Fig. 2: Characterization of the rAAV-miniBEND system for gene delivery to brainECs.

a, Schematic diagram of the injection strategy. b, Images of EGFP expression illustrating the transduction efficiency of AAV-BR1 viruses. Ef1α-EGFP, AAV-BR1-Ef1α-EGFP; miniBEND-Cre, AAV-BR1-miniBEND(mPro1576-mCis700)-Cre. c,d, Density (per mm² per 10¹¹ AAV particles injected intravenously) of labelled ECs (d) and non-ECs (c) in b. e, Images displaying the transduction efficiency of AAV-BI30 viruses and AAV-PHP.eB-Ef1α-Cre, AAV-BI30-Ef1α-Cre; miniBEND-Cre, AAV-PHP.eB-miniBEND(mPro723-mCis700)-Cre. f,g, Density of non-ECs (f) and ECs (g) labelled in e. h, Schematic diagram of the strategy for local injection. i, Images showing the transduction efficiency of each group of viruses. BR1-Ef1α-EGFP, AAV-BR1-Ef1α-EGFP; BI30-Ef1α-EGFP, AAV-BI30-Ef1α-EGFP; PHP.eB-miniBEND-Cre, AAV-PHP.eB-mPro723-Cre-mCis700; 9P13-miniBEND-Cre, AAV-9P13-mPro723-Cre-mCis700. White arrowheads, neurons. j, Schematic of local injection of viruses in Glut1^fl/fl mice (P20). k, Labelling for GLUT1 and CD31 in the Glut1^fl/fl mice after local injection of AAV-miniBEND (mPro723-mCis700)-Cre (cKO) and AAV-Ef1α-EGFP (Ctrl) in the left and right hemispheres, respectively. White arrowheads, brainECs. l, Cell density (the number of GLUT1⁺ EC cells per mm² area) of GLUT1⁺ brainECs. m, Efficiency of AAV-miniBEND (AAV-PHP.eB-mPro723-mCis700)-Cre for cKO (that is, percentage of GLUT1⁻ cells among CD31⁺ cells) after local injection of Glut1^fl/fl. n, Schematic of viral injection into the lateral ventricle of neonatal mice (P0–4). o, BrainECs were labelled after injection of AAV-BR1-CAG-EGFP, AAV-BI30-Ef1α-Cre and AAV-BI30-miniBEND (mPro723-mCis700)-Cre viruses, as indicated. AAV-BI30-miniBEND-Cre, AAV-BI30-miniBEND (mPro723-mCis700)-Cre. p,q, Density (the number of labelled cells per mm² per 10¹⁰ AAV particles) of labelled non-ECs (p) and ECs (q). r, Images of brainECs after injection of AAVs into the lateral ventricle of wild-type (WT) or Ai neonatal mice. CAG-mScarlet, AAV-PHP.eB-CAG-mScarlet; miniBEND-Cre, AAV-PHP.eB-miniBEND(mPro1576/mPro973-mCis700)-Cre. s,t, Density of labelled non-ECs (s) and ECs (t). NS, not significant. HO, Hoechst 33342. n = 10 regions from 2 mice (c,d); n = 10 regions from 2 mice (f,g); n = 20 regions from 4 mice (l); n = 20 regions from 4 mice (m); and n = 18 regions from 3 mice (p,q). Data indicate the mean ±s.e.m. along with individual data points; unpaired two-sided Welch’s t-test; **P < 0.001; ****P < 0.0001. Diagrams in panels a, j and n created with BioRender.com.

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