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Directed evolution of adeno-associated virus for efficient gene delivery to microglia

Nature Methods volume 19, pages 976–985 (2022)Cite this article

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Abstract

As the resident immune cells in the central nervous system (CNS), microglia orchestrate immune responses and dynamically sculpt neural circuits in the CNS. Microglial dysfunction and mutations of microglia-specific genes have been implicated in many diseases of the CNS. Developing effective and safe vehicles for transgene delivery into microglia will facilitate the studies of microglia biology and microglia-associated disease mechanisms. Here, we report the discovery of adeno-associated virus (AAV) variants that mediate efficient in vitro and in vivo microglial transduction via directed evolution of the AAV capsid protein. These AAV-cMG and AAV-MG variants are capable of delivering various genetic payloads into microglia with high efficiency, and enable sufficient transgene expression to support fluorescent labeling, Ca2+ and neurotransmitter imaging and genome editing in microglia in vivo. Furthermore, single-cell RNA sequencing shows that the AAV-MG variants mediate in vivo transgene delivery without inducing microglia immune activation. These AAV variants should facilitate the use of various genetically encoded sensors and effectors in the study of microglia-related biology.

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Fig. 1: AAV-cMG mediates efficient gene transduction in cultured microglia.
Fig. 2: Directed evolution generates AAV-MGs that mediate efficient gene transduction in microglia in vivo.
Fig. 3: In vivo transduction of microglia by AAV-MGs does not induce microglia activation.
Fig. 4: AAV-MGs enable in vivo two-photon imaging of microglial Ca2+ signal and ATP transmission.
Fig. 5: AAV-MGs mediate efficient genome editing of microglia in vivo.
Fig. 6: AAV-MG delivery of SaCas9 for in vivo genome editing in microglia.

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Data availability

All numerical source data are provided with this paper. The next-generation sequencing datasets reported in this work are available under the GEO accession number GSE197743. The GRCm38 reference genome assembly was downloaded from https://nov2020.archive.ensembl.org/Mus_musculus/Info/Index. The following plasmids are deposited to Addgene: rAAV2/cMG (184539), rAAV2/MG1.1 (184540), rAAV2/MG1.2 (184541), rAAV2/cMG.WPP (184542) and rAAV2/cMG.QRP (184543). The raw images generated in this study are not suitable for distribution through public repositories due to the large file size and are available from the corresponding authors upon request. Source data are provided with this paper.

Code availability

The codes and scripts for analysis of the sequencing data included in this paper are available at https://github.com/RuiyuRayWang/Lin_AAV_Microglia_public.

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Acknowledgements

The authors thank all members of M.L.’s laboratory for their assistance in this study. M.L. is supported by Ministry of Science and Technology China Brain Initiative Grant (2021ZD0202803), the Research Unit of Medical Neurobiology, Chinese Academy of Medical Sciences (2019RU003), and the Beijing Municipal Government. The funder had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Author notes

  1. These authors contributed equally: Rui Lin, Youtong Zhou, Ting Yan, Ruiyu Wang.

Authors and Affiliations

  1. National Institute of Biological Sciences (NIBS), Beijing, China

    Rui Lin, Youtong Zhou, Ting Yan, Ruiyu Wang, Heng Li & Minmin Luo

  2. Chinese Institute for Brain Research, Beijing, China

    Youtong Zhou, Xinshuang Zhang, Xiangyu Zhou, Fei Zhao, Li Zhang, Yulong Li & Minmin Luo

  3. School of Life Sciences, Tsinghua University, Beijing, China

    Ting Yan & Heng Li

  4. Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China

    Ting Yan

  5. School of Life Sciences, Peking University, Beijing, China

    Ruiyu Wang, Zhaofa Wu & Yulong Li

  6. Tsinghua Institute of Multidisciplinary Biomedical Research (TIMBR), Beijing, China

    Heng Li & Minmin Luo

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Contributions

R.L. and M.L. designed the experiments. R.L. and Y.Z. performed most of the experiments. T.Y. performed the experiments that involved cultured mouse microglia. R.W., T.Y., X.Zha., X.Zho. and L.Z. performed scRNA-seq and analysis. Z.W. and Y.L. developed the ATP fluorescent sensor, GRABATP. H.L. performed the in vivo imaging. Y.Z. and F.Z. packaged AAV viruses. R.L. and M.L. wrote the manuscript with contributions from all of the authors.

