Soluble fibroblast growth factor receptor 3 (FGR3) polypeptide for use in the prevention or treatment of skeletal growth retardation disorders
11702642 · 2023-07-18
Assignee
- INSERM (Institut National de la Santé et de la Recherche Médicale) (Paris, FR)
- Université Paul Sabatier Toulouse III (Toulouse, FR)
Inventors
Cpc classification
C12Y207/10001
CHEMISTRY; METALLURGY
A61P19/08
HUMAN NECESSITIES
C12N9/12
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to the prevention or treatment of skeletal growth retardation disorders, in particular skeletal diseases developed by patients that display abnormal increased activation of the fibroblast growth factor receptor 3 (FGFR3), in particular by expression of a constitutively activated mutant of FGFR3. More particularly, the present invention relates to a soluble FGFR3 for use in the prevention or treatment of achondroplasia.
Claims
1. A method for treating an FGFR3-related skeletal growth retardation disorder in a subject in need thereof, the method comprising administering to the subject a soluble Fibroblast Growth Factor Receptor 3 (sFGFR3) polypeptide, wherein the sFGFR3 polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
2. The method of claim 1, wherein the FGFR3-related skeletal growth retardation disorder is thanatophoric dysplasia type I (TDI), thanatophoric dysplasia type II (TDII), severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN), hypochondroplasia, achondroplasia, Muenke syndrome, or Crouzon syndrome with acanthosis nigricans.
3. The method of claim 2, wherein the FGFR3-related skeletal growth retardation disorder is achondroplasia.
4. The method of claim 2, wherein the FGFR3-related skeletal growth retardation disorder is hypochondroplasia.
5. The method of claim 1, wherein the subject is a human.
6. The method of claim 1, wherein the FGFR3-related skeletal growth retardation disorder is caused by expression in the subject of an FGFR3 variant that exhibits ligand-dependent overactivation.
7. The method of claim 6, wherein the FGFR3 variant comprises an amino acid substitution of a glycine residue at position 380 of wild-type FGFR3 with an arginine residue (G380R).
8. The method of claim 1, wherein the subject is administered 0.0002 mg/kg/day to 20 mg/kg/day of the sFGFR3 polypeptide.
9. The method of claim 1, wherein the subject is administered 0.001 mg/kg/day to 7 mg/kg/day of the sFGFR3 polypeptide.
10. The method of claim 1, wherein the sFGFR3 polypeptide is administered subcutaneously.
11. The method of claim 1, wherein the sFGFR3 polypeptide is administered intravenously.
Description
FIGURES
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EXAMPLE
(5) Material & Methods
(6) sFGFR3 subcloning and recombinant protein production: To facilitate sub-cloning, full-length cDNA sequence encoding the FGFR3 ATM (2.1 kb) (35), a generous gift from Dr. Kurokawa-Seo, Kyoto Sangyo University, Japan, was optimized to decrease GC content while encoding for the original protein sequence (GeneOptimizer® process, GeneArt). The synthesized fragment was subcloned into pFLAG-CMV3_G727 (Sigma Aldrich) using HindIII and KpnI cloning sites. Plasmid DNA was purified from transformed bacteria and concentration determined by UV spectroscopy. The final construct was verified by sequencing. The sequence homology within the used restriction sites was 100%.
(7) Recombinant FLAG-sFGFR3 protein was produced by transient transfection using GeneJuice transfection reagent (Merck Millipore) in HEK 293 cells allowing all necessary post-translational modifications. Each transfection was performed in a cell factory (High flask T600, Merck Millipore) with 80% confluent HEK 293 in 100 ml DMEM without phenol red (Gibco, Life Technologies) supplemented with glutamine 2 mM (Gibco, Life Technologies) and 1% antibiotics (Gibco, Life Technologies). 600 μl GeneJuice and 240 μg pFLAG-sFGFR3 were resuspended in 30 ml OptiMEM (Gibco, Life Technologies), incubated 30 min at room temperature, and then incubated for 4 h onto the cells at 37° C. in 5% CO.sub.2. Medium was then replaced by 120 ml DMEM without phenol red, supplemented with glutamine 2 mM and 1% antibiotics. After 72 h, production medium was filtrated using 0.22 μm filters and concentrated on Amicon Ultra-15 60 kDa (Merck Millipore). Recombinant protein was then purified on an affinity column (ANTI-FLAG M2 Affinity Gel. Sigma Aldrich) according to the manufacturer's instructions. FLAG-sFGFR3 amounts were measured by specific ELISA (R&D Systems) according to the manufacturer's instructions. FLAG-sFGFR3 was then stored at a concentration of 0.5 μg/ml in 50% glycerol solution.
