues.. ................................ ............................. 106 3 7 Stabilizing interactions for the K531 loop.. ................................ ........................ 108 4 1 Crystal structure of AAV1 ................................ ..................... 123 4 2 Superposition of AAV1 SIA crystal structure with other AAV structures ........... 124 4 3 Molecular docking model of AAV9 crystal structure with GAL using patch DOCK. ................................ ................................ ................................ .............. 125 4 4 Surface trimer representation of AAV2 and AAV6 showing in silico calculation of HS interaction region on AAV2 and AAV6 trimer molecules using DOCK6... ................................ ................................ ................................ .......... 126 4 5 Superposition of AAV6 HS in silico model with other AAV structures. ............ 127

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11 5 1 Silver stain SDS PAGE of purified r AAV1 and rAAV6 reciprocal mutants. ...... 131 5 2 Negative stain electron microscopy (EM) of purified rAAV1 and rAAV 6 wild type s and reciprocal mutants. ................................ ................................ ........... 132 5 3 Green Fluorescence Protein (GFP) Infectivity Assay using HEK293 cells.. ..... 133 6 1 Structural alignment of crystallographic ordered VP amino acid sequences (~217 736) of AAV1, AAV2, AAV4 and AAV6. ................................ ................. 138 6 2 Trimer surface representation of AAV6 crystal structure showing glycan interacting regions.. ................................ ................................ .......................... 139

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12 LIST OF ABBREVIATION S AAV A deno associated virus ADV A leutian mink disease virus BPV1 bovine parvovirus 1 CAR coxsackie and adenovirus receptor CFG consortium for functional glycomics Cryo EM cryo electron microscopy CPV canine parvovirus DMEM DNA deoxyribonucleic acid EDTA ethylene diaminetetraacetic acid EGFR epidermal growth factor receptor ELISA Enzyme Linked Immunosorb e nt Assay EM electron microscopy FGFR1 fibroblast growth factor receptor 1 FPV feline panleukopenia virus H 1PV human tumor isolated 1 parvovirus HBoV human bocavirus HEK Human Embryonic Kidney HGFR hepatocyte growth factor receptor HIV human immuno deficiency virus HS heparin sulfate HSP G heparan sulfate proteoglycan HSV herpes simplex virus ITR inverted terminal repeats

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13 LamR laminin receptor mRNA messenger ribonucleic acid Neu5Ac N acetylneuraminic acid NLS nuclear localization signal NT nucleotides PBS phosphate buffer saline PDGFR plat elet derived growth factor receptor PEG polyethylene glycol PLA 2 phospholipase A 2 PPV porcine parvovirus QPCR quantitative PCR RFU relative fluorescence units RNA ribonucleic acid SIA sialic acid ssDNA single stranded deoxyribonucleic acid TfR transferrin receptor VLPs virus like particle VP viral protein

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14 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy STRUCTURAL CHARACTERIZATION OF A DENO ASSOCIATED VIRUS SEROTYPES 1 AND 6 GLYCAN INTERACTIONS By Robert Ng August 2012 Chair: Mavis Agbandje McKenna Major: Medical Science Biochemistry and Molecular Biology Adeno Associated Viruses (AAVs) are small ssDNA viruses with the ability to package non genomic DNA for therapeutic gene delivery. Due to their non pathogenicity and ability to transduce non dividing and dividing cells, these viruses have generated significant interest in their development as therapeutic vecto rs. These properties dictate the tissue of choice for a particular gene delivery application. Towards understanding the capsid determinant(s) of these functions, we have employed AAV1 a nd AAV6, which differ by just 6 of 736 VP residues yet exhibit tissue and transduction differences, as models for receptor attachment site characterization. AAV1 binds sialic acid (SIA), while AAV6 binds both SIA and heparan sulfate (HS). The crystal structure s of AAV1 and AAV6 at 2.5 an d 3.0 resolution showed 5 of 6 differing AAV1/AAV6 amino acids within the ordered VP structure localized proxima l to the icosahedral three fold axis and identified this capsid region as dictating important functions during infection. A series of reciproca l single residue mutations (AAV1 to AAV6 and AAV6 to AAV1) were thus generated to interrogate the role of the interior and exterior residues in dictating transduction efficiency. Quantitative comparisons of virus titers using ELISA, qPCR and a HEK293 GFP infectivity assay show no significant

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15 differences in capsid as sembly, genome packaging and cellular transduction respectively, among recombinant wild type AAV1 and AAV6 and their mutation s. In addition, to structur ally characterizing the glycan receptor interaction of AAV1 with SIA, X ray crystallography was used to map binding site on the AAV1 capsid to a region conserved in AAV6, suggesting that these two viruses utilize the same capsid re gion for this interaction. Site directed mutagenesis and bio chemical studies are underway to confirm this finding. With respect to HS binding, the docking algorithm DOCK6 was used as to localize a potential binding site to a region on the AAV6 caps id that contains an AAV1/AAV6 E5 31K residue difference consistent with previous mutagenesis and biochemical data. Data arising from these studies will aid the targeting of the AAV capsid to specific tissues or receptor populations for improved targeted gene delivery through recombinant DNA engineering

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16 CHAPTER 1 BACKGROUND AND INTRO DUCTION Gene Therapy and Gene Delivery Vectors Gene therapy can be defined as the transfer of new functional genetic material to the cells of an individual resulting in therapeutic benefit ( 216 ) Since the first successful gene therapy trial by Rosenberg et al. using retrovirus to deliver the genetic marker neomycin into melanoma patients in 1990 ( 253 ) gene therapy has developed immensel y. Within a decade, there were more than 550 clinical trials underway (http://www.wiley.com/legacy/wileychi/genmed/clinical). In 2012, over 1600 clinical trials had been completed or underway ( 99 ) However, there are several challenges remain ing that need to be overcome. These include: complexity of multi gene disorders, the design of an effective delivery vector, tissue specificity, the control of transgene expression, patient immune rejection, and vector pathogenicity ( 54 ) In order to overcome these challenges, more efforts are required to understand the basic molecular biology of the vectors which can be used to improve safe and efficiency. One ke y determinant for the clinical success of gene therapy is the efficiency of gene transfer in to the cells of patient s The gene delivery vector must possess special characteristics and mechanisms that allow it to pass through apolar and hydrophobic cellular membranes and be stable enough to deliver its cargo into the nucleus ( 116 ) In order these include engineering the vector from immune rejection in the extracellular milieu carrying DNA to penetrate through cellular membrane s protecting the DNA from cytoplasmic (enzymatic) degradation factors, and effectively expressing the transgene product in the nu cleus. Moreover, host safety and high production yield also play major

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17 roles in the development of a gene therapy system. Generally, gene therapy vector can be divided into non viral and viral vectors ( 116 ) Non viral Delivery Vectors Besides naked DNA injection which is generally facilitated by high pressure injection or electroporation ( 140 159 ) current ongoing non viral gene delivery involves either a cationic polymer system, a nanoparticle system, lipoplex, or a multifunctional envelope type nano device (MEND) as which described below ( 145 ) Cationic Polymer System Based on their natural positively charged properties, various cationic polymers have been shown to effectively condense anion rich DNA, termed polyplexes, and attach to highly sulfated glycosaminoglycans (GAG) on the cell surface ( 162 ) This i s then followed by endocytosis and plasmid expression. Examples of cationic polymers used for gene therapy include polyethylenimine (PEI), polypropyleniminedendrimers, poly L lysine (PLL), chitosan, and polyethylene glycol (PEG). Since its initial use in 1 995, PEI has been the most extensively used cationic polymer system for gene delivery and it performs the best among cationic polymers ( 40 162 ) PEI has a high transfection efficiency that can be attributed to th e buffering capacity of its amine groups spon ( 13 26 ) However, the tendency of PEI/DNA mixtures to form aggregates and accumulate in lung can cause cellular toxicity which re duces their potential applicability as a gene delivery vector in vivo ( 226 ) To overcome this setback, various formulations of PEG, amino acids, cholesterol deri vatives, and PEI molecules have been shown to create amphiphilic polymer carriers which result in increase of DNA condensation and

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18 biocompatibility as well as a 3 to 11 fold increase in transfection efficiency ( 15 46 280 ) Several imp ortant factors affecting efficiency/ cytotoxicity of pol y plexes are ionic strength, zeta potential molecular weight, degree of branching, and particle size. Efficiency of polymer DNA condensation depends largely on the +/ charge ratio and zeta potential o f the polyplexes. The closer the +/ ratio is to 1, the lower the zeta potential is, indicating an increase in the tendency of aggregation and cellular toxicity. Most PEI formulations studied were prepared using PEI with molecular weight of 10 800 kDa ra ng e and consist of linear and branched PEIs ( 162 ) In order to improve in vivo specific targeting and transgene efficiency, small ligand and glycan modifications (e.g. transferrin and glucose) on the functional group of cationic polymers have been studied ( 177 ) Inorganic Nanoparticles Recent advances in nanotechnology have inspired the application of nanoparticles (NPs) as gene delivery systems ( 290 ) Upon administration, specific proteins attach and adsorb nanoparticles based on their size, hydrophobicity, and surface characteristic. Adsorption of proteins to the nanoparticle surface changes its overall physicochemical properties including hydrodynam ic diameter and surface charges ( 154 173 ) These specific proteins may then determine the transfer efficiency of the nanoparticles. Another term used in nanoparticle delivery system is magnetofection which involves ds magnetic nanoparticles ( 239 ) This application has allowed for a safe and effective in vivo delivery system using superparamagnetic iron oxide nanoparticles (SPIONs) with a combination of polyplexe s (SPIONs PAA PEI) to treat adenocarcinoma ( 242 ) In general gene delivery using nanoparticles provides several attractive features which are not present in other non

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19 viral systems. First, nanoparticles provide much lower cytotoxicity, genotoxicity, and immunogenicity. Second, when combined with liposomes, they show much higher gene transfer efficiency than liposomes alone. Third, due to their small size, nanoparticles can travel with blood circulation and pass through the blood brain b arrier. This makes them a perfect vector for central nervous system (CNS) gene delivery. Iron oxide nanoparticles coated with PLL serves as a good example as in vivo studies show high gene transfer efficiency for glia and brain targeting following intraven ous injection ( 308 ) Other examples are calcium phosphate and organically modified silica (ORMOSIL) ( 33 ) Lipids Similar to polyplexes, this delivery system can self assembled based on the electrostatic interactions between lipids and DNA, termed lipoplexes. Based on lipid types, formulations of ongo ing lipofection studies involve cationic (1,2 dioleoyl 3 trimethylammonium [N dimethylaminoethane) carbamoyl] cholesterol (DC Chol)) and neutral (dioleophosphatidylethanolamine (DOPE)) liposomes ( 296 ) Similar to polyplex, various lipid formulations have been shown to affect DNA condensation, packaging size, cellular toxicity, and transfection efficiency. The physicochemical characterist ics of lipoplexes range between 190 240mV surface potential and pH 10 11.5. The +/ ratio and zeta potential of lipoplexes should be above 1 ( 176 238 ) While lipofection offers an attractive attribute due to its less limited packaging size, studies have also shown that in vivo transfection efficiency in vivo and in vitro transfection is 40 80nm and 200 400 nm, respectively ( 322 ) Studies have shown that PLL or antibody modifications of lipoplex surface will reduce aggregation and improve lipofection efficiency. Lipofe ction efficiencies depend not only o n the cellular plasma

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20 membrane permeability of the liposomes, but also on their endosome destabilizing activity. Certain lipoplexes (glycyrrhizin (GA) and tocopherol ester succinic acid (TS)) are attractive due to their pH sensitivity which has been shown to improve gene transfer efficiency in CV 1 cells (monkey kidney fibroblast) by 100 fold ( 64 ) An example of lipoplexes that is commercially available is lipofectamine which is widely used in mammalian cell plasmid transfections ( 277 ) Multifunctional Envelope type Nano Device (MEND) MEND, introduced by Hideyoshi Harashima ( 166 ) is a novel delivery system composed of the condensed core of polyplexes containing nucleic acid which is encapsulated by a lipid envelope (liposome). While in vivo reporter gene administration using either positively charged lipoplex or polyplex have dem onstrated limited liver delivery efficiency due to high tendency of aggregation and accumulation in lung cells, the MEND delivery system provides a higher level of luciferase activity in liver ( 314 ) It is shown that this improved liver delivery profile is attributed to the lower accumulation tendency of the MEND system in lung. Modifications o f MEND systems have also been studied which include the incorporation of pH sensitive membrane lytic GALA (Glu Ala Leu Ala) and other cell targeting proteins in the system ( 257 ) Curr ent Progress in Non viral Gene therapy Compared to viral gene delivery (described below), non viral gene delivery offers a more controlled production, relatively safer delivery profile and low or no immune rejection. In addition, its flexible modification s, highly assorted formulations, and ease of manipulations have resulted in significant increase in cell targeting, endosomal destabilization, and biocompatibility which has drawn huge attention to investigating this system for the development of gene deli very systems ( 184 ) However, there is a low

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21 level of transfection efficiency due to structural polymorphism or heterogeneity associated with preparations of lipoplexes and polyplexes. Thus much effort will be required to attain the high level of transfection efficiency a nd transgene expression obtainable by viral delivery approaches. Studies have been performed combining these systems with viral proteins which has resulted in a significant increase of transfection efficiency ( 20 227 297 ) which can be implemented for the development of better and promising approaches for gene d elivery. Viruses as natural nucleic acid delivery system s Since the first gene transformation s in the 1970s using DNA, RNA, or retroviruses ( 109 219 ) these discoveries have inspi red researchers to utilize viruses for development as vector s for gene therapy technology. While gene delivery using non viral vector s has the advantages of larger production yield and low immunogenicity, gene delivery using viral vectors has been shown to have significant ly higher transduction efficiency into patient cells. The difference in transduction efficiencies results from the natural life cycle properties of virus es which have evolved to infect and replicate very effectively in their natural specif ic hosts and cell types ( 117 125 ) Viruses have been defined as obligate intracellular parasites that infect all domains of life, from bacteria and archaea to eukaryotes and may cause severe disease in their host ( 74 169 ) Basic virus structure, called nucleocapsid, consists of the genomic material (DNA or RNA) which is encapsulated and protected by a protein shell (capsid). Some viruses, so called envelope d viruses, have an exte rnal lipid membrane envelope to protect the nucleocapsid and carry glycoproteins which serve as the ligand for receptor attachment on cell surfaces. The viral genome encodes various multifunctional regulatory, replication, and assembly viral proteins which are mostly toxic for host cells. Moreover,

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22 in order to undergo efficient replication, most viruses have evolved to exploit host cell machineries for their successful replication. Viruses can be classified based on their route of transmission, life cycle, genomic structures, and capsid morphologies ( 74 ) The most recent International Committee on Taxonomy of Viruses (ICTV) report classifies viruses into 6 orders, 94 families, 395 genera and 2475 identified species, with 72 virus families not assigned to an order ( 29 ) (http://www.ictvonline.org/). Despite the differences in life cycle among various species, most vi ruses are known to share common properties including viral genome packaging, specific host / c ell targeting properties, and efficiency of cellular internalization. Hence, the overall idea in viral gene therapy is to genetically engineer viruses to carry therapeutic genes and effectively deliver them into the target cells. This strategy substitutes most of the wild type viral genome with the gene of interest (including promoter and polyadenylation signal s ) resulting in the delivery of the target gene into patient cells without viral replication. In the case of recombinant viral vector production, mai ntenance of cis acting elements (packaging signals and viral specific replication elements) are required for viral replication and are transiently expressed in the packaging cells ( 117 125 ) Currently there more than 65% of ongoing clinical trials (n=1222/1786 in 2011) utilizi ng viral vectors as the delivery system, with examples including gammaretrovirus, lentivirus, adenovirus and Adeno associated virus (AAV) ( http://www.wiley.com/legacy/wileychi/genmed/clin ical ) (Figure 1 1). Gammaretroviral Vectors The most clinically utiliz ed prototype of retroviruses is Moloney murine leukemia gammaretrovirus (MuLV) ( 118 ) Due to its natural tropism to cause and infect cancer cells in host (murine) cells, it has gained popula rity as a viral vector for human cancer

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23 therapy. MLV is a positive single stranded (ss) RNA enveloped virus with the particle size of approximately ~ 90nm. MLV infects and replicates within its host cells through reverse transcription to produce double stra nded (ds) DNA (provirus), which is then integrated into the host chromosomes. The wild type gammaretrovirus genome ( ~ 9 11kb) consists of three open reading frames (ORF); gag po l and env (which code for capsid proteins, reverse transcriptase polymerase, a nd envelope proteins, respectively) vector, the gene of interest can be inserted by removing the three ORFs. However five cis replicating elements are indispensable, the p shown that several accessory elements can increase the transgene expression and/or regulate neighboring gene expression caused by insertion mutation, e.g. woodchuck hepatitis post transcriptional regulatory element (WPRE) and removal of the enhancer inactivatin g (SIN) vector. The production of recombinant retroviral vector s was achieved by transfecting the recombinant plasmid into transformed murine cells expressing retroviral Gag, Pol and Env. Within the packaging cells, the recombinant therapeutic gene, using the packaging signal, is inserted into newly assembly recombinant gammeretroviral virions. Upon administration, these replication defective virions will release and integrate the therapeutic gene into the host chromosome and express the transgene In ord er to design more improved tissue targeting and specificity, much effort has been put toward the modification of the Env proteins of retroviral vectors. An example is

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24 the VSG G pseudotype retroviral vector, in which genetically engineered vesicular stomati tis virus (VSV) G protein is used to replace retrovirus Env proteins in the packaging cells, These recombinant virions display VSV G proteins on their envelope membrane which wa s shown to have higher tissue transduction efficiency in various cells ( 9 317 ) Lentiviral Vectors While gammaretroviral vectors are known to only infect replicating cells, lentiv iruses can infect both replicating and non replicating cell lines ( 108 174 ) This striking feature is attributed to the presence of accessory proteins (integrase, matrix and Vpr) which form a pre integration complex (PIC) with the nuclear pore ( 278 ) Human Immuno deficiency Virus (HIV) type 1 is the prototype member of Lentivirus genus in Retrovir idae family. In addition to the ORFs of gammaretrovirus, the HIV 1 genome encodes for two regulatory ( Tat and R ev ) and four accessory proteins (N ef, V pr, V pu and V if) which are important for the wild type HIV life cycle. Similar to the design of the gammar etroviral vectors, the production of lentiviral vectors requires the maintenance of cis replicating elements. Since the first recombinant lentivirus used for in vivo gene transduction, there are currently three generations of lentiviral vectors available ( 267 ) In the first generation of the HIV delivery system ( 221 ) all HIV genes were expresse d in the provirus, except for env which was complemented by an additional plasmid expressing VSV G protein. Since the packaging cells express and carry ~80% of the viral genome, this approach raises concerns regarding cellular toxicity during human gene de livery. Hence, second generation vectors were designed by removal of accessory genes; include vpr vif and nef and carried only LTR, gag pol tat and rev However, these vectors still exhibited

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25 cellular toxicity. The current generation of lentiviral ve ctors are less complex and expresses only three ( gag pol and rev ) out of nine HIV wild type genes ( 198 ) thus offering safer gene delivery. The most recent progress on the production of lentiviral vector in clinical studies involves transfection of packaging cells with four different plasmids. The first plasmid inactivating driven by a heterologous promoter (RSV). T he remaining three plasmids encode Gag and Pol, Rev, and VSV G, respectively. Within the packaging cells, recombinant lentiviral vectors will replicate and package the gene of interest. Adenoviral Vectors Adenovirus is a ds DNA ( ~ 36kb) non enveloped isocah e d ral virus with a particle size of ~90nm in diameter ( 30 302 ) Currently there are more than 100 members of Adeno virinae family which infect a broad range of vetebrates, including humans. Fifty seven different serotypes have been isolated from humans and most of these are associated with upper respiratory infections in children. These different serotypes can be class ified into 7 subgroups (A G). Serological studi e s show that ~60% of children populations are seropositive for Adenovirus (Ad) subgroup C ( 69 ) and Ad5 (subgroup C) is the most commonly used for gene delivery vector design. Due to its natural host tropism in humans, administration of adenoviral vectors el icits pre existing cytotoxic T and B cell responses which limits transduction efficiency during gene delivery applications. In order to overcome this barrier, much effort has been directed toward the development of less toxic, low immunogeni c and specific cell targeted Ad vectors. The wild type Ad genome consists of five early transcription units (E1A, E1B, E2, E3 and

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26 E4), two delayed early transcription units (IX and IVa2), and one major late (ML) transcription unit. In the first generation Ad vectors ( 81 ) wild type Ad E1 and E3 genes were removed from the recombinant virus with the goal to ensure that the adenoviral vector was replication defective and to increase the packaging size to 8.3 kb, respectively. However in vivo administration using this recombinant vector resulted in high inflammatory responses ( 190 ) The next generation of Ad vectors which provided larger packaging size (14kb) by removal of two more genes (E2 and E4) still did not overcome the problem of sign ificant immune response and, moreover, transgene expression using this vector is much reduced compared to the previous E2 or E4 containing vector ( 293 ) The current generation of Ad vectors provide the largest packaging size (37kb) and safest delivery vector by replacing the complete genome of Ad virus with therapeutic genes, which are called gutless or gutted vectors ( 14 47 ) During vector production, the replication defective property of this gutless vector is complemented by the expression of a replication competent helper vector in the packaging cells ( 235 ) Adeno associated Viral (AAV ) Vectors Adeno associated virus is a small ssDNA (~5kb) parvoviruses with a particle size of ~26nm in diameter ( 31 42 ) It belongs to Dependovirus genus and requires helper viruses (e.g. Ad virus and Herpes Simplex Virus (HSV)) to undergo successful lytic infection. Since its first discovery as contaminant with an Ad virus preparation in 1965, no disease or pathogenicity has been reported associated with AAV infection. Due to its non pathogenicity, AAV has drawn huge attention for the development as a gene delivery vector. To date, over 100 gene sequences have been isolated from human/non human primate tissues and twelve serotypes have been described (AAV1

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27 AAV12), in which AAV2 is the most clinically utilized serotype ( 113 ) However, differential tissue tropism and transduction efficiencies among various serotypes have inspired the development of other serotypes for as more tissue specific vectors ( 326 ) AAV1 and AAV6 for muscle and cardiac gene delivery, and AAV9 for brain delivery. The wild type AAV genome contains two ORFs; rep and cap which encode for viral proteins important for v iral replication and capsid assembly, respectively. These ORFs are flanked by two inverted terminal repeats (ITR) which contain hairpin shaped sequences important for viral replication and genome packaging. These repeats are the only cis acting elements pr esent in AAV. AAV vectors are generated by co transfection of two or more plasmids into packaging cells; a common example is transformed HEK293 cells. The first plasmid carries the recombinant therapeutic gene flanked by two ITRs. The other remaining plasmids carry rep cap and Ad helper genes. Recombinant virions produced can be isolated and purified from the cell lysates. Compared to retroviral and adenoviral vectors, AAV vectors show no proto oncogene / cellular toxicity and relatively less immunog enicity, respectively. Examples of Viral Vector Mediated Gene Delivery Therapy By 2012, more than 1200 clinical trials were underway using viral vector gene therapy (http://www.wiley.com/legacy/wileychi/genmed/clinical). There were several issues encounter ed during these years, e.g., including the death of a patient in the trial using an adenoviral vector to treat ornithine transcarbamy lase (OTC) deficit ( 25 178 201 ) the devel opment of leukemia in two patients treated wi th gammaretroviral vector for X linked SCID ( 51 ) Despite the problems mentioned, gene therapy has been in the spotlight due to the successful development of treatments for inherited diseases, e.g.

