Fig 2: CRISPR/SpCas9-mediated NOTCH2 knockdown led to efficient trans-differentiation of club cells into ciliated cells(A) Schematic of Notch2 genomic targeting sites by sgRNA pairs (upper panel) and the AAV vector design carrying the paired sgRNA cassette and an mCherry reporter (lower panel). (B) The sgRNA pairs effectively downregulated NOTCH2 expression in MLE-12-SpCas9 cells. Cells were infected by a single AAV at an MOI of 1 × 106 or three AAVs combined (each at an MOI = 1 × 106). Proteins were extracted from the pooled cell population (without selection) on day 7 after infection and subjected to capillary protein analysis. (C) Schematic of animal study design. Three sequential doses of AAV-Notch2-sgRNA pairs at 4 × 1012 vg/mouse/round were delivered to Rosa26-CAG-SpCas9-eGFP transgenic mice at 7-day intervals. Animals were taken down for analysis at weeks 4 or 8 after the first round of infection. (D) AAV transduction efficiency in cell types of interest as measured by %mCherry expression. FACS analysis of %mCherry expression at week 4 (n = 13) and week 8 (n = 16) after the first round of sgRNA pair delivery in AECII and distal airway epithelial cells. Each dot represents one animal. Error bars represent SEM. ***p < 0.001; n.s., not significant. (E) Representative NOTCH2 IHC staining of the paraffin-embedded lung tissue from the control group (left picture) and treatment group (right picture) at week 4 time point. Scale bars, 200 µm. (F) Quantification of NOTCH2 expression by IHC at weeks 4 and 8 time points. Data represented the NOTCH2 protein level of the whole lung tissue (not specific to AECII, club, and ciliated cells). Data were normalized to the PBS control animals. Each dot represents one animal. Error bars represent SEM. p values are indicated. (G) Representative IF images of club and ciliated cells at the week 4 time point. Club cells stained in white (CC10: a cell marker for club cells); ciliated cells stained in green (acetylated alpha-tubulin: a cell marker for ciliated cells). mCherry stained red. mCherry expression was an indicator of AAV-infected cells. Cell nuclei stained by DAPI in blue. Scale bars, 200 µm. (H) Quantification of club cells as stained by the cell marker CC10 (CC10+ area). Data were pooled from weeks 4 and 8 time points. Each dot represents one animal. Error bars represent SEM. ***p < 0.001, ****p < 0.0001; n.s., not significant. (I) Quantification of ciliated cells as stained by the nuclear marker FoxJ1 (FoxJ1+ area). Data was pooled from weeks 4 and 8 time points. Each dot represents one animal. Error bars represent SEM. ****p < 0.0001; n.s., not significant.

Fig 3: Subcutaneous transplantation of human fetal lung tissues into SCID mice to develop the human lung xenograft mouse model. (A) Scheme of construction of the human lung xenograft mouse model. Small fragments (~10 mm3) of human fetal lung tissues were transplanted subcutaneously (s.c.) into the right flank of SCID mice. After 8 weeks, the lung xenografts can grow and develop mature lung structures. (B) Gross view and (C) histological analysis of the human lung xenograft at 8 weeks post-transplantation. (D) Immunohistochemistry staining of lung marker expression in the human lung xenograft. Sections of human lung xenografts at 8 weeks post-transplantation were immunostained for human CK-19 (hCK-19), human vimentin (hVimentin), human PDPN (hPDPN), human SP-C (hSP-C), human CC10 (hCC10) to visualize the lung epithelium and mesenchyme, type I and type II pneumocytes, and bronchiolar Clara cells, respectively. Tissue sections were also stained for human ACE2 (hACE2) expression. The lower panels represent higher magnification images of the areas outlined by the dotted lines in the upper panels. Scale bars in the left and right panels in (C) represent 1 mm and 200 µm, respectively. Scale bars for 100 × and 400 × magnification in (D) represent 200 µm and 50 µm, respectively. Staining was performed on tissue sections from 4 lung xenografts obtained from two donors, and representative images are shown.

Fig 4: Profiling AAV6 transduction in murine lung(A) Quantification of GFP expression within cells dosed with increasing titers of AAV6-CAG-GFP. IF staining of GFP and various cell markers were performed on paraffin-embedded lung tissue harvested at week 1 after delivery of AAV6-CAG-GFP. Lung sections containing five lung lobes were quantified for each animal. n = 5. Error bars represent SEM. (B) Representative IF images showing GFP stained in green, mCherry in red, cell markers (SPC, CC10, and acetyl-alpha tubulin, respectively) in white, and nuclei (DAPI) in blue on paraffin-embedded lung sections. Animals were co-transduced with AAV6-CAG-GFP and AAV6-CAG-mCherry at 4 × 1012 vg/virus/mouse. Lung tissues were harvested at week 1 after infection. Scale bars, 500 µm. The yellow box shows the zoomed-in region. Scale bars, 50 µm (inset). (C) Quantification of the colocalization of GFP, mCherry, and cell markers. IF staining of GFP, mCherry, and various cell markers was performed on paraffin-embedded lung tissue harvested at week 1 after co-transduction of AAV reporter viruses (GFP and mCherry). Lung sections containing five lung lobes were quantified for each animal. n = 5. Error bars represent SEM. (D) The composition of lung cell types presented in GFP– versus GFP+ samples as shown in the pie charts. (E) The percentage of each lung cell type presented in GFP– versus GFP+ samples.