(8) FLAG-sFGFR3 incubation with FGF: Fixed amounts of human or murine FGFb (100 μg) (R&D Systems) were incubated for 2 h at 37° C. with increasing doses of FLAG-sFGFR3 (0 to 250 ng/ml) in PBS 1% BSA. Specific commercial ELISA kits (R&D Systems) were used to quantify remammg unbound FGFs. All experiments were performed m triplicates and repeated five times.
(9) Immunoblotting analysis: Immunoblotting was performed following incubation of several doses of FLAG-sFGFR3 on ATDC5 cells. For this, ATDC5 cells were plated at a density of 2×10.sup.6 in 6 well plates and, following adhesion, cultured for 48 h in 0.5% BSA in DMEM-F12 (Gibco, Life Technologies) containing 1% antibiotics. Cells were then cultured for 10 min with 100 μg/ml murine FGF pre-incubated for 2 hat 37° C. with increasing doses of FLAG-sFGFR3 (0, 12.5, 125, 1250, 12500 μg/ml). At the end of the incubation period, remaining unbound FGFs were measured by specific ELISA (R&D Systems). Cells were then solubilized in lysis buffer (20 mM Tris. pH 7.4, 150 mM NaCl, 10 mM EDTA, 150 mM NaF, 2 mM sodium orthovanadate, 10 mM pyrophosphate, proteases inhibitors, and 1% Triton X-100) for 45 min at 4° C. Lysates were cleared (14 000 rpm, 10 min) and proteins were separated by SDS-PAGE and immunoblotted as previously described (36). The proteins were probed with anti-phospho p42/44 MAPK (4370S, Cell Signaling), anti-total p42/44 MAPK (4696S, Cell Signaling) and anti-hsp60 (scl 722, Santa Cruz Biotechnology) antibodies (1 μg/ml). All experiments were performed six times.
(10) Immunohistochemistry of FLAG-sFGFR3: Immunohistochemistry of FLAG-sFGFR3 was performed on tibiae of 3 day old Fgfdachl+ mice and their wildtype littermates. For this, following decapitation of newborn mice, tibiae were carefully harvested and incubated in 24 well plates in presence of 5 ng FLAG-sFGFR3 for 24 hat 37° C. in 5% CO2. Tibiae were then rinsed in PBS and fixed in 10% formalin for 24 h. Following decalcification in EDTA for 2 days, bones were paraffin embedded and 5 μm sections were incubated with 5 μg/ml anti-FLAG M2-FITC monoclonal antibody (Sigma Aldrich). Sections were counterstained with Hoechst solution and visualized under fluorescent microscopy. An anti-IgG antibody was used as negative control.
(11) Animals and treatments: The Principles of Laboratory Animal Care (NTH publication no. 85-23, revised 1985; grantsl.nih.gov/grants/olaw/references/phspol.htm) and the European commission guidelines for the protection of animals used for scientific purposes (europa.eu/environment/chemicals/lab_animals/legislation_en.htm) were followed at all times. All procedures were approved by the Institutional Ethic Committee for the use of Laboratory Animals (CIEPAL Azur) (approval #NCE-2012-52).
