Type
Text
Type
Dissertation
Advisor
Carrico, Isaac S. | Parker, Kathlyn | Schärer, Orlando | Hearing, Patrick.
Date
2015-05-01
Keywords
Biochemistry | Bioorthogonal chemistry, Gene therapy, Metabolic labeling, Viral vector
Department
Department of Chemistry.
Language
en_US
Source
This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree.
Identifier
http://hdl.handle.net/11401/77070
Publisher
The Graduate School, Stony Brook University: Stony Brook, NY.
Format
application/pdf
Abstract
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-α-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. | 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-α-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. | 113 pages
Recommended Citation
Chu, Yanjie, "Surface Remodeling of Lentiviral and Adeno-Associated Viral Vectors via Metabolically Introduced Bioorthogonal Functionalities" (2015). Stony Brook Theses and Dissertations Collection, 2006-2020 (closed to submissions). 2907.
https://commons.library.stonybrook.edu/stony-brook-theses-and-dissertations-collection/2907