Corresponding authors

Correspondence to Rui Lin or Minmin Luo.

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Competing interests

The National Institute of Biological Sciences (NIBS), Beijing, China has filed patent applications related to this work with R.L. and M.L. listed as inventors. All other authors have no competing interests.

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Nature Methods thanks Sandra Siegert, Amanda Sierra and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Nina Vogt, in collaboration with the Nature Methods team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 AAV-cMG transduction does not activate microglia and does not blunt microglial responses to lipopolysaccharide (LPS) and interleukin-4 (IL4).

a, Principal component analysis of the transcriptomes of cultured mouse microglia from four treatment groups: control untransduced, lipopolysaccharide (LPS)-treated, interleukin-4-treated (IL4) and AAV-cMG-transduced group (n = 3 replicates for each group). b, Hierarchical clustering performed on marker genes of microglial states for different treatment groups as shown in (a). The color bar represents the z-score of the relative gene expression. c, Principal component analysis of the transcriptomes of cultured mouse microglia from five treatment groups: untransduced microglia (n = 3), untransduced microglia treated with LPS (n = 1), untransduced microglia treated with IL4 (n = 3), AAV-cMG-transduced microglia treated with LPS (n = 3), and AAV-cMG-transduced microglia treated with IL4 (n = 3). d, Hierarchical clustering performed on LPS- or IL4-treated microglia as shown in (c). The color bar represents the Euclidean distance between samples.

Extended Data Fig. 2 Representative images of serial coronal sections of the striatum from Cx3cr1CreER mice injected with AAV9-, AAV6TM-, AAV8-, AAV-cMG.QRP-, AAV-cMG.WPP-, AAV-MG1.1-, or AAV-MG1.2-SFFV-DIO-mScarlet.

Scale bar, 500 µm.

Extended Data Fig. 3 Schematic of in vivo selection of AAV-cMG.WPP variants library by the Cre recombination-based AAV targeted evolution (CREATE) strategy.

The AAV-cMG.WPP variants library was injected into the striatum and the midbrain of Cx3cr1CreER mice, which selectively express Cre recombinase in microglia. For those variants that successfully transduced microglia, Cre flipped the double-floxed inverse open reading frame (DIO) on their AAV genomes, allowing the selective amplification and recovery of the heptamer insertion sequences using a pair of primers.

Extended Data Fig. 4 AAV-MGs mediate efficient microglial transduction in vivo.

a, Representative images showing the mScarlet expression patterns in the orbitofrontal cortex (OFC) of Cx3cr1CreER mice injected with AAV-cMG.WPP-, AAV-MG1.1-, or AAV-MG1.2-SFFV-DIO-mScarlet. Scale bar, 500 µm. b, Cell counts of mScarlet and Iba double-positive cells in the OFC of Cx3cr1CreER mice injected with AAV-cMG.WPP- (n = 3), AAV-MG1.1- (n = 5), or AAV-MG1.2-SFFV-DIO-mScarlet (n = 5) (two-way ANOVA with Tukey’s post-hoc test; cMG.WPP vs. MG1.1: P < 0.0001; AAV-cMG.WPP vs. AAV-MG1.2: P < 0.0001). Data are presented as mean ± standard error of the mean (s.e.m.). c, Quantification of the percentage of Iba-positive microglia that are labeled by mScarlet in the OFC of Cx3cr1CreER mice injected with AAV-MG1.1- or AAV-MG1.2-SFFV-DIO-mScarlet (n = 5 mice for each group). The quantifications were conducted over an area of 1 × 1 mm2. Data are presented as mean ± s.e.m. d, Representative images showing the mScarlet expression patterns in the midbrain of Cx3cr1CreER mice injected with AAV-cMG.WPP-, AAV-MG1.1-, or AAV-MG1.2-SFFV-DIO-mScarlet. Scale bar, 500 µm. e, Cell counts of mScarlet and Iba double-positive cells in the midbrain of Cx3cr1CreER mice injected with AAV-cMG.WPP- (n = 3), AAV-MG1.1- (n = 5), or AAV-MG1.2-SFFV-DIO-mScarlet (n = 5) (two-way ANOVA with Tukey’s post-hoc test; cMG.WPP vs. MG1.1: P < 0.0001; AAV-cMG.WPP vs. AAV-MG1.2: P < 0.0001). Data are presented as mean ± s.e.m. f, Representative images showing the mScarlet expression patterns in the hippocampus and the thalamus of Cx3cr1CreER mice injected with AAV-MG1.1- or AAV-MG1.2-SFFV-DIO-mScarlet (right). Scale bar, 500 µm.