(12) Experiments were performed on transgenic Fgfr3.sup.ach/+ animals in which expression of the mutant FGFR3 is driven by the Col2a1 promoter/enhancer (22). Mice were exposed to a 12 h light/dark cycle and had free access to standard laboratory food and water. All measurements and analyses were performed blinded and genotypes were analyzed after all analyses were done by PCR of genomic DNA which amplify 360 bp of the FGFR3 transgene (22). Two doses of FLAG-sFGFR3 (0.5 ng and 5 ng in 10 μl PBS with 50% glycerol) were tested. At day 3, all newborn mice from a single litter received the same dose. Control litters received 10 μl of PBS containing 50% glycerol. Subcutaneous injections were thereafter done twice a week for three weeks, alternatively on the left and right sides of the back. Mice were observed daily with particular attention to locomotion and urination alterations. At day 22, all animals but two litters per group were sacrificed by CO.sub.2 asphyxation; genus and genotypes were determined. Body weights were measured. Blood was harvested by cardiac puncture and mixed with 50 μl 0.5M EDTA; half of the samples were centrifuged for a biochemical assessment using a Beckman AU 2700 Analyzer (electrolytes (Na.sup.+, K.sup.+, Cl.sup.−), lactate dehydrogenase (LDH), cholesterol, creatinin, creatinin kinase (CK), aspartate aminotransferase (AST), alanine aminostransferase (ALT), amylase, total bilirubin (BLT)); the other half was analyzed without centrifugation for blood numeration (Hemavet 950FS, Mascot Hematology). Cadavers were carefully skinned and eviscerated and skeletal measurements (body and tail lengths) were obtained using an electronic digital caliper (Fisher Scientific). Total body length was measured from the nose to the end of the last caudal vertebra; tail was measured starting at the first caudal vertebra. Organs (heart, lungs, liver, kidneys, spleen) were harvested, weight and stored in 10% formalin for further histological analysis using standard paraffin-embedded techniques. X-rays of all skeletons were taken using a Faxitron X-ray machine (Edimex). Using an established method (28), kyphotic index were measured for each animals on the X-rays. Cleared skeletons were then stained simultaneously with alcian blue and alizarin red using standard procedures and stored in glycerol prior to analysis. Stained long bones (tibiae, femurs, humerus) were dissected and measured using an electronic digital caliper; vertebrae and skulls were also dissected and analyzed.
(13) Breeding was set up to theoretically generate litters with half wildtype and half heterozygous Fgfr3.sup.ach/+ mice. To avoid bias due to variations of phenotype penetrance, experiments were performed on at least 2 litters (one treated and one control) arising from the same breeders. A total of 15, 9 and 11 litters representing a total of 312 pups were treated with PBS, 0.5 ng or 5 ng FLAG-sFGFR3, respectively. The n per group is presented in Table 1.
(14) Effect of FLAG-sFGFR3 on the fertility of treated animals: Animals from the litters that were not used for skeletal measurements were kept until breeding age was reached. At age 8 week, they were then mated with 8 week old FVB/N mice from Charles River. Newborn mice were counted at birth for each treated and control male and female and compared with fertility statistics of the previous generation. At age 22, offspring were euthanized and growth was evaluated as described above.
(15) Statistical analysis: All experiments and data measurements were performed by blind experimenters at all times. Statistical analyses were performed with GraphPad Prism 6.0 software. To determine the statistical tests to be used, necessary assumptions were verified. To verify normality and equal variance, an Agostino and Pearson omnibus normality test and a Brown-Forsythe test were performed, respectively. Because all skeletal measurements data sets fulfilled normality and equal variance requirements, two-tailed Student's t test for comparisons of two independent groups were used in the different statistical analyses. Comparison of mortality data between treated and control groups was done using a Kruskal-Wallis test. Comparison of FLAG-sFGFR3 binding to human and murine FGFs was done by linear regression. Immunoblotting data distribution followed normality and were thus analyzed using a one-way ANOVA using a Holm-Sidak's multiple comparisons test. For organ weight correlation analyses, Pearson or Spearman tests were used when data sets followed or not normal distribution, respectively. All statistical tests were considered significant at a p<0.05 level of error. In all figures, values of p are shown as follows: *p<0.05; **p<0.01; ***p<0.001. Data are presented as means±SD.
(16) Results
(17) FLAG-sFGFR3 effectively binds FGFs and decreases MAPK signaling in ATDC5 cells: In order to detect recombinant soluble FGFR3 in vivo, the inventors used a soluble form of FGFR3 labeled with a FLAG tag. This tag was used because of the availability of reagents for its purification and its detection (23, 24). It has also already been used in vivo without inducing premature elimination of the tagged protein by the immune system (25, 26). In the present experiments, recombinant FLAG-sFGFR3 was produced by transient transfection, purified using affinity column and stored at a concentration of 0.5 μg/ml in 50% glycerol.