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28 including immuno deficiency and bone marrow transfer ( 10 ) Recently, more and more application oriented AAV vectors have been successfully used for treatment of retinal diseases and several neurodegenerative diseases ( 32 50 250 ) Since around 2005, the number of gene therapy clinical trials has increased back to ~100 per year hemophilia, and cardiovascular diseases. Notably, less than 10% of the trials nowadays are aim ed at monogenic inherited disorders, which initially inspired the whole idea of gene therapy ( 116 ) The Wiley group ( http://www.wiley.com/legacy/wileychi/genmed/clinical ) has reported that more than 1780 gene therapy clinical trials have been undertaken worldwide in which ~64% are performed for the treatment of cancer related diseases and that over two thirds of clinical trials are based on viral vector administration Gene Therapy for Severe Acquired Disease: Cancer Currently, the alternatives for the treatment of tumors include the introduction of a tumor suppressor gene f or cell apoptosis, the introduction of suicide genes, using anti angiogenesis factors and immune regulatory genes, or the application of onc olytic viruses. The most common loss of tumor suppressor activity arises from a defect in p53, RB1 and BRCA1. Previous studies had indicated that reactivation of these tumor suppressors in tumor cells res ulted in apoptosis and elimination of tumors; as we ll as causing the cells to be more sensitive to chemotherapy. An example of a clinical trial for cancer treatment was the administration of an adenoviral vector expressing normal p53 protein into 25 non small cell lung cancer (NSCLC) patients ( 279 ) The outcome of this study was a 28 64% decrease in tumor progression up to 14 months.

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29 Alternative treatment s for cancer cells include using a suicide gene. This concept exploits the capability of herpes simplex viral thymidine kinase (HSV TK) to phosphorylate and conver t ganciclovir (GCV) or acyclovir (ACV) into toxic drugs which will terminate DNA replication ( 241 ) Selective infection of viral vectors into cancer cells will then eliminate T K+ cancer cells. Examples of viral vectors using this strategy are an AAV vector for bladder cancer, an Ad viral vector for breast and colorectal cancers, a lentiviral vector for prostate cancer, and a HIV vector for T cell leukemia ( 16 194 215 233 294 ) Immune regulatory factors also represent good candidates for the treatment of cancer ( 181 299 ) This concept is exploited by gene thera py to inhibit cancer cell differentiation through immune stimulatory factors, for examples cytokine (IL 2, IL 12, GM CSF), co stimulatory molecules (B7), major histocompatibility (MHC) ( 136 189 321 ) Studies have shown that administration of viral vectors resulted in gene modification o f tumor cells, stimulation of reactivation of the host's immune system and prevention of tumor recurrence ( 273 ) However, this treatment is less effective in later stage cancer patients. This might be due to immunosuppressive factors released by cancer cells to suppress immune response. To overcome this barrier, future progress is directed toward the development of geneticall y modified dendritic cells (DC) which express higher level of cytokines and promote T cell activation ( 104 ) The most recent cancer cell killing strategy involves the use of the E1B deficient Ad vector, called ONYX 15 ( 139 254 ) It was reported that the Ad E1B protein can inactivate the p53 tumor suppressor gene which is mutated in most cancer patients. In normal cells, p53 will limit Ad replication through interaction with E1B protein ( 206 )

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30 Using the ONYX 15 Ad vector, selective oncolytic strategy was feasible and resulted in relatively safe and significant (>50%) tumor regression ( 112 164 218 ) In addition to Ad several viral vectors which have different oncolytic mechanisms were also studied, e.g. Herpes Simplex Virus (HSV), Newcastle disease virus, Reovirus, and Vesicular Stomatitis Virus (VSV). Anti angiogenesis can be def ined as the application of specific compound to reduce or eliminate formation of blood vessels (vasculogenesis) ( 107 ) Most tumors secrete various growth factors to induce capillary growth in order to supp ort thei r expansion. Studies have shown that Ad vector s expressing the extracellular domain of human vascular endothelial growth factor receptor (hVEGFR) were able to infect human lung cancer cells and inhibit VEGF stimulated DNA synthesis. Following the study, in vivo administration of treated cells into a mouse model decreased colorectal tumor size 10 days post injection and enhanced apoptosis in vivo ( 259 ) Another in vivo gene delivery using an Adenoviral vector was performed to investigate the treatment of Transitional Cell Carcinoma (TCC) in the bladder o f a mouse. This study utilized A d mediated an antisense bFGF ( bFGF AS) strategy to reduce microvessel density and enhanced endothelial cell apoptosis ( 146 ) Gene Therapy for Severe Acquired Disease: Acquired Immune Deficiencies Syndrome (AIDS) One alternative of gene therapy applications for AIDS patients is to generate genetically modified hematopoietic cells that may exhibit intracellular expression of anti HIV genes to inhibit HIV replication and pathogenicity. To date, most AIDS therapeutic strategies involve anti HIV drugs that target Gag and Pol. Two regulatory proteins (Tat and Rev) and four accessory proteins (Nef, Vif, Vpu, Vpr) have been identified to be

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31 important for HIV replication ( 249 ) Rev responsive element (RRE) transcripts were identified to suppress HIV replication through interaction with Rev. When applied into human CD4+ cells, expression of RRE (RRE decoys) resulted in no detectab le HIV replication ( 19 ) Other promising approaches a s to treat HIV are RNA interference (RNAi) tools which target and breakdown viral RNA coding for accessory proteins ( 249 ) Gene Therapy for Genetic Disorder: Severe Combined Immuno deficiencies Severe Combined Immuno deficiencies (SCID) is a class of inherited immune disorders in which the functions of both T cells and B cells of the adaptive immune response system are impaired. The t wo most common SCIDs are X linked SCID and adenosine deaminase (ADA) deficiency In X linked SCID patients, mutations in the functional chain which is shared by various cytokine receptors. In healthy patients, interactions between cytokines and their receptors tri gger cell signaling which are important for T and B cells differentiation. Failure in interleukin signaling will result in low expression or absence of T cells and natural killer (NK) cells and non functional B cells. In 1990, gene therapy introduced a new era of medicine by using a retroviral vector to and NK cells leading to the recovery of immuno compromised abnormalities. The second common SCID is caused by ADA de ficiency ( 281 ) Adenosine deaminase is a zinc metalloenzyme whose function is important for purine breakdown. In ADA SCID patients, non functional ADA results in the accumulation of deoxyadenotide (dATP) which inhibits the activity of ribonucleotidereductase to generate deoxynucleotide (dNTs) ( 100 ) Imbalance of dNT synthesis results in limited lymphocyt e proliferation causing the immune system to be compromised. Clinical

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32 studies using retroviral vectors have shown long term (2 years) ADA expression in T and B cells of SCID patients ( 11 ) Gene Therapy for Genetic Disorder: Lysosomal Storage Disease (LSD) Gaucher disease (GD), named after the founder Phillipe Gaucher in 1882 ( 115 ) is the most common type of genetic lysosomal storage disorder (LSD) caused by deficient glucocerebrosidase (GCase) gene expression resulting in accumulation of glycolipids (glucocerebroside) in macrophages and enlargement of the liver and spleen ( 44 ) To date, researchers have pursued the cure of GD using retro viral, lentiviral, and AAV vectors packaging the glucocerebrosidase gene. Using a GD mouse model, preclinical studies ha ve shown promising and relative ly long (20 weeks) in vivo ge ne expression which results in no dete ctable of Gaucher cells in spleen and liver ( 103 143 ) Gene Therapy for Genetic Disorder: Cystic Fibrosis Cystic Fibrosis (CF) is an autosomal recessive disorder of secretory glands mainly in the lungs ( 248 ) This inherited disorder is caused by mutations in the gene coding for the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein (located on q31 .2 locus of chromosome 7) which is important for the balance of sodium and chloride ions across epithelial membranes. In most CF patients, the mutated CFTR gene expresses a mis folded CFTR which results in an imbalance of electrical potential inside and ou tside the cell and blockage of ion channels that cause remodeling and infection in the lung. Early clinical studies were attempted to deliver the normal CFTR gene using Ad vector s to the epidermal lung cells ( 121 122 ) It was shown that first administration gave low or limited gene expression. Hence, second and additional attempts were aimed at increasing the amount and duration of gene expression. However, due to pre

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33 existing immune responses the therapeutic effects became very limited. The majority of CF patients also have pre existing neutralizing antibodi es against AAV making them also ineffective as delivery vectors ( 131 ) Recently, repeat administration of lentiviral vectors had been shown to potentially evade host immune rejection and exhibit long term gene expression ( 121 ) Leber's Congenital Amaurosis type 2 (LCA2) is an inherited eye disease characterized by vision loss, retinitis pigmentosa, and severe retinal dysfunction. Visual loss in LCA2 is caused by the mutation in the RPE65 gene (located on chromosome 1 locus p31) which results in the combination of a biochemical blockade of the retinoid cycle and degeneration of retinal photoreceptors ( 200 ) In the normal photo transduction process of retinal epithelium cells, the Retinal Pigment Epithelium 65kDa protein (RPE65) functions in a multistep process (visual cycle). This process converts retinols between trans and cis conformations which is important for electric signa l transmission and visual pigment regeneration in photoreceptor (rods and cones) cells ( 55 ) RPE65 gene therapy using AAV2 vector has yield significant visual improvement among three tested patients in 2008. Visual perf orma nces were analyzed 90 days post treatment and demonstrated an increase of 50 fold for cones and up to 63,000 fold for rods photoreceptor cell regenerations ( 72 138 196 ) Gene Therapy for Genetic Disorder: Hemophilia B Hemophilia B is an X linked blood clotting disorder caused by mutation at gene coding for Factor IX ( 35 90 ) Factor IX deficiency leads to an increased propensity for hemorrhage. After g etting injured, hemophilia patients will ble ed for a longer time than healthy patients. Factor IX deficiency may also lead to internal bleeding, in which,

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34 otherwise treated, will lead to organ damage. Recently, it was reported that AAV gene therapy has tre ated six hemophilia B patients who produce less than 1% than normal levels of Factor IX. Studies resulted in spontaneous blood clotting and sustained expression of Factor IX in four out of six patients ( 240 ) While high dose s of vector administration resulted in elevated levels of transaminase and liver enzymes, consumption of steroids has been shown to ameliorate the side effect of high dose vector application. These striking results suggest the potential for the use of a similar approach for the treatment of hemophilia A (deficiency in Factor VIII) ( 319 ) Lessons and Future Perspectives in Viral Vector Gene Therapy Gammaretroviral vectors accounted for about 20% of the trial s in the 1990s. However, due to its capability to only infect dividing cells and its cellular toxicity caused by insertional mutagenesis, the use of these viral vectors is now less widespread The PIC of these virus, inc luding viral cDNA and a number of viral and host proteins, as well as viral integrase enzyme cannot get access to the nucleus except when the nuclear membrane breaks down during mitosis ( 210 ) In our body, most neurons, skeletal muscle cells, cardiomyocytes, endothelial cells, and peripheral blood lymphocytes are classified as non dividing cells. Hence, the use of gammaretroviral vectors is limited to ex vivo delivery to cells that are activel y dividing. In the case of insertional mutagenesis into the human chromosome, mounting evidence has demonstrated that these viruses preferentially target the promoter region of actively transcribed genes in human genome ( 60 80 ) In 2002, two children treated with gammaretroviral vector for X linked SCID developed acute lymphoblastic leukemia (T ALL) ( 128 167 ) This mutation has been shown to be due to the insertion of the retrovi r al vector in to the L MO2 (LIM domain 2) proto oncogene ( 129 205 ) This attracted the attention and concern of the scientific

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35 community and medical world to analyze gammaretroviral insertional muta genesis properties. Subsequently, two other children from the same group in Paris and London also developed T ALL. In all these patients, the gene coding for LMO2 protein was mutated due to the insertion of retrovi r al vector ( 127 ) Since then, insertion mutagenesis was reported in other disease models such as Wiskott Aldrich Syndrome (WAS) and X linked chronic granulomatous disease, as well as gene transfer in various animal models ( 43 59 82 ) Ho wever, despi te the issue of insertional muta genesis, the most successful hematopoietic gene transfer comes from gammaretrovi r al vector for ADA SCID patient s ( 10 ) Lentiviral vectors began to be used as gene therap y vector s in 2003 especially suiting the need for transducing non dividing cells, both in vivo and ex vivo The first lentiviral related clinical trial was approved for HIV 1 infection ( 195 ) And ever since, 40 more clinical trials using lentiviral vectors have been approved or are in the process of approval. These tria ls include studies for monogenic disorders ( X linked SCID, sickle cell anemia, Fanconi anemia, X linked thalassemia and mucopolysaccharidosis), cancer s (metastatic melanoma, non Hod gkin lymphoma and leukemia), and neurodege nerative diseases (Parkinson disease) ( 182 ) There are three generations of lentiviral vectors already developed (mentioned above). The first two generations raised safety concerns which have been addressed in the third generation of lentiviral vectors by limiti ng the viral protein production. However, whether the insertion of viral DNA in to the host genome would activate oncogenic genes is still under debate. Ex vivo transduction studies have showed that these vectors resemble wild type HIV 1 in genome integrat ion efficiency in the transduced cells. However,

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36 lentiviruses integrate t heir viral genome into the entire gene transcription unit, whereas retroviruses preferentially integrate into the transcription started site, such as the promoter and first intron ( 61 80 ) In a recent clinical study for adrenoleukodystrophy (ALD), the efficacy and safety of lentiviral gene transfer were verified in hematopo ei tic stem progenitor cells. Several common insertion sites (CIS) have been identified in the patients' cells, suggesting a selective advantage associated with lentiviral integration ( 34 58 ) Accounting for about 25% of gene therapy clinical trials, the first and second generations of Ad vectors have raised safety concerns due to immune and inflammatory responses ( 87 220 ) which was responsible for the death of an 18 year old patient in a gene therapy trial for the he reditary deficit of ornithine transcarbamylase (OTC), at the University of Pennsylvania, Philadelphia in 1999 ( 25 178 ) These vectors activated a series of inflammatory cytokines, which recruit macrophages, neutrophils, and NK cells to eliminat e transduced cells. In liver, 80 90% of vectors are eliminated by inflammatory res ponses within 24 h ours after administration. Subsequently, after 4 7 days of post administration antibody production wa s activated. T his response was due to the presence of A d vectors themselves, not against the gene expressed from the virus. Learning from such an unexpected experience, a gutless adenoviral vector was then introduced into the field of gene therapy. While less immune response was detected against transduced ce lls with this new vector, a transient inflammatory response and liver abnormalities, resulting from contamination of replication competent helper virus during vector preparation, was observed ( 70 ) Notably, growing interests have been focused on the development of ONYX 15 vector (men tioned above) ( 36 ) This viral vector has been approved for phase I and II clinical trials for the treatment of recurrent

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37 squamous cell carcinoma of the head and neck (SCCHN) ( 112 224 ) Administration using this viral vector in combination with chemotherapy had also been shown to be safe and efficient ( 161 ) To date, AAV vectors represent the most promisin g delivery tool for in vivo gene therapy and the clinical trial number has been increasing substanti ally over recent years (Table 1 1). Their success is attributed to the biological characteristics of AAV vectors; simple genome organization (prevent contam ination of wild type viral protein with the vector which can lead to inflammation), limited genome integration (prevent the possibility of proto oncogene caused by non specific locus integration), they can infect both dividing and non dividing cells, and s erotypes derived differential tissue specificities ( 52 ) Over 80 clinical trials have been conducted over past few years and are underway for phase I, II and III ( 116 213 ) (www.clinicaltrial.org) Two examples of current e xamples of AAV vector gene delivery success are the treatments of LCA2 and H emophilia B. Following studies from Hauswirth and Cicdeyan, visual assessment was performed for another group of 12 patients (8 44 age year old) who received RPE65 gene therapy. Re sults have shown this intervention can increase visual light response by at least 20 times and works even better in children ( 196 ) Introduction to Parvoviruses The Parvoviridae family consists of small ( ~ 26nm) non enveloped icosahedral viruses that package a linear ssDNA genome of ~5kb ( 31 ) Based on the host range, these viruses are divided into two subfamilies: Parvovirinae and Densovirinae ( 29 ) The Parvovirinae, which infect vertebrates, is further subdivided (based on genome architecture) into five genera: Amdovirus, Bocavirus, Dependovirus, Erythrovirus, and Parvovirus. Members of the Denso virinae (subdivided into four genera: Iteravirus,

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38 Brevidensovirus, Densovirus, Pefudensovirus) infect only insects and art hropods. This study will focus on the members of Parvovirinae and especially the members in Dependovirus genus. The type species of e ach of the Parvovirinae genera are; amdovirus: Aleutian mink Disease Virus (ADV); bocavirus: Bovine Parvovirus (BPV); erythrovirus: Human Parvovirus B19V (B19V); parvovirus: Minute Virus of Mice (MVM) and dependovirus: Adeno Associated Virus serotype 2 (AA V2). While the first four genera contain pathogenic viruses that can replicate independently of helper virus function and are thus referred to as autonomous parvoviruses ( 45 236 ) members of the dependovirus genus rely on co infection with a complex helper virus (such as Ad or HSV) for a successful lytic life cycle ( 42 ) Viral Genome and Capsid: Architectures and Functions The common genomic structure of parvoviruses consists of two open reading frames (ORFs) flanked by two inverted terminal repeats (ITR rep or ns ) encodes non structural proteins (referred to Rep in the dependoviruses and NS in the autonomous parvoviruses) which are important for genome replication and cap ) encodes structural viral proteins (VP) w hich assemble the capsid ( 77 ) (Figure 1 1). Members of the bocavirus genus have a third ORF between rep and cap that codes for a non structural protein, NP 1, required for genome replication ( 65 276 ) The ITRs are palindromic sequences (1 20 to ~550 nucleotides are essential for replication. The parvovirus VP is a multifunctional protein capable of performing a wide variety of structural and biologic al functions during the viral life cycle, including host cell surface

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39 receptor recognition, intracellular trafficking, nuclear entry, capsid self assembly, genome encapsulation, maturation to produce infections virus progeny, nuclear exit, and host immune response detection and evasion ( 5 6 ) The relatively small parvoviral genome has allowed the use of genetic manipulation to identify functional domains/regions of the VPs/capsid. These studies show that the parvoviruses have evolved to utilize the VP1u for a phospholipase A2 (PLA2) function required for endosomal escape and nuclear entry during infection ( 320 ) regions of VP2 for nuclear entry (in dependoviruses) and for nuclear exit (in some autonomous viruses), and amino a cid stretches within the VP3 common region for receptor attachment, tropism and host range determination, capsid ass embly, DNA packaging, and host antibody recognition ( 5 6 ) The number of VPs encoded by the cap gene and used to assemble the capsid differs between members of the Parvovirinae. The depend oviruses (Adeno associated virus (AAV)) contain VP1, VP2, and VP3 formed by alternative mRNA splicing of the transcript and alternative translation initiation codon usage, whereas the autonomous parvoviruses are assembled from VP1 and VP2 translated from a lternatively spliced mRNAs. For the parvovirus genus, a VP3 is generated by post translational cleavage of ~20 amino acids from the N terminus of VP2 following genome packaging ( 77 ) Thus, ADV, B19V, and BPV contain only VP1 and VP2, while AAV2 and MVM virions contain VP1, VP2, and VP3. The amino acid sequences of the VPs are overlapping, with th e entire sequence of VP3 (when present) contained within VP2 which is in turn contained within VP1. Sixty copies of these VPs, in a predicted ratio of 1:1:10 for VP1:VP2:VP3 (when present) or 1:10 for VP1:VP2, assemble the capsid with T=1 icosahedral

