Fig 2: scRNA-seq reveals EC subpopulations in skeletal muscle(A) Visualization of mEC populations on a t-SNE plot, color-coded for the identified subpopulations, including venous ECs (vEC), arterial ECs (aEC), arteriolar ECs (arlEC), capillary ECs (CapEC 1 and CapEC 2), and an unknown EC population (xEC).(B) Heatmap with Z scores showing top 10 enriched marker genes per cell type relative to other subpopulations.(C) Violin plots of normalized scRNA expression profiles of known EC subtype markers in each mEC cluster.(D–F) Representative images of GAS cross-sections, stained for (D) an EC marker (CD31, red) combined with a smooth muscle actin (aSMA) (orange), SELP (green), and VWF (gray) (enriched in venous ECs); (E) EFNB2 (green) (enriched in arteries and arterioles) and AQP1 (gray) (low expression in arteries and arterioles; and (F) CAR4 (green) and AQP1 (gray) (enriched in capillaries). Arrows indicate SELP+VWF+ veins in (D) and EFNB2+AQP1- arteries in (E). Scale bars, 50 µm.See also Figure S1.

Fig 3: Previously unidentified receptors enhance infectivity of engineered AAVs in vitro.(A) Plot of membrane protein differential expression score in endothelial cells versus mean transcript abundance in endothelial cells constructed from single-cell RNA sequencing of C57BL/6J cortex. Proteins selected for cell culture screening (table S1) are indicated by red circles, and three hits are marked with red crosses. (B) Potential receptors were screened in cell culture by comparing AAV fluorescent protein transgene levels at low multiplicity of infection (MOI) in cells transfected with the cDNA for each potential receptor and in mock-transfected cells. (C) Dose dependence of AAV9 and PHP.eB packaging CAG-mNeonGreen in HEK293T cells in 96-well plates. At 1 × 109 v.g. per well, PHP.eB shows Ly6a-enhanced infection. Scales show extent of infection (max, 0.75; min, 0.03) and total brightness per signal area (max, 0.79; min, 0.39). (D) Potency of engineered AAVs for HEK293T cells transfected with the potential receptor cDNA panel. Extent of infection (left: max, 0.33; min, 0.001; right: max, 0.55; min, 0.03) and total brightness per signal area (left: max, 0.55; min, 0.06; right: max, 0.61: min, 0.11). (E) Representative images of HEK cells cultured in 96-well plates, either mock-transfected (None) or transfected with a potential receptor, infected with 1 × 109 v.g. per well (yellow label background indicates LY6A binding) packaging CAG-mNeonGreen and imaged 24 hours after transduction. An overlay of bright-field and fluorescence images is presented. Scale bar, 200 µm. (F) Amino acid frequencies by position among engineered AAVs found to have infectivity enhancements with Ly6a (six viruses), Ly6c1 (nine viruses), and Car4 (two viruses). (G) Potency of engineered AAVs for HEK293T cells transfected with LY6 and CAR family potential receptors. Extent of infection (left: max, 0.51; min, 0.03; right: max, 0.52; min, 0.05) and total brightness per signal area (left: max, 0.75; min, 0.13; right: max, 0.79; min, 0.34).

Fig 4: CA-IV is required for the CNS potency of Car4-dependent AAV.(A) Immunostaining for CA-IV in the brains of WT/WT and KO/KO Car4 mice. Magnified regions from WT/WT demonstrate endothelial expression across diverse brain regions. (B) AAV-PHP.eB, 9P31, and 9P36 packaging mNeonGreen under the control of the ubiquitous CAG promoter were intravenously administered to WT/WT Car4 mice at a dose of 3 × 1011 v.g. per animal (n = 3 per condition). Three weeks after administration, transgene expression was assayed by mNeonGreen fluorescence throughout the brain and liver. (C) AAV-PHP.eB, 9P31, and 9P36 packaging mNeonGreen under the control of the ubiquitous CAG promoter were intravenously administered to KO/KO Car4 mice at a dose of 3 × 1011 v.g. per animal (n = 3 per condition). Three weeks after administration, transgene expression was assayed by mNeonGreen fluorescence throughout the brain and liver. Scale bars, 2 mm.