(18) To verify that FLAG-sFGFR3 effectively bound free FGFs, fixed amounts of human FGFb were incubated with increasing quantities of FLAG-sFGFR3. As seen in
(19) The inventors then verified that the complexation of FGF with FLAG-sFGFR3 resulted in a decreased intracellular FGF signaling on Erk phosphorylation. ATDC5 cells were used as a murine chondrocytic cell line to study chondrocyte biology (27). As seen
(20) Soluble FGFR3 effectively restores bone growth in Fgfr3.sup.ach/+ mice: Prior to test FLAG-sFGFR3 treatment effect in vivo, the inventors verified that it could penetrate the dense cartilaginous matrix of the growth plate and reach target chondrocytes. Long bones isolated from three day old Fgfr3.sup.ach/+ mice and their wildtype (wt) littermates were incubated for 24 h in presence of 5 ng FLAG-sFGFR3. As seen in
(21) To evaluate the biological effects of FLAG-sFGFR3 treatment on skeletal bone growth in Fgfr3.sup.ach/+ mice, all newborn mice from one litter received the same treatment without knowing their phenotype. They received a subcutaneous injection of 0.5 or 5 ng FLAG-sFGFR3, or PBS in control groups, twice a week during 3 weeks. The first observation was the significant reduction in mortality in treated compared to untreated litters. At the end of the treatment period, control groups contained about a third of transgenic animals alive while in both treated groups, there were approximately 50% wt mice and 50% Fgfr3.sup.ach/+ (Table 1). Moreover, in the control litters, 31% of animals died prior to the end of the experiments compared to 11.8% and 6.7% in the 0.5 ng and 5 ng FLAG-sFGFR3 treated litters, respectively. This reduction in group size was due to premature death or euthanasia of paraplegic animals. When it was possible, autopsy was performed and confirmed death due to respiratory failure as seen by the presence of blood within the lungs. Two animals died consequently to bowel obstruction. All of these animals were Fgfr3.sup.ach/+, a confirmed by genotyping. No wild-type animal died prematurely. It is noteworthy to emphasize that in the control group, the majority of affected Fgfr3.sup.ach/+ mice died from respiratory failure, while in the 5 ng FLAG-sFGFR3 treatment group, they mostly suffered from paraplegia and only few had respiratory distress (Table 1). Altogether these data indicate that with treatment fewer animals died, and those who died had a less severe phenotype.
(22) TABLE-US-00004 TABLE 1 Number of pups in the different treatment groups at day 3 and day 22. Litters were considered as single entities and all newborn mice from the same cage received the same treatment. Dead and alive animals were counted daily. Autopsy revealed death by respiratory failure and bowel occlusion for 2 animals. Animals with paraplegia were euthanized upon discovery and recorded in the dead animal group. All dead animals were Fgfr3.sup.ach/+. Statistical comparison versus control group was done using the Kruskal-Wallis test. Number of litters Number of pups % dead animals before day 22 per group Day 3 Day 22 (cause of death) PBS 15 132 91 31% (23 by respiratory failure, (wt: 67; ach: 24) 2 by bowel occlusion, 16 by paraplegia) 0.5 ng SFGFR3 9 76 67 11.8% ** (4 by respiratory (wt: 31; ach: 36) failure, 5 by paraplegia) 5 ng sFGFR3 11 104 97 6.7% ** (1 by respiratory failure, (wt: 47; ach: 50) 6 by paraplegia) ** p < 0.01. wt: wildtype mice; ach: Fgfr3.sup.ach/+ mice.
(23) At day 22, time of weaning, animals were sacrificed and their growth was evaluated. The inventors first confirmed that there was no statistical difference between males and females (Table S1) and regrouped them for all subsequent analyses. As illustrated in
(24) Altogether, these results show that following chronic subcutaneous administration of FLAG-sFGFR3 to neonate Fgfr3.sup.ach/+ mice, normal bone growth was restored and that it was also effective on skeletal growth of animals that do not trigger an FGFR3 activating mutation.
