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Cited by 12Virus-based gene therapy researchRNA Interference and Gene DeliveryBarrier Structure and Function Studies

AAV vectors for specific and efficient gene expression in microglia

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Aoki, Ryo, Konno, Ayumu, Hosoi, Nobutake, Kawabata, Hayato, Hirai, Hirokazu·2025·Cell Reports Methods
DOI 10.1016/j.crmeth.2025.101116PMC PMC12461629
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Abstract

Abstract Microglia play a critical role in diseases such as Alzheimer’s and stroke, making them a significant target for therapeutic intervention. However, due to their immune functions in detecting and combating viral invasion, efficient gene delivery to microglia remains challenging. We achieved specific and efficient gene delivery to microglia using an adeno- associated virus (AAV) vector designed for this purpose. This microglia-targeting AAV vector includes the mouse microglia/macrophage-specific ionized calcium-binding adaptor molecule 1 (mIba1) promoter, green fluorescent protein (GFP), microRNA target sequences (miR.Ts), woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and polyadenylation (polyA) signal, positioned between inverted terminal repeats. When miR.Ts were placed downstream of WPRE (between WPRE and polyA), gene expression occurred not only in microglia but also in a substantial number of neurons. However, when miR.Ts were positioned upstream of WPRE (between GFP and WPRE) or on both sides of WPRE, neuronal expression was significantly suppressed, resulting in selective GFP expression in microglia. Notably, positioning miR.Ts on both sides of WPRE achieved over 90% specificity and more than 60% efficiency in transgene expression in microglia three weeks after viral administration. This vector also enabled GCaMP expression in microglia, facilitating real-time monitoring of calcium dynamics and microglial process activity in the cortex. Additionally, intravenous administration of this vector with the blood-brain barrier-penetrant AAV-9P31 capsid variant resulted in extensive GFP expression selectively in microglia throughout the brain. These findings establish this AAV vector system as a robust tool for long-term, specific, and efficient gene expression in microglia.

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  • The IBA1 promoter paired with an artificial miRNA scaffold is a well-established strategy for microglia-specific gene manipulation.

    “This promoter, paired with an artificial miRNA scaffold embedded in the miR‐RNA backbone, is a well‐established strategy for microglia‐specific gene manipulation. 33 , 34 , 35”

    From Aberrant phase separation from a rare <i>ABI3</i> mutation drives microglial dysfunction and Alzheimer's risk (2026) · cites this paper as [35]

    The Results section (‘The Iba1 promoter is indispensable for transgene expression in microglia’; page not provided) supports the role of the mIba1 promoter in microglia-selective expression. But the Methods describe pairing it with miRNA target sequences, not an artificial miRNA scaffold embedded in a miRNA backbone. This paper therefore does not support the specific pairing claimed. (LLM, conf=0.89)

    Experimental — accuracy not yet measured

  • The IBA1 promoter has been validated extensively for microglial specificity, with reported transduction specificity of up to 95% in resident microglia.

    “The IBA1 promoter used in this study has been validated extensively for microglial specificity, with reported transduction specificity of up to 95% in resident microglia. 33 , 34 , 35”

    From Aberrant phase separation from a rare <i>ABI3</i> mutation drives microglial dysfunction and Alzheimer's risk (2026) · cites this paper as [35]

    The Results section on striatal and cerebellar expression reports 96.7% microglial specificity in mouse striatum, supporting a figure of roughly 95%. But that result is for an optimized AAV vector combining the mIba1 promoter with microRNA-target sequences, not for the promoter alone; the paper does not establish that the promoter has been validated extensively or that this specificity applies generally to resident microglia. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

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Cited by

Sorted by citations

Authors
Aoki, R., Konno, A., Hosoi, N., Kawabata, H., & Hirai, H.
Affiliations
Gunma University (JP)
Corresponding author
  • Konno, Ayumu
  • Hirai, Hirokazu
ORCID
Venue
Cell Reports Methods
Publisher
Elsevier BV
Published
2025-08
Volume
5
Issue
8
Pages
101116
ISSN
2667-2375 (ISSN-L)
DOI
10.1016/j.crmeth.2025.101116
PMC
PMC12461629
OpenAlex
W4412750599
Cited by
12
References
8
Citations by year
2026: 9 · 2025: 3
FWCI
6.24
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Reports Methods (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Japan Agency for Medical Research and Development (JP24wm0625103)
  • Ministry of Education, Culture, Sports, Science and Technology
  • Japan Society for the Promotion of Science (23H02791, 20K06906 / 24K10022, 22K06454 / 24H01221)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
Keywords
adeno-associated virus vectors, microglia, gene delivery, polyadenylation signal, blood-brain barrier penetration, GCaMP calcium imaging, intravenous administration, Alzheimer's disease, stroke
MeSH terms
Dependovirus, Animals, Blood-Brain Barrier, Genetic Vectors, Humans, Promoter Regions, Genetic, Gene Expression, Microglia, Gene Transfer Techniques, MicroRNAs, Green Fluorescent Proteins, Mice

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