{"@context":"http://iiif.io/api/presentation/2/context.json","@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/manifest.json","@type":"sc:Manifest","label":"Surface Remodeling of Lentiviral and Adeno-Associated Viral Vectors via Metabolically Introduced Bioorthogonal Functionalities","metadata":[{"label":"dc.description.sponsorship","value":"This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree."},{"label":"dc.format","value":"Monograph"},{"label":"dc.format.medium","value":"Electronic Resource"},{"label":"dc.identifier.uri","value":"http://hdl.handle.net/11401/77070"},{"label":"dc.language.iso","value":"en_US"},{"label":"dc.publisher","value":"The Graduate School, Stony Brook University: Stony Brook, NY."},{"label":"dcterms.abstract","value":"As the most effective means to transfer genetic material into cells and tissues, viral vectors have been widely used for both therapeutic and basic biological applications. Extensive effort has been invested in the modification of viral surfaces to generate vectors with enhanced properties such as restricted cell tropism, easier purification and reduced immunogenicity. In particular, targeted gene transduction of specific cells has been a highly desirable goal and attempted by various strategies. Bioorthogonal chemical reactions such as Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC) and Staudinger ligation have significantly facilitated the study of biomolecules in their native environment due to the fast rates and superior selectivity under physiological conditions. We modified the surface of two frequently used viral gene delivery vehicles, lentiviral and adeno-associated viral (AAV) vectors, by a two-step labeling method. The viral particles were first metabolically labeled during production with unnatural substrates carrying the bioorthogonal azide group and subsequently modified via click reactions, allowing the attachment of a variety of ligands. This strategy exhibited minimal perturbation on virus physiology and demonstrated remarkable flexibility. For lentiviral vectors, the unnatural sugar N-azidoacetyl sialic acid (SiaNAz) was metabolically incorporated into viral envelope glycoproteins simply by addition of the precursor peracetylated N-\u00ce\u00b1-azidoacetylmannosamine (Ac4ManNAz) into the cell culture medium during virus production. Ligands targeting high-profile cancer associated receptors were functionalized with the strained alkyne bicyclo-[6.1.0]-nonyne (BCN) and introduced onto the viral surface through SPAAC, leading to enhanced transduction toward targeted cells in vitro. Furthermore, this general, versatile labeling technique is expected to be easily extended to the surface modification of other retroviruses. For AAV vectors, the unnatural amino acid azidohomoalanine (Aha), a methionine surrogate, was added to the methionine-depleted cell growth medium during virus production for metabolic incorporation into the viral capsid. The impact of Aha on viral production and transduction efficiency was assessed by analyzing viruses produced in the presence of Aha in various concentrations using quantitative real time PCR assays and transduction assays. Successful incorporation of Aha onto viral surface was suggested by SDS-PAGE and western blotting analysis on virus samples modified with a reporter ligand through CuAAC."},{"label":"dcterms.available","value":"2017-09-20T16:51:51Z"},{"label":"dcterms.contributor","value":"Parker, Kathlyn"},{"label":"dcterms.creator","value":"Chu, Yanjie"},{"label":"dcterms.dateAccepted","value":"2017-09-20T16:51:51Z"},{"label":"dcterms.dateSubmitted","value":"2017-09-20T16:51:51Z"},{"label":"dcterms.description","value":"Department of Chemistry."},{"label":"dcterms.extent","value":"113 pg."},{"label":"dcterms.format","value":"Application/PDF"},{"label":"dcterms.identifier","value":"http://hdl.handle.net/11401/77070"},{"label":"dcterms.issued","value":"2015-05-01"},{"label":"dcterms.language","value":"en_US"},{"label":"dcterms.provenance","value":"Made available in DSpace on 2017-09-20T16:51:51Z (GMT). No. of bitstreams: 1\nChu_grad.sunysb_0771E_12553.pdf: 2177896 bytes, checksum: 28efcef209d5167d59ae4d2dec09edd3 (MD5)\n  Previous issue date: 2015"},{"label":"dcterms.publisher","value":"The Graduate School, Stony Brook University: Stony Brook, NY."},{"label":"dcterms.subject","value":"Biochemistry"},{"label":"dcterms.title","value":"Surface Remodeling of Lentiviral and Adeno-Associated Viral Vectors via Metabolically Introduced Bioorthogonal Functionalities"},{"label":"dcterms.type","value":"Dissertation"},{"label":"dc.type","value":"Dissertation"}],"description":"This manifest was generated dynamically","viewingDirection":"left-to-right","sequences":[{"@type":"sc:Sequence","canvases":[{"@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/canvas/page-1.json","@type":"sc:Canvas","label":"Page 1","height":1650,"width":1275,"images":[{"@type":"oa:Annotation","motivation":"sc:painting","resource":{"@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/99%2F72%2F21%2F99722137342993145030670259794613179528/full/full/0/default.jpg","@type":"dctypes:Image","format":"image/jpeg","height":1650,"width":1275,"service":{"@context":"http://iiif.io/api/image/2/context.json","@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/99%2F72%2F21%2F99722137342993145030670259794613179528","profile":"http://iiif.io/api/image/2/level2.json"}},"on":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/canvas/page-1.json"}]}]}]}