(25) TABLE-US-00005 TABLE S1 Statistical comparison of body measurements between male and female in the different treatment groups. Following 3 weeks of treatments (PBS, 0.5 ng or 5 ng FLAG-sFGFR3), animals were sacrificed at age 22 days. Body weight, body length and tail length were measured. Genus and genotypes were determined. Following verification of normality variation in each data set, measurements were compared between males and females within the same treatment and genotype group. Data followed normal distribution; a Student's t test was used to compare data to measurements obtained on untreated mice. No statistical difference was found in any group. Body weight Body length Tail length male vs n (g) (mm) (mm) female PBS wt male 41 11.09 ± 1.26 132.43 ± 5.45 70.49 ± 3.65 ns female 26 10.67 ± 1.99 134.04 ± 6.76 71.76 ± 4.60 ach male 13 9.18 ± 1.82 120.56 ± 5.71 64.36 ± 2.52 ns female 11 8.14 ± 1.55 113.95 ± 8.22 60.03 ± 3.79 0.5 ng wt male 12 12.31 ± 1.27 134.55 ± 7.98 73.94 ± 6.35 ns sFGFR3 female 19 12.03 ± 1.18 134.59 ± 6.46 72.25 ± 4.51 ach male 19 10.50 ± 1.19 126.78 ± 9.38 69.48 ± 6.64 ns female 17 9.34 ± 1.47 121.49 ± 10.01 65.33 ± 7.96 5 ng wt male 23 13.29 ± 1.48 141.47 ± 6.77 74.76 ± 5.42 ns sFGFR3 female 24 12.13 ± 1.85 138.73 ± 8.21 74.01 ± 6.01 ach male 13 11.41 ± 3.14 130.43 ± 13.11 69.15 ± 9.07 ns female 37 10.72 ± 2.19 127.59 ± 11.58 67.43 ± 7.95 ns = non significant. wt: wildtype mice; ach: Fgfr3.sup.ach/+ mice.
(26) FLAG-sFGFR3 treatment decreases spinal and skull deformities associated with achondroplasia in Fgfr3.sup.ach/+ mice: in Fgfr3.sup.ach/+ mice, spinal abnormalities are recognized in particular by the presence of a kyphosis that can be characterized by the calculation of a kyphotic index (KI). In this scoring system, established by Laws et al. (28), mice with a KI<4.0 present a kyphosis (for more details, please see legend of
(27) Similar to achondroplasia patients that typically have enlarged heads, Fgfr3.sup.ach/+ mice suffer from skull deformities. While cranium width (W) is not statistically different between transgenic and wt mice (10.35±0.28 mm vs 10.17±0.32 mm, respectively), the length (L) is significantly shorter in Fgfr3.sup.ach/+ mice (18.11±0.75 mm vs 20.05±0.51 mm in wt mice, respectively). This leads to a L/W ratio equal to 1.75±0.77 in untreated Fgfr3.sup.ach/+ mice and equal to 1.94±0.05 in control wt mice (
(28) Altogether, these results show that FLAG-sFGFR3 treatment is effective at preventing the development of skeleton deformities associated with achondroplasia.
(29) No toxicological effects are detected in treated animals: Because the inventors are using a systemic approach to deliver recombinant soluble FGFR3, they paid a particular attention to possible unwanted side effects. They analyzed organs of all 255 animals, performed biochemical and numeration blood tests and verified fertility of some treated animals (two litters per group) including normality of their offspring.
(30) Potential treatment side effects were first evaluated on several organs (liver, lung, heart, spleen, kidneys) at the time of sacrifice. Organs were observed macroscopically, weighted and randomly analyzed microscopically by histology. None of the 255 animals that received chronic subcutaneous injections of FLAG-sFGFR3 or PBS presented macroscopic abnormalities. Histology was performed on randomly selected organs in all groups and data were analyzed blindly by an anatomopathologist. No signs of toxicity were observed on any histological slides. In all control groups, organ weights were correlated with the mouse body weight (Table 2). In the treated groups, organs increased with enhanced bone growth. As an example, the lungs of untreated Fgfr3.sup.ach/+ mice were 156.0±88.7 mg. They increased to 172.5±67.5 mg in the 5 ng treatment group, reaching the weight of lungs in untreated wt mice (170.5±36.3 mg). Similar results were found for all organs and this weight augmentation was statistically correlated with body weight increase in all groups (Table 2). To evaluate organ functions, they performed biochemical blood tests including electrolytes titration, liver, kidney and spleen enzymes assays. All tests proved to be statistically identical between Fgfr3.sup.ach/+ and wt animals in the treated and control groups (Table S2). Blood counts were also analyzed and similarly, no differences between blood formulations were noticed between treated and control groups (Table S3).
