Research ArticleGENE EDITING

In utero gene editing for monogenic lung disease

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Science Translational Medicine  17 Apr 2019:
Vol. 11, Issue 488, eaav8375
DOI: 10.1126/scitranslmed.aav8375
  • Fig. 1 Intra-amniotic delivery of CRISPR-Cas9 results in pulmonary gene editing.

    (A) Schematic representation of intra-amniotic route of fetal lung gene editing. (B) Experimental design of gene editing in R26mTmG/+ mice. (C) Fluorescent stereomicroscopy, using a filter to detect tdTomato and EGFP, of lungs from R26mTmG/+ mice injected with Ad.Cre, Ad.mTmG, or Ad.Null. (D) IHC for EGFP and tdTomato expression in the proximal airway and distal air saccules of lungs from R26mTmG/+ mice injected with Ad.Cre, Ad.mTmG, or Ad.Null. White arrowheads indicate EGFP staining. (E) PCR assay using primers to detect the on-target editing in DNA isolated from E19 lungs of R26mTmG/+ mice injected with Ad.Cre, Ad.mTmG, or Ad.Null. Edited band, 545 base pairs (bp); unedited band, 2951 bp. n = 2 to 6 per group. One fetus that was injected with Ad.mTmG and lacked notable EGFP fluorescence (GFP) was also negative for gene editing by PCR, indicating a likely technical failure at the time of injection. (F) Sanger sequencing of the 545-bp edited mTmG PCR product from an R26mTmG/+ mouse injected with Ad.mTmG. (G) Sanger sequencing of the 545-bp Cre-recombined mTmG PCR product from an R26mTmG/+ mouse injected with Ad.Cre. Scale bars, 1000 μm (C) and 50 μm (D). IA, intra-amniotic; E, gestational day.

  • Fig. 2 Intra-amniotic delivery of CRISPR-Cas9 targets pulmonary epithelial cells for gene editing.

    (A) FACS plots of lungs harvested at E19 after intra-amniotic injection of Ad.mTmG, Ad.Cre, or Ad.Null at E16. Each row shows representative FACS plots from a single lung. (B) Quantitation of cell type–specific gene editing using FACS analysis for EGFP+ cells within each major pulmonary cell type after intra-amniotic injection of Ad.mTmG and Ad.Cre. n = 5 per group. (C) EGFP+ gene-edited and Cre-recombined cells depicted by white arrowheads within subsets of pulmonary epithelial cells marked by AQP5, SFTPC, SCGB1A1, and FOXJ1. (D) Quantification of gene-edited airway and alveolar epithelial cells after Ad.mTmG intra-amniotic delivery. (E) Quantification of Cre-recombined airway and alveolar epithelial cells after Ad.Cre intra-amniotic delivery. n = 2 to 5 per group. Epi, epithelial; Endo, endothelial; Mes, mesenchymal. Scale bars, 50 μm.

  • Fig. 3 Pulmonary epithelial cell gene editing is stable over time.

    (A) Experimental design for longer-term analysis of pulmonary epithelial cell gene editing after intra-amniotic Ad.mTmG delivery at E16. (B) Quantification of edited pulmonary epithelial, endothelial, and mesenchymal cell types at E19, P7, P30, and 6 months by FACS analysis. (C) Quantification of gene editing in individual pulmonary cell types at E19, P7, P30, and 6 months by IHC. (D) Schematic summary of fetal pulmonary cells that underwent gene editing after intra-amniotic delivery of CRISPR-Cas9 targeting the mT gene. n = 3 to 5 per group; **P < 0.01 and *P < 0.05 by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.

  • Fig. 4 Prenatal gene editing in SftpcI73Tmice decreases mutant SP-CI73Tproprotein and improves lung alveolarization.

    (A) Schematic representation of SftpcI73T mutation causing intracellular accumulation of SP-CI73T proprotein resulting in AT2 cell injury and potential cell rescue with CRISPR-Cas9–mediated excision of SftpcI73T. (B) Fluorescent stereomicroscopy, using a filter to detect EGFP, of an E19 fetus (outlined by white dashed line) after intra-amniotic injection of Ad.Sftpc.GFP at E16 shows green fluorescence in the chest region. (C) Fluorescent stereomicroscopy, using a filter to detect EGFP, of lungs at E19 after E16 intra-amniotic injection of Ad.Sftpc.GFP. (D) IHC for EGFP of lung parenchyma at E19 after E16 intra-amniotic injection of Ad.Sftpc.GFP. (E) FACS analysis to assess EGFP expression in all pulmonary cells and pulmonary epithelial cells (EPCAM+ cells) from E19 fetuses after E16 intra-amniotic injection of Ad.Sftpc.GFP. n = 10 to 11 per group. (F) PCR analysis of DNA from E19 lung epithelial cells (EPCAM+-sorted cells) of E16 Ad.Sftpc.GFP intra-amniotic injected fetuses. Edited Sftpc band, 605 bp; −C and +C, negative and positive controls consisting of nontransfected mouse neuro-2a cells and mouse N2a cells cotransfected with plasmids containing spyCas9 or sgRNA1-A and sgRNA5-B, respectively. (G) Schematic of SftpcI73T experimental design. (H) Excision of the mutant Sftpc allele in AT2 cells was assessed by IHC. Lungs of E19 SftpcI73T/WT mice were assessed for expression of surfactant protein B (SFTPB) and hemagglutinin (HA) after E16 intra-amniotic injection of Ad.Null.GFP or Ad.Sftpc.GFP. SFTPB+HA (yellow arrowheads indicate representative cells), excision; SFTPB+HA+ (white arrowheads indicate representative cells), no excision; control, uninjected WT E19 lungs. (I) The percentage of SFTPB+HA cells on IHC was quantified. (J) Lung IHC for homeodomain only protein X (HOPX) at E19 to assess AT1 cell morphology and spreading in SftpcI73T/WT mice injected with Ad.Null.GFP or Ad.Sftpc.GFP at E16. (K) The internuclear distance was measured to quantify AT1 spreading. (L) Hematoxylin and eosin (H&E) staining of lungs from E19 SftpcI73T/WT mice injected at E16 with Ad.Null.GFP or Ad.Sftpc.GFP to assess alveolarization and sacculation. (M) The MLI was calculated to assess alveolarization. n = 3 to 4 per group; ##P < 0.0001, **P < 0.01, and *P < 0.05 by one-way ANOVA followed by Tukey’s multiple comparison test. WT, wild-type. Scale bars, 50 μm.

