Journal: Advanced Science

Article Title: Endothelial Retargeting of AAV9 In Vivo

doi: 10.1002/advs.202103867

Figure Lengend Snippet: Coating of rAAV2/9 with PAMAM dendrimers. A) Generation 2 polyamidoamine dendrimers modified stepwise by PEGylation with NHS‐PEG 2K ‐OPSS and conjugated to peptides via the N terminal cysteine residue. B) Representative transmission electron micrograph of AAV9 (left) or AAV9 coated with PEGylated G2 PAMAM (right). (Scale bars: 100 nm). C) Comparison of particle diameters of uncoated ( N = 153) and coated rAAV ( N = 69, p < 0.0001). D) Zeta potential of uncoated or G2 Cys coated AAV9 particles. E) Transduction efficacy of AAV9‐GFP on a nonendothelial (Human embryonic kidney = HEK293T cells) and an endothelial cell line (human umbilical vein endothelial cells = HUVECs) at MOI of 1 × 10 6 coated by incrementally increasing doses (pg/cell) of G2 Cys PAMAM (green: GFP, Scale bars: 100 µm).

Article Snippet: The following antibodies were used for immunofluorescence staining: EGFP (CAB4211, Thermo Fisher), murine CD31 (BM4086, Origene), porcine CD31 (LCI‐4, Bio‐Rad), CRISPR‐Cas9 (7A9‐3A3, Novus Biologicals), AAV9 intact capsid (ADK9, Origene).

Techniques: Modification, Residue, Transmission Assay, Comparison, Zeta Potential Analyzer, Transduction

Journal: Advanced Science

Article Title: Endothelial Retargeting of AAV9 In Vivo

doi: 10.1002/advs.202103867

Figure Lengend Snippet: Modulation of in vivo AAV9‐Cre transduction by G2 CNN coating in mTmG mice. Examples of hindlimb skeletal muscle transduction of mTmG mice expressing membrane dTomato (red) unless Cre recombinase activity enables expression of enhanced green fluorescence protein (EGFP, green). Cre was transduced by A) an unmodified rAAV2/9 vector with CMV promoter, B) unmodified AAV9 pCMV Cre, or C) G2 CNN coated AAV9 pEndo. Cre. Transduction with D) G2 CNN coated AAV9 with endoglin promoter was also carried out 30 min after preinjection with isotype control IgG k or E) anti‐ Stab2 antibody. Top: EGFP alone, bottom: merge of EGFP, dTomato and DAPI (blue). Scale bars represent 25 µm.

Article Snippet: The following antibodies were used for immunofluorescence staining: EGFP (CAB4211, Thermo Fisher), murine CD31 (BM4086, Origene), porcine CD31 (LCI‐4, Bio‐Rad), CRISPR‐Cas9 (7A9‐3A3, Novus Biologicals), AAV9 intact capsid (ADK9, Origene).

Techniques: In Vivo, Transduction, Expressing, Membrane, Activity Assay, Fluorescence, Plasmid Preparation, Control

Journal: Advanced Science

Article Title: Endothelial Retargeting of AAV9 In Vivo

doi: 10.1002/advs.202103867

Figure Lengend Snippet: Endothelial transduction of mTmG pigs with AAV9‐Cre. mT mG −1 reporter pigs were transduced with either unmodified AAV9.pCMV.Cre (left) or G2 CNN coated AAV9.pEndo.Cre (right) via local application into A) hindlimb muscle and B) left ventricle. Top: EGFP alone (green), bottom: merge of EGFP, dTomato (red), CD31 (white), and DAPI (blue). Scale bars represent A) 100 µm or B) 25 µm.

Article Snippet: The following antibodies were used for immunofluorescence staining: EGFP (CAB4211, Thermo Fisher), murine CD31 (BM4086, Origene), porcine CD31 (LCI‐4, Bio‐Rad), CRISPR‐Cas9 (7A9‐3A3, Novus Biologicals), AAV9 intact capsid (ADK9, Origene).

