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Bilge Esin Ozturk, Timothy P Day, Serhan Turunc, Sara Jabalameli, John Gerard Flannery, Jose Alain Sahel, Leah Byrne; A modular linked-AAV building block system for gene and protein delivery. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2901.
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© ARVO (1962-2015); The Authors (2016-present)
Adeno-associated viruses (AAV) are the leading method for in vivo gene transfer to the retina, but they carry only 4.7 kB and result in permanent expression of the encoded gene product, significantly restricting their use. We have created a modular AAV-based delivery system to increase the carrying capacity of AAV, enable delivery of specific ratios of multiple genes, and transiently deliver protein to cells. This AAV building-block system may expand the potential of AAV to treat inherited retinal disease.
Linker molecules were engineered onto surface exposed regions of the AAV capsid to create AAV building blocks. Optimal locations to insert the linker peptides were determined. Binding conditions were optimized to link AAV vectors. Purification strategies were tested using size exclusion and affinity chromatography. Microscopy, histology and western blotting were used to evaluate linkage of viruses, and gene and protein delivery.
AAV building blocks package transgenes efficiently and are stable and infectious. We tested their ability to bind protein or another AAV vector carrying complementary linker molecules. Western blot and electron microscopy show that AAV vectors successfully bind therapeutic protein and form stable linked vector dimer particles.
This modular linked AAV system overcomes the packaging size limitation of AAV, one of the most significant barriers to in vivo gene and protein delivery and expands the utility of AAV-mediated gene delivery for biological research and gene therapy. Importantly, this system is modular and highly flexible, and could be modified for a wide variety of experimental purposes.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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