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40 symm etry ( 62 ) VP1, containing a unique N terminal region (VP1u), is always the minor component i n all virus capsids while the smallest VP is always the major component. The capsid, with its packaged ssDNA genome, has to traverse two cellular barriers during infection, the plasma and nuclear membranes, for replication in the cell nucleus (Figure 1 2). Like most other viruses, attachment to a cell surface receptor is an essential first step of parvovirus infection ( 23 75 ) Extensive biochemical and molecular characterizations have led to the proposal that follo wing initial attachment the parvovirus capsid is internalized through clathrin coated pits ( 95 234 ) and trafficked through the endocytic pathway to the nucleus for genome replication and subsequent transcription of the g enome and translation of the message for production of the Rep / NS and VP proteins required for the formation of progeny virus. In vitro and biochemical studies using lysosomotropic drugs (bafilomycin A1 and chloroquine) is shown to interfere with endosom al pH and block parvovirus infection, suggesting that endosomal acidification is essential for the viral infection ( 23 24 ) For the dependoviruses, there is evidence that some AAV serotypes are trafficked through the Golgi and endoplasmic retic ulum (ER) enroute to the nucleus, and for both dependovirus and autonomous members, cellular components, such as actin and dynein as well as microtubules, are proposed to be involved in cellular trafficking ( 94 133 135 234 252 289 ) Parvovirus Capsid Structure Towards correlating the capsid structure with its various functions during cellular infection the three dimension al structures for several Parvovirinae members have been determined using X ray crystallography and/or cryo electron microscopy and image reconstruction (cryo reconstruction) ( 7 8 92 119 126 157 158 171 172 180 191 207 223 225 232 266 284 291 304 312 313 ) In all these structures only ~520

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41 residues of the common VP2 or VP3 C terminal region (depending on viruses) are resolved leaving the N terminal extensions of the larger VP(s), which are proposed to be disordered, un observed. Low copy numbers of the minor capsid prot eins or differential conformations adopted by the N termini, which is inconsistent with the 60 fold icosahedral averaging applied during structural determination, could result in the lack of N terminal VP ordering ( 5 6 62 ) Cryo reconstruction studies of AAV capsids in which medium resolution structures for capsids assembled with or without VP1 in addition to those containing only VP3 suggest that the VP1u is located in the interior of the capsid underneath the icosahedral twofold axis ( 171 ) For B19V, cryo EM reconstruction of wild type virions and empty particles showed VP2 exposed on the capsid surface ( 157 ) The VP structure (the ordered ~520 residues) is highly conserved, even for members that are only ~20% identical at the amino acid sequence level, such as AAV2 and B19V The core consists of a conserved eight the C 3). An additional Parvovirinae structures. The remaining VP structure consists of loops inserted between strands that contribute to the capsid surface topology. These loops also contain strand structure. The loops are named after the strands betw een structurally varied between members in the same and different genera, and dictate host s pecific tropism, antigenic response, and transduction efficiency in viral vectors. The

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42 sheet forms the interior surface of the capsid while the inserted loops form characteristic features at and around the icosahedral two three and five fold sy mmetry axes (Figure 1 4). There is (I) a depression at each two fold axis, (II) a single protrusion at the three fold axis of members of the parvovirus genus (e.g. MVM, Canine Parvovirus (CPV), Feline Panleukopenia Virus (FPV), Porcine Parvovirus (PPV)) o r three separate protrusions surrounding the three fold axis (which thus appears as a (AAVs), and erythrovirus (B19V) genera, (III) a cylindrical channel at each five fold axis, and five fold depressions ( 5 62 ) The protrusions are more pronounced in ADV and the AAVs compared to B19V and HBoV. B 19V and HBoV appear to share characteristics of members of the parvovirus and dependovirus genera and have flatter protrusions. The two symmetry related VP monomers; t he single three fold protrusion in members of the parvovirus genus are created from six loops (within the GH loop), two from each three fold symmetry related VP monomer; each of the three separate protrusions in the other Parvovirinae members are created b y three loop regions (also within the GH loop) from two VP monomers; the five fold channel is created by five symmetry related DE loops and the surface of the depression surrounding this axis is lined by the HI loop. Dependovirus: Overview of Adeno associa ted viruses (AAVs) These viruses are non pathogenic and able to package foreign DNA, resulting in intense efforts to develop several members for therapeutic gene delivery applications. Currently clinical trials are underway with AAV vectors packaging the rapeutic genes for the treatment of several diseases, including alpha1

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43 se and HIV infection ( 57 217 ) (Table 1 1) Challenges for these clinical studies include the need to (I) improve viral tissue sp ecificity and (II) decrease the detrimental effects of the host immune response against the vector (especially for treatments that may require vector re administration) ( 41 209 ) Efforts to overcome these issues have included the isolation and characterization of novel AAV serotypes and/or variants to exploit their tissue tropisms, transgene expression efficiencies, and hopefully lack of human immune system recognition. Amino aci d sequence (VP1) comparison between the twelve serotypes (mentioned in previous section) shows ~60 99% identity, with AAV4 and AAV5 being the most different ( 113 ) In addition, AAV sequences have been isolated from several other mammalian hosts, including caprine, mouse, bovine, snake, lizard, avia n tissues ( 17 147 156 192 245 ) which share different levels of sequence similarity with the isolates from the human and non human primate sources. AAV Capsid Structure and Capsid Cell Surface Receptor Interactions Currently, atomic structures of nine serotype members, AAV1 9, serving as the representative membe rs of the AAV antigenic clades and clonal isolates ( 113 ) have been determined using X ray crystallography and/or cryo reconstruction ( 92 180 223 225 232 291 311 312 ) are superimposable while the tops of the loops between these conserved regions are varied in sequence and structure and defined as variable regions (VRs) I IX ( 119 ) Mutagenesis, biochemical, and st ructural studies have demonstrated that residues in these VRs are crucial in viral life infection, including viral receptor binding ( 6 )

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44 Biochemical studies have id entified primary receptors and co receptors utilized for initial cellular recognition and internalization, respectively, during infection by the AAVs (Table 1) ( 12 18 28 37 88 89 91 153 155 183 188 228 244 263 264 274 275 292 298 307 ) With respect to primary recognition several glycans are utilized by the AAVs. Heparan su lfate (HS) proteoglycan serves as the cell surface receptor for AAV2 and AAV3b (closely related to AAV2), terminal N acetyl neuraminic acid (sialic acid (SA)) for AAV1, AAV4, AAV5, and galactose for AAV9 (Table 1) ( 6 91 228 263 264 275 292 306 307 ) AAV6 is able to utilize HS or SA depending on the cell type being infected (Table 1) ( 306 30 7 ) The primary receptor for the other human and non human primate AAV serotypes, AAV7, AAV8, AAV10 AAV12, are yet to be determined. 1 4 linked N acetyl glucosamine found on gp96, for cellular transcytosis ( 89 ) The residues involved in glycan interaction s have been characterized for AAV2, AAV5, and AAV6 as discussed below. Mutagenesis of AAV2 followed by structure determination of AAV2 HS complexes mapped VP re sidues involve in the AAV2 HS interaction to several structurally adjacent basic residues R484, R487, K532, R585, and R588 (AAV2, VP1 numbering) (Figure 4), consistent with the highly acidic nature of HS molecules ( 160 183 228 230 ) These AAV2 residues are located at the inner wall of the protrusions surrounding threefold axis (Figure 2B and 4). They are located in three VRs, VRV, VRVI, and VRVIII. Residues in VRV from one AAV VP interacts with VRIV and V RVIII from another three fold symmetry related VP to assemble the top of the protrusions surrounding the three fold axis while VRVI forms the base of the protrusions facing the two fold axis ( 183 228 ) Thus the binding site is only present on assembled capsids. The structure of AAV2

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45 complexed with a HS oli gosaccharide ( 183 228 ) showed the location of the b ound HS molecules adjacent to the residues mapped by mutagenesis. Levy et al ( 183 ) also reported structural rearrangements of the HI loop on the canyon floor surrounding the five fold channel which they proposed could be related to the opening of the channel to prime the capsid for (I) externalization of the VPIu for endosomal escape or (II) genome release following nuclear entry. The capsid amino acids involved in glycan recognition have been reported for 3 N linked SA for infection ( 153 292 ) A single residue mutation, A581T (AAV5, VP1 numbering), affects airway cellular transduction shown to require SA binding ( 105 ) This residue, structurally equivalent to AAV2 A591, is also located on the inner wall of the threefold protrusion in the AAV5 capsid structure. AAV6 and the closely relate 6 N linked SA for cellular infection, with AAV6 also able to utilize HS as a cellular receptor ( 306 307 ) Mutagenesis studies, in which the six amino acids (129, 418, 531, 584, 598, 642, AAV1/6 VP1 numbering) that di stinguish the two viruses were reciprocally changed, identified K531 in AAV6 as playing an important role its HS recognition with a change to the E531 present in AAV1 abolishing this interaction ( 306 ) This residue, structurally equivalent to E530 in AAV2, is located on the AAV6 capsid surface at the base of the threefold protrusion within the vicinity of the AAV2 HS residues, but is on the wall facing the twofold depression not the threefold depression ( 225 ) Recently, a second mutagenesis study identified another AAV1/6 residue, K459, as being important for AAV6 HSPG recognition ( 312 ) This residue is structurally equivalent to AAV2 S458 located at the plateau close to the top of the threefold protrusion and is not adjacent to K531. The AAV2 HS mutagenesis and

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46 structure data combined with the mutagenesis data fo r AAV5 and AAV6 suggests that the AAVs commonly utilize amino acids in the three fold region for recognition of different carbohydrate moieties Although the galactose binding site of AAV9 capsid surface is not yet structurally characterized, several mutag enesis and transduction studies have shown the roles of residues on AAV9 variable regions corresponding with cardiac, liver, and muscle cellular transductions ( 170 185 243 ) Pulicherla et al have shown mutations on residues Trp 503 and Pro 504 on AAV9 VRV is important for liver cellular transductions ( 243 ) Using capsid genome shuffling, a new chimera virus was generated from combination of AAV1, AAV2, AAV8 and AAV9 which has improved cardiac transduction. Regions of AAV9 that contributes to this chimera virus are VRIV (456 479) and VRVII (550 568) ( 185 ) Another region which is identified to be important for delayed blood clearance in ca r diac transduction using AAV9 is 699 735, located in VRIX ( 170 ) Altogether, these results suggest that VRIV, VRV, VRVII and VRIX are the potential determinant for AAV9 superi or liver, cardiac and muscle cellular transduction. Following the recognition of a cell surface receptor by the AAVs, an interaction with cell membrane proteins, which serve as co receptors, are required for capsid internalization. For AAV2, several intern alization co receptors have been identified, hepatocyte growth factor receptor (HGFR), and 37/67 kDa laminin receptor (LamR) ( 12 244 274 298 ) LamR was actually identified as a co receptor for AAV8 that could also serve a similar function for other AAVs, including AAV2, AAV3b and AAV9 ( 12 ) and similar to AAV2, AAV3b also utilizes LamR, HGFR and FGFR1 as its co receptors ( 12

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47 37 188 ) The interaction between AAV3 and HGFR appears to be specific for human cell derived HGFR ( hHGFR), while AAV2 is also able to bind the murine derived receptor, mHGFR. Sequence differences between the murine and human receptors are suggested as a source of the differential AAV3 and AAV2 recognition ( 188 ) Other proteins required for internalization of other AAV serotypes include platelet derived growth factor receptor (PDGFR) and epider mal growth factor receptor (EGFR), for AAV5 and AAV6, respectively ( 88 298 ) For the AAV co receptors, the VP residues important fo have been identified ( 12 18 ) the binding site for the other co receptors are unknown An 511 NGR 513 motif (AAV2 VP1 numbering), was reported to play a role its binding ( 18 ) These residues are located close to the base of the three fold protrusion (two/ fivefold wall) proximal to the HS binding residues with G512 exposed on the capsid surface. The NGR sequence is conserved in most of the AAV serotypes, with the exception of AAV4, AAV5, and AAV11. This motif is partially conserved in B19V that 1 as a receptor. Due to the close proximity of this region to the HS binding site in AAV2, it has been postulated that the primary HS receptor binding might promote this AAV2 capsid co receptor interaction or serve as a pre requisite. The binding site for LamR on the AAV8 capsid was mapped to two large peptide regions containing residues 491 547 and 593 623 (AAV8, VP1 numbering) ( 12 ) These residues are located on the inner surface of the AAV8 thr ee fold protrusions facing the three fold axis ( 223 ) and include GH loop amino acids in VRV and VRVI in the first stretch of amino acids and residues in a structurally conserved VP region past VRVII strand in the second stretch. The report that this receptor is utilized by several AAVs

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48 would thus suggest that second stretch of residues, 593 623, conserved in sequence and structure ( 223 ) may be more important for this recognition. These residues, structurally equivalent to residues 590 620 in AAV2, are close to those reported to bind HS in AAV2 and SA in AAV5, with surface amino acids clustered around the three fold axis. The again suggests the possibility that the initial cell surface recognition leads to a subsequent engagement of LamR for internalization. AAV Capsid Internalization and Post Entry Ev ents The interaction of the AAV capsid with internalization receptors results in host cell surface rearrangement which is a prerequisite for endocytosis, a clathrin mediated process ( 285 ) Several immunofluorescence microscopy and chemical inhibition studies showed AAV capsids co localized with dynamin and microtubules as well as markers from early, late, and recycling endos omes, and lysosomes ( 135 ) In addition, endosomal processing is reported to be the rate limiting step in viral transduction in several cell lines ( 6 96 ) Biochemical experiments confirmed that the AAV VP1u (amino acids 1 137, VP1 numbering) is exposed during endosomal trafficking ( 39 272 ) Once exposed, the VP1u PLA2 activity hydrolyzes membrane phospholipids to cause the endosomal membrane rearrangement required to facilitate viral capsid release ( 320 ) During the transition from early to late endosomes, acidification induced capsid structural changes, including the externalization of the VP1u, is reported to be essential for viral trafficking, uncoating, and genome release in n ucleus. However, pH alone is not sufficient to mediate VP1u externalization for the AAVs, suggesting a requirement for yet to be identified cellular factors in this capsid transition ( 171 ) Artificial heat treatment (65 0 C) alone can mimic t he endosomal conditions required for VP1u externalization in the

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49 presence of viral genome ( 171 ) Higher temperatures (75 C) are required to release the VP1u in capsids that are devoid (empty) of DNA ( 171 ) As discussed above the structure of VP1u inside the capsid or in its externalized state has not been structurally helical in nature as reported for other PLA2 domains. Mutagenesis and biochemical studies suggest that the fivefold channel might play a role in its extrusion ( 39 ) Confocal microscopy studies have shown that in addition to the trafficking through the endocytic pathway, AAV2 can localize to the secretory pathway enroute to the nucleus ( 149 ) while electron and immunofluorescence microscopy have observed AAV5 in the Golgi compartment ( 21 22 ) Further studies are required to characterize the role of these alternative trafficking routes and the role of capsid interactions in facilitating them. The AAV8 capsid structure is the best characterized serotype with respect to the effect of the pHs e ncountered in the endocytic pathway. Structures determined by X ray crystallography to 2.7 resolution for capsids incubated at pH7.5, pH6.0, pH5.5, and pH4.0 identified two regions affected by pH that are conserved among the AAVs ( 222 ) and Y707 (AAV8, VP1 numbering), and is located close to the icosahedral two fold axis, with residue Y707 (equivalent to AAV2 Y704) visible on the capsid surface viewed down the axis. The amino acid side chain transitions occurring as pH decreases result in a reduction in the number of interactions between two fold related VP monomers and ( 222 ) It was thus proposed that amino acids at the two fold interface may be involved capsid destabilization events that enable AAV VP1u externalization without capsid disassembly. The second region is located on capsid

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50 interior surface under the three fold axis and involves residues F631 and H632 (AAV8 VP1 numbering). Previous structural studies have assigned this region as the nucleic acid binding pocket for AAVs ( 119 180 223 225 ) and implied a role in AAV genome stabilization. A low pH induced H632 side chain conformational change disrupts the interaction of the VP with the ordered nucleic acid dens ity observed in crystal structures. The loss of this density was suggested to be a priming of the capsid for genomic uncoating. Thus similar to other viruses ( 229 251 ) this study suggests that the AAV viral genome undergoes rearrangement inside the capsid prior to its release and that altered capsid genome interactions, triggered by pH, are important aspects of cytoplasmic pre processing prior to nuclear entry for genomic uncoating. Several cellular host protein machineries have been reported to interact with the AAV capsid during trafficking to the nucleus that is detrimental to infection. As an example, targeting of AAV2 capsids to the proteasome in the cytoplasm is reported to limit viral transduction efficiency ( 323 324 ) Phosphorylation of capsid surface exposed tyrosines followed by ubiquitination is reported to be the signal for recognition by proteasome and subsequent capsid degradation. Mutation of the seven AAV2 capsid surface exposed tyr osine residues, Y252, Y272, Y444, Y500, Y700, Y704, and Y730 (AAV2 VP1 numbering) to phenylalanine resulted in significant (up to ~700 fold) increase in transduction efficiency dependent on cell line s ( 324 ) Significantly, AAV8 Y707, equivalent to AAV2 Y704, clustered at the icosahedral twofold axis with Y700 and Y730, undergoes the pH mediated transition discussed above, consistent with a role in capsid cellular interactions during trafficking.

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51 The exact mechanism of AAV genome translocation into the host cell nucleus following escape from cytoplasmic compartments is poorly understood as are the determinants of capsid uncoating in the nucleus. The VP1/VP2 N terminal regions contain nuclear locali zation signals (NLSs) which when mutated significantly reduce AAV infectivity ( 120 150 269 305 ) These N terminal VP regions can be detected by antibodies in the cytoplasm of infected cells indicating that they become externalized prior to nuclear entry ( 269 ) Capsid processing prior to cytoplasmic release and su bsequence nuclear entry, as suggested by the pH mediated structural changes described above and the reported release of the VP1u N terminus, is consistent with the observation that microinjection of virions into the cytoplasm does not confer a nuclear tran slocation phenotype, even if the N terminal regions are pre exposed by heating ( 269 ) While the size of the AAV capsid at ~260 in diameter is small enough to be translocated through the nuclear pore complex (NPC), there are conflicti ng reports on the issue. Some reports say that it can while others say that this may not be the case ( 134 310 ) The exact mechanism remains to be elucidated. The majority of studies indicate that AAV genome uncoating occurs in the nucleus ( 150 151 256 269 310 ) However there are reports that uncoating may occur before or during nuclear entry. Regardless of the uncoating site, the capsid transitions resulting in genome release following nuclear entry remains poorly understood and likely requires cytoplasmic pre processi ng, some discussed above, prior to nuclear entry ( 150 ) Following viral genome release into the nucleus, rolling hairpin replication (RCR) is proposed as the mechanism for parvovirus genome replication ( 76 ) However, there little is known about the role of the capsid protein in this process. Two elements of the

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52 AAV genome shown to be indispensable for replication are the ITRs and p5 rep transcript. The two large replic ation proteins, Rep78 and 68 (Rep78/68), encoded by the rep ORF and translated from the p5 transcript, possess terminal resolution site (trs) Viruses with mutations in Rep7 8 and Rep68 are defective for viral DNA replication ( 268 295 ) In vitro studies show that after successful genome replication, the viral ssDNA genome is packaged into pre assembled empty capsids in the nucleus ( 97 163 ) Expression of the AAV VPs alone is sufficient for capsid assembly suggesting that genome packaging is not required for this process (e.g. ( 38 ) ), although the observation of a nucleotide in most AAV capsid structures determined to date, including virus like particles expressed in heterologous systems in the absence of viral genome, suggests otherwise. Recent studies identified a transiently expressed 23kDa prote in, assembly activating protein (AAP), which is required for capsid assembly ( 270 271 ) This protein, nucleolus where capsid assembly is prop osed to occur. Interestingly, comparison of capsid assembly for AAV1, 2, and 5 showed that AAV5 requires its own AAP and cannot be complemented by the AAV2 AAP. AAV5 is one of the most sequence diverse serotypes AAV1 12 ( 113 ) and this observation suggests that AAP engages in specific interactions w ith the VP during assembly. The step in capsid assembly requiring AAP is yet to be determined. Site directed mutagenesis studies have identified several AAV VP residues that are important for capsid assembly. These are mostly charged residues involved in s ymmetry interface interactions ( 6 ) Significantly, residues in the HI loop,

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53 which forms the majority of the fivefold symmetry related interactions in assembled AAV capsids, plays a role in capsid assembly as well as genome packaging ( 9 3 120 ) The AAVs package both strands of their ssDNA genome with equal frequency into different capsids in a process that is highly dependent on the small replication prot eins, Rep52 and Rep40, which are encoded by the rep ORF. A reduction in expression of these proteins results in a significant decrease in the packaging efficiency ( 63 ) Biochemical studies have shown t ( 73 ) which is believed to interact with the assembled empty capsid and unwind the replicative products of AAV genome for packaging into t he capsid. The interactions between Rep 52/40 and the AAV capsid have been reported to be both DNA dependent and independent ( 199 202 204 231 316 318 ) Post capsid assembly and genome packaging, wil d type AAV virions must exit the nucleus and traffic to the cell surface for a second round of infection. Significance Variable residues on the capsid surface have been shown to dictate differential functional properties among the AAV serotypes e.g. recep tor binding, transduction efficiency, and antigenicities. Recently, there has been a lot of interest in the use of AAV1 and AAV6 as gene delivery vectors to transduce cardiac, muscle, and lung cells compared to AAV2 because of their superior transduction p roperties in these tissues. These two serotypes on ly differ in 6 of 736 residues in VP1, with 5 of these located within the overlapping VP3 region which assembles the capsid shell and is responsible for essential capsid functions associated with infection, such as receptor attachment and internalization. Furthermore, this small number of amino acid differences between AAV1 and AAV6 has been reported to confer receptor attachment and transduction

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54 differences. We hypothesize d that the comparison of AAV1 and A AV6 and their receptor interactions will aid dissection of the contribution of specific capsid residues / regions to AAV receptor attachment and transduction phenotypes. In this study, we utilized structural, biochemical, and molecular approaches to characterize these properties for these two viruses.The data obtained will be applicable to the development of cell/tissue targeted AAV gene delivery vectors by enabling the engineering of surface residues for specific receptor recognition.