(31) TABLE-US-00006 TABLE 2 Coefficient correlation (r) between organ and body weight in the different treatment groups. Treatment Liver Heart Lung Spleen Kidney PBS 0.892 *** 0.748 *** 0.857 *** 0.655 *** 0.877 *** 0.5 ng 0.883 ** 0.777 ** 0.731 * 0.774 * 0.777 ** sFGFR3 5 ng 0.881 *** 0.794 *** 0.720 *** 0.584 ** 0.531 * sFGFR3 PBS 0.941 *** 0.943 *** 0.886 ** 0.726 ** 0.848 *** 0.5 ng 0.921 *** 0.758 * 0.709 ** 0.650 * 0.828 *** sFGFR3 5 ng 0.957 *** 0.983 *** 0.885 *** 0.883 *** 0.850 *** sFGFR3 Pearson or Spearman tests were used for statistical analysis of organ/body weights correlations in each treatment group; * p < 0.05; ** p < 0.01; *** p < 0.001. wt: wildtype mice; ach: Fgfr3.sup.ach/+ mice.
(32) TABLE-US-00007 TABLE S2 Blood biochemical parameters were not modified by FLAG-sFGFR3 treatment. To evaluate treatment toxicity, at time of sacrifice, plasma from the PBS and 5 ng FLAG-SFGFR3 groups were analyzed using a Beckman AU 2700 Analyzer. Overall health was evaluated by measurements of electrolytes (Na.sup.+, K.sup.+, Cl.sup.−), lactate dehydrogenase (LDH) and cholesterol. Kidney function was assessed by creatinin and creatinin kinase (CK) assays. Liver and pancreas functions were assessed by aspartate aminotransferase (AST), alanine aminostransferase (ALT), total bilirubin (BLT) and amylase, respectively. Statistical comparisons were performed using a one way ANOVA. No statistical difference was found in any group. Na K Cl LDH Cholesterol Treatment (mmol/L) (mmol/L) (mmol/L) (UI/L) (mmol/L) wt PBS 810.8 ± 80.8 9.26 ± 0.51 75.20 ± 3.81 1672 ± 124 0.99 ± 0.15 5 ng 818.3 ± 69.4 8.75 ± 0.59 72.83 ± 1.14 2059 ± 478 0.90 ± 0.16 sFGFR3 ach PBS 709.7 ± 90.5 8.97 ± 0.98 81.79 ± 3.79 1675 ± 228 1.06 ± 0.11 5 ng 706.2 ± 57.0 9.20 ± 0.77 76.33 ± 0.88 1738 ± 402 1.02 ± 0.08 sFGFR3 Creatinine Total Creatinine kinase AST ALT Amylase bilirubine Treatment (μmol/L) (UI/L) (UI/L) (UI/L) (UI/L) (μmol/L) wt PBS 9.00 ± 1.26 1389 ± 679 155.1 ± 27 65.5 ± 16.5 138.1 ± 4.7 22.8 ± 1.7 5 ng 10.20 ± 0.95 655 ± 180 137.0 ± 24.6 42.3 ± 4.2 112.0 ± 8.0 20.8 ± 0.4 sFGFR3 ach PBS 9.18 ± 1.60 551 ± 127 140.1 ± 22.9 48.2 ± 14.6 129.8 ± 21.4 21.8 ± 1.1 5 ng 12.33 ± 1.43 943 ± 261 196.4 ± 51.2 77.7 ± 14.5 192.0 ± 33.3 20.3 ± 0.6 SFGFR3 wt: wildtype mice; ach: Fgfr3.sup.ach/+ mice.
