A large animal model of heritable pulmonary arterial hypertension using gene-edited BMPR2 sheep

Figures

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  Figure 1. Agarose gel of BMPR2 gene–edited lamb genotypes. A 431 bp WT amplicon of BMPR2 was amplified by primers Intron2-F2 and Intron-3R and visualized on a 1.75% agarose gel to reveal obvious insertions and deletions in several of the 8 lambs. Neg, negative control.

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  Figure 2. Edited BMPR2 alleles identified by Amplicon-EZ sequencing. Alleles shown were observed in more than 1% of Illumina Amplicon-EZ reads. Positions are on Ovis aries Chr2 (NC_056055.1). Red highlighting indicates deleted sequence, blue indicates inserted sequence. Protospacer-adjacent motifs (PAMs) are underlined, with disrupting sequences bolded, and highlighted yellow when repaired using the single-stranded oligodeoxyribonucleotide (ssODN) template. The Cas9-induced double-stranded break 3 bp downstream of the PAM is identified in gray; resulting frame-shifted nucleotides are indicated in italics. The sgRNA1 PAM (CCT→CAT) was disrupted in at least 1 allele. The additional silent PAM disruption by the ssODN (CGG→CGA) at 204745563 was to disrupt the PAM for sgRNA2; the C/T variation at 204745602 is a known SNP (rs404303969). DSB, double stranded break; WT, wildtype.

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  Figure 3. Relative BMPR2 mRNA expression in WT and BMPR2-edited F0 lambs quantified by qPCR. (A) Expression analysis of live-born lamb no. 4 ear (square), heart (triangle), and lung (circle) samples compared with WT controls (n = 3–5). Lamb 4 was compared to WT using an unpaired, 2-tailed Welch’s t test. (B) Expression analysis of live-born lamb 5 and 6 lung samples compared with WT controls (n = 3). Mean and SEM values are shown with individual data points overlaid.

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  Figure 4. Expression of BMPR2 transcripts in ear skin of WT and BMPR2-edited F0 lambs 4 and 8. The WT BMPR2 allele (435 bp), 49 bp deletion allele (386 bp), and 94 bp insertion allele (529 bp) are easily visualized. ACTB expression (157 bp) was used as a housekeeping control.

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  Figure 5. Pulmonary hypertension evaluation. Echocardiography at 3 weeks of age, with representative images of (A) control versus (B) BMPR2+/– female (no. 8). RV, right ventricle; LV, left ventricle; V, ventral. Right pulmonary angiography of (C) control and (D) BMPR2+/– lamb no. 8. H&E staining (original magnification, ×60) of pulmonary arterioles from BMPR2+/– lambs no. 5 (F) and 6 (H) associated with medium-sized airways. (E and G) Arterioles from age-matched control lambs.

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  Figure 6. Histopathology of pulmonary arteries from lungs from BMPR2+/– lambs 5 and 6 compared with control. (A–C) Pentachrome staining identifies arterial wall hypertrophy in BMPR2+/– lambs. Scale bars: 50 μm. (D–F) Immunofluorescent staining highlights expansion of both endothelial (von Willebrand factor [VWF], green) and smooth muscle (α-smooth muscle actin [SMA], red) layers. Scale bars: 25 μm. (G–I) MKI67 staining: Open arrowheads mark MKI67+ proliferating cells in the smooth muscle layer and the closed arrowheads mark MKI67+ proliferating cells in the endothelium. a, pulmonary artery. Scale bars: 25 μm. (J–M) For morphometry, 5 vessels each were analyzed for the small- and medium-sized arteries in each animal, with 3 each for the large-sized arteries. Differences in intimal and medial thickness (J and K) within each arterial size range were determined using 1-way ANOVA with Tukey’s correction for multiple comparisons. For endothelial (L) and smooth muscle cell (M) proliferation, Kruskal-Wallis non-parametric tests were used to compare lambs. Mean and SEM values are shown with individual data points overlaid.

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  Figure 7. Editing sequence design. Single guide RNA 1 (sgRNA1) (green) and sgRNA2 (blue) were targeted to exon 3 of BMPR2. sgRNA1 was used in production of gene-edited lambs, and sgRNA2 was used solely for in vitro testing (indicated by dotted box). Protospacer-adjacent motifs (PAMs) are indicated by underlining; mismatches relative to the Oar_v3.1 reference genome are indicated in bold. The location where sgRNA1 would be predicted to introduce a double-stranded break (DSB) is indicated by the green dashed line. Non-homologous end joining (NHEJ) repair of the DSB will repeatedly cleave and introduce indel mutations until the guide sequence is disrupted, while homology-directed repair (HDR) using the 99 bp single-stranded oligodeoxyribonucleotide (ssODN) will introduce a synonymous silent mutation, disrupting the PAM of sgRNA1 (CTT→CAT) and preventing further Cas9 activity. The additional PAM-disrupting mutation (CGG→CGA) towards the 3′ end of the ssODN was designed to disrupt the PAM of sgRNA2.