Techniques: Transduction

Journal: Advanced Science

Article Title: Endothelial Retargeting of AAV9 In Vivo

doi: 10.1002/advs.202103867

Figure Lengend Snippet: Endothelial S1FG delivery via endothelial retargeted AAV9. A) AAV9 encoding S1FG, an artificial leukocyte adhesion molecule consisting of SDF‐1, the mucin domain of CX3CL1, and a GPI‐anchor, was injected to tail veins of wild type mice. B) Intravital microscopy of cremaster was carried out. C) Muscle venules (red outline) revealed enhanced adhesion of leukocytes (black dots) upon S1FG expression. D) Adhesion of leukocytes showed a significant increase with endothelial retargeting (G2 CNN coated AAV9.pEndo.S1FG) compared to untargeted delivery (unmodified AAV9.pCMV.S1FG) or control (unmodified AAV9.pCMV.lacZ); with cotreatment with CXCR4 antagonist AMD3100 abrogating this effect. Scale bars: 100 µm. Values represent the mean ± SEM. n = 4, 1‐way ANOVA with Dunnett post‐test.

Article Snippet: The following antibodies were used for immunofluorescence staining: EGFP (CAB4211, Thermo Fisher), murine CD31 (BM4086, Origene), porcine CD31 (LCI‐4, Bio‐Rad), CRISPR‐Cas9 (7A9‐3A3, Novus Biologicals), AAV9 intact capsid (ADK9, Origene).

Techniques: Injection, Intravital Microscopy, Expressing, Control

Journal: Advanced Science

Article Title: Endothelial Retargeting of AAV9 In Vivo

doi: 10.1002/advs.202103867

Figure Lengend Snippet: Endothelial retargeted of Annexin A1 abrogates leukocyte recruitment in chronic atherosclerotic mouse model. A) Endothelial activation and subsequent macrophage recruitment is a crucial part of atherosclerotic plaque formation and progression. B) Unmodified or endothelial retargeted Annexin A1 encoding AAV9 were tail vein injected to ApoE −/‐ mice on high fat diet. Ly6C + , Ly6G + , and CD11b + leukocyte subpopulations were stained by intravenous injection of respective antibodies and visualized by intravital microscopy of carotid arteries. C) Adhesion of all three subtypes showed a significant decrease with endothelial retargeting (G2 CNN coated AAV9.pEndo.Anxa) in comparison to untargeted delivery (unmodified AAV9.pCMV.Anxa) or control (unmodified AAV9.pCMV.EGFP). Scale bars: 100 µm; n = 12, 1‐way ANOVA with Dunnett post‐test.

Article Snippet: The following antibodies were used for immunofluorescence staining: EGFP (CAB4211, Thermo Fisher), murine CD31 (BM4086, Origene), porcine CD31 (LCI‐4, Bio‐Rad), CRISPR‐Cas9 (7A9‐3A3, Novus Biologicals), AAV9 intact capsid (ADK9, Origene).

Techniques: Activation Assay, Injection, Staining, Intravital Microscopy, Comparison, Control

Journal: Advanced Science

Article Title: Endothelial Retargeting of AAV9 In Vivo

doi: 10.1002/advs.202103867

Figure Lengend Snippet: Blood pressure alteration by Cas9 mediated eNOS deletion with endothelial retargeted AAV9. A) Vasodilative effect of nitrous oxide synthesis by endothelial cells was targeted by deletion of exons 6 through 10 of the eNOS gene via CRISPR/Cas9, rendering overlaying smooth muscle cells incapable of relaxation. Conditional Cas9 knock‐in mice were transduced by unmodified AAV9.pEndo.Cre or G2 CNN coated AAV9.pEndo.Cre with sgRNA targeting eNOS. B–E) Noninvasive blood pressure measurements were taken at baseline, 4 and 8 weeks; along with cardiac catheterization at week 8. Systolic, diastolic, mean arterial, and developed pressure values were plotted ( n = 6, 1‐way ANOVA with Dunnett post‐test). F) Cas9 expression (green) was observed in CD31 + cells (red) in both heart (left) and muscle (right) (Scale bars: 25 µm). G) Edited alleles (indicated by arrows) in magnetically sorted CD31 + cells of skeletal muscle were evident upon agarose gel separation (NEB 1kb+ ladder).

Article Snippet: The following antibodies were used for immunofluorescence staining: EGFP (CAB4211, Thermo Fisher), murine CD31 (BM4086, Origene), porcine CD31 (LCI‐4, Bio‐Rad), CRISPR‐Cas9 (7A9‐3A3, Novus Biologicals), AAV9 intact capsid (ADK9, Origene).

Techniques: CRISPR, Knock-In, Expressing, Agarose Gel Electrophoresis