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Microglia-specific targeting by novel capsid-modified AAV6 vectors
Awilda M Rosario1, Pedro E Cruz1, Carolina Ceballos-Diaz1
1Department of Neuroscience, Center for Translational Research in Neurodegenerative Disease, University of Florida , Gainesville, Florida, USA.
Abstract:
Recombinant adeno-associated viruses (rAAV) have been widely used in gene therapy applications for central nervous system diseases. Though rAAV can efficiently target neurons and astrocytes in mouse brains, microglia, the immune cells of the brain, are refractile to rAAV. To identify AAV capsids with microglia-specific transduction properties, we initially screened the most commonly used serotypes, AAV1-9 and rh10, on primary mouse microglia cultures. While these capsids were not permissive, we then tested the microglial targeting properties of a newly characterized set of modified rAAV6 capsid variants with high tropism for monocytes. Indeed, these newly characterized rAAV6 capsid variants, specially a triply mutated Y731F/Y705F/T492V form, carrying a self-complementary genome and microglia-specific promoters (F4/80 or CD68) could efficiently and selectively transduce microglia in vitro. Delivery of these constructs in mice brains resulted in microglia-specific expression of green fluorescent protein, albeit at modest levels. We further show that CD68 promoter-driven expression of the inflammatory cytokine, interleukin-6, using this capsid variant leads to increased astrogliosis in the brains of wild-type mice. Our study describes the first instance of AAV-targeted microglial gene expression leading to functional modulation of the innate immune system in mice brains. This provides the rationale for utilizing these unique capsid/promoter combinations for microglia-specific gene targeting for modeling or functional studies.
Insights
Researchers developed novel recombinant adeno-associated virus (rAAV) capsid variants to target microglia, the brain's immune cells. This breakthrough enables microglia-specific gene expression for studying central nervous system diseases.
Area of Science:
- Neuroscience
- Immunology
- Gene Therapy
Background:
- Recombinant adeno-associated viruses (rAAV) are key tools for gene therapy in the central nervous system.
- Microglia, the brain's resident immune cells, are typically resistant to rAAV transduction.
- Targeting microglia is crucial for understanding and treating neuroinflammatory and neurodegenerative diseases.
Purpose of the Study:
- To identify adeno-associated virus (AAV) capsids capable of microglia-specific gene delivery.
- To develop novel rAAV vectors for functional studies and gene therapy targeting microglia.
- To investigate the potential of engineered AAV capsids for modulating microglial activity.
Main Methods:
- Screening of common AAV serotypes (AAV1-9, rh10) for microglial transduction.
- Testing modified rAAV6 capsid variants, including a Y731F/Y705F/T492V mutant, on primary mouse microglia cultures.
- In vivo delivery of rAAV constructs with microglia-specific promoters (F4/80, CD68) in mouse brains.
- Assessment of gene expression and downstream effects like astrogliosis.
Main Results:
- Standard AAV serotypes showed limited transduction of primary mouse microglia.
- Engineered rAAV6 capsid variants, particularly the Y731F/Y705F/T492V mutant, efficiently and selectively transduced microglia in vitro.
- In vivo studies demonstrated microglia-specific expression of reporter genes and interleukin-6.
- CD68 promoter-driven IL-6 expression induced astrogliosis, indicating functional modulation of the innate immune system.
Conclusions:
- Novel rAAV6 capsid variants enable targeted gene delivery to microglia.
- This represents the first successful instance of AAV-mediated gene expression and functional modulation in microglia.
- These engineered vectors offer a promising platform for microglia-specific gene targeting in research and therapeutic applications for CNS diseases.
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