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Abstract

In recent years, the adeno-associated virus (AAV) has developed as the most widely-used vector for clinical gene therapy and basic biology research. AAV capsid has been extensively modified via different approaches to create the vectors with desired transduction efficiency and specificity in the target tissues. Nowadays, the majority of new capsids are developed from directed evolution in animal models because it does not require preliminary knowledge of the interaction between AAVs and target cells. However, in recent years, studies in different animal models have revealed that, variants selected in animals often fail to translate across species. One of the main reasons is the difference of receptors across species. Therefore, to develop AAV capsids that can be used in human for gene therapy, it is important to know the receptors that mediate the transduction. In the study, I aimed to rationally design AAV vectors to transduce target cells by binding to cell-specific receptors. I worked on two major projects: (1) targeting to hACE2-expressing cells; and (2) penetration of the blood-brain-barrier (BBB) followed by targeting to glioblastoma (GBM). To achieve these goals, I displayed already known receptor-binding ligands (peptides and proteins) on the AAV capsid with three methods, namely, genetic insertion into the AAV capsid protein, chemical/covalent conjugation to the assembled AAV capsid, and non-covalent binding to the assembled AAV capsid. Here, I developed a site-specific chemical conjugation method, which allowed chemical conjugation of any kind of ligands on the desired site by introducing a cysteine residue. Furthermore, I adjusted the number of ligands displayed on the AAV capsid by producing mosaic capsids composed of wild-type VPs and ligand-displaying VPs. Thirdly, I displayed two ligands on the same capsid by producing mosaic capsids composed of two ligand-displaying subunits. In the hACE2-targeting project, display of receptor binding domain of SARS-CoV-2 on the AAV capsid via non-covalent binding was successful and achieved the desired specific transduction of hACE2-expressing cells in vitro. In the BBB-penetration and GBM-targeting project, I identified two variants, AAV9-V4-FAL and AAV9-V8-FAL, displaying the EGFR and EGFRvIII binding peptide FALGEA, which transduced GBM efficiently in U87-transplanted nsg mice after intravenous administration and which were depleted from the major off-target tissue liver. Meanwhile, from the analysis of off-target tissues, I identified another variant, AAV9-V8-RTD, displaying the integrin αvβ6/αvβ8 binding peptide RTDLDSLRT, which transduced heart and muscle with high efficiency.