Biology subjects

Bennett, M. L.

Publications and source records attributed to Bennett, M. L..

4 recordsLinked to original sources

bioRxiv2026

Comparative analysis of microglia-targeted AAVs reveals capsid choice drives efficiency in vitro but has limited impact in vivo

Microglia play key roles in brain development, homeostasis, and neurodegeneration. Although multiple strategies for viral gene delivery to microglia have been reported, they have not been directly compared. Here, we developed microglia-targeting AAV capsids and benchmarked them against existing approaches. The novel capsids exhibit improved transduction efficiency in cultured mouse and human microglia, as well as neurons and astrocytes. However, when we compared microglial transduction efficiency of the novel capsids with published engineered and naturally occurring capsids after intracranial injection, all capsids achieved efficient and specific transduction when paired with a genome incorporating IBA1 promoter and miR-124 target sites. In contrast, CAG promoter did not support efficient microglial transduction. Moreover, blood-brain barrier- crossing capsids carrying IBA1 promoter and miR-124 target sites efficiently transduced microglia at high doses but exhibited off-target expression. Together, our work provides improved capsids for in vitro manipulation of microglia and establishes viral genome design, not capsid identity, as the principal determinant of efficient in vivo microglial targeting.

neuroscience↗

bioRxiv2025

Targeting DNA-LNPs to Endothelial Cells Improves Expression Magnitude, Duration, and Specificity

DNA-lipid nanoparticles (DNA-LNPs) loaded with inhibitors of the cGAS-STING pathway enable safe and effective delivery of DNA in vivo. Herein, we report the first instances of extrahepatic DNA-LNP targeting. DNA-LNPs conjugated to antibodies against PECAM-1 or VCAM-1 target the endothelium of the lungs and brain/spleen, respectively. These LNPs drive robust transgene expression in their target organs, with greater magnitude and duration than untargeted LNPs. Lung specificity of PECAM-targeted transgene expression increases over two weeks, resulting in markedly higher lung-to-liver expression ratios than our previous PECAM-targeted mRNA-LNPs. Off-target liver DNA expression declines to undetectable levels but persists in the lungs, while mRNA expression uniformly decreases due to its short half-life. We further improve this expression specificity by replacing full-length antibodies with Fab fragments. Single-cell analysis reveals a key mechanism underlying the improvements in organ-specificity: target organ expression is dominated by long-lived endothelial cells, while off-target liver delivery and expression are in non-endothelial cells with shorter half-lives. Collectively, these studies demonstrate that targeted DNA-LNPs achieve high levels of organ- and cell-type-specific transgene expression and thus provide a therapeutic platform for dozens of endothelial-centric diseases.

bioengineering↗

bioRxiv2024

Microglia replacement by ER-Hoxb8 conditionally immortalized macrophages provides insight into Aicardi-Goutieres Syndrome neuropathology

Microglia, the brains resident macrophages, can be reconstituted by surrogate cells - a process termed "microglia replacement." To expand the microglia replacement toolkit, we here introduce estrogen-regulated (ER) homeobox B8 (Hoxb8) conditionally immortalized macrophages, a cell model for generation of immune cells from murine bone marrow, as a versatile model for microglia replacement. We find that ER-Hoxb8 macrophages are highly comparable to primary bone marrow-derived (BMD) macrophages in vitro, and, when transplanted into a microglia-free brain, engraft the parenchyma and differentiate into microglia-like cells. Furthermore, ER-Hoxb8 progenitors are readily transducible by virus and easily stored as stable, genetically manipulated cell lines. As a demonstration of this systems power for studying the effects of disease mutations on microglia in vivo, we created stable, Adar1-mutated ER-Hoxb8 lines using CRISPR-Cas9 to study the intrinsic contribution of macrophages to Aicardi-Goutieres Syndrome (AGS), an inherited interferonopathy that primarily affects the brain and immune system. We find that Adar1 knockout elicited interferon secretion and impaired macrophage production in vitro, while preventing brain macrophage engraftment in vivo - phenotypes that can be rescued with concurrent mutation of Ifih1 (MDA5) in vitro, but not in vivo. Lastly, we extended these findings by generating ER-Hoxb8 progenitors from mice harboring a patient-specific Adar1 mutation (D1113H). We demonstrated the ability of microglia-specific D1113H mutation to drive interferon production in vivo, suggesting microglia drive AGS neuropathology. In sum, we introduce the ER-Hoxb8 approach to model microglia replacement and use it to clarify macrophage contributions to AGS.

neuroscience↗

bioRxiv2018

Developmental heterogeneity of microglia and brain myeloid cells revealed by deep single-cell RNA sequencing

Microglia are increasingly recognized for their major contributions during brain development and neurodegenerative disease. It is currently unknown if these functions are carried out by subsets of microglia during different stages of development and adulthood or within specific brain regions. Here, we performed deep single-cell RNA sequencing (scRNA-seq) of microglia and related myeloid cells sorted from various regions of embryonic, postnatal, and adult mouse brains. We found that the majority of adult microglia with homeostatic signatures are remarkably similar in transcriptomes, regardless of brain region. By contrast, postnatal microglia represent a more heterogeneous population. We discovered that postnatal white matter-associated microglia (WAM) are strikingly different from microglia in other regions and express genes enriched in degenerative disease-associated microglia. These postnatal WAM have distinct amoeboid morphology, are metabolically active, and phagocytose newly formed oligodendrocytes. This scRNA-seq atlas will be a valuable resource for dissecting innate immune functions in health and disease.\n\nHighlightsO_LIMyeloid scRNA-seq atlas across brain regions and developmental stages\nC_LIO_LILimited transcriptomic heterogeneity of homeostatic microglia in the adult brain\nC_LIO_LIPhase-specific gene sets of proliferating microglia along cell cycle pseudotime\nC_LIO_LIPhagocytic postnatal white matter-associated microglia sharing DAM gene signatures\nC_LI

neuroscience↗