Source data

Extended Data Fig. 5 Selective Cre-dependent transgene expression in microglia by AAV-MGs.

a, Representative images showing the striatum of mice injected with AAV-MG1.1- or AAV-MG1.2-SFFV-DIO-mScarlet. The left panel shows the striatum of C57BL/6N mice received intrastriatal virus injection together with i.p. tamoxifen injection. The right panel shows the striatum of Cx3cr1CreER mice received intrastriatal virus injection without tamoxifen administration. Scale bars, 500 µm. b, Representative images showing the colocalization of mScarlet and Iba+ immunosignals in the OFC and the midbrain of Cx3cr1CreER mice injected with AAV-MG1.1- or AAV-MG1.2-SFFV-DIO-mScarlet. Scale bars, 50 µm. c, Representative images showing the colocalization of mScarlet and Iba+ immunosignals in the striatum of Tmem119CreER mice injected with AAV-MG1.1- or AAV-MG1.2-SFFV-DIO-mScarlet. Scale bars, 50 µm. d, Quantification of the percentage of mScarlet and Iba double-positive cells among total mScarlet-positive cells in the striatum of Tmem119CreER mice injected with AAV-MG1.1- or AAV-MG1.2-SFFV-DIO-mScarlet. Data are presented as scatter and mean.

Source data

Extended Data Fig. 6 AAV-MGs effectively transduce neurons and astrocytes in vivo but do not efficiently transduce cultured microglia.

a, Representative images showing the mScarlet expression patterns in the striatum of C57BL/6 N mice injected with a virus mixture of AAV-Ef1a-Cre and AAV-MG1.1-SFFV-DIO-mScarlet or a virus mixture of AAV-Ef1a-Cre and AAV-MG1.2-SFFV-DIO-mScarlet. Yellow: mScarlet, blue: DAPI. Scale bars, 500 µm (slide scanning) and 100 µm (confocal images). b, Representative images showing the colocalization of mScarlet and NeuN+/GFAP+ immunosignals in the striatum of C57BL/6 N mice injected with a virus mixture of AAV-Ef1a-Cre and AAV-MG1.1-SFFV-DIO-mScarlet or a virus mixture of AAV-Ef1a-Cre and AAV-MG1.2-SFFV-DIO-mScarlet. Scale bars, 50 µm. c, Representative fluorescent and bright-field images of cultured mouse microglia transduced with mScarlet reporter rAAVs packaged using AAV-MG1.1 or AAV-MG1.2. Scale bars, 200 µm.

Extended Data Fig. 7 Reference (10x) and query (Smart-seq2) single-cell RNA sequencing datasets characterizing the single-cell transcriptomes of microglia.

a,d, Quantification of gene counts, UMI counts, the percentage of mitochondria RNA, and the percentage of ribosome RNA in the query (a) and the reference (d) datasets. Red dots represent sequenced microglia that failed to pass the quality check and were removed from subsequent analysis. b,e, UMAP plot of all cells (left and middle) and filtered microglia (right) sequenced using the Smart-seq2 protocol (b) or 10x Genomics platform (e). The blue color in the left panel indicates Hexb expression level. Cells with high Hexb expression (red in the middle panel) were identified as microglia and retained for subsequent analysis. c, Violin plots showing the UMI counts and the number of detected genes in the reference and the query datasets. f,g, Gene expression analysis of the query (f) and the reference (g) datasets. The heatmap shows the z-score of relative gene expression. The violin plots show the expression level of homeostatic marker genes (P2ry12, Cx3cr1, Sall1 and Csf1r) and reactive marker genes (Il1b, Spp1, Ifitm3 and Ccl5).