(33) TABLE-US-00008 TABLE S3 Blood counts were not modified by FLAG-sFGFR3 treatment. The effects of FLAG-SFGFR3 treatment on blood counts were evaluated on plasma samples at the time of sacrifice. Analysis included hemoglobin (Hb), hematocrit (Ht), white blood cells (WBC), red blood cells (RBC) and platelets (PLT) counts. The percentages of the different leukocyte populations were evaluated (NE: neutrophil; LY: lymphocyte, MO: monocyte, EO: eosinophil; BA: basophil). Statistical comparisons were performed using a one way ANOVA. No statistical difference was found in any group. Treatment Hb (g/dL) Ht (%) WBC (K/μl) RBC (K/μl) PLT (K/μl) wt PBS 8.11 ± 0.11 16.82 ± 1.72 4.31 ± 0.39 5.63 ± 0.02 471.1 ± 26.9 5 ng 8.56 ± 0.06 23.21 ± 1.33 5.28 ± 0.54 5.81 ± 0.01 447.0 ± 32.2 sFGFR3 ach PBS 8.32 ± 0.26 22.5 ± 4.74 3.37 ± 0.49 5.47 ± 0.18 472.0 ± 112.8 5 ng 8.25 ± 0.15 17.44 ± 1.12 5.86 ± 5.81 5.67 ± 0.04 620.0 ± 28.8 sFGFR3 Treatment NE (%) LY (%) MO (%) EO (%) BA (%) wt PBS 30.76 ± 1.86 53.56 ± 2.49 7.52 ± 0.59 5.89 ± 0.44 2.27 ± 0.27 5 ng 25.54 ± 2.37 56.90 ± 3.15 9.78 ± 0.67 5.75 ± 0.54 1.83 ± 0.24 sFGFR3 ach PBS 24.25 ± 6.08 60.02 ± 8.86 6.56 ± 1.48 6.59 ± 1.72 2.56 ± 0.59 5 ng 29.25 ± 1.47 51.88 ± 2.39 10.16 ± 0.82 6.99 ± 1.20 1.85 ± 0.33 sFGFR3 wt: wildtype mice; ach: Fgfr3.sup.ach/+ mice.
(34) To evaluate unwanted side effects on fecundity, after the three week treatment, animals were weaned and were mated at age 8 weeks with wt FVB males or females from Charles River. As seen in Table 3, all treated animals were fertile and their offspring were of normal size with an approximate 50% wt/50% Fgfr3.sup.ach/+ descendants. While Fgfr3.sup.ach/+ females usually have a first litter slightly reduced in size compared to that of wt females, it is interesting to note that primiparous treated transgenic females had litters that were identical in size to wt primiparous females, confirming enlargement of their pelvis following treatment (Table 3).
(35) TABLE-US-00009 TABLE 3 FLAG-sFGFR3 treatment did not affect fertility of the treated mice. Pups from two litters per group were weaned after the three week treatment. They were mated at age 8 weeks with wt animals from Charles River. The number of pups of the first litters was counted for each primiparous female. At age 22 days, animals were euthanized. Their body growth was evaluated as previously. A Student's t test was used to compare data to measurements of untreated wt animals generated from control genitors. % of wt and Body weight Body length Litter size ach at day 22 (g) (mm) Control ach ♂ × wt ♀ 9.2 ± 0.9 74% wt, 26% wt, 10.88 ± wt, 133.24 ± genitors ach 1.62 6.11 wt ♂ × ach ♀ 7.7 ± 0.7 *** ach, 8.66 ± ach, 117.25 ± 1.68 *** 6.96 *** sFGFR3 treated ach ♂ × wt ♀ 10.2 ± 1.9 % wt, % ach treated treated wt ♂ × wt ♀ 9.7 ± 0.9 N/A wt ♂ × treated ach ♀ 10.6 ± 1.5 % wt, % ach wt ♂ × treated wt ♀ 9.8 ± 1.9 N/A *** p < 0.001 versus untreated wt. N/A: not applicable, wt: wildtype mice; ach: Fgfr3.sup.ach/+ mice.
(36) Altogether these experiments did not highlight any complications from the FLAG-sFGFR3 treatment itself neither on blood formulation, organ function and development nor fertility of the treated animals, suggesting that the use of a soluble form of FGFR3 may be a viable treatment approach for clinical applications.
DISCUSSION
(37) The present study validates the proof of concept that a therapeutic strategy based on the use of a soluble form of FGFR3 can prevent abnormal bone growth in mice carrying the achondroplasia mutation. Treatment was administered twice a week by subcutaneous injections to the animals throughout the growth period. Following this three week treatment period, ensuing endochondral bone growth led to normal, harmonious stature. Importantly, these effects were dose-dependent; the dose of 0.5 ng FLAG-sFGFR3 was sufficient to induce body weight and length that were identical to that of untreated wt mice and at the dose of 5 ng, treated dwarf mice were even heavier and had longer long bones than untreated wt animals. Foremost, the present results emphasize the notion that the achondroplasia mutation requires ligand binding to be activated. Indeed, it has been demonstrated that in the case of the G380R mutation, FGFR3 activation is ligand-dependent (12), but there is still no clear consensus in the literature (29-31), suggesting not one but multiple mechanisms leading to prolonged intracellular signaling.
