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Chu Thi Hien Thu 1,2,3

Investigating the effect of HSPGs primary receptors on retinal transduction using Adeno -Associated viral vectors. Chu Thi Hien Thu 1,2,3. 1 Hanoi University of Science, Vietnam National University 2 Massachusetts Eye and Ear Infirmary/ Schepens Eye Research Institute (MEEI/SERI)

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Chu Thi Hien Thu 1,2,3

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  1. Investigating the effect of HSPGs primary receptors on retinal transduction using Adeno-Associated viral vectors Chu ThiHien Thu1,2,3 1Hanoi University of Science, Vietnam National University 2Massachusetts Eye and Ear Infirmary/Schepens Eye Research Institute (MEEI/SERI) 3Harvard Stem Cell Institute Internship Program (HIP 2014)

  2. Introduction • In vivo gene therapy • Direct injection of genetic materials into patient for therapeutic effect • Adeno-associated Viral Vector (AAV) • Most efficient and most commonly used vector for in vivo gene therapy • Virus: non-pathogenic, ssDNA non-enveloped parvovirus • Vector: many target tissues, dividing and non-dividing  cells, long term stable transgene expression • Retinal gene therapy • Demonstrated to effective and safe in clinical trials for inherited forms of blindness • Small dose, accessible organ, limited bio-distribution http://webvision.med.utah.edu/book/part-i-foundations/simple-anatomy-of-the-retina/

  3. Effective Possible damages Macular surgery Injection position uncertainty… Best: AAV8 • Novel synthetic AAV vectors (Ancs) • Designed, constructed in Vandenberghe lab • Minimally susceptible to AAV immunity in humans Safe but not really high efficiency Best: AAV2 Dalkara and Sahel et al., 2014 • ILM: high in HSPG • HSPG is receptor for AAV2 through Arg-rich capsid motif • AAV2 is only AAV that transduces following intravitreal injection ILM Hypothesis: HSPG binding on novel synthetic AAVs will increase intravitreal transduction

  4. Methodology 1. Mutagenesis of Ancestral AAVs 2. Virus production Restriction enzyme digestion bands 3. Viral transduction efficiency 4. Heparin competition assay and enzymatic removal Luciferase assay

  5. Experimental results 1. Mutagenesis of Ancestral AAVs 2. Virus production Sequencing check Restriction enzyme digest Anc80L0027_mut Anc27 Anc65 Anc80L0065_mut 5600 bp AAV2 L65WT L65H+ 4000 bp 1500 bp 810 bp 700 bp 450 bp 3. Viral transduction efficiency 4. Conclusion • Structural modeling predicts altered HSPG binding pocket • HSPG+ Ancs produce albeit at low titers • HSPG+ Anc65 transduces in vitro albeit at reduced efficiency • Additional structural modeling needs to be done to introduce HSPG binding onto Ancs

  6. Acknowledgements Dr. LukVandenberghe PhD LiviaCarvalho MEEI/SERI HSCI

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