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55 Table 1 1. Current Ongoing Clinical Trials using Adeno associated Virus Vectors Disease AAV A Serotype Transgene Product Phase deficiency 1, 2 I/ II 2, rh10 B CLN2 C I 2 Aspatoacylase I Cystic Fibrosis 2 CFTR D I/ II Hemophilia B 2, 8 Factor IX I/ II 2 RPE65 E I/ II/ III LPL F deficiency 1 LPL I/ II/ III 2 AADC G GAD H Neurotrophin I/ II 1 GAA I I/ II Muscular dystrophy 1, 2.5 Microdystrophin, sarcoglycan I Macular degeneration 2 sFLT01 J I Severe heart failure 1, 6 SERCA2a K I/ II Rheumatoid arthritis 2 TNFR Fc L I A AAV=Adeno associated Virus; B rh10= rhesus macaque variant 10; C CLN2= ceroid lipofuscinosis type 2 ; D CFTR=Cystic Fibrosis Transmembrane Regulatory Receptor; E RPE65=retinal pigmentosa epithelium 65kDa; F LPL=Lipoprotein lipase; G AADC= Aromatic L amino acid decarboxylase ; H GAD= Glutamic acid decarboxylase ; I GAA= acid alpha glucosidase ; J sFLT01=platelet derived growth factor receptor; K SERCA2a= sarcoplasmic reticulum Ca2+ ATPase; L TNFR Fc=Tumor Necrosis Factor Receptor Fc. (Table modified from ( 213 ) )

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56 Table 1 2. Adeno associated virus: Cellular Receptors, and Host Range Virus Receptors Co receptors Host AAV1 6 N linked sialic acid Human AAV2 HSPG a FGFR1 b HGFR c LamR d Humans AAV3 HSPG a HGFR c LamR d FGFR1 b Humans AAV4 2 3 O linked sialic acid NHP e AAV5 6 N linked sialic acid PDGFR f Humans AAV6 6 N linked sialic acid, HSPG a EGFR g Humans AAV8 LamR d NHP e AAV9 Galactose LamR d Humans Bovine AAV Gangliosides, Chitotriose Bovine a HSPG=heparin sulfate proteoglycan; b FGFR1=fibroblast growth factor receptor1; c HGFR=hepatocyte growth factor receptor; d LamR=37/67 kDa laminin receptor; e NHP=non human primate; f PDGFR=platelet derived growth factor receptor; g EGFR=epidermal growth factor receptor. (Table modified from ( 132 ) )

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57 Figure 1 1. Genome architecture of (LEFT) gammaretrovirus (9 11kb), lentivirus (9 11kb), Adenovirus (36kb), and Adeno associated virus (AAV) (5kb) and (RIGHT) the corresponding recombinant viral vectors for gene therapy. Genes coding for the protein for viral replica tion, viral capsid assembly, accessory proteins and toxic proteins are labeled (described in the text). LTR= Long Terminal Repeats, PBS= Primer Binding Site, PPT= PolyPurine (reverse tra nscriptase), env=envelope, ITR=Inverted Terminal repeats, E=Early transcript, ML=Major Late transcript, VP=Viral Protein (Figure modified from ( 117 ) ).

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58 Figure 1 2. Schematic of the life cycle of Adeno associated Viruses (figure modifi ed from ( 132 ) ).

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59 Figure 1 3. Conserved secondary structure superposition of VP for one member from every genus in Parvovirinae subfamily is shown: ADV (red),HBoV (yellow), AAV2 (blue), B19V (green) and MVMp (orange). Atomic coordinates for AAV2, MVMp, and B19V were obtain ed from RCSB protein database (PDB ID numbers 1lp3, 1z14, and 1s58, respectively). The ADV and HBoV images were generated from pseudo atomic coordinates built into cryo reconstructions ( 126 207 ) The N terminus (N), C terminus (C), variable regions (VRI IX, VR1 8), DE, and HI loops are labeled. The boxed region is shown below, depic conserved in all parvovirus VP structures determined to date (Figure adapted from ( 132 ) ).

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60 Figure 1 4. Surface representation of AAV2 used to illustrate the topological features of the parvovirus capsid surface as discussed in the text. The different colored protrusion facing the icosahedral twofold axis (black ), icosahedral threefold axis (blue), and fivefold axis (green). The image is depth cued (blue red yellow white) to show regions at the shortest radial distance to capsid center in blue and those at the furthest radial distance in white (see Figure 3 for r adial distances) (Figure adapted from ( 132 ) ).

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61 CHAPTER 2 MATERIAL AND METHODS This chapter describes common experimental procedures and materials utilized throughout this thesis. The studies can be divided into two main sections: the first section involves the purification and structure determination of virus like particles (VLPs) and the second section involves the purification (Figure 2 1) and biophysical characterization of mammalia n expressed recombinant AAV virions (Figure 2 2). Production and Purification of AAV Virus Like Particles (VLPs) A recombinant baculovirus encoding the AAV6 capsid proteins (VP2 and VP3 ORFs) or AAV1 capsid proteins (VP1, VP2 and VP3 ORFs) were provided by R. Jude Samulski (University of North Carolina, Chapel Hill (UNC)) and Sergei Zolotukhin (University of Florida (UF)), respectively. These constructs were generated using the Bac to Bac system (Gibco/Invitrogen Corporation). The DH10Bac competent cells co ntaining the baculovirus genome were transformed with pFastBac transfer plasmids containing the AAV component insert. Bacmid DNA purified from recombination positive white colonies was transfected into Sf9 cells using the TransITInsecta reagent (Mirus). Th ree days post transfection, media containing recombinant baculovirus with VLPs ORF inserted were harvested (P0) and plaque assays were conducted to prepare independent plaque isolates. Several individual plaques were propagated to passage one (P1) to evalu ate the level of VP expression using Western blot against the anti AAV B1 antibody ( 301 ) The clone with the highest level of protein expression was propagated to P2 and then P3 using Multiplicity of Infection (MOI) of 0.1 plaque forming units (pfu)/cell.

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62 Sf9 insect cells cultured in Sf900 II SFM m edia (Gibco/Invitrogen Corporation) were infected with a titered P3 baculovirus stock, at an MOI of 5.0 pfu/cell. VLPs were expressed and purified from Sf9 cells as depicted in Figure 2 1. VLPs were released from infected cells by three freeze thaw cycles in lysis buffer (50 mM Tris HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.2% Triton X 100), with the addition of benzonase (Merck, Germany) after the second cycle. The sample was clarified by centrifugation at 12,100 x g at 4 0 C for 15 minutes. Next, the cell lysa te was pelleted through a 20% (w/v) sucrose cushion in TNET buffer (25 mM Tris HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.06% Triton X 100) by ultracentrifugation at 149,000 x g at 4 0 C for 3 h. The pellet from the sucrose cushion was resuspended in TNTM buff er (50 mM Tris HCl pH 8.0, 100 mM NaCl, 0.06% Triton X 100, 30 mM MgCl2,) overnight at 4 0 C. The sample was clarified by several rounds of centrifugation at 5,000 x g to remove insoluble material. The clarified sample was loaded onto a sucrose step gradien t (5 40% (w/v)) and spun at 151,000 x g at 4 0 C for 3 h. A visible blue virus band was extracted from the 20/25% sucrose layer and dialyzed into 2 0 mM Tris HCl pH 7.5, 2mM MgCl2, 35 0 mM NaCl at 4 0 C. The approximate VLP concentration in mg/ml was calculate d based on optical density measurements at 280 nm, assuming an extinction coefficient of 1.7. The purity and integrity of the VLPs were analyzed using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE) and negative stain electron micros copy (EM), respectively. VLP and Vector Concentration T h e VLP or vector was concentrated and buffer exchanged in a Biomax 100 K concentrator (Millipore, Bedford, MA). Three times the sample volume of desired buffer was added to wash the membrane on the retentate vial by centrifugation at ~ 1,933 xg at

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63 4 0 C.In the last wash, sample was added into the retentate vial and centrifu g ed until the desired sample volume or concentration was reached. Buffer exchange was done by adding three times the final sample volume of the desired buffer onto the retentate vial and continued centrifugation at ~1,933 xg at 4 0 C. Negative Stain Electron Microscopy Five microliters (L) of purified samples was loaded onto carbon coated copper grids for two minutes and blotted dry using Whatman filter paper. The sample was then negatively stained twice with 5 L of 2% Uranyl Acetate (UA) for 20 seconds and 7 seconds. The grids were air dried and then examined in a JOEL 1200 EX transmission electron microscope (TEM). The instrument was set to collect images at 50,000 x magnification and on film. AAV6 VLP Structure Determination by Cryo Reconstruction Small (3 .5 l) aliquots of purified VLPs (~10 mg/ml) were vitrified via standard rapid freeze plunging procedures ( 4 98 ) Samples were applied to glow discharged (~15 s in an Emitech K3 50 glow discharge unit) Quantifoil holey grids, blotted for ~5 s, plunged into liquid ethane, transferred to liquid nitrogen and then into a pre cooled Gatan 626 cryo specimen holder. Data was collected with an FEI Sphera microscope (200 kV, equipped with a LaB6 electron gun) under low dose conditions (24 e / 2 ) at 50,000x nominal magnification and with a defocus range of 1.0 to 2.5 m. Eighteen micrographs with minimal astigmatism and specimen drift were digitized at 7 m intervals (representing 1.4 pixe ls) on a Zeiss SCAI scanner. A total of 1870 particles were extracted, pre processed, and their defocus levels estimated using the RobEM program (http://cryoEM.ucsd.edu/programs.shtm) for reconstructing the structure of the AAV6 VLPs using the AUTO3DEM pro gram ( 315 ) To compensate for the effects of

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64 phase reversals in the contrast transfer function of the images, phase corrections were performed but no ampl itude corrections were applied. A Fourier Shell Correlation of 0.5 was used as the threshold for estimating the resolution of the reconstruction ( 288 ) The available coordinates for an AAV1 VP3 poly alanine capsid model (aa218 736, VP1 numbering) extracted from X ray crystal structure (PDB accession No. 3NG9) was docked into the AAV6 reconstructed density map using the Situs Package (colores; with angular degree of 30 0 and resolution of 9.7 ) for pseudo atomic model interpretation of the structure ( 303 ) Structure Determination of AAV6 VLP using X ray Crystallography Crystals were grown from purified VLPs in 10 0 mM HEPES pH7.3 at a concentration of ~10.0 mg/ml using hanging drop vapor diffusion, wi th 60 mM MgCl2 and 100 mM NaCl as additives, 4% polyethylene glycol (PEG) 6000 as a precipitant, and 25% glycerol as the cryo protectant. X ray diffraction data were collected from a single crystal at the Cornell High Energy Synchrotron Source (CHESS) with a crystal to detector distance of 350 mm, oscillation angle of 0.30 per image, and exposure time of 50 seconds. The data were indexed, processed, scaled, and reduced using the HKL 2000 package ( 214 ) The crystal diffracted X rays to 3.0 resolution and was in the rhombohedral crystal system, space group R32 with unit cell par ameters a = 262.6, c = 609.9 in the hexagonal setting. The orientation of the AAV6 VLPs in the crystal unit cell were determined using the self 180 o 120 o and 72 o searchin g for icosahedral 2 3 and 5 fold symmetry axes with observed data in the 10.0 ( 282 ) The crystallographic 2 and 3 fold symmetry operators were observed to be coincident with icosahedral symmetry

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65 operators, resulting in 10 VPs (non crystallographic symmetry (NCS) operators) per crystallographic asymmetric unit of the R32 space group, sim ilar to the structural determination of AAV1. The diffraction data was phased using molecular replacement method in the AMoRe program ( 283 ) The or ientation and position of the AAV6 10 mer in the crystal unit cell was determined by cross rotation and translation searches using atomic coordinates for 10 AAV1 VP3 monomers (a 10 mer of residues 218 to 736, VP1 numbering) from AAV1 crystal structure (PDB accession No. 3NG9) with the amino acids that differ to AAV6, i.e. E418, E531, F584, A598, and N642, within VP3 mutated to alanine to eliminate phase bias. This VP3 10 mer model (generated with VIPER ( 56 ) ) was oriented and positioned into the AAV6 crystal unit cell based on the output rotation angles and positioned at (0, 0, 0), based on sp ace group packing considerations, to calculate initial phases. The phases were improved by refinement using the Crystallography and NMR System (CNS) package ( 48 49 ) using simulated annealing, atomic position energy minimizat ion, and atomic displacement parameter (ADP) refinement, with the application of strict 10mer NCS operators. A single cycle of electron density Fourier map (2F o F c and F o F c in which F o represents the observed structure factors and F c the calculated from the model) averaging was carried in CNS, while maintaining strict NCS operators, using the experimentally measured amplitudes and the improved phases following each model refinement cycle. The refinement and averaging procedures were a lternated with model building, using the Coot program, into averaged electron density maps ( 101 102 )

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66 Following the building of VP3 common amino acids 218 736 into the averaged density maps, two re gions of unassigned densities were observed in the Fourier F o F c purine nucleotide (NT) and a pyrimidine base could be modeled. The identity of the purine and pyrimidine bases could not be unambiguously determined from the averaged densities at the 3.0 resolution of the map. However, there was no amino side group density at the C2 position of the purine ring and no methyl group density at the C5 position of the pyrimidi ne ring, thus the base densities were interpreted as adenosine and cytosine, respectively. An occupancy of 0.5 was determined for each of the two bases by an empirical approach in which values (0.3 to 1.0) were applied to achieve temperature factors that w ere comparable with those of the average VP3 amino acid atoms. Finally, 12 solvent molecules were built into remaining positive Fo Fc density. To improve the quality of the maps between refinement steps, density map modification was carried using the Densi ty Modification (DM) subroutine in CCP4 ( 79 300 ) which performed histogram matching, solvent flattening and NCS averagi ng. The refined structure has an R cryst (where R cryst obs | |F calc obs |x100, in which F o represents the observed structure factors and F c the calculated from the model) and R free (obtained from 5 % subsets of reflections that are not used in refi nement) values of 27.5% and 28.8%, respectively, with final root mean square deviations (r.m.s.d.) of 0.009 for bond lengths and 1.48 o for bond angles. These values are within the range for structures reported at comparable resolution as calculated by th e Polygon subroutine in the program PHENIX (Python based Hierarchical Environment for Integrated Xtallography) ( 1 3 286 ) The quality of the refined structure was analyzed using the

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67 Coot program and MOLPROBITY ( 66 83 84 ) A Ramachandran plot showed 92.5% and 7.5% for residues in the most favorably and additionally allowed regions, respectively ( 144 ) Structure Determination of AAV1 VLPs with Sialic Acid (SIA) using X ray Crystallography Crystals were grown from purified VLPs in 100 mM HE PES NaOH pH7.3 at a concentration of 4.0 mg/ml using hanging drop vapor diffusion, with 50 mM MgCl 2 7 % polyethylene glycol 6000 as a precipitant, and 25% glycerol as cryo protectant. Forty eight hours prior to data collection, a crystal was soaked into c ryo protectant solution ray diffraction data were collected from a single crystal at Cornell High Energy Synchrotron Source (CHESS) with a crystal to detector di stance of 300 mm, oscillation angle of 0.30 per image, and exposure time of 70 seconds. A total of 226 images were collected and used for data reduction. The data were indexed, processed, scaled, and reduced using the HKL 2000 package ( 214 ) The crystal diffracted X rays to 3.0 resolution and was in the monoclinic crystal sys tem, space group C2 with unit cell 0 The orientation of the AAV1 VLPs in the crystal unit cell were determined using the self o 120 o and 72 o sear ching for icosahedral 2 3 and 5 fold symmetry axes with observed data in the 10.0 resolution range ( 282 ) The diffraction data was phased using the molecular replacement method in the AMoRe program ( 283 ) The orientation and position of two AAV1 30 mers in the crystal unit cell was determined by cross rotation and transla tion searches using the atomic coordinates for 30 AAV1 VP3 monomers (a 30 mer of

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68 residues 218 to 736, VP1 numbering) from AAV1 alone crystal structure (PDB accession No.3NG9). This VP3 30 mer model (generated with VIPER ( 56 ) ) was oriented and positioned into the AAV1 crystal unit cell based on the output rotation angles and positioned at (0, 0, 0), based on space group packing considerations, to calculate initial phases. The phases were improved by refinement using Refmac v5.5 in CCP4 package ( 79 300 ) using simulated annealing, atomic position energy minimization, and atomic displacement parameter (ADP) refinement, with the application of restrained by 60 NCS operators. A single cycle of electron density Fouri er map (defined as above) averaging was carried in Refmac v5.5, with NCS restraints, using the experimentally measured amplitudes and the improved phases, following each model refinement cycle. The refinement and averaging procedures were alternated with m odel building, using the Coot program ( 85 101 102 ) into averaged electron density maps. Following the building of VP3 common amino acids 218 736 into the averaged density maps, an unassigned positive region of density was observed in the Fourier F o F c N acetyl neuraminic acid (sialic acid / SIA) molecule could be modeled. This SIA molecule was generated suing PRODRG ( 262 287 ) (Figure 2.3A). Currently, the refined structure has an R cryst (where R cryst obs | |F calc obs |x100, where F obs and F calc are the amplitudes for the observed and calculated reflections, respectively) and R free values of 2 6.3% and 27.0 %, respectively, with root mean square deviations (r.m.s.d.) of 0.01 for bond lengths and 1.55 o for bond angles. Sit e directed M utagenesis of AAV1 and AAV6 A series of single mutations for the six residues that differ between AAV1 and AAV6 (in the pXR1 and pXR6backgrounds, respectively) as well as the wild type

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69 plasmids were provided by R. Jude Samulski (UNC) ( 306 ) Plasmids expressing these mutants were extracted and purified using the QIAGEN DNA Mini Prep kit. Purified plasmids were then subjected to OD 260 spectrophotometry to analyze the quantity of the plasmids. The samples were loaded onto 0.8% agarose gels with 1x Syber Safe stain and analyzed using Gel Doc (Biorad). A total of 7 primers were designed using Vector NTI and polymerase chain reaction (PCR) sequencing were performed to validate the AAV capsid sequences for the plasmids (Table 2 1). In order to confirm structurally mapped SIA binding site on the AAV1 capsid, mutagenesis w as performed on the AAV1 ORF. Structural comparison between the AAVs was done using the SSM subroutin e in the Coot package ( 85 101 102 ) Based on the transduction phenotype studies, we decided to mutate the SIA interacting residues to AAV2 corresponding residues, except Trp 503. However, recent mutagenesis study had showed this residue (W503A) plays a role in AAV9 Gal interaction. The residues selected for mutagenesis were N447S, S472R, V473D, N500E, T502S and W503A (AAV1 VP numbering). Due to 100% a.a. identity between AAV1 and AAV6 at these residues positions, a similar series of mutations were also generated for AAV 6. Side directed mutagenesis was performed using the Quick Change XLII mutagenesis kit (Strategene). Ten nanograms (ng) of purified wild type plasmids were used as the template for each mutagenesis with 125ng each of the forward and reverse primers and a t otal PCR reaction volume of 51 L. The following cycling conditions were used: one cycle of 95 0 C for 1 min, 18 cycles of 95 0 C for 50 seconds, 60 0 C for 50 seconds, 68 0 C for 8 minutes, one cycle of 68 0 C for 7 minutes, and product was kept at 4 0 C. Following t he PCR reaction, 10 U of Dpn I enzymes was added to each product and incubated at