(38) While it was essential that long bone growth be restored for the treatment to be effective, it was critical to also significantly impact the onset of complications, due to skeletal deformities. For the inventors, this is indispensable if one wants to develop a treatment for the clinic. Restoration of vertebra maturation and normal closure of cranial synchondroses in treated Fgfr3.sup.ach/+ mice had numerous effects on these animals. The first consequence was the reduced mortality among the transgenic population. As stated in the result section, autopsy revealed respiratory failures in the majority of the cases. Based on the anatomical characteristics of the skull and vertebrae of the dwarf mice, the inventors believe that these were consequent to brainstem compression, similar to what can be observed in achondroplasia patients.
(39) A second outcome of treatment effect was a shift in the penetrance of the phenotype. While measurements could appear as though treatment effects were not totally dose-dependent and more importantly not as effective as could be expected based on body weights, the inventors believe that at the highest dose, treatment saved the smallest Fgfr3.sup.ach/+ mice from brainstem compression and respiratory failure. Untreated, these animals would not have survived past week 1 or 2. In this treatment group, the inventors hypothesize that these animals are those that remain very small even though they have less severe complications.
(40) Despite the increasing number of reports studying the mechanisms underlying achondroplasia and related skeletal dysplasia, only three studies have been published showing therapeutic strategies effectively tested in mice with chondrodysplasia. Xie et al. recently published a report that intermittent PTH treatment partially rescues bone growth in mice with achondroplasia (32). In their study. PTH was administered subcutaneously at the dose of 100 μg/kg body weight per day for 4 weeks after birth. Although the mechanism by which PTH affects FGFR3 intracellular signaling is not clearly established, bone growth was partially rescued in treated transgenic mice; PTH treated transgenic mice were still smaller than their wt littermates. In this study, only very little information was mentioned related to achondroplasia complications, except for the partial rescue of cranial synchondrose. The lethal phenotype of TDI mice was also rescued with chronic PTH treatment of pregnant females. Another potential therapeutic antagonist of FGFR3 signaling is the C-natriuretic peptide (CNP) (33). In this paper, the authors treated transgenic mice with achondroplasia by continuous intravenous infusion of synthetic CNP for 3 weeks starting at age 4 week. It is believe that CNP increases the width of the growth plate accelerating growth plate activity. The major obstacle for use in human is the very short half-life of CNP, estimated to be 2.6 min in plasma (34).
(41) A study has been very recently published describing the use of a new FGFR3-binding peptide that rescues the lethal phenotype and partially restores the structural distortion of growth plates in TDII mice, observed on 12 pups (19). In this study, effects on MAPK signaling and bone growth correction were only partial and daily administration was required probably due to the short half-life of the peptide. Here, the effects of SFGFR3 were of higher magnitude, with complete restoration of normal stature. The inventors believe that the half-life of the soluble form of FGFR3 containing IgG like domains is significantly prolonged. Indeed, only 6 injections (between birth and weaning) were necessary to completely restore bone growth in the 86 treated Fgfr3ach/+ mice. They can imagine that only a few injections would be necessary to treat achondroplasia children as out-patients, starting during the first year until puberty. This would substantially impact occurrence of injection side effects, typically found with daily injection regimens. The use of a soluble recombinant protein also allows for rapid termination during treatment if safety issues are raised and at puberty when bone growth ceases. If necessary, it is also possible to alternate between treatment and resting periods. By preventing the complications, sFGFR3 treatment would avoid the necessity of surgical interventions and also reduce any stress due to hospitalization. Furthermore, our study did not reveal any toxicological effect on blood or fertility and offspring of treated animals. Current studies are ongoing to evaluate if the three week sFGFR3 treatment had an effect on the long-term health of the treated mice. As of today, treated mice are 6 month old and no apparent side effects are visible and blood tests are normal.
(42) In conclusion, the present study demonstrates the viability of targeting FGFR3 in the extracellular compartment as an effective treatment to restore growth plate maturation and induce normal bone growth in achondroplasia. The absence of unwanted side effects validates its use as a promising therapy for this and related chondrodysplasia caused by activating mutation in FGF receptors. Furthermore, in the present study, the inventors also report a positive effect of sFGFR3 treatment on the growth of wt animals. This is of importance suggesting its possible interest for the treatment of idiopathic growth retardations, or to prevent severe complications in other rare diseases such as hypophosphatasia.
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