Fig 2: Effects of AAV gene therapy in human FOP iPSCs and mouse Acvr1(R206H)KI cells.a Human FOP iPSCs were treated with PBS or 5 × 1010 genome copies (GCs) of 15 different AAV capsids packaged with the same CBA-Egfp transgene. 2 days later, EGFP expression was assessed by immunoblotting with an anti-GFP antibody. Anti-HSP90 antibody was used for loading control. b Genome integrity of rAAV6.2 carrying amiR-RH6.ACVR1opt or amiR-RH7.ACVR1opt was assessed by electrophoresis in the native gel. ssFL single-stranded full-length. c, d 5 × 1010 GCs of rAAV6.2 carrying EGFP control (ctrl), amiR-RH6.ACVR1opt, or amiR-RH7.ACVR1opt were transduced to human FOP iPSCs, cultured under osteogenic conditions for 4 days, and subjected to next-generation sequencing (NGS) for ratio expression: ACVR1R206H vs. ACVR1WT (c, top) or RT-PCR for ACVR1opt expression (c, bottom, n = 4). Alternatively, total RNA was subjected to bulk RNA sequencing (d, n = 2). A volcano plot showing the gene expression for up/downregulated genes in the cells expressing amiR-RH6.ACVR1opt or amiR-RH7.ACVR1opt relative to control-expressing cells is displayed. A volcano plot was generated from multiple t-test. e–g AAV-treated, human WT or FOP iPSCs were cultured under osteogenic conditions and alkaline phosphatase activity (ALP) and alizarin red staining were performed to assess early and late osteoblast differentiation, respectively (e, n = 9). Alternatively, osteogenic gene expression (RUNX2) was assessed by RT-PCR (f, n = 4). AAV-treated, human FOP iPSCs were incubated with PBS or Activin A (100 ng/ml) for 6 h, and ID1 mRNA levels were measured by RT-PCR (g, n = 4). h, i PDGFRa+Sca1+CD31-CD45- FAPs were sorted by FACS from the digested skeletal muscle of 4-week-old Acvr1(R206H)Fl;PDGFRa-cre mice and transduced with 5 × 1010 GCs of AAV6.2 carrying EGFP control, amiR-RH6.ACVR1opt, or amiR-RH7.ACVR1opt. 2 days later, AAV-treated FAPs were cultured under osteogenic conditions with PBS or Activin A (50 ng/ml) for 6 days, and ALP activity (h, n = 6) and osteogenic gene expression (BGLAP, IBSP, i, n = 4) were assessed for osteoblast differentiation. Data are representative of three independent experiments (a, b). Values represent mean ± SD by an unpaired two-tailed Student’s t-test or one-way ANOVA test (e–i).

Fig 3: Development of AAV vector targeting the human ACVR1R206H receptor.a Schematic diagram of the plasmids expressing a codon-optimized version of the human ACVR1 complementary DNA (ACVR1opt) with the CBA promoter and three different introns (created with biorender.com). CBA: CMV enhancer/chicken β-actin promoter. b Validation of the expression of the ACVR1opt receptor. Plasmids expressing ACVR1opt were transiently transfected into HEK293 cells and cell lysates were subjected to immunoblotting with anti-ACVR1 antibody. Anti-HSP90 antibody was used as a loading control. c Schematic diagram representing 12 amiRs that target different sequence sites of human ACVR1R206H mRNA (amiR-ACVR1R206H). The red box indicates the R206H mutation site (c.617G>A). Adenine (green) was mismatched to increase the selectivity of gene silencing (created with biorender.com). d Plasmids encoding amiR-ctrl or 12 different amiRs were transiently transfected into HEK293 cells along with amiR-sensor plasmids (sensor-Luc) that contain Renilla luciferase and aimR complementary sequences for human ACVR1R206H, ACVR1WT, and ACVR1opt (n = 3). One day later, a luciferase assay was performed to measure Renilla luciferase and normalized it to firefly luciferase. Lower activities indicate higher silencing efficacy of amiRs. e Plasmids encoding amiR-ctrl or 12 different amiRs were transiently transfected into HEK293 cells along with a plasmid expressing human ACVR1R206H, ACVR1WT, or ACVR1opt cDNA and immunoblotted for ACVR1. Anti-HSP90 antibody was used for loading control. f Schematic diagram of the combination gene therapy constructs expressing amiR-ACVR1R206H (RH6 or RH7) and ACVR1opt cDNA under the CBA promoter and MBL intron (MBLi) or synthetic intron (Syni, created with biorender.com). g, h Plasmids encoding amiR-ctrl (ctrl), Syni.amiR-RH6.ACVR1opt, Syni.amiR-RH7.ACVR1opt, MBLi.amiR-RH6.ACVR1opt, or MBLi.amiR-RH7.ACVR1opt were transiently transfected into HEK293 cells along with sensor-Luc plasmids. Luciferase assay (g, n = 3) or immunoblotting analysis for ACVR1 (h) was performed. Anti-HSP90 antibody was used for loading control. i Plasmids were transiently transfected into HEK293 cells along with the BMP SMADs-responsive reporter gene (BRE-luc) and treated with Activin A (100 ng/ml, n = 4). 24 h later, Activin A signaling activity was measured by luciferase assay. Data are representative of three independent experiments (b, e). Values represent mean ± SD by an unpaired two-tailed Student’s t-test or one-way ANOVA test (i).