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70 37 0 C for 1 hour. The DpnI treated product was then analyzed using 0.8% agarose gel E.Coli competent cells. Tr E.Coli competent cells Two L of PCR product (for each wild type and mutant virus) was added to 50 L pre chilled 1.5 milliliter (mL) microcentrifuge tube, the cells were gently mixed and then incubated on ice for 30 minutes. The cells and DNA mixture were heat shocked for 45 seconds at 45 0 C then immediately transferred into ice and chilled for 2 minutes. Five hundred L of Luria broth (LB) medium was added to the newly transformed cells. The tube containing the transformants was shaken at 225 rotations per minute (rpm) for 1 hour at 37 0 C. The transformation mix was then plated on the LB agar containing 100g/mL of ampicillin and incubated at 37 0 C overnight. Cesium Chloride Plasmid Purification For large scal e purification of supercoiled plasmid DNA, cesium chloride sedimentation was performed. At least 30 hours prior to purification, an E.coli colony containing plasmid of interest was inoculated into 5 mL LB containing 100 g/mL ampicillin and incubated at 37 0 C with 225 rpm shaking for at least 8 hours. Log phase growing bacteria was added into 1L LB containing 100 g/mL ampicillin and continue grow overnight at 37 0 C. The bacteria were harvested by centrifugation at 4,45 0 xg for 20 minutes at 4 0 C and resuspend ed with 20 mL resuspension buffer (25mM Tris Cl pH 8.0, 1 0 mM EDTA, 15 % sucrose, and 100 g/mL RNaseA). After the pellet was entirely resuspended, 50 mg of lysozyme was added to the resuspension. Forty eight mL of freshly made lysis buffer (1% SDS, 0.2N N aOH) was added to the mixture, gently mixed and incubated on ice for 10 minutes. To precipitate high molecular weight DNA and

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71 proteins, 36 mL of 3M NaOAc pH 5.2, followed by 200 L chloroform were added into the lysed cells. The slurry is then incubated on ice for 20 minutes and centrifuged at 14,30 0 xg for 20 minutes at 4 0 C. To remove any cellular debris, the supernatant was filtered using cheese cloth and collected into a clean 500 mL bottle. The plasmid DNA was precipitated using an equal volume of isopropanol on ice for 30 minutes and centrifuged at 14,30 0 xg for 20 minutes at 4 0 C. The DNA pellet was gently resuspended with 8 mL of sterile H 2 O. Eight point four gram of cesium chloride (CsCl) was added into DNA solution and dissolved completely at 4 0 C, followed by addition of 125 L 10 mg/mL ethidium bromide. DNA sample was then subjected to ultracentrifugation at 361,80 0 xg at 12 0 C overnight. Following sedimentation, a pink colored band was observed which contains supercoiled DNA. To remove ethidium bromide, the sample was diluted and washed several times with equal volumes of isoamyl alcohol until the DNA sample no longer looked pink. The aqueous fraction was transferred into a new centrifuge tube in each extraction. After final extraction, the CsCl DNA mixture was diluted with 2.5X the volume of sterile H 2 O, followed by 2X the volume of 95% ethanol (EtOH) to precipitate DNA. The DNA precipitant was centrifuged at 14,30 0 xg for 15 minutes at 4 0 C. The pellet was then washed with 1 mL sterile H 2 O and ex tracted twice with equal volume of 25:24:1 phenol/chloroform/isoamyl alcohol. Final precipitation was performed by addition of 10% (v/v) 3M NaOAc pH 5.2, followed by 2.5 times volume of 95% EtOH. The DNA precipitant was pelleted twice using a bench top cen trifuge at maximum speed (11,000 xg) for 5 minutes, with a single 75% EtOH wash in between. The DNA pellet was then air dried and resuspended overnight at 4 0 C with 1 mL sterile H 2 O. The concentration of purified DNA was analyzed using optical density readi ngs at OD 260

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72 Production of Mammalian Expressed Recombinant Virions To produce recombinant AAV (rAAV) with wild type or the mutated cap ORFs, transformed human embryonic kidney (HEK) 293 cells were triple transfected with 18g pXR plasmid which contains AA V rep and cap ORFs, 18g of pTRUF11which contains the green fluorescence protein (GFP) gene driven by Cytomegalovirus (CMV) promoter, packaging, and 54g of pXX6 which contains adenovirus helper genes. Transfection was performed using 190 L of 1mg/ml polyethylenemine (PEI) at pH4.0 onto 75% confluent cells, incubated for 48 hours at 37 0 C with 5% CO 2 and the transfection efficiency was analyzed using a UV microscope. The cells we re then harvested by centrifugation at 1,140x g for 20 minutes and resuspended in 1 mL lysis buffer (50 mM Tris HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.2% Triton X 100). The recombinant viruses were released by three freeze thaw cycles. The lysate were clari fied by centrifugation at 3,70 0 xg for 20 minutes and the rAAV virions were purified using 20% sucrose cushion in TNET buffer and ion exchange chromatography. Purification of rAAV using Ion Exchange Chromatography For ion exchange purification of the mammalian cell expressed rAAV1 and rAAV6 vectors, a 5 ml HiTrap Q column (Pharmacia) is washed with 25mL of distilled deionized H 2 O and equilibrated at 5 ml/min with 25 ml of binding buffer (20 mM Tris Cl pH 8.5, 15 mM NaCl), then 25 ml elution buffer (20 mM Tris Cl pH 8.5, 500 mM NaCl), followed by 25 ml of binding buffer using a GE ATKA FPLC system. Ten mL samples of viruses resuspended in TNET (from the sucrose cushion pellet) were diluted 5 times with binding buffer (containing 5 L phenol red) and appl mL/min. After the sample is loaded, the column was washed with 50 mL of binding

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73 buffer. The vector was eluted with a gradient concentration (100% in 30 minutes) of the elution buffer and fractions were collected into 1. 5mL microcentrifuge tubes. Eluted fractions were then subjected to 10% SDS PAGE to verify the quality of the sample. Bio chemic al Characterization of Recombinant Virions Total capsid titer was determined by an ADK1a enzyme linked immunosorbe nt assay (ELISA) PRAAV1). The clarified cell lysate were serially diluted (1:100, 1:500 and 1:1000) and 100 L was added to the 96 kit well. The readings that were within the range of detection limit co mpared to the standards were used to calculate the numbers of capsids. Total packaged genome or copy number was determined by real time or quantitative PCR (qPCR). Ten L of crude lysate was treated with benzonase for 1 hour at 37 0 C (in 5 0 mM Tris Cl pH7.5 1 0 mM MgCl 2 ) to degrade non encapsidated nucleic acids. Each benzonase treated samples was digested with proteinase K (Roche #1373196) in 1 0 mM Tris Cl pH8.0, 1 0 mM EDTA, 1% SDS) and incubated in 37 0 C for 1 hour. The mixture was then treated twice with equal volume of 25:24:1 phenol:chloroform:isoamyl alcohol, and the upper aqueous solution were transferred into a new 1.5mL microcentrifuge tube after each extraction. The aqueous solution was washed with equal volume of chloroform and transferred into a n ew 1.5mL microcentrifuge tube. The DNA fraction was precipitated overnight with 10% (v/v) of 3M NaOAc and twice the volume of EtOH and incubated at 20 0 C overnight. The sample was then pelleted for 20 minutes at 13,050 xg, air dried for 5 minutes and resus pended with 10 L water. One L of extracted viral DNA, 5 M of primers (forward and reverse) of UF11, 12.5 L of iQ SYBR Green supermix which contains Taq DNA polymerase and

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74 fluoresein (Biorad #170 8882) was combined to a total volume of 25 L with water. The sample was run on the Bio Rad MyiQ v2.0. In vitro GFP Infectivity Assay The infectivity phenotype was determined using a GFP expression assay and measured by FACS Calibur (BD Biosciences). Approximately 1x10 4 HEK293 cells were seeded with complete Dul 96 well plates overnight in 5% CO2 at 37C. Approximately 5.8x10 9 of purified r AAV vectors ( wild type and mutant) containing UF11 were mixed with 2x10 4 infectious units (i.u.) of Ad5 (MOI of 1) in DME M w/o fetal bovine serum (FBS), then used to infect HEK293 cells. Forty eight hours post infection, cells well harvested, washed, and expressed GFP was analyzed using a FACS Cal ibur (BD Biosciences ) implying the relative transduction efficiency of rAAVs UF11. The number of cells analyzed was ~4x10 3 Mock infections, Ad infected HEK293 cells without rAAVs UF11 were also analyzed. In silico modeling and calculation of ligand binding to the AAV capsid Molecular docking of the interaction between a heparan sulfate (HS) molecule and AAV1 (PDB accession No. 3NG9), AAV2 (PDB accession No. 1LP3), AAV5 (PDB accession No.3NTT), and AAV6 (PDB acces sion No. 3AOH) capsid was performed using the DOCK6 package v6.1 as described in program tutorial ( 175 ) The orientation and position of the VP monomers were moved to the Viper ( 56 ) standard orientation using SSM program, and an icosahedral trimer symmetric molecule was generated for AAV1, AAV2, AAV5 and AAV6 usin g Viper oligomer generator ( 56 ) A HS dissacharide (consisting of 2 sulfamido glucopyranosyl 6 O sulfate and 2 O sulfo iduronic acid) PDB

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75 c oordinate was obtained and modified from the crystal structure of the C1 complement protein complexed with heparan sulfate determined to 2.3 resolution (PDB accession No. 2WNU) ( 114 ) The atomic position of HS was translated closed to the adjacent region of AAV 3 fold wall surface. Hydrogen atoms were added to AAV VPand HS molecule coordinates using Dock Prep (in Chimera). The solvent accessible molecular sphere for all ato ms was calculated and generated using the dmsprogram (implemented in Chimera v1.3 or later) with a water probe radius of 1.4 A radius range of 15 from the initial HS molecule coordinate was used to filter and select the calculated spheres. An extra mar gin of 10 box was generated to enclose the calculated solvent accessible spheres. Scoring grid energy was calculated using the default parameter values including an attractive exponential of 6, a repulsive exponential of 12, and a dielectric factor of 4. In addition, a default 0.4 bump overlap filter was included in the calculation. Based on a series of energy scores calculated, rigid body fitting was performed on the solution with the lowest grid score for the HS molecule docked onto AAV VP trimer. Str uctural Comparison among AAV Serotype Structures The VP crystal structure was compared between AAV1 (PDB accession No. 3NG9), AAV2 (PDB accession No. 1LP3), AAV3b (PDB accession No. 3KIC), AAV4 (PDB accession No. 2G8G), AAV5 (PDB accession No. 3NTT), AAV6 (PDB accession No. 3OAH) AAV8 (PDB accession No. 2QA0), and AAV9 (PDB accession No. 3UX1), by structural alignment with the secondary structure matching (SSM) subroutine in the Coot program ( 85 101 102 ) information on residues that are structurally equivalent, inserts gaps when the atoms

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76 positions. Variable regions (VRs) between the AAV structures were identified as previously defined ( 119 ) stretches of more than two amino acids with are >1.0 apart between two serotypes.

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77 Table 2 1.Nucleotide sequence of primers used in this study. Primer Sequence VP a.a. S472R S472R S472R S472R V473D V473D V473D V473D N500E N500E N500E N500E T502S T502S T502S T502S N447S N447S W503A W503A 129 129 418 418 531, 584, 598 531, 584, 598 642 642 Primers used for screening AAV1 and AAV6 reciprocal mutants

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78 Figure 2 1.Schematic flow chart of AAV1 and AAV6 VLP expressions, purifications and structural determination.

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79 Figure 2 2. Schematic flow chart of mutagenesis and transduction phenotype studies of AAV1 and AAV6 SIA interaction residues mutants.

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80 Figu re 2 3. Stick representations of (A) a sialic acid (SIA) and (B) a heparan sulfate (HS) building block; GlcNS(6S) IdoA(2S) molecule. Molecules are colored based on the elements; green for carbon (C), red for oxygen (O), blue for nitrogen (N), and orange fo r sulfur (S). The numberings on the carbon atom of the neuraminic acid, iduronic acid and glucopyranosic acid molecules are labeled.

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81 CHAPTER 3 STRUCTURE DETERMINAT ION OF ADENO ASSOCIATED VIRUS SEROTYPE 6 Introduction AAVs have shown significant potential as clinical gene delivery vectors (discussed in chapter 1). To date, more than 100 AAV isolates have been identified ( 113 ) Among the human and nonhuman primate AAVs isolated, 12 serotypes (AAV serotype 1 (AAV1) to AAV12) have been descri bed and are classified into six phylogenetic clades on the basis of their VP sequences and antigenic reactivities, with AAV4 and AAV5 considered to be clonal isolates ( 113 ) AAV1 and AAV6, which represent clade A, differ by only 6 out of 736 VP1 amino acids (5 amino acids within VP3) and are antigen ically cross reactive. Other clade representatives include AAV2 (clade B), AAV2 AAV3 hybrid (clade C), AAV7 (clade D), AAV8 (clade E), and AAV9 (clade F) ( 113 ) The AAVs are under development as clinical gene delivery vectors (e.g., ( 57 71 78 123 124 240 ) ), with AAV2, the prototype member of the genus, being the most extensively studied serotype for this application. Th ough AAV2 has been used to treat several disorders ( 213 ) it has the disadvantage of broad in vitro tissue tropism and its naturally acqu ired neutralizing antibodies makes this vector less effective for re administration compared to other serotypes ( 69 ) Therefore, other serotypes have been studied and utilized to transduce specific tissues. Efforts have thus focused on characterizing the capsid associated tissue tropism and transduction properties conferred by the capsid of representative serotypes of other clades ( 113 ) Outcomes of these studies include the observation that AAV1 and AAV6, for example, transduce cardiac, muscle, and airway epithelial cells more efficiently (e.g., up to 200 fold) than AAV2 ( 130 137 148 ) In addition, the six residues (Table 3 1) that differ between the VPs of AAV1 and AAV6 (a

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82 natural re combinant of AAV1 and AAV2 ( 255 ) ) confer functional disparity between these two viruses. For example, AAV6 shows ~3 fold higher lung cell epithelium transduction than AAV1 ( 130 ) and AAV1 and AAV6 bind terminally sialylated proteoglycans as their primary receptor, whereas AAV6 additionally binds to heparan sulfate (HS) proteoglycans with moderate affinity ( 306 307 ) Therefore, a comparison of the AAV1 and AAV6 serotypes and, in particular, their c apsid structures can help pinpoint the capsid regions that confer differences in cellular recognition and tissue transduction. The structures of AAV1 AAV5 and AAV7 AAV9 have been determined by X ray crystallography and/or cryo electron microscopy and im age reconstruction (cryo EM) ( ( 92 119 172 180 223 232 291 311 ) and unpublished data). The capsid VP structures contain a conserved eight barrel core and large loop regions between the strands that form the capsid surface. The capsid surface is characterized by depressions at th e icosahedral two fold axes of symmetry, finger li ke projections surrounding the three fold axes, and canyon like depress ions surrounding the five fold axes. A total of nine variable regions (VRs; VRI to VRIX) were defined when the two most disparate struc tures, AAV2 and AAV4, were compared ( 119 ) The VRs contain amino acids that contribute to slight differences in surface topologies and distinct functional phenotypes, such as in receptor binding, transduction efficiency, and antigenic reactivity ( 5 6 2 ) The structure of AAV6 was determined to complete the structural library for the representative members of the AAV clade and clonal isolates and was used to further annotation the differential properties of the AAVs when correlated with the available functional data.

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83 Results and Discussions Structure of AAV6 VLP The structure of the AAV6 VLP was determined to 9.7 resolution by cryo EM and X ray crystallography to 3.0 resolution. The capsid surface of the cryo reconstructed AAV6 structure exhibits the previously defined characteristic features of AAV capsids, with a depression at each 2 fold axis, protrusions surrounding each 3 fold axis, and a canyon like depression surrounding the channel at each 5 fold axis (Figure 3 1 A and B). Consistent with t he high degree of structural similarity between AAV1 and AAV6, a correlation coefficient of 0.94 was calculated for the fit between the cryo reconstructed density map and a map generated from structure factors calculated from the docked AAV1 crystal struct ure (PDB accession No. 3NG9) based on a polyalanine model generated using the Mapman program ( 152 ) The docked model provided 642 ) that differ between AAV1 and AAV6 in the C terminal regions of VP3 (Figure 3 1 C and D). The side chain orientations and potential interactions of these residues could not be determined from the cryo reconstructed structure but were obtained from the X r ay crystal structure. The AAV6 crystal structure was determined to 3.0 resolution (Table 3 2). The refinement and molecular geometry statistics are consistent with those reported for other members of the Parvoviridae as well as structures determined for other virus families at comparable resolution, as reported on the VIPERdb website (http://viperdb.scripps.edu/). As has been previously reported for other AAV capsid structures, the N terminal region of VP2 and the first 15 amino acids of VP3 were

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84 unresol ved in both the cryo EM and X ray structures ( ( 92 119 172 180 223 232 291 311 ) ). In all of these AAV structures, only the overlapping C terminal VP (~520 a.a.) region common to the VP1, VP2, and VP3 sequence is unambiguously resolved. T he lack of ordered density for the VP N termini in the AAV6 VLPs used in this study likely results from the low copy number of VP2 (~5 copies) and the fact that the VP3 N terminus likely adopts different conformations, two factors which are incompatible wi th the icosahedral symmetry imposed during the structure determination procedures. The C terminal 519 a.a. (residues 218 to 736, VP1 numbering) common to VP2 and VP3 (hereafter referred to as VP3) were unambiguously assigned in the averaged AAV6 electron d ensity map (Figure3 2 A and B). This same stretch of amino acids was fitted into the cryo reconstructed density map (Figure 3 1 B to D). The structural topology of the AAV6 VP3 is similar to that previously reported for other parvoviruses, with a conserved eight the wall of the depression surrounding the icosahedral 2 fold axes ( ( 92 119 172 180 223 232 291 311 ) ) (Figure 3 was also observed in the cryo reconstructed density map (data not shown). The capsid 2 C). 2 C). Comparisons of the previously determined AAV crystal structures identified regions of variable conformation (VRI to VRIX) in these loops ( 119 ) The VRs are spread throughout the VP3 structure but are clustered on the capsid surface. These regions also differ between AAV6 and the other AAVs, in particular, AAV4 (Figure 3 A; see T able 3 3).