  • Fig. 5 Prenatal gene editing in SftpcI73Tmutant mice improves survival.

    (A) Schematic of experimental design for survival analysis of SftpcI73T mutant mice. (B) Survival of C57BL/6 mice injected at E16 with Ad.Sftpc.GFP (blue), gene-edited SftpcI73T/WT mice injected with Ad.Sftpc.GFP at E16 (red), SftpcI73T/WT mice injected with Ad.Null.GFP at E16 (green), and uninjected SftpcI73T/WT mice (purple). (C) The survival frequency of Ad.Sftpc.GFP-treated SftpcI73T/WT mice was normalized to the survival rate of control C57BL/6-treated mice at 1 week of age. n = 20 to 87 per group; **P < 0.01 by log-rank (Mantel-Cox) test for comparison of survival curves. (D) H&E staining of lungs from 1-week-old SftpcI73T/WT mice and C57BL/6 mice injected with Ad.Sftpc.GFP at E16 and uninjected WT C57BL/6 mice was performed to assess alveolarization. (E) The MLI was calculated to assess alveolarization. (F) IHC for SFTPB and HA was performed on lungs from 1-week-old SftpcI73T/WT mice and C57BL/6 mice injected with Ad.Sftpc.GFP at E16 and uninjected WT C57BL/6 mice to assess AT2 cell morphology and excision of the mutant Sftpc allele in AT2 cells. SFTPB+HA (yellow arrowheads indicate representative cells), excision; SFTPB+HA+ (white arrowheads indicate representative cells), no excision. (G) The percentage of SFTPB+HA cells, indicative of gene-edited cells in Ad.Sftpc.GFP injected SftpcI73T/WT mice, was quantified on IHC. (H) IHC for HOPX was performed to assess AT1 cell morphology in 1-week-old SftpcI73T/WT mice and C57BL/6 mice injected with Ad.Sftpc.GFP at E16 and uninjected WT C57BL/6 mice. (I) The internuclear distance was calculated to assess AT1 cell spreading. n = 3 to 4 per group; ##P < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparison test. Scale bars, 50 μm.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/488/eaav8375/DC1

    Fig. S1. R26mTmG gene locus of interest and lack of gene editing in nonpulmonary organs after intra-amniotic delivery.

    Fig. S2. Distribution of gene-edited cells in the lung.

    Fig. S3. Gene editing in pulmonary cell types.

    Fig. S4. Selection of sgRNAs for excision of the Sftpc gene and in vivo gene editing in C57BL/6 and SftpcI73T/WT mice.

    Fig. S5. Transmission electron microscopy of gene-edited lungs of SftpcI73T/WT mice.

    Fig. S6. Lung morphology of gene-edited SftpcI73/WT mice in adulthood.

    Table S1. mTmg and Sftpc sgRNAs for in vitro and in vivo editing.

    Table S2. Primers used for surveyor assay for Sftpc sgRNAs.

    Table S3. Primers used for PCR and Sanger sequencing.

    Table S4. Primers used for qPCR for Sftpc gene deletion.

    Table S5. Primer sequences for NGS of Sftpc off-target sites.

    Table S6. Analysis of 20 off-target sites.

    Movie S1. Intra-amniotic injection of dilute trypan blue at E16.

    Movie S2. In utero gene-edited Sftpc173T/WT mouse at P7.

    Data file S1. Original data.

  • The PDF file includes:

    • Fig. S1. R26mTmG gene locus of interest and lack of gene editing in nonpulmonary organs after intra-amniotic delivery.
    • Fig. S2. Distribution of gene-edited cells in the lung.
    • Fig. S3. Gene editing in pulmonary cell types.
    • Fig. S4. Selection of sgRNAs for excision of the Sftpc gene and in vivo gene editing in C57BL/6 and SftpcI73T/WT mice.
    • Fig. S5. Transmission electron microscopy of gene-edited lungs of SftpcI73T/WT mice.
    • Fig. S6. Lung morphology of gene-edited SftpcI73/WT mice in adulthood.
    • Table S1. mTmg and Sftpc sgRNAs for in vitro and in vivo editing.
    • Table S2. Primers used for surveyor assay for Sftpc sgRNAs.
    • Table S3. Primers used for PCR and Sanger sequencing.
    • Table S4. Primers used for qPCR for Sftpc gene deletion.
    • Table S5. Primer sequences for NGS of Sftpc off-target sites.
    • Table S6. Analysis of 20 off-target sites.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Intra-amniotic injection of dilute trypan blue at E16.
    • Movie S2 (.mp4 format). In utero gene-edited SftpcI73T/WT mouse at P7.
    • Data file S1 (Microsoft Excel format). Original data.

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