Evaluation of Adeno-associated virus trafficking and transduction in the retina
Public Deposited- Last Modified
- March 19, 2019
- Creator
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Woodard, Kenton
- Affiliation: School of Medicine, UNC Neuroscience Center, Neuroscience Curriculum
- Abstract
- Gene delivery by viruses, specifically AAV, offers an efficient way to safely supply exogenous DNA to cells long term in vivo. The ability of AAV to infect post-mitotic neurons makes it a great vector for neuroscience applications. The route of delivery into the eye largely dictates the tropism and distribution of AAV, but never has the numerous routes been compared. We injected scAAV2 containing CMV-GFP transgenes into the various compartments (intrastromal [IS], intracameral [IC], intravitreal [IVIT], subretinal [SR], suprachoroidal [SC]) within the eyes of mice. Anterior chamber injections lead to fluorescence mainly in the anterior portion while the posterior injections lead to transduction of the ciliary body and retina. IC and IVIT injections led to transduction of the inner retina and SR and SC injections transduced the outer retina, with the best fluorescence detected by either SR or IVIT delivery. Many AAV serotypes are known to efficiently transduce the retina when delivered to the SR space, but show limited success when delivered to the vitreous due to the inner limiting membrane (ILM). Transduction of AAV by SR delivery occurred by a heparan sulfate (HS) independent mechanism whereas IVIT transduction was highly dependent on HS binding. This was due to the accumulation of vector at the ILM where HS proteoglycan (HSPG) was abundantly found. For clinical relevance, this mechanism was tested in human ex vivo retinas and determined to be conserved. The HS-binding motif enhanced the transduction of other AAV serotypes (AAV1, AAV8) by the same mechanism. To determine the extent of HS-binding on tropism, capsid motifs from other AAV serotypes were used to create chimeric AAV2 capsids. A motif from AAV1 enhanced the transduction along the retinal vessels whereas the galactose motif from AAV9 lead to Müller glial specific transduction. The double chimera showed characteristics of both motifs that completely ablated native AAV2 transduction. Through rational engineering of capsid motifs, we have identified motifs involved in retinal trafficking and generated a novel capsid variant for targeted and enhanced transduction within the retina by IVIT delivery.
- Date of publication
- December 2016
- Keyword
- DOI
- Resource type
- Rights statement
- In Copyright
- Advisor
- Asokan, Aravind
- Maness, Patricia
- Samulski, Richard
- Weiss, Ellen
- Budenz, Donald
- Degree
- Doctor of Philosophy
- Degree granting institution
- University of North Carolina at Chapel Hill Graduate School
- Graduation year
- 2016
- Language
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