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85 These VRs contribute to phenotypic differences, such as receptor attachment, transduction efficiency, and antigenic reactivity, between the AAVs ( 5 62 ) The conserved DE and HI loops (Figure 3 2 C and 3 respectively, play essential structural and functional roles in the life cycle of the AAVs and other parvoviruses. The DE loops in five (symmetry related) monomers interact and form the channel at the 5 fold axis throu gh which genomic ssDNA is postulated to be packaged ( 171 ) This is also where a phospholipase A2 (PLA2) domain, located within the VP1 unique N termini, is proposed to be externalized during cellular trafficking ( 171 ) Structu ral variation is observed at the top of the DE loop (Figure3 3 A, VRII), consistent with dynamics which might be required for genome packaging or the PLA2 externalization ( 168 171 ) The HI loop lines the floor of the depression around the icosahedral 5 fold axes and is implicated in ca psid assembly as well as capsid dynamics associated with receptor attachment ( 93 183 ) In addition to the VP3 structure and solvent molecules, densities consistent with a purine NT and a pyrimidine base were observed in the AAV6 VLP capsid structure, despite the fact that these particles should be empty (devoid of DNA). The purine base, assigned as an adenine due to the lack of a C 2 amino group density, is located in a conserved DNA binding region and occupies the same position as the bases previously reported i n the crystal structures of AAV3, AAV4, and AAV8 ( 119 180 223 ) (Figure 3 4; the structure of AAV8 is not shown). The conserved binding region co ntains amino acids E417, V419, P420, D609, H630, P631, and S632, which are capable of forming polar and hydrophobic interactions with the NT and sandwich the bases between the two prolines (Figure 3 4; residue D609 is not shown). However, in AAV6, the orie ntation

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86 of the base as modeled into the F o F c map is rotated 180 about the plane of the base relative to the bases built into the other AAV structures due to the position of the density interpreted as the deoxyribose sugar (Figure 3 4). In the 2F o F c Four ier map (calculated before initial NT model building), a dual position of the density interpretable as the deoxyribose was observed, whereas the densities interpretable as the base and phosphate groups overlap with positions observed for the other AAV NTs (Figure 3 4). The dual orientation of the sugar portion of this ordered NT suggests that both orientations can occur with equal probability. The overlapping position of the base within the conserved binding pocket suggests that its interactions with the su rrounding amino acids dictate the ordering of this nucleotide. The observation of a single sugar conformation in the F o F c Fourier density map (Figure 3 4) suggests a higher propensity for the orientation modeled inside the AAV6 VLPs. Five angstroms from t he purine base, a second base, cytosine (assigned on the basis of the absence of methyl group density at the C 5 position of the pyrimidine ring), is ordered in a position proximal to the 3 fold axis (Figure 3 4). No sugar or phosphate groups were observed for this base, which interacts with the main chain of H630. This Histidine is conserved in representative clade members of the AAVs (Figure 3 4), but despite this conservation, the density for this base was not reported in the AAV3 crystal structure ( 180 ) nor was it observed in our structures of AAV4 and AAV8 ( 119 223 ) There is no indication that this cytosine base and the purine NT are components of a single DNA chain. Significantly, like the AAV6 structure, that of AAV8 was also determined from baculovirus/Sf9 expressed VLPs produced in the absence of the rep ORF. Th ese observations support a proposal that the AAVs are able to package

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87 fragments of host cellular DNA in the absence of Rep proteins ( 183 ) A similar packaging of cellular genomic material is commonly observed for RNA viruses expressed in a heterologous system, most likely due to a requi rement for interaction with nucleic acid for capsid assembly ( 106 ) For bacteriophage virus that packages a genome similar in size to those of parvoviruses, in virions, subgenomic pieces of DNA are also observed in empty capsids and are also likely required to facilitate capsid assembly ( 208 ) DNA packaging is n ot required for the assembly of autonomous parvovirus VLPs, as reported for minute virus of mice ( 141 168 ) but the AAV observations suggest that it may play a role for the dependoviruses, though this remains to be v erified. The lower occupancy (0.5) of the AAV6 bases, relative to the surrounding protein (as was reported for AAV8 ( 223 ) ), is consistent with the expected lack of icosahedral symmetry for NTs order ed within VLPs. Indeed, only a single copy of the ssDNA genome is packaged into wild type virions, and thus, the same NT/base cannot be ordered in all 60 sites within the capsid, unless it is part of a conserved DNA sequence repeated 60 times and forming s pecific interactions with the capsid. Such a conserved DNA sequence has not been reported for the AAVs. Given occupancy of less than one, the strict NCS utilized for electron density averaging during the structure determination would be expected to result in reduced sigma for NT/base density at each averaging cycle and eventual loss of signal. Thus, the unexpected observation of ordered DNA density inside AAV6 and other AAVs suggests the presence of a common DNA recognition motif inside the capsid directly under the 3 fold axes (Figure 3 2 C). The fact that the recognition site amino acids are conserved in most AAV sequences and all the

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88 structures determined to date suggests an important function for this DNA interaction in the life cycle of the viruses, suc h as a role in capsid stabilization or assembly. Comparison of AAV6 Structure to Those of Other AAVs Pinpoints C apsi d Regions That Control Differential Tissue Transduction P roperty Superposition of the AAV6 structure with those available for AAV1 to AAV5, AAV8 and AAV9 using the SSM application in the Coot program ( 101 102 ) shows differences two sero types ( 119 ) ) with (i) AAV2 at VRI, VRII, VRIV, VRV, and VRVII; (ii) AAV3 at VRI, VRII, VRIV, VRVI, VRVII, and VRIX; (iii) AAV4 at VRI to VRIX; and (iv) AAV8 at VRI, VRII, VRIV, VRV, and VRVII (Figure 3 3). Variable regions I and IV were commonly divergent in conformation between AAV6 and t hese four AAVs (Table 3 3; Figure 3 3 B and C). The amino acids that form these two surface loops are also highly divergent between the representative members of the AAV clades. AAV1 and AAV6 are 99% identical and superimpose with an RMSD of 0.33 and th ey exhibit the lowest 3; Figure 3 3). AAV4 is the most structurally diverse from AAV6, with which it shares the lowest degree of sequence homology (59% compared to 80 to 99% wit h the other AAVs; Table 3 though the structures superimpose with an overall RMSD of 0.94 AAV6 and AAV2 itions in VRIV differ between 1.3 and 4.9 (Table 3 3). The AAV VRs cluster on the capsid surface in the raised regions between the icosahedral 2 and 5 fold axes (VRs I, III, and IX) and on the wall (VRs VI and VII) and top (VRs IV, V, and VIII) of the p rotrusions surrounding the icosahedral 3 fold axes ( 5 62 ) Significantly, VRI and VRIV (Figure 3 3 B and C)

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89 have been shown to play a role in AAV tissue transduction and antigenic recognition ( 192 265 ) These reports suggest that structural heterogeneity, in addition to sequence variation, confers these capsid associated functions. The observation that VRIV adopts slightly different conformations in AAV1 and AAV6 may be related to this loop being the least ordered VP3 common region in the AAV6 structure. Atoms in the amino acids at the top of the loop exhibit high temperature factors, consistent with high thermal motion and minor conformation variation compared to those for AAV1. To obtain a more detailed analysis of the structural determinants that dictate differences in receptor attachment and tissue transduction in the highly homologous AAV1 and AAV6, we superimposed the AAV1 crystal structure (PDB accession No.3NG9) onto the re fined AAV6 structure in the electron density map. This enabled the visualization of the positions of five of six amino acids (AAV1 and AAV6 amino acids E418D, E531K, F584L, A598V, and N642H) that differ between the AAV1 and AAV6 VPs (F584L and N642H are sh own in Figure 3 2 A and B, respectively). Amino acid 129 (VP1 numbering) in the VP1 unique region was not present in the VP2/VP3 VLP construct used for this structure determination. Three of the ordered residues (531, 584, and 598) are located on the capsi d surface, at (V598) or close to (K531 and L584) the icosahedral 3 fold symmetry axes (Figure 3 5), whereas D418 and H642 are located on the interior surface of the capsid, below the same capsid region (Figure 3 5). Residues D418 and H642 are located in st ructurally conserved VP regions, whereas surface residue 531 is located in VRVI, residue 584 is located in VRVIII, and residue 598 is close to VRVIII. The localization of these five amino acids at or surrounding the icosahedral 3 fold axes, with K531 being near the icosahedral 2 fold axes in VRVI,

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90 highlights the importance of these capsid regions in AAV biology with respect to receptor attachment and transduction efficiency. The surface exposed amino acid differences indicate potential sites for conferring the differential receptor recognition and transduction properties of AAV1 and AAV6 that are associated with entry or pre uncoating interactions. The residues on the inside, with 418 in the vicinity of the DNA binding region (Figure 3 5) are unlikely to pla y a role in receptor attachment but could be involved in post entry / uncoating events that affect transduction efficiency. With respect to receptor attachment, residue K531 in AAV6 (E531 in AAV1) has been reported to be important for its HS binding proper ties, and an E531K mutant of AAV1 shows strong affinity for HS and also confers liver cell transduction ( 305 ) identifying a position at the base of the protrusions and close to the depression at the icosahedral 2 fold axes (Figure 3 5) that confers this phenotype. None of the mutations in AAV1 converting the remaining five amino acids which differ from those in AAV6 to the type found in the latter virus conferred an HS binding ph enotype ( 306 ) A novel primate AAV variant, AAV(VR 942), which also uses HS as a primary receptor, contains a K528 residue that is predicted to be structurally equivalent to the AAV6 K531 residue ( 258 ) (Table 3 5). AAV2 also binds HS ( 275 ) but lacks this basic residue and, instead, utilizes two critical residues, R585 and R588, along with R484, R487, K527, and K532 (minor contributors) (AAV2 VP1 numbering) (Table 3 4) for this interaction ( 160 183 228 230 ) Except for R487, these residues form a basic footprint on the surface (Figure 3 6 B) of the AAV2 capsid on the inner face of the protrusions surrounding the 3 fold axes. Interestingly, AAV1 and AAV6 contain basic residues R485, R488, K528, and K533 (equivalent to R484, R487, K527, and K532, respectively, in AAV2) in the

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91 equivalent region of the mapped AAV2 HS binding site, plus R576 and H597 (Figure 3 6 A, B, and D). A ba sic region is created on the AAV1 and AAV6 capsid surface by residues R485, R576, and H597 close to the 3 fold axis and is missing in AAV2, which contains R484, Q575, and N596 at the structurally equivalent positions (Figure 3 6 A, B, and D). However, a ro le in HS binding has not been reported for R576 and H597. On the other hand, in AAV6, K531 is located adjacent to R488 (equivalent to AAV2 R487, but is now surface exposed), K528, and K533, which creates a second continuous basic patch on the capsid surfac e, whereas in AAV1, E531 creates a gap in this patch (Figure 5A and D). Hence, this continuous, basic region is likely sufficient for and the determinant of HS binding by AAV6. AAV5, which binds sialic acid, is missing all these basic residues, except for R471, which is equivalent to AAV6 R485 (Table 3 4). In addition to these basic residues, the juxtaposition of acidic residues on the capsid surface and their interactions with amino acids in the vicinity of the mapped basic HS binding residues appears to b e important for the binding of this glycan by AAV serotypes. Mutation of an acidic residue, D532 to N532, adjacent to K533 on the AAV6 capsid surface (Figure 3 4 D and 3 7), in an AAV variant derived by directed evolution from AAV libraries, shH10, was rep orted to confer HS binding dependence and sialic acid binding independence (Table 3 1) for cellular transduction by the variant ( 165 ) Though wild type AAV6 binds HS, it can transduce cells in the absence of HS but not in the absence of sialic acid (33, 58, 70 ). Residue D532 is predicted to stabilize the surface loop containing K531by means of electrostatic interactions with H527 and D562 (Figure 6A), which are likely to be disrupted if the acid group at position 532 is lost. This loop also contains K528 and K5 33, which flank K531 on the capsid surface, as

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92 discussed above (Figure 5D and 6A). The D532N mutation reduces the negative charge on the capsid and likely disrupts the D532 H527 D562 interaction; consequently, it could alter the conformation of this loop a nd thus the cellular interactions of constituent residues. The predicted loop stabilizing interaction is conserved in HS binding AAV2 through the interactions of E531 H526 D561 and also E563 (Figure 3 7); thus, the stabilization is expected to be tighter i n this virus. The side chain of AAV6 residue E564, participate in the stabilizing interactions (Figure 3 7). The shH10 mutant has improved HS binding compared to that o f wild type AAV6 and exhibits an improved transduction phenotype. AAV6, on the other hand, binds HS more weakly than AAV2 but also exhibits better transduction properties. Thus, binding affinity alone does not control transduction efficiency ( 165 ) Also, improved HS binding of the AAV6 D532N m utant sialic acid. These observations highlight the complex nature of cellular interactions that control cellular transduction mechanisms. Further support for the r ole of acidic residues in HS binding was reported by Wu et al ., who found that alanine scanning mutagenesis of acidic AAV2 residues 561 DEEE 564 to 561 AAAA 564 resulted in a noninfectious HS negative (HS ) mutant ( 305 ) AAV6 residue D562 is not on the capsid surface but is structurally equivalent to AAV2 D561, which along with E563 and E531 participates in interactions which stabilize the basic residues involved in AAV HS binding (Figure 3 7), as discussed above. A disruption of the D561 and E563 interactions with neighboring residues, which is predicted to occur when these acidic residues are mutated to alanine in AAV2, is likely

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93 involved in the HS phenotype of the 561 AAAA 564 mutant. Interestingly, AAV2 E531, D561, and E563 are contained in two highly conserved acidic stretches of amino acids in the representative AAV clade members, with the exception of clonal isolates AAV4 and AAV5. The above observations suggest that their i nteractions are important for stabilizing the configuration of HS binding regions on the AAV2 capsid as well as the AAV6 capsid. The region of the AAV capsid required for interaction with sialic acid has not been structurally mapped, but mutagenesis studie s with AAV5 suggest the involvement of A581 at the icosahedral 3 fold axes ( 105 ) (Figure 3 6 C). An alanine is conserved at the equivalent positions in AAV1, AAV2, and AAV6 (A592, A591, and A592, respe ctively) (Table 3 4; Figure 3 6). Of note, the region at or immediately adjacent to the icosahedral 3 fold axis is hydrophobic in AAV1, AAV5, and AAV6, which bind sialic acid, and polar in AAV2, which does not (Figure 3 6). Thus, if this is a conserved, si alic acid recognition site among the AAVs, the V598A difference between AAV6 and AAV1, which, along with V582 and A592, forms a continuous hydrophobic surface at the 3 fold axes (Figure 3 6 A and D), may be involved in the sialic acid binding interaction o f both viruses. Both viruses are reported to have common sialic acid linkage recognition ( 307 ) and thus, if residue 598 exhibits a serotype specific phenotype in transduction, it is unlikely to be due to this interaction. Efforts to engineer AAV variants with improved/tissue specific transduct ion properties have led to chimeric AAV1 and AAV6 vectors that show differential lung epithelial cell transduction efficiency dependent on which residues in the two serotypes are located at VP1 unique position 129 and within the common VP3 sequence ( 186 187 ) The AAV6 F129L mutation (AAV6.2 in Table 3 1) confers 2 fold better transduction in airway epithelium compared

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94 to the parental serotype and AAV6 K531E (AAV6.1 in Table 3 1), which eliminates HS binding, confers an AAV1 transduction phenotype that is reduced compared to that of the parental AAV6 ( 187 ) This observation suggests that amino acids at both the 129 a nd 531 positions affect cellular transduction. Residue 129, located in the VP1 unique region, is part of a PLA2 domain in the parvoviruses that is predicted to be located inside the assembled capsid but that later becomes externalized through the 5 fold ch annel during capsid trafficking through the endocytic pathway. This is purported to aid endosomal escape for nuclear localization and subsequent genome replication ( 171 ) Residue 531, as discussed above, facilitates HS binding in AAV6. Thus, residues 129 and 531 are likely involved in post entry events and receptor recognition, respectively. Consistent with this suggestion, a mutant containing F129L and K531E (Table 3 1, AAV6R2) had the reduced transduction phenotype of the virus with the sin gle K531E mutation ( 187 ) indicating that the K531E mutation functions e arly in infection, prior to the step affected by amino acid F129L. Li et al used directed evolution from an AAV library to identify chimeric human airway epithelia (HAE) transducing vectors, HAE 1 and HAE 2 (Table 3 1), which contain mostly AAV1 and AAV6 sequences and which have improved transduction efficiency relative to that of the parental serotypes ( 186 ) HAE 1 contains AAV1 residues 1 to 583/641 to 736 and AAV6 residues 584 to 640, and HAE 2 contains AAV9 residues 1 to 30/104 to 193, AAV1/AAV6 residues 31 to 103, AAV6 residues 194 to 641, and AAV1 residues 642 to 736. The reported transduction efficiencies for these viruses compared to those of the parental serotypes were in the order AAV1/AAV9 < AAV6 < HAE 1 < HAE 2, with HAE 1 and HAE 2 showing ~3 to 4 fold and ~2 fold improved transduction compared to that of AAV1 and AAV6,

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95 respectively. HAE 1 contains AAV1 residue L129 (not observed in the crystal structures), capsid surface residue E531, and interior residues E418 and N64 2, with AAV6 contributing surface residues L584 and V598 (Table 3 5). Thus, L584 and V598 confer the ~3 fold improvement in HAE 1 transduction compared to that of AAV1. The improvement in transduction relative to that in AAV6 could be due to the L129 from AAV1, as discussed above. HAE 2 contains the equivalent of AAV1 L129 (contributed from AAV9) and interior residue N642 from AAV1 and AAV6 capsid surface residues, K531, L584, and V598, as well as interior residue D418 from AAV6 (Table 3 1). For this vector the AAV6 K531 residue likely combines with the L129, L584, and V598 residues of HAE 1 to achieve the further improvement in transduction compared to that of the parental viruses and HAE 1. Both chimeras were observed to bind equally to the apical surface of HAE, suggesting that their difference in transduction was post entry, consistent with an intracellular step in the viral life cycle at which L129 is critical. A functional role for the interior residues, 418 and 642, is yet to be defined. In summary, t his comparative analysis of AAV1 and AAV6 highlights key AAV residues that control host interactions, including receptor recognition and attachment as well as post entry events, which enable successful infection and improved cellular transduction. These re sults should facilitate further molecular characterization and manipulation of AAV vectors for improved tissue specific targeting.

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96 Table 3 1. Amino acid differences between AAV1 and AAV6 and their reported mutants AAV 129 418 531 532 584 598 642 Glycan T arget AAV1 L E E D F A N S AAV1 E/K L E K D F A N HS + (and S) c AAV6 F D K D L V H HS and S AAV6.1 F D E D L V H HS (and S) c AAV6.2 L D K D L V H HS (and S) c AAV6R2 L D E D L V H HS (and S) c HAE1 L E E D L V N (HS and S) d HAE2 L D K D L V N (HS and S) d shH10 F D K N L V N HS (and S ind) e a = Mutant residues in bold face have an AAV6 parental original; those underlined have an AAV1 parental origin; b = S: sialic acid; HS: heparan sulfate; HS + : HS positive; HS : HS negative; c = The sialic acid binding phenotypes of these mutants were not discussed in the respective publications but are assumed to be still present; d = The glycan targets for these mutants were not discussed in this publication; thus, the phenotypes indicated ar e assumed; e = This mutant is sialic acid independent (S ind) for cellular transduction. Table was adapted from ( 2 25 )

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97 Table 3 2. Data Collection, Reduction and Refinement Statistics a Data Collection CHESS F1 0.917 Space group R32:H Unit cell parameters () a = 262.6, c = 609.9 Resolution 40.0 3.0 (3.1 3.0) No. of unique reflections 119,617 (8,285) Completeness (%) 72.3 (50.5) Average I/sigma 6.5 (2.5) Rmerge (%) 15.4 (44.3) Refinement CNS v1.2 No. of atoms (protein/solvent/DNA) 4,117/ 12 /25 Average B factors ( 2 ) 61.87 Rcryst / Rfree (%) 27.5 / 28.8 RMSD bonds () and angles ( 0 ) 0.009 / 1.48 Ramachandran plot Most favorable allowed (%) 92.5 Additionally allowed (%) 7.5 a Values in the parenthesis are for the highest resolution shell; b CNS = Crystallography and NMR System; c I hkl < I hkl I hkl | ) x 100, where I hkl is the intensity of an individual hkl reflection and < I hkl > is the mean intensity for all measured values of this reflection; d F obs | | F calc F obs |) x 100, F obs and F calc are the amplitudes for the observed and calculated reflections, respectively; Rfree was calculated with the 5% of reflections excluded from the data set during refinement. Table was adapted from ( 225 )

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98 Table 3 structures overall and for VRI and VRIV AAV Serotype a.a. % identity Overall VRI VRIV AAV1 99 0.33 1.3 1.8 AAV2 83 0.67 1.2 5.7 1.3 4.9 AAV3 86 0.57 1.7 5.8 1.3 6.4 AAV4 59 0.94 0.9 6.8 1.0 14.6 AAV8 80 0.57 1.3 7.4 1.4 2.4 AAV9 80 (The t able was adapted from ( 225 ) )

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99 T able 3 4. Comparison of residues reported to be involved in heparan sulfate and sialic acid binding for AAV6, AAV1, AAV2, AAV5, andAAV VR942 and the amino acids at equivalent positions in each virus AAV Amino acid at residue( AAV6 VP numbering) 485 488 528 531 533 586 589 592 6 R R K K K S T A 1 R(485) R(488) K(528) E(531) K(533) S(586) T(589) A(592) 2 R(484) R(487) K(527) E(530) K(532) R(585) R(588) A(591) 5 R(471) G(474) L(515) S(518) N(519) S(575) T(578) A(581) VR942 R(482) R(485) K(525) K(528) K(530) N(583) A(586) T(589) *; Numbers in parentheses are based on VP1 numbering for the respective serotypes.Table was ada pted from ( 225 )

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100 Figure 3 1.AAV6 structure. (A) Surface representation of the AAV6 cryo reconstructed image at 9.7 resolution. The capsid surface density is shown as a radially colored, depth cued image (low to high radii, pink to gray). Selected icosahedral 2 fold (2f), 3 fold (3f), and 5 fold (5f) axes of the capsid are indicated by arrows. (B) Cross sectioned slab from the cryo the polyalanine model (residues 218 to 736, pink) derived from the AAV1 crystal structure (PDB accession No. 3NG9). Dashed arrows indicate the approximate locations of icosahedral axes of symmetry. (C) Coil representation of an AAV6 VP backbone trace (pink) showing the locations of the five amino acids (pink spheres) within VP3 that d iffer between AAV1 and AAV6. The first and last letters in each residue label refer to AAV6 and AAV1, respectively. (D) A trimer of AAV6 VPs (pink, green, and blue) showing the symmetry related clustering of the differing residues (spheres) shown in panel C and colored according to the monomer in which they reside. These residues cluster near the icosahedral 3 fold axes in both the interior (residues 418 and 642) and exterior (residues 531, 584, and 598) surfaces of the capsid. The view is approximately dow n the icosahedral 3 fold axis. Approximate positions of icosahedral 2 3 and 5 fold symmetry axes of the capsid are depicted as filled ovals, triangles, and pentagons, respectively, in panels C and D. Panels A and B were generated using the Chimera prog ram ( 237 ) and panels C and D were generated using the PyMol prog ram ( 86 ) and adapted from ( 225 )

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101

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102 Figure 3 2.Crystal structure of AAV6. (A and B) Sections of the 2Fo Fc electron density 584 in panel A and residue 642 in panel B) that differ between AAV6 and AAV1. The AAV1 (pur ple) and AAV6 (atom type) coordinates are shown in stick form. (C) Ribbon diagram representation of AAV6 VP3 monomer barrel regions program ( 86 ) and adapted from ( 225 )

