Fgfr3 enhancer deletion markedly improves all skeletal features in a mouse model of achondroplasia
Marco Angelozzi, … , Andrew M. Bloh, Véronique Lefebvre
Marco Angelozzi, … , Andrew M. Bloh, Véronique Lefebvre
Published January 16, 2025
Citation Information: J Clin Invest. 2025;135(2):e184929. https://doi.org/10.1172/JCI184929.
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Research Article Bone biology Genetics

Fgfr3 enhancer deletion markedly improves all skeletal features in a mouse model of achondroplasia

Abstract

Achondroplasia, the most prevalent short-stature disorder, is caused by missense variants overactivating the fibroblast growth factor receptor 3 (FGFR3). As current surgical and pharmaceutical treatments only partially improve some disease features, we sought to explore a genetic approach. We show that an enhancer located 29 kb upstream of mouse Fgfr3 (–29E) is sufficient to confer a transgenic mouse reporter with a domain of expression in cartilage matching that of Fgfr3. Its CRISPR/Cas9-mediated deletion in otherwise WT mice reduced Fgfr3 expression in this domain by half without causing adverse phenotypes. Importantly, its deletion in mice harboring the ortholog of the most common human achondroplasia variant largely normalized long bone and vertebral body growth, markedly reduced spinal canal and foramen magnum stenosis, and improved craniofacial defects. Consequently, mouse achondroplasia is no longer lethal, and adults are overall healthy. These findings, together with high conservation of –29E in humans, open a path to develop genetic therapies for people with achondroplasia.

Authors

Marco Angelozzi, Arnaud Molin, Anirudha Karvande, Ángela Fernández-Iglesias, Samantha Whipple, Andrew M. Bloh, Véronique Lefebvre

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Figure 3

–29E deletion substantially lowers Fgfr3 expression in cartilage without impacting health.

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–29E deletion substantially lowers Fgfr3 expression in cartilage withou...
(A) Strategy used to generate mice with –29E deletion. EP, electroporation. Inset, adapted UCSC genome browser representation of the mouse chromosome 5 segment containing –29E and the gRNAs used for –29E deletion. The plot shows placental mammal conservation of the enhancer compared with flanking sequences. (B) Weight curves of –29E+/+, –29E+/–, and –29E–/– mice (n = 5–17). Symbols and brackets represent means and SDs, respectively. Individual mouse weight values are shown in the Supporting Data Values file. Statistical analysis was performed in this panel and others using 1-way ANOVA followed by Tukey’s multiple comparison tests. No significant differences among genotypes were detected (ns). (C) X-rays of representative 8-week-old males. Similar results were obtained with females. Scale bar: 1 cm. (D) Naso-anal lengths of 3- and 8-week-old mice. Each symbol corresponds to a distinct mouse. Blue dots, males; pink triangles, females. (E) Long bone lengths of the same mice as in D. The percentages of average values obtained for –29E+/– and –29E–/– mice relative to –29E+/+ mice are indicated at 8 weeks. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). P values slightly above significance are indicated. (F) Pictures of H&E-stained sections through tibial proximal GPs of representative 3-week-old mice. POC, primary ossification center; SOC, secondary ossification center. Scale bar: 50 μm. (G) Quantification of GP zone heights in the same group of mice (n = 5–6) as in F. No statistically significant difference (ns) was detected for mutant versus control GP zones. (H) RT-qPCR assays of Fgfr3 RNA levels in skeletal and nonskeletal tissues. Only females were used. Percentages of average control values are indicated. (I) Representative Fgfr3 RISH of sections through tibia proximal GPs. The magenta color (RNA signal) was saturated, and the blue color (hematoxylin) desaturated using Adobe Photoshop. Scale bar: 50 μm. (J) Representative Fgfr3 RISH of sections through vertebral GPs. Images were processed as in I. Scale bar: 50 μm.