Cross-species tropism of AAV.CPP.16 in the respiratory tract and its gene therapies against pulmonary fibrosis and viral infection

Figures

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  Figure 1. AAV transduction of airway cells in cell culture and mouse models (A) Schematic diagram showing experimental design. (B and C) Images showing transduction of cultured human nasal epithelial cells RPMI 2650 in vitro (multiplicity of infection, MOI, 1 × 105) by AAV6-CAG-GFP, AAV9-CAG-GFP, and AAV.CPP.16-CAG-GFP (CPP.16) (B) and quantification of GFP fluorescence intensity normalized as fold change relative to AAV6. (C) Mean ± SEM, n = 3. Scale bar: 100 μm. (D–I) Representative fluorescence images and quantitative analysis showing AAV-mediated GFP expression in the nasal cavity (D and E), trachea (F and G), and lung (H and I) in C57BL/6J mice 21 days after intranasal delivery of 1 × 1011 vg of AAV6, AAV9, or CPP.16. Mean ± SEM, n = 6–7 each treatment. Scale bar in (D): 500 μm. Scale bar in (F) and (H): 100 μm. Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparison test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

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  Figure 2. Characterization of AAV-transduced cell types in the mouse respiratory tract and lungs (A) Schematic diagram illustrating major airway cell types, their anatomical locations, and antibody markers. Mouse tissues were collected 21 days after intranasal administration of 1 × 1011 vg of AAV-CAG-GFP. (B) Representative images showing transduced goblet cells (MUC5A+) in the nasal cavity. Scale bar: 25 μm for low-magnification images and 5 μm for enlarged images. (C) AAV-mediated transduction of club cells (CC10+) in the trachea. Arrows point to cells co-labeled with GFP and CC10 antibodies. Scale bar: 25 μm. (D) AAV-mediated transduction of alveolar type I (AT1) (RAGE+) in lung parenchyma tissues. Scale bar: 25 μm. (E–G) Quantifications on the percentage of GFP+ goblet cells in the nasal cavity (E), club cells in the trachea (F), and AT1 cells in the lung (G). Mean ± SEM, n = 3–4 for each group. Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparison test (∗∗p < 0.01, ∗∗∗p < 0.001).

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  Figure 3. Intranasal AAV.CPP.16-mediated gene delivery in non-human primates (A, C, and E) Representative images showing AAV-mediated GFP and RFP co-expression in the nasal cavity (A), trachea (C), and lung (E) in three adult non-human primates (NHPs). DAPI nuclear staining was shown in blue. In NHP #1, AAV9-GFP and AAV9-RFP were pre-mixed and co-administered intranasally. In NHPs #2 and 3, CPP.16-GFP and AAV9-RFP were co-administered. AAV9-RFP serves as an internal control. 4 × 1012 vg of AAVs per AAV per animal were applied. Tissues were collected 28 days after AAV administration. Scale bar: 20 μm. (B, D, and F) Quantification of GFP fluorescence intensity measured as the ratio of GFP/RFP in the nasal cavity (B), trachea (D), and lung (F). Values were normalized to those in NHP#1 to show the fold changes of CPP.16-mediated GFP expression relative to AAV9.

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  Figure 4. An AAV.CPP.16-mediated gene therapy for idiopathic pulmonary fibrosis (A) Schematic diagram illustrating design of a bifunctional fusion protein that inhibits VEGF and TGF-β1 and construction of an AAV vector expressing such a protein. (B) Experimental design for testing the AAV.CPP.16 gene therapy (CPP.16-IPF trap) in a bleomycin-induced IPF mouse model. i.t., intratracheal; inhal., Inhaled; i.g., intragastric. (C) Representative H&E staining on lung sections in mice with different treatments. Scale bar: 200 μm for low-magnification images and 50 μm for enlarged images. (D) Quantification of lung cell areas in H&E-stained lung sections. n = 3–6 per group, mean ± SEM, one-way ANOVA with Tukey’s post-test. (E) Representative Masson, picrosirius red, and αSMA immunohistochemical staining of lung sections in mice with different treatments. Masson (blue) and picrosirius red (red) stainings label collagen expression. Scale bar: 200 μm. (F) Quantification of fibrotic areas on lung sections. n = 3–6 per group, mean ± SEM, one-way ANOVA with Tukey’s post-test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. PBS, phosphate-buffered saline; BLM, bleomycin; IPF, idiopathic pulmonary fibrosis; PFD,…

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  Figure 5. Inhibition of VEGF and TGF-β1 pathways in the mouse lung treated with AAV.CPP.16-IPF trap gene therapy (A) Transgene expression in the mouse lung treated with AAV.CPP.16-IPF trap. Reverse-transcription PCR (RT-PCR) was performed using primers that targeted the two fragments of the fusion protein. n = 4 per group, mean ± SEM, one-way ANOVA with Tukey’s post-test. (B) Concentration of VEGFα in the mouse serum measured by ELISA. n = 3–8 per group, mean ± SEM, one-way ANOVA with Tukey’s post-test. (C) Concentration of TGF-β1 in the mouse serum measured by ELISA. n = 3–8 per group, mean ± SEM, one-way ANOVA with Tukey’s post-test. (D) mRNA expression levels of fibrogenic genes Acta2, Postn, and Fn in the lung tissues. n = 4 per group, mean ± SEM, one-way ANOVA with Tukey’s post-test. (E) mRNA expression levels of inflammation genes Tnfa, Il6, and Il1b in the lung tissues. n = 4 per group, mean ± SEM, one-way ANOVA with Tukey’s post-test. (F–H) Immunoblot quantification of key fibrogenic and inflammation proteins. n = 3 per group. mean ± SEM, one-way ANOVA with Tukey’s post-test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. PBS, phosphate-buffered saline; BLM, bleomycin; IPF, idiopathic…

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  Figure 6. An AAV.CPP.16-mediated gene therapy against SARS-CoV-2 using an all-in-one CRISPR-CasRx RNA editing system (A) Schematic diagram showing an all-in-one AAV vector expressing both CasRx and four gRNAs that target the SARS-CoV-2-derived RNA-dependent RNA polymerase (Rdrp) gene. (B) Workflow for testing Rdrp RNA knockdown in 293T cells by co-transfection with CasRx-gRNA and Rdrp plasmids. (C) Quantification of Rdrp knockdown after co-transfection in 293T. Abbott et al.: gRNA from a published study.40n = 6–9 per group, mean ± SEM, one-way ANOVA with Tukey’s post-test. (D) Workflow for testing Rdrp RNA knockdown in RPMI 1650 cells by co-transduction with AAV.CPP.16-CasRx-gRNA and Ad5-Rdrp. (E) Quantification for knockdown of Ad5-expressed Rdrp transcript by AAV.CPP.16-CasRx-gRNA in RPMI 1650 cells. n = 3 per group, mean ± SEM, one-way ANOVA with Tukey’s post-test. (F) Timeline for studying intranasal AAV.CPP.16-CasRx-gRNA.Interface in Ad5-Rdrp-challenged mice. (G) Quantification of Rdrp knockdown in the nostril, trachea, and lung of Ad5-Rdrp-challenged mice treated with AAV.CPP.16-CasRx-gRNA.Interface or AAV.CPP.16-CasRx-scramble control. n = 6 each group, mean ± SEM, two-way ANOVA…