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103 Figure 3 3.Comparison of available AAV crystal structures. (A) Superposition of the VP3 monomer structure of AAV1 (purple), AAV2 (blue), AAV3 (yellow), AAV4 (red), AAV5 (gray), AAV6 (pink), AAV8 (green) and AAV9 (brown). Common variable regions VRI to VRI X are labeled with roman numerals. The DE and HI loops are labeled. Approximate positions of the icosahedral 2 3 5 fold axes are depicted as described in the legend to Figure 1. (B and C) Close up views of VRI and VRIV, respectively. This figure was ge nerated using Pymol program ( 86 ) and adapted from ( 225 )

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104 Figure 3 4. AAV6 DNA binding site. A. The conserved nucleotide binding pocket, showing the ordered Fo interpreted as a deoxyadenylate and cytosine (labeled). This orientation shows position of the density interpreted as the deoxyribose sugar, wh ich is rotated approximately 180 relative to the positions of the sugars in the NT models built for other available AAV structures. B. The dual conformation of the deoxyadenylate NT observed in the 2Fo Fc density map. Refinement of two models (I and II) b uilt into this density indicated that the model I conformation had the highest occupancy (based on temperature factor comparison), consistent with the orientation that was dominant in the Fo Fc difference density map. (C) 900 rotation from panel (A); resid ue D609 has been omitted for clarity. (D) Comparison of nucleotide binding pocket in AAV3 (yellow), AAV4 (black), and AAV6 (pink) crystal structures. This region (structure and DNA) is also conserved in AAV8 (data not shown). AAV6 amino acid positions are labeled. This figure was generated using the PyMol program ( 86 ) AAV6 amino acids wit hin 2.4 to 5.0 of the ordered density are shown and labeled. This figure was mod ified from ( 225 )

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105 Figure 3 5. Locations of amino acid differences in AAV1 / AAV6 capsids. (A) Surface representation of an AAV6 trimer viewed from outside approximately along a 3 fold axis (middle) with the white boxed section r otated 900 (top). The monomers are colores pink (reference), green (3 fold) and blue (3 fold), with the differing AAV1/AAV6 amino acids colored in yellow. Residues K531E, L584F (3f L584 is from a 3 fold related monomer) and V598A (first letter AAV6 and sec ond letter, AAV1) are located on the capsid surface. The panel on the left shows the close proximity of residue 531 and 584 at the base of the 3 fold protrusions facing icosahedral 2 fold axis. (B) Same as panel A, but rotated 1800 to show the location of the residues D418E and H642N on the interior surface of the capsid. The approximate position of the 3 fold axis (3f axis) is indicated with a solid arrow in all three panels. Approximate positions of icosahedral and 2 and 5 fold symmetry axes on the capsi d are depicted as in Figure 1. This figure was generated using Pymol program ( 86 ) and mod ified from ( 225 )

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106 Figure 3 6. Comparison of AAV surface residues. Schematic Roadmap projections ( 309 ) of surface residues in a portion of the icosahedral asymmetric unit for crystal structures of AAV1 (A), AAV2 (B), AAV5 (C), and AAV6 (D) are shown. The are a occupied by each amino acid residue correlates to surface exposure when the capsid is viewed down an icosahedral 2 fold axis. The boundary for each residue is shown in black, and the colors correspond to acidic (red), basic (blue), polar (yellow), and hy drophobic (green) residues. Dashed outlines highlight regions proposed to play a role in glycan binding by the respective serotypes. Residues are labeled by type and number. The icosahedral 3 fold axis is depicted by the filled triangle. Figure is ad apted from ( 225 )

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107

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108 Figure 3 7 Stabilizing interactions for the K531 loop. (A) Residues D532, H527, and D562 that form electrostatic interactions at the base of the loop containing basic residues K528, K531, and K533 are shown along with neighboring residues R485 and R488, equivalent to R484 and R487, respectively involved in HS binding by AAV2. 3f L584 is contributed from a 3 fold (3f) related VP3 monomer. The residues (in stick form) are colored according to atom type: carbon, yellow; nitrogen, blue; and oxygen, red. Dashed lines indicate the distance between in teracting residues. Disruption of the D532 H527 D562 interaction by a D532N mutation is predicted to alter the conformation of the basic loop and in turn could alter AAV6 HS binding properties. (B) AAV2 residues E531, H526, D561, and E563 that form electro static interactions which stabilize an equivalent surface loop in this serotype containing residues K527, E530, and K532 and the neighboring amino acids, R484 and R487, involved in HS binding. Mutation of D561 and E563 to alanine disrupts HS binding in AAV 2. 3f L583 is contributed from a 3f VP3 monomer. Residues are colored as in panel A.A superposition of the residues shown panel A with AAV2 (PDB accession No. 1LP3) using the AAV6 amino acids (labeled in black) shown according to atom type and those for AA V2 are colored blue (labeled in blue). This figure was generated using the PyMol program ( 86 ) and adap ted from ( 225 )

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109 CHAPTER 4 STRUCTURALLY ANNOTAT ING AAV1 AND AAV6 GLYCAN BINDING INTERACTIONS I ntroduction One key feature of the viral capsid that determines cell type specificity or tissue tropism is its interaction with a specific host receptor (glycoproteins or glycolipids) ( 260 ) Several receptors have been identified to be involved in AAV infection and these receptors can function either as the primary receptor or the co receptor (Table 1 2) ( 12 37 53 88 153 155 188 244 263 264 274 275 298 306 307 ) The primary receptor, also called the attachment r eceptor, is the first receptor recognized by the virus capsid and initiates the interaction of viral capsid surface with the cellular membrane. On the other hand, co receptors are cell membrane associated molecules that are critical for viral internalizati on. Several AAVs utilize sialic acid (SIA) as their primary receptor and, for these serotypes, neuraminidase treatment abolishes cellular transduction ( 153 263 307 ) AAV2 and AAV3 utilize heparan sulfate proteoglycan (HSPG) as the primary receptor for the liver cells ( 37 275 ) AAV4 utilizes O linked sialylated oligosaccharides and AAV5 utilizes N linked sialylated oligosaccharides to infect cells ( 153 ) Glycan microarray screening (by the Consortium for Functional Glycomics (CFG)) and biochemical assays showed N linked sialic acid as the primary receptor for AAV1 ( 307 ) AAV6, which belongs to the same phylogenetic clade as AAV1 and differs by only 6 of 736 residues on the VP1, can bind both SIA and HS molecules as the primary receptors ( 306 ) Recently, biochemical and in vitro has identified terminal galactose containing glycans as receptor for AAV9 ( 264 ) The cell surface glycans utilized by the other AAV

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110 phylogenetic clade representatives, AAV7 AAV8 are currently unknown. Othe r than AAV2, AAV3, AAV5, and AAV9 ( 27 91 105 160 179 183 228 230 ) the region on the remaining AAV capsid surfaces required for the binding of HS / SIA glycans are yet unknown Regions on the 3 fold axis of capsid surface have been shown to be the critical for HS binding by AAV2 as well as SIA binding by AAV5. In order to improve the efficacy of AAV gene delivery with respect to cardiac, muscle and lung cellular targeting, we need to structurally and biochemically annotate the recept or binding sites of AAV1 and AAV6 capsids and characterize the difference role in receptor recognition and tissue specificity. This chapter will focus on the study to identify and pinpoint residues on the AAV1 and AAV6 capsids utilized for glycan recogniti on. Results and Discussions Crys tal Structure of AAV1 omplex To better understand the interaction between AAV1 and SIA, we soaked AAV1 VLP crystal in cryo collection. Diffraction data sets were obtained for the complex and processed to 3.0 resolution in the monoclinic system with C2 space group and completeness of 60.5%. Data collection and refinement statistics are shown in Table 3 1. These statistical values and quality of the data sets was consistent with those used to solve the crystal structures of other parvoviruses (e.g., ( 191 212 ) ). Using the method of molecular replacement with the AAV1 crystal structure (PDB accession No.3NG9) to solve the phase problem, we were able to assign 519 residues (218 736) of the VP3 common C terminal region using averaged 2F o F c electron density map. To identify the glycan binding site on AAV1 capsid, an averaged F o F c difference map was calculated and a

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111 shallow pocket on the outside wall of 3 fold protrusion facing the 5 fold cylindrical pore. 3 4GlcNAc ) was utilized for this crystal structure which implies that only the terminal SIA moiety was structurally stabilized upon capsid interaction (Figure 4 1) The orientation of the SIA molecule was assigned into the density in which the extension of the GalNac and GlcNAc was not sterically clashing with VP coordinates. The modeled SIA molecule was refined with occupancy of 1.0 and resulted in the averaged B f actor of ~ 2 0 3 2 The crystal structure reveals a 1:1 stoichiometry of binding between AAV1 VP and SIA molecule and thus suggests a total of 60 SIA binding sites on AAV capsid. The glycan interacts (contact distance less than 4.0 ) with residues S268, D27 0, N271, N447, S472, V473, N500, T502 and W503. This binding pocket is contributed by two interacting VP monomers Variable regions involved in SIA interactions are VRI (268, 270 and 271), VRIV (447, 472 and 473), and VRV (500, 502, and 503). Other than N4 47, which interacts with SIA via a main chain atom (carboxyl oxygen to SIA C11) and ND with SIA O4, the other interactions involve hydrogen and hydrophobic bonding interactions between the VP a.a. side chains and SIA molecule. Glycerol group of the SIA int eracts with side chains of residues S268, D270, N271, S472, V473 and W503. Side chain of residues N500 and T502 interact with SIA carboxylate group. These similar SIA interacting residues were also observed in the influenza virus hemaglutinin (HA) complex structure (PDB accession No.1R VZ), which involves residues V135, T136, A137, W153, L194, and Q226 ( 111 ) Structural comparison between the

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112 AAV1 (PDB accession No.3NG9) and AAV 1 SIA crystal structures reveals no dramatic residues on the tip of VRIV. RMSD between the C alpha positions of this region (residue 453 456) between two structures were calcu lated to be in range of 0.6 1. 1 These residues are known to have the highest B factor range within VP (more than 2 fold of the averaged thermal factor for protein atoms) among all available AAV structures. This suggests that the variability might due to the inherent flexibility of VRIV within the VP monomers. In addition to VP amino acids assignment, averaged positive F o F c difference density was also observed in the interior surface surrounding the center of 3 fold symmetry axis which was been modeled and proposed as density for nucleotide molecules in several available AAV crystal structures (discussed in Chapter 3). Structural Comparison of AAV S erotypes at AAV1 SIA Interacting R esidues Superposition of the AAV1 crystal structure with those available for AAV2 to AAV6, AAV8, and AAV9 using the Coot program ( 101 102 ) AAV1 SIA interacting regions (AAV1 SIAIR = AAV1 SIA contact residues 2a.a) (Fi gure 4 3) (Table 4 2) ( ( 5 6 62 92 119 180 223 225 232 291 311 312 ) and unpublished data). Among the serotypes compared, AAV4 (PDB accession No. 2G8G) and AAV5 (PDB accession No. 3NTT), which have 61.8 and 60.0 % a.a. identity to AAV1 respectively, possess the most diverse conformations within these variable regions which create steric clashes into the SIA electron density map. Amino acid structural alignment of SIA interacting residues on VRI among AAVs shows 100% identity (ex cept AAV4 and AAV5). This implies contact residues on VRI might not be the potential important residues for AAV1 SIA interaction.

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113 AAV1 (PDB accession No.3NG9) shows a 99.2% ident ity and small r.m.s.d. (0.05 0.78 ) within these interacting regions suggesting that AAV6 might utilize this same capsid region for SIA interaction. On the other hand, structural alignment between AAV1 and AAV 6 and most clinically studied serotype AAV2 (b inds HSPG in different region) within this capsid region identified five different residues: N447S, S472R, V473D, N500E, and T502S (AAV1/6 to AAV2 a.a.) (Table 4 4). While a series of single mutations of AAV2 residues equivalent to AAV1 SIA binding site to alanine have no significant effect in capsid assembly and genome packaging properties, these single mutations significantly increase (3 4 fold) or knockdown (~20 fold) HeLa and HepG2 cellular transduction (compared to wild type AAV2 at 100%). In addition to the VRI SIA interacting region, W503 in the VRV interacting region is also conserved among AAVs. Recent biochemical, in vitro and in vivo studies showed that AAV9 utilize s the similar capsid region (D271, N272, Y446, N470 and W503 (AAV9 VP numbering)) for its interaction with terminal galactose (GAL) glycans ( 27 264 ) Galactose differs from SIA molecule in that it does not possess the carboxylate and glycerol chain in SIA ; instead four carbons (C1, C3 C5) are occu pied by oxygen atoms This implies that the AAV1 residues that interact with SIA glyce rol and carboxylate chains and are different to AAV9 are the potential AAV1 and AAV9 res idues that dictat e receptor binding specificity. Using molecular docking with Pat chDock ( 261 ) the potential interaction site between GAL mol ecule on AAV9 trimer molecule was calculated and modeled into the similar binding pocket ( 27 ) Superposition of the AAV1 and AAV9 glycan binding region highligh ted differences in capsid residue side chains that might be

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114 affecting their receptor binding properties. The side chain of AAV9 N470 (equivalent to G470 in AAV1) sterically clash es into the AAV1 SIA binding pocket The O3 and O4 of GAL interact (2.6 3.3 ) with side chain of N470 and the GAL binds deeper in the pocket and involves residues D271, N272, and W503 Although mutagenesis study had showed a mutation in Y446 is critical for AAV9 GAL binding ( 27 ) the molecular docking result indicates the distance between Y446 is more than 4.5 away from the GAL molecule. Previous mutagenesis studies on the Y444F in AAV2 (equivalent to AAV9 Y446), which is proposed to avo id ubiquitin mediated proteasome degradation, resulted in significant increase (~1 3x compared to wild type AAV2) in hepatocyte transduction ( 323 324 ) .However, an AAV3 Y444F mutation does not show similar transduction properties, which implies diverged functional roles of tyrosine residue in this capsid region amon g different AAVs ( 67 ) In s ilico Docking Model of AAV6 Hepara n Sulfate While efforts are ongoing to determine a crystal structure for AAV6 bound to HS, a molecular docking approach using Dock6 has been used to obtain a low and stable interaction energy for HS binding to the AAV6 capsid surface ( 175 ) In order to validate the quality of thi s computational approach, the AAV2 crystal structure (PDB accession No.1LP3) was used as a positive control. A HS molecule (obtained and modified from 2WNU PDB file) was initially modeled onto the AAV2 capsid surface adjacent to the previously characterize d HS binding region: R484, R487, H509, K527, R585 and R588. Using the default parameters (as mentioned in chapter 2), DOCK6 was able to calculate the lowest interaction energy for the HS molecule in which the final orientation and location is shifted compared to the initial modeled position re lative to the AAV2 capsid

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115 surface (data not shown). The AAV2 capsid surface basic residues identified to be within interacting distance with HS are R484, R487, H509, K527, R585 and R588 (Figure 4 A C); consistent with the previously identified binding resi dues using mutagenesis studies and cryo electron microscopy and image reconstruction (cryo reconstruction) ( 160 183 228 230 ) A similar approach was undertaken for analyzing a potential AAV6 HS complex w ith DOCK6 which was able to calculate the lowest interaction energy between these molecules. The surface basic residues identified for the AAV6 HS interaction are R485, R488, K528, K531, and R576 (Figure 4 D F). The position of this calculated model is con sistent with previous mutagenesis studies in AAV1 and AAV6, in which a single mutation K531E was able to abolish AAV6 HS binding property ( 306 ) These residues are located on AAV6 VRV (R485 and R488), VRVI (K528 and K 531), and VRVIII (R576). The HS model utilized in this study consists of two core carbohydrate domains; monosulfatediduronic acid (IdoA) and bisulfatedglucopyranosic acid (GlcNS). The sulfate group from IdoA is shown to interact with side chains of R485 an d R576, and the sulfate group from GlcNS interacts with R488 and K531. In addition to the sulfate group inte ractions, the side chain of K 5 2 8 is shown to bind the HS molecule via the carboxyl chain of IdoA. Another HS carboxyl chain is shown to interact wit h main chain atom from G513 (which is nota residue of any VR). Besides the hydrogen bonding interactions, L584 (F584 i n AAV1) is shown to involve in van der W aal interaction with the HS. This predicted interactin g region is assembled from two VP monomers a nd located on the shoulder of the protrusion facing the capsid 5 fold symmetry axis.

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116 In silico modeling using AAV1 and AAV5 trimer molecules also localized the HS molecule in proximity with AAV2 and AAV6 HS interaction region (data not shown). Grid scores calculated from DOCK6 suggested relative AAV HS interaction energies; AAV6= 64.4, AAV2= 54.6, AAV5= 50.1, and AAV1= 44.8. Structural Comparison of AAV S erotypes at AAV6 HS Interacting R esidues Structure alignment and superposition of the AAV6 structure with those available for AAV1 to AAV5, AAV8, and AAV9 shows the degree of variability (r.m.s.d) within the AAV6 HS interacting VR among different serotypes ( ( 5 6 62 92 119 180 223 225 232 291 311 312 ) and unpublished data) and identifies five AAV2 / AAV6 HS interacting regions (HSIR) (483 490, 508 515, 526 535, 574 578,and 584 591) (Figure 4 5 and Table 4 3). Residues R585 and R588 (AAV2 VP n umbering) are unique to AAV2 HS interacti on and none of the compared se rotypes have basic amino acids at this o n position present in residues 526 531 (VRVI) (Table 4 3). Except for AAV4 and AAV5 (which are shown to have the highest variability in these IRs), the r.m.s.d. among serotypes was calculated to be much smaller than for the SIA interacting regions. Sequence alignment within HS contact residues among different serotypes showed that AAV4 and AAV5 do not have similar basic amino acids at these position s (Table 4 4), except for AAV6 R485 (K479 and R471 in AAV4 and AAV5, respectively). In addition to R585 and R588, and H509 are unique for AAV2 HS interaction and AAV6 utilizes an alternate residue R576 (equivalent to Q575 in AAV2). The calculated model of AAV6 HS interaction also involve residue G513 which is conserved among AAV serotypes and the HS interaction is shown to be contributed by main chain.

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117 Besides the involvement of HSIR for AAV2 / AAV6 HS binding, several studies have identified this region a s also being important for AAV serotype specific transduction phenotype. AAV9 is a unique serotype due to its capability to surpass blood brain barrier (BBB) and has been shown in vivo to transduce neurons in brain and spinal cord ( 197 ) Zhonget. al. has isolated a new AAV variant (CLvD8) from chimpanzee which differs by only four a. a. (I647T, Y445H, H527Y, and R533S (AAV9 to CLvD8)) in VP3 protein compared to wild type AAV9 and has lost the potential to cross vascular barrier ( 325 ) Site directed mutagenesis was perform ed within these four residues to generate four single mutant vectors carrying luciferase reporter gene. Comparisons of in vivo luciferase expression after intravenous, intramuscular and intranasal administrations showed that two single mutants (H527Y and R 533S) were not able to perform similar transduction phenotype as wild type AAV9. This data suggests the role of residues H527 and R533 in VRVI for surpassing vascular barrier during systemic delivery. Previous studies had demonstrated that mutations of sur face exposed tyrosine residues (Y F) on AAV capsid are able to protect AAV delivery vectors against ubiquitin mediated proteasome degradation ( 323 324 ) Ubiquitination is a post translation modification process in which the activated ubiquitin enzyme creates an amide bond via the lysine residue in the protein and targets it for proteasomal degradation. Gabriel et.al. had perf ormed site directed mutagenesis on surface exposed lysine residues on AAV capsid and shown that AAV2 single K532R (equivalent to K533 in AAV6) mutation can increase HEK293 and HeLa cellular transductions by 9x and 18x, respectively ( 110 )

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118 This study suggests the important role of residue 533 in VRVI in AAV2 c ellular transduction properties. In summary, the crystal structure of AAV1 3.0 resolution and an in silico method was utilized to predict the HS binding site on the AAV6 capsid surface. Variable regio ns important for AAV1 / AAV6 SIA binding are VRI, VRIV, and VRV, and for AAV2 / AAV6 HS interaction are VRV, VRVI and VRVIII. Structural superposition of the receptor binding sites on AAV1 and AAV6 onto different AAV serotypes have provided useful informat ion to identify and pinpoint specific regions on AAV1, AAV2, and AAV6 capsid surface required for glycan receptor binding. Results from these studies will be applicable for the modification of glycan contact residue to engineer recombinant vectors with spe cific receptor targeting properties which will be the first step towards increasing the transduction efficiency of AAV vector.

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119 Table 4 1. Data Collection, Reduction and Refinement Statistics a Data Collection CHESS F2 0.979 Space group C2 Unit cell parameters () Resolution 50.0 3.0 (3.1 3.0) No. of unique reflections 593,542 (45,233) Completeness (%) 60.5 (46.3) Average I/sigma 4.0 (1.6) R merge (%) 16.6 (45.3) Refinement Refmac No. of atoms (protein/SIA/DNA) 4,117/ 21 /18 Average B factors ( 2 ) 31.0 Rcryst / Rfree (%) 2 6 3 / 2 7 0 RMSD bonds () and angles ( 0 ) 0.014 / 1.37 Ramachandran plot Most favorable allowed (%) 92.1 Additionally allowed (%) 6.0 a Values in the parenthesis are for the highest resolution shell; b CNS = Crystallography and NMR System; c R merge I hkl < I hkl I hkl | ) x 100, where I hkl is the intensity of an individual hkl reflection and < I hkl > is the mean intensity for all measured values of this reflection; d R cryst F obs | | F calc F obs |) x 100, F obs and F calc are the amplitudes for the observed and calculated reflections, respectively; Rfree was calculated with the 5% of reflecti ons excluded from the data set during refinement.

