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Houston, TX May 2019 ABSTRACT Houston, TX May 2019 ABSTRACT Engineering Adeno-Associated Virus for Protease Targeted Gene Therapy and Immune Avoidance by Tawana Michelle Robinson Adeno-associated virus (AAV) has earned significant attention as a safe and efficient gene therapy tool. AAV has been used in over 100 clinical trials to treat a variety of human diseases. However, non-specific targeting to diseased cells and activation of the host immune response hinder its therapeutic efficacy. To address these challenges, genetic modification of the AAV capsid can lead to an improved gene delivery platform. Therefore, capsid-engineering strategies may be necessary to develop optimized vectors for clinical progress. This present work reveals design rules governed by amino acid properties for engineered AAV to become activated by upregulated proteolytic biomarkers in diseased sites. AAV constructs with varying chemical properties were synthesized and characterized for functional behavior. In parallel, a Nature- inspired strategy was employed to create an immune-evasive AAV vector. A ii panel of AAV vectors with inserted stealth peptides in the AAV capsid wasgenerated to study immune cell uptake. Finally, to gain a better understanding of AAV intracellular trafficking, we found several amino acid residues that are necessary for viral infectivity. The ultimate goal for my research contributions is to develop and to advance AAV vectors for future clinical applications. iii Acknowledgments First, I will like to thank Junghae Suh for your mentorship in shaping me to become a scientist. I am grateful for you pushing me to prepare me for the future that you envisioned for me - towards academic leadership and a stellar research career. Thank you for always coaching me and allowing me to pursue my research passion. Thank you Jeffrey Hartgerink for your guidance and scientific advice throughout the years. Your dedication in supporting my research goals during challenging circumstances is unforgettable. Thank you Dean Matsuda for your endless support, advice, and guidance. You have inspired me to pursue excellent research, but also the importance of being involved in serving others in the Rice community. Thank you Jane Grande-Allen for mentoring me and being a supportive anchor throughout my predoctoral career. You made tremendous efforts to be available for me to talk about my research and career opportunities. Finally, I give a special thank you to all of my friends and family who fed me, supported me, and loved me endlessly through such a challenging journey. iv Table of Contents Chapter 1: Introduction......................................................... 2 Gene Therapy.......................................................................... 2 Adeno-Associated Virus (AAV)…………………………………………………………… 4 AAV Structure and Genome……………………………………………………… 4 AAV Intracellular Trafficking…………………………………………………… 5 AAV Immune Response…………………………………………………………… 7 AAV Transgene Immune Response………………………………… 7 AAV Capsid Immune Response……………………………………… 8 AAV Capsid Engineering…………………………………………………………………… 9 Rational insertion of defined peptide/protein………………………… 10 Rational insertion of random peptide……………………………………… 12 Random insertion of defined peptide/protein………………………… 13 Tweaking viruses through point mutations…………………………… 15 Random point mutations………………………………………………… 15 Rational point mutations………………………………………………… 16 v Thesis Overview………………………………………………………………………………… 17 Chapter 2: Role of Tetra Amino Acid Motif Properties on the Function of Protease-Activatable Viral Vectors………………… 22 Introduction.......................................................................... 22 Results……………………………………………………………………………………………… 24 Production of Viruses with Peptide Lock Variants……………………… 24 Capsid Stability of Viruses with Peptide Lock……………………………. 27 Impact of Peptide Lock Variants on Heparin Binding………………… 28 Ability of Peptide Lock Variants to Block Cellular Transduction……………………………………………………………………………….. 29 Ability of Peptide Locks to be Cleaved by Extracellular Proteases……………………………………………………………………………………. 32 Discussion……………………………………………………………………………………………. 34 Conclusions…………………………………………………………………………………………. 37 Materials and Methods………………………………………………………………………… 39 References…………………………………………………………………………………………… 44 Chapter 3: Engineering Adeno-Associated Virus with a Self-Peptide for Immune Evasion………………………………………………………... 49 vi Introduction.............................................................................. 49 Results…………………………………………………………………………………………………… 51 Synthesis of AAV-SP variants……………………………………………………… 51 Infectivity of AAV-SP variants……………………….……………………………. 53 Phagocytic Susceptibility of AAV-SP variants………..……………………. 54 Discussion and Conclusions………………………………………………………………….. 