Extended Data Fig. 8 The expression levels of homeostatic or reactive microglia marker genes are not correlated with AAV-MGs-mediated transgene expression.

a,b, Violin plots showing the expression of classical homeostatic (a) and reactive (b) marker genes in the Smart-seq2-sequenced microglia from Cx3cr1CreER mice injected with AAV-MG1.1- or AAV-MG-1.2-SFFV-DIO-mScarlet. Microglia that have no mScarlet transcript detected (normalized mScarlet expression = 0) are annotated as ‘untransduced’. c,d, Scatter plots showing the expression levels of classical homeostatic (c) and reactive (d) marker genes against the expression levels of mScarlet in AAV-MGs-transduced microglia sequenced using Smart-seq2. The Spearman correlation coefficient and the significance level are shown in each panel.

Extended Data Fig. 9 AAV-MG1.2 enables in vivo two-photon imaging of microglia extracellular ATP changes following acute laser ablation.

a, Representative images showing the colocalization of jGCaMP8s (HA-tag immunosignals) and Iba+ immunosignals in the primary somatosensory (S1) cortex of Cx3cr1CreER mice injected with AAV-MG1.2-SFFV-DIO-jGCaMP8s. Scale bar, 50 µm. b, Representative images showing the colocalization of GRABATP1.0 (HA-tag immunosignals) and Iba+ immunosignals in the S1 cortex of Cx3cr1CreER mice injected with AAV-MG1.2-SFFV-DIO-GRABATP1.0. Scale bar, 50 µm. c, Quantification of GRABATP1.0 fluorescence signals at microglial somata in the control mice (n = 10 cells; black) or in the mice that received laser ablation (n = 16 cells; red) (two-way ANOVA; P values as listed in the figure). Data are presented as mean ± s.e.m. d, Image showing GRABATP1.0 expression in microglia and heatmaps showing the GRABATP1.0 fluorescence signals at 10, 20, and 40 min in the imaging session. For the laser ablation group, the laser was applied at the center of field of view at the beginning (0 min) of the imaging session. Scale bar, 100 µm.

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Extended Data Fig. 10 In vivo microglia-specific gene knockout mediated by AAV-MGs.

a, Representative images of serial coronal sections of the striatum from a Cx3cr1CreER:Rosa26-LSL-Cas9-GFP mouse injected with AAV-MG1.1-sgRNA-LacZ or AAV-MG1.1-sgRNA-Tmem119. The brain sections were immunostained against Tmem119. Scale bar, 500 µm. b, Representative images of serial coronal sections of the striatum from a Cx3cr1CreER:Rosa26-LSL-Cas9-GFP mouse injected with AAV-MG1.2-sgRNA-LacZ or AAV-MG1.2-sgRNA-Cd68. The brain sections were immunostained against Cd68. Scale bar, 500 µm. c, Representative images of the S1 cortex of Cx3cr1GFP mice injected with AAV-MG1.2-CMV-SaCas9 or AAV-MG1.2-CMV-SaCas9-U6-sgRNA-P2ry12. The brain sections were immunostained against P2ry12. Scale bar, 100 µm. d, Quantification of the percentage of P2ry12-positive pixels in a 1 × 1 mm2 region in the S1 cortex of Cx3cr1GFP mice injected with AAV-MG1.2-CMV-SaCas9 (n = 2) or AAV-MG1.2-CMV-SaCas9-sgRNA-P2ry12 (n = 3) (two-way ANOVA; P values as listed in the figure). Data are presented as mean ± s.e.m.

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Lin, R., Zhou, Y., Yan, T. et al. Directed evolution of adeno-associated virus for efficient gene delivery to microglia. Nat Methods 19, 976–985 (2022). https://doi.org/10.1038/s41592-022-01547-7

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