Fgfr3 enhancer deletion markedly improves all skeletal features in a mouse model of achondroplasia
Marco Angelozzi, Arnaud Molin, Anirudha Karvande, Ángela Fernández-Iglesias, Samantha Whipple, Andrew M. Bloh, Véronique Lefebvre
Marco Angelozzi, Arnaud Molin, Anirudha Karvande, Ángela Fernández-Iglesias, Samantha Whipple, Andrew M. Bloh, Véronique Lefebvre
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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 2

In vitro and in vivo assessment of –29E activity.

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In vitro and in vivo assessment of –29E activity. (A) Reporters used in ...
(A) Reporters used in assays in vitro in B. Four putative enhancers were cloned in the multiple cloning site (MCS) of reporters driven by a Col2a1 or Fgfr3 promoter. polyA, polyadenylation sites. (B) Reporter activities assessed in transiently transfected RCS and MC3T3-E1 cells. Averages with SDs of firefly luciferase values normalized with NanoLuc values are shown for triplicate cultures from a representative experiment. Individual values are shown in the Supporting Data Values file. Numbers above bars indicate fold increases relative to promoter alone activities. (C) –29E-lacZ transgene flanked with H19 insulators. Two copies of –29E precede the Hsp68 promoter (with transcription start site indicated), an optimized lacZ sequence, and IRES-EGFP and polyadenylation sequences. (D) Left, sections through the tibial proximal epiphysis of P5 –29E-lacZ and nontransgenic littermates stained with Xgal (blue) and counterstained with Nuclear Fast Red (pink). The position of the epiphysis (Ep), CZ, and HZ are indicated. Right, Fgfr3 RISH of an equivalent section from a P0 WT mouse. The magenta color (RNA signal) was saturated and blue color (hematoxylin) desaturated using Adobe Photoshop. AC, articular cartilage; M, meniscus; POC, primary ossification center. Scale bars: 100 μm (X-gal staining images); 80 μm (right image). (E) X-gal staining of coronal and transverse sections through the vertebral column of same mice as in D. AF, annulus fibrosus; CEP, cartilaginous end plate; NCS, neurocentral synchondroses; NP, nucleus pulposus; Sp, spinal cord; VB, vertebral body. X-gal staining in ossification centers of transgenic and nontransgenic mice reflects endogenous β-galactosidase expressed in osteoclasts (66). Scale bars: 100μm (top); 150 μm (bottom). (F) X-gal staining of a sagittal section through the head of a P5 –29E-lacZ mouse. Colored-box areas in each image are shown at higher magnification on the right. BO, basioccipital bone; BS, basisphenoid bone; CV, cervical vertebrae; Et, ethmoid bone; MC, Meckel’s cartilage; NB, nasal bone; NS, nasal septum cartilage; OB, occipital bone; OBC, occipital bone cartilage; PaB, palatine bone; PS, presphenoid bone; SOS, spheno-occipital synchondrosis. Scale bar: 100 μm (left image) 200 μm (right image). (G) Whole-mount X-gal staining of the ventral portion of thoracic cages from same mice as in D. Scale bar: 2 mm. (H) Left, X-gal staining of tibia proximal epiphysis sections from P21 –29E-lacZ and nontransgenic littermates. Right, Fgfr3 RISH of a P21 WT mouse section matching those used for X-gal staining. The image was processed as in D. Black-boxed areas are shown at higher magnification below. SOC, secondary ossification center. Scale bars: 150 μm (top); 50 μm (bottom). (I) Top, X-gal staining of vertebral column sections from the same mice as in H. Bottom, Fgfr3 RISH of a P21 WT mouse section matching those used for X-gal staining. The image was processed as in D. Scale bars: 100 μm.