56 Materials and Methods………………………………………………………………………….. 59 References……………………………………………………………………………………………… 61 Chapter 4: Investigation of an Essential Serine-Rich Motif for AAV Intracellular Trafficking to the Nucleus Introduction............................................................................. 67 Results…………………………………………………………………………………………………. 69 Bioinformatic Alignment of N-Terminal S/T Motif in AAV VP1 and VP2 Subunits……………………………………………………………………… 69 Production of AAV2 Vectors with Alanine Substitutions in the N-Terminal S/T motif………………………………………………………………… 71 Transduction Efficiency of AAV2 N-Terminal S/T Motif Mutants………..…………………………………………………………………………… 72 vii Cellular Internalization of AAV2 N-Terminal S/T Motif Mutants………..…………………………………………………………………………… 74 Nuclear Uptake of AAV2 N-Terminal S/T Motif Mutants………..…………………………………………………………………………… 74 Nuclear Uncoating of AAV2 N-Terminal S/T Motif Mutants………..…………………………………………………………………………… 75 Transcriptional Levels of AAV2 N-Terminal S/T Motif Mutants………..…………………………………………………………………………… 77 Discussion and Conclusions………………………………………………………………. 77 Materials and Methods………………………………………………………………………… 80 References…………………………………………………………………………………………… 86 Chapter 5: Conclusions and Future Directions……………………. 94 viii List of Figures Figure 1-1 Timeline for 50 years of AAV vector technology……………… 3 Figure 1-2 AAV Structure and Genome ……………………………………………. 5 Figure 1-3 AAV Cellular Trafficking…...……………………………………………… 6 Figure 2-1 Protease-activatable virus (provector) design and concept………………………………………………………………………………………………. 23 Figure 2-2 Titers of provector candidates with different tetra amino acid (4AA) motifs………………………………………………………………………………………. 25 Figure 2-3 Detection of viral capsid proteins (VPs) and peptide lock incorporation of provector candidates via Western blot………………….. 26 Figure 2-4 Capsid stabilities of provector candidates as assessed by their abilities to protect their genomes from nuclease digestion…………….. 28 Figure 2-5 Ability of various 4AA motifs to disrupt virus-heparin binding……………………………………………………………………………………………….. 29 Figure 2-6 Transduction assay to test if the various 4AA motifs are able to inhibit cellular transduction by provector candidates in the absence of proteases……………………………………………………………………………………….. 30 Figure 2-7 Detection of proteolyzed VP fragments of Provector-D4 ix and Provector-E4 in the presence of MMP7………………………………………. 33 Figure 2-8 Recovery of cellular transduction ability of Provector-D4 and Provector-E4 in the presence of MMP7………………………………………. 34 Figure 3-1 Design of SP insertion into AAV capsid……………………………. 51 Figure 3-2 Structural characterization of AAV-SP…………………………….. 52 Figure 3-3 Transduction of AAV-SP mutants…………………………………….. 54 Figure 3-4 Macrophage uptake of AAV-SP mutants…………………………. 55 Figure 3-5 Macrophage uptake of AAV-SP mutants with anti- SIRPα…………………………………………………………………………………………………. 56 Figure 4-1 Multiple sequence alignment of 167 AAV VP1 sequences……………………………………………………………………………………………. 70 Figure 4-2 Production of AAV2 vectors with alanine substitutions in the N-terminal S/T motif……………………………………………………………….. 72 Figure 4-3 Transduction efficiencies of AAV2 S/T motif mutants in HeLa and HEK293T cells…………………………………………………………………….. 73 Figure 4-4 Cellular uptake of AAV2 S/T motif mutants in HeLa cells……………………………………………………………………………………………………… 74 Figure 4-5 Nuclear uptake of AAV2 S/T motif mutants in HeLa cells……………………………………………………………………………………………………… 75 x Figure 4-6 Nuclear capsid uncoating of AAV2 S/T motif mutants in HeLa cells………………………………………………………………………………………… 76 Figure 4-7 GFP mRNA expression levels of AAV2 S/T motif mutants……………………………………………………………………………………………... 77 xi This page was intentionally left blank 1 INTRODUCTION Gene Therapy Gene therapy began almost fifty years ago as a treatment strategy for genetic human diseases (Figure 1-1) 1. Initially conceived as a treatment solely for inherited diseases, gene therapy is now being applied to acquired conditions as well, such as genetic engineering of T cells for cancer immunotherapy. In the 1990s, gene therapy was tested in human clinical trials 1. Unfortunately, one of the trials to deliver the ornithine transcarbamylase (OTC) gene via an adenoviral vector produced no therapeutic benefits and resulted in the unexpected death of Jesse Gelsinger 2. One primary explanation for disappointing clinical results was insufficient knowledge of viral vector biology. Success was finally achieved for the gene therapy of an X-linked inherited form of severe combined immunodeficiency3. At least 90% of the patients were effectively treated for immunodeficiency 4. For another hematopoietic disorder, chronic granulomatous disease (CGD), researchers observed clinical progress in diminished immune infections 3. Efficient genetic modification
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