Open Access Te Herenga Waka-Victoria University of Wellington
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Construction and optimization of novel recombinant Adeno-Associated Virus rAAV2/5 for targeting microglia to regulate immune responses during neuroinflammation

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posted on 2021-11-14, 10:37 authored by Belhoul-Fakir, Hanane

Activated microglia promote central nervous system (CNS) inflammation through antigen presentation and secretion of pro-inflammatory cytokines and chemokines. Although this activation is necessary to protect the brain during infection, aberrant release of pro-inflammatory and/or cytotoxic factors such as tumor necrosis factor-α, interleukin-1β, nitric oxide and reactive oxygen substances may lead to neuronal damage and degeneration.  Targeting microglia during neuroinflammation to regulate the expression of cytokines without affecting other cell types in the CNS is challenging since no specific microglial markers have yet been established that distinguish microglia from infiltrating, peripheral myeloid cells. Therefore, we propose that a viral-based gene delivery system might be a better strategy to regulate gene expression in microglia. Using the recombinant Adeno-associated virus (AAV) vector pseudotype 2/5, which preferentially infects microglia (, we constructed a plasmid backbone which contains GFP under the control of the F4/80 promoter, a macrophage-specific marker. In order to demonstrate the specificity of this promoter for macrophages, we transfected human kidney cells HEK 293 cells, mouse leukemic macrophages RAW 264.7 cells, human hepatocytes cell line (HepG2) and human ovarian carcinoma cell line (1A9) with the AAV-F4/80-eGFP construct or the control plasmid AAV-CAG-eGFP. Our results indicate that the rAAV-F4/80-GFP construct is selective for macrophages.  To begin to assess the usefulness of this system to alter microglia function, we have cloned the Membrane Associated Ring-CH protein (MARCHI) into the rAAV-F4/80-eGFP vector that has been shown earlier to regulate antigen presentation by inducing the intracellular sequestration of MHC class II. We were able to confirm this finding by transfecting interferon gamma stimulated macrophages cell line RAW 264.7 cells via our constructed AAV-F4/80-MARCHI-eGFP vector and demonstrate the ability of our recombinant AAV vector that is driven by specific promoter to deliver and express MARCHI to induce MHC class II sequestration. Together this work will lead to the development of tools that will allow us to dissect the pathways by which microglia promote neuroinflammation.


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Te Herenga Waka—Victoria University of Wellington

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Author Retains Copyright

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Degree Grantor

Te Herenga Waka—Victoria University of Wellington

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Degree Name

Master of Science

ANZSRC Type Of Activity code

970111 Expanding Knowledge in the Medical and Health Sciences

Victoria University of Wellington Item Type

Awarded Research Masters Thesis



Victoria University of Wellington School

School of Biological Sciences


La Flamme, Anne