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120 Table 4 structures overall and for SIA interacting regions (SIAIR) Sero Type a.a. % identity numbering) Overall 266 273 445 449 470 475 498 505 AAV2 83.7 0.63 0.28 3.19 0.44 0.86 0.48 1.02 0.18 1.18 AAV3 85.8 0.61 0.28 3.66 0.40 0.90 0.32 0.92 0.13 0.59 AAV4 61.8 0.92 0.45 3.25 0.46 1.39 0.25 1.05 1.15 3.11 AAV5 60.0 1.00 0.56 2.63 0.71 1.78 0.32 2.01 0.90 3.25 AAV6 99.2 0.33 0.09 0.77 0.05 0.32 0.05 0.42 0.20 0.79 AAV8 80.0 0.54 0.29 3.47 0.20 0.63 0.11 0.40 0.63 0.99 AAV9 79.8 0.50 0.18 3.20 0.76 0.99 0.16 0.33 0.40 0.78 R.m.s.d was calculated using SSM superposition in Coot program. SIAIR = SIA contact residues 2 a.a.

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121 Table 4 structures overall and for HS interacting regions Sero Type a.a. % identity numbering) Overall 483 490 510 515 526 535 574 578 584 591 AAV1 99.2 0.33 0.26 0.55 0.09 0.43 0.15 0.72 0.06 0.32 0.22 0.62 AAV2 83.7 0.67 0.26 0.91 0.35 0.90 0.21 0.84 0.26 0.80 0.35 0.84 AAV3 85.6 0.60 0.06 0.24 0.25 0.70 0.30 1.24 0.40 0.89 0.37 0.68 AAV4 62.0 0.94 0.30 1.44 0.23 1.02 1.21 3.99 0.13 1.21 1.36 2.94 AAV5 59.4 1.05 0.63 1.78 0.30 1.15 0.41 3.16 0.67 0.90 0.71 2.49 AAV8 80.0 0.54 0.16 0.51 0.36 0.64 0.19 0.52 0.12 0.52 0.34 0.98 AAV9 79.6 0.50 0.13 0.52 0.38 0.66 0.13 1.03 0.16 0.34 0.23 0.82 R.m.s.d was calculated using SSM superposition in Coot program. SIAIR = SIA contact residues 2 a.a.

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122 Table 4 4. Structure alignme n t of AAV residues involve in SIA and HS interaction. Amino Acids a AAV Serotypes References AAV1 AAV2 AAV3 AAV4 AAV5 AAV6 AAV8 AAV9 268 S S267 S267 Q259 S258 S268 T270 S269 ( 193 ) 270 D D269 D269 S260 A260 D270 D272 D271 ( 27 193 ) 271 N N270 N270 N261 N261 N271 N273 N272 ( 27 193 ) 345 S T344 T346 A335 T335 S345 T347 T346 ( 211 ) 444 Y Y443 Y443 W437 Y436 Y444 Y446 Y445 ( 325 ) 445 Y Y444 Y444 G438 R437 Y445 Y447 Y446 ( 27 67 323 324 ) 447 N S446 N446 Q440 V439 N447 S449 S448 ( 193 ) 470 G D469 S470 N464 R456 G470 T472 N470 ( 27 193 ) 472 S R471 S472 S466 A458 S472 A474 A472 ( 193 ) 473 V D472 L473 N467 N459 V473 N475 V473 ( 193 ) 485 R R484 R485 K479 R471 R485 R487 R485 ( 160 183 193 228 230 ) 488 R R487 R488 G482 G474 R488 R490 R488 ( 160 183 193 228 230 ) 500 N E499 N500 I493 S487 N500 N502 E500 ( 193 ) 502 T S501 P502 L501 A488 T502 A504 A502 ( 193 ) 503 W W502 W503 I502 F489 W503 W505 W503 ( 193 ) 505 S G504 A505 Y504 T491 S505 A506 G505 ( 211 ) 510 N H509 H510 T509 E496 N510 H512 A510 ( 160 183 193 228 230 ) 513 G G512 G513 G512 G499 G513 G515 G513 ( 18 193 ) 527 H H526 H527 A526 N514 H527 H529 H527 ( 193 325 ) 528 K K527 K528 G527 L515 K528 K530 K528 ( 193 ) 531 E E530 E531 D530 S518 K531 E533 E531 ( 193 306 ) 533 K K532 K533 S531 N519 K533 R535 R533 ( 110 193 ) 576 R Q575 Q576 M574 V565 R576 E578 S576 ( 193 ) 586 S R585 S586 S584 S575 S586 Q588 S586 ( 160 183 193 228 230 ) 589 T R588 T589 N587 T578 T589 T591 A589 ( 160 183 193 228 230 ) 592 A A591 T592 T590 A581 A592 Q594 Q592 ( 91 105 193 ) a = AAV1 VP numbering

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123 Figure 4 1.Crystal structure of AAV1 complex (A) Surface representation of AAV1 colored in depth cue rendering (from blue (in) to red (out). The black open circle represents the location of AAV1 SIA interaction. Lower panel is the close up window of the AAV1 SIA interaction region. Differen t colors (purple and blue) represent different VP monomers The SIA molecule (shown as stick representation) was modeled onto the shoulder of the protrusion surrounding 3 fold symmetry axis. Variable regions involved in these intra monomer interactions were labeled (VRI, VRIV, VRV and VRVIII). White mesh represents the averaged 2F o F c level. The SIA molecule is colored based on the atom types (green for carbon, red for oxygen, and blue for nitrogen ). (B) Stick representa tion of AAV1 colored based on atom types (yellow for carbon) showing the potential SIA interacting residues (except F501) which are within a distance of 4.0 from the 2F o F c density shown in blue mesh. These SIA interacting residues are located on VRI, VR IV, and VRV. Figures were generated using the PyMol program ( 86 )

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124 Figure 4 2. Superposition of AAV1 SIA crystal structure with other AAV structures in VRI: 259 275 (A), VRIV: 442 477 (B ), and VRV: 496 508 (C). (A spheres. (D) Overall superposition of panel A C. Purple mesh r epresents the averaged 2F o F c representations and color coded as purple: AAV1, blue: AAV2, yellow: AAV3, red: AAV4, grey: AAV5, pink: AAV6, green: AAV8 and b rown: AAV9. Figures were generated using Pymol program ( 86 )

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125 Figure 4 3.Molecular docking model of AAV9 cr ystal structure with GAL using patch DOCK. (A) Stick representation of AAV9 crystal structure colored based on atom types (brown for carbon, red for oxygen and blue for nitrogen) (PDB accession No. 3UX1) showing the location and po sition o f modeled GAL (brown) using patch DOCK. Residues in the vicinity of the GAL are labeled and located on VRI, VRIV, and VRV. (B) Superposition of AAV1 SIA crystal structure (as shown in Figure 4 1B) onto AAV9 GAL model. Residues with similar amino ac ids are labeled as inpanel A. Figures were generated using Pymol program ( 86 )

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126 Fi gure 4 4. Surface trimer representation of AAV2 (A C) and AAV6 (D F) showing in silico calculation of HS interaction region on AAV2 and AAV6 trimer molecules using DOCK6. Different colors represent different VP monomers. (A and D) Surface trimer representa tion showing the solvent accessible surface area (yellow spheres) calculated and generated by INSPH in DOCK6 with the site box (10) shown in black line. (B and E) Surface trimer representation as panel A and D (for AAV2 and AAV6, respectively) showing the location of DOCK6 modeled HS molecules shown as stick representations. Basic residues on the AAV capsid surface are colored green. Dashed line boxes show the close up window for panel C and F. Figures were generated using Pymol program ( 86 )

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127 Figure 4 5. Superposition of AAV6 HS in silico model with other AAV structures in VRVI: 524 537 (A), VRV: 483 493 (B), and VRVIII: 574 578 and 584 591from two different VP monomers (C). (A C) AAV6 HS HS unique residues (R585 and R588) are shown as blue spheres. (D) Overall superposition of panel A C. HS molecule is shown in stick representation. IdoA =iduronic acid and GlcNS= Glucopyranosic acid. coded as purple: AAV1, blue: A AV2, yellow: AAV3, red: AAV4, grey: AAV5, pink: AAV6, green: AAV8 and brown: AAV9. Figures were generated using Pymol program ( 86 )

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128 CHAPTER 5 CHARACTERIZING THE T ISSUE TRANSDUCTION D ETERMINANTS IN AAV1 AND AAV6 Introduction The crystal structures of AAV1 and AAV6, determined to 2.5 and 3.0 resolution (PDB accession No. 3NG9 and 3AOH), respectively, showed 5 of the 6 differing AAV1/AAV6 amino acids within the ordered VP structure (~218 736) localized proximate to the icosahedral 3 fold axis. This observation suggested that this caps id region plays an important role in dictating the differences in tissue transduction observed for these two closely related viruses (reviewed in ( 5 6 62 132 ) and C hapter 1, 3 and 4). Two of the residues (418 and 642) were located in the interior surface of the capsid and three residues (531, 584 and 598) were located on the exter ior capsid surface ( 225 ) Thus a series of reciprocal single residue mutations (AAV1 to AAV6 and AAV6 to AAV1) were generated to interrogate the role of the interior and exterior residues in dictating the AAV1 and AAV6 transdu ction efficiency ( 306 ) Data arising from these studies will provide clues on the role of these residues in initial cell surface recognition, post entry transitions, capsid trafficking, and possibly capsid processing f or efficient transduction. Results and Discussion To pinpoint critical residues dictating differential lung and muscle transductions between AAV1 and AAV6, series of single reciprocal mutants were generated (as described in ( 247 306 ) ) and expressed using human embryonic kidney (HEK) 293 cells. Polymerase chain reactions (PCR) were performed using sets of reverse and forward primers to confirm the mutated codons in the

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129 cap ORF. Capsid titers (determined using a commercially available ADK1a ELISA) for recombinant AAV1s (rAAV1) and rAAV6 are in the range from 2.4 x 10 13 to 6.6 x 10 13 and 2.5 x 10 12 to 5.9 x 10 13 caps id s/ml, respectively (Table 5 1). With respect to genome packaging, for the rAAV1s and rAAV6s, despite the fact that rAAV1 and r AAV6 constructs carried rep ORFs from different serotypes (rAAV1 with AAV2 Rep and rAAV6s with AAV6 Rep), the viral genome titers were similar at approximately ~1x10 11 vg/ml. This result is consistent with the previous studies which showed the complementary Rep function between AAV serotypes in genome replication and packaging ( 68 142 ) Recombinant AAV1 and rAAV6s were purified using ion exchange chromatography ( 327 ) and subjected to negative stain electron microscopy (EM) (Figure 5 1 and 5 2). Intact rAAV capsids (~25nm) were observed in the EM (Figure 5 2). The preliminary transduction efficiencies among recombinant virions were a ssessed using the commonly used HEK293 cell (Figure 5 3). When the percentages of green cells were compared to the wild type virions (normalized to 100%), no significant differences (n=3, P value < 0.05) were observed among the single site mutations (Figur e 5 3B). In summary, mutagenesis and biochemical characterization of the twelve reciprocal single mutations between AAV1 and AAV6 show no significant difference in the capsid assembly and genome packaging. A preliminary GFP infectivity assay showed no sig nificant difference in the transduction phenotypes of the mutants in HEK293 cells.

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130 Table 5 1. Biochemical characterization of AAV1 and AAV6 reciprocal mutants a Recombinant AAV b capsids/mL c genome/mL d AAV1 wild type 3.45 x 10 13 4.05 x 10 11 AAV1.L129F 2.40 x 10 13 5.40 x 10 11 AAV1.E418D 4.40 x 10 13 4.60 x 10 11 AAV1.E531K 2.55 x 10 13 3.83 x 10 11 AAV1.F584L 6.80 x 10 13 3.93 x 10 11 AAV1.A598V 3.45 x 10 13 4.74 x 10 11 AAV1.N642H 3.95 x 10 13 4.65 x 10 11 AAV6 wild type 3.49 x 10 12 3.49 x 10 11 AAV6.F129L 3.58 x 10 12 3.58 x 10 11 AAV6.D418E 4.41 x 10 13 4.41 x 10 11 AAV6.K531E 4.37 x 10 13 4.37 x 10 11 AAV6.L584F 7.40 x 10 12 7.40 x 10 11 AAV6.V598A 2.52 x 10 12 2.52 x 10 11 AAV6.H642N 5.89 x 10 13 5.89 x 10 10 a Averaged from three independent repeats b Recombinant virions were generated as previously described ( 306 ) c Determined using ELISA (Progen # PRAAV1) d Determined using BioRAD SYBR Green against UF11

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131 Figure 5 1.Silver stain SDS PAGE of purified r AAV1 and rAAV6 reciprocal mutants showing the presence of VP1 (81kDa), VP2 (72kDa), and VP3 (63kDa).

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132 Figure 5 2. Negative stain electron microscopy (EM) of purified rAAV1 and rAAV6 wild type s and reciprocal mutants. Figures were obtained from an FEI Spirit microscope.

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133 Figure 5 3. Green Fluorescence Protein (GFP) Infectivity Assay using HEK293 cells. (A) Representative FACS analysis of the GFP expression by recombinant AAV1 and AAV6 reciprocal mutants carrying pTRUF11 (GFP reporter gene). (B) The averaged result from the three independent GFP infectivity assays shown in panel A.

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134 CHAPTER 6 SUMMARY AND FUTURE D IRECTIONS The focuses of this study were to structurally characterize the glycan receptor interaction on the capsid surface of two related AAV serotypes, AAV1 and AAV6, and to identify the potential functional role of the si x differing residues between these serotypes. In the effort to characterize the AAV capsid glycan receptor interaction, the structure of AAV6 was initially determined to 9.7 using cryo electron microscopy and image reconstruction (cryo reconstruction). T he AAV6 reconstruction map shows the characteristic features of AAV capsid topology; a depression at each 2 fold axis, protrusions surrounding each 3 fold axis, and a canyon like depression surrounding the channel at each 5 fold axis. In this study, we wer e able to identify the location of five out of six AAV1/AAV6 different residues. However to better determined the orientation of a.a. side chains and potential interactions differ between these serotypes, we undertook X ray crystallography approach. The cr ystal structure of AAV6 VLP was determined at 3.0 resolutions and this allows the completion of crystal structural library of the representative members of AAV phylogenetic clades (A F and clonal isolates) ( 92 119 180 223 225 232 246 291 311 312 ) Structural super position and comparisons of crystallographic ordered VP region (218 736) between AAV6 and other serotypes variable regions (VRI VRIX) which spread across the VP3 common region b ut are clustered on the AAV capsid surface. Comparison between AAV1 and AAV6 crystal structures enables us to annotate five of six different amino acids (E418D, E531K, F584L, A598V and N642H). The ordered five different residues are clustered surrounding t he virus icosahedral three fold axis, residue 418 and 642 are located in the

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135 interior surface and residue 531 (on VRVI) 584, and 598 (on VRVIII) are located on the exterior capsid surface (Figure 6 1 and Figure 6 2 ). Based on previous mutagenesis, bioche mical and in vivo studies, regions on the three fold symmetry of AAV capsid have been shown to exhibit functional role in receptor interaction, tissue transduction and antigenicities ( 5 7 ) For the assessment of cap sid associated differential receptor interaction between these serotypes, efforts have been underway to obtain crystal diffraction data for AAV1/ AAV6 glycan complex. Currently, we have obtained crystal structure of AAV1 4GlcNAc ) complex at 3.0 resolution. Using this X ray data, we were able to calculated positive F o F c difference electron density map and a SIA molecule was modeled and refined into the density map located in a pocket closed to the plateau at the outside wall of the 3 fold protrusion. These potential interacting residues are S268, D270 and N271 on VRI; N447, S472 and V473 on VRIV; and N500, T502 and W503 on VRV (Figure 6 1). Structural alignment between AAV1 and AAV6 shows 100% a.a. identity on this region sugges ting that these serotypes utilize similar region for SIA interaction (Figure 6 2). In the case of AAV6 HS interaction, in silico DOCK6 program we were able to calculate the lowest and most stable interaction region between HS molecule onto AAV6 trimer c rystal structure. The location of modeled HS molecule was calculated at proximity to basic residue patches, containing R485 and R488 on VRIV; R528 and K531 on VRVI; and R576 on VRVIII (Figure 6 1). This HS interacting capsi d region is located on the outsid e shoulder of the p rotrusion surrounding the three fold axis (Figure 6 2).

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136 Mutagenesis studies of the AAV1 SIA contact residues to AAV2 corresponding a.a.; N447S, S472R, V473D, N500E, and T502S, are underway. In addition to these mutations, we also propos e to generate W503A mutation which is shown to be important for AAV9 GAL interaction. Following the generation and expression of these mutations, immediate future of the project will focus on the biochemical characterization of these mutants using ELISA an d qPCR to calculate and examine the possible effect of mutation on capsid assembly and genome packaging. In order to assess potential role of glycan interaction in cellular transduction, we propose to perform GFP infectivity and cell binding assays on thes e recombinant mutant AAVs against different cell lines (e.g., muscle, lung and liver), as well as on stably transformed CHO cell lines which are deficient in a series of glyco 1, Lec 2, Lec 8, pgs D667, and A745. Based on the results of these assays, future efforts might be inevitable to generate double and triple mutations of the SIA contact residues to assess the cooperative properties of this SIA interacting region. In addition to SIA interacting residues, a series of reciprocal mutation s between AAV1 and AAV6 on their six differing residues were successfully expressed in HEK293 cells and purified using ion exchange chromatography. Using ELISA, qPCR and GFP infectivity assays, we were able to show that the recombinant w ild type AAV1 and AAV6 and their corresponding singletons exhibit no significant differences in capsid assembly, genome packaging, and HEK293 transduction properties. Provided with the current HEK293 transduction data, more efforts are necessary to better characterize the functional role of individual AAV1/AAV6 different residues in cellular interaction and transduction using different tissue types, including muscle, heart and lung cell line. In

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137 addition, double and triple residue mutagenesis and biochemica l studies are also necessary to address the role of the six AAV1/AAV6 differing residues in AAV cellular transduction. In addition to receptor targeting or de targeting, vector stability and antigenicity properties are also major concerns in vector generation for clinical application. Hence, comparative analysis of the thermal stability of the recombinant wild type s and al l mutants (12 AAV1 / AAV6 SIA and 12 AAV1 / AAV6 reciprocal mutants) will be tested. Overall, data from these studies will provide the structural information on capsid regions involved in receptor binding, genome packaging, capsid assembly and stability as well as antigenicity which will aid in the development of superior AAV vectors with improved tissue specificity and transduction efficiency.

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138 Figure 6 1. Structural alignment of crystallographic ordered VP amino acid sequences (~217 736) of AAV1, AAV2, AAV4 and AAV6. Non identical amino acids (AAV1, AAV2 and AAV6) are highlighted in the figure. Secondary structure more than 1.0 compared to AAV1 are subs c ripted. Roman numerals indicate residues on variable regions (6). Capsid regions identified for sialic acid (SIA) and heparan sulfate (HS) interactions are labeled (S and H, respectively). A sterisk s indicate the different residues between AAV1 and AAV6.

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139 Figure 6 2. Trimer surface representation of AAV6 crystal structure showing glycan interacting regions. Different colors (cyan, purple and light purple) represent different VP monomers. Re sidue 584 and 598 are colored in orange. K531 is colored green. Heparan sulfate (HS) and sialic acid (SIA) interacting residue are colored as blue and red, respectively. Approximate positions of icosahed ral 2 3 and 5 fold symmetry axes on the capsid are depicted as oval, triangle and pentagon, respectively.

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169 BIOGRAPHICAL SKETCH Robert Ng was born in 1984 in Medan, Indonesia. He spent most of his childhood and schooling years in Medan. He completed his high school education at Sutomo 1, Medan in 2002. Towards the later years of his high school education, he became very interested in biotechnology, and wanted to study abroad and become a scientist. After graduating from high school, he enrolled in Bachelors in Science (B.Sc.) at National Taiwan University, Taipei, Taiwan and graduated in 2006 with an honor in agricultural chemistry. During this time he conducted two year research at National Taiwan University, Taipei, taiwan gaining experience in molecular biology and proteomi cs techniques under the guidance of Dr. Whi Fin Wu. This experience kindled within him a keen interest for further research. He then continued to do his master in biochemistry and molecular biology at University of Florida, Gainesville, FL. During his mast er's program Robert developed deep passion for serious scientific research and work under interactions between mitochondria ribosomal proteins. Without completion of his maste decided to apply to the PhD degree in Interdisciplinary program (IDP) in University of Florida. In the fall of 2007 he started the IDP program and spent his first year complet ing the core courses and lab rotations, finally began research as a graduate assistant in Agbandje McKenna Laboratory in summer 2008. While pursuing his PhD program Robert started working under the supervision of Dr. Mavis Agbandje McKenna (Professor, Depa rtment of Biochemistry and Molecular Biology, UFL). Robert was initiated to some very interesting and challenging problems in the field of virus crystallography and cryo electron microscopy reconstruction.