Viral delivery of tissue nonspecific alkaline phosphatase diminishes craniosynostosis in one of two FGFR2C342Y/+ mouse models of Crouzon syndrome

Abstract

Craniosynostosis is the premature fusion of cranial bones. The goal of this study was to determine if delivery of recombinant tissue nonspecific alkaline phosphatase (TNAP) could prevent or diminish the severity of craniosynostosis in a C57BL/6 FGFR2C342Y/+ model of neonatal onset craniosynostosis or a BALB/c FGFR2C342Y/+ model of postnatal onset craniosynostosis. Mice were injected with a lentivirus encoding a mineral targeted form of TNAP immediately after birth. Cranial bone fusion as well as cranial bone volume, mineral content and density were assessed by micro CT. Craniofacial shape was measured with calipers. Alkaline phosphatase, alanine amino transferase (ALT) and aspartate amino transferase (AST) activity levels were measured in serum. Neonatal delivery of TNAP diminished craniosynostosis severity from 94% suture obliteration in vehicle treated mice to 67% suture obliteration in treated mice, p<0.02) and the incidence of malocclusion from 82.4% to 34.7% (p<0.03), with no effect on cranial bone in C57BL/6 FGFR2C342Y/+ mice. In contrast, treatment with TNAP increased cranial bone volume (p< 0.01), density (p< 0.01) and mineral content (p< 0.01) as compared to vehicle treated controls, but had no effect on craniosynostosis or malocclusion in BALB/c FGFR2C342Y/+ mice. These results indicate that postnatal recombinant TNAP enzyme therapy diminishes craniosynostosis severity in the C57BL/6 FGFR2C342Y/+ neonatal onset mouse model of Crouzon syndrome, and that effects of exogenous TNAP are genetic background dependent.

Fig 1. Neonatal coronal suture fusion in C57BL/6 but not BALB/c FGFR2^C342Y/+ mice.

Representative images of dissected C57BL/6 FGFR2^+/+ (A), C57BL/6 FGFR2^C342Y/+ (B), BALB/c FGFR2^+/+ (C) and BALB/c FGFR2^C342Y/+ (D) post-natal day 3 mouse skulls are shown. Parietal with frontal bone overlap is evident in all shown mice. Coronal suture fusion is only present in C57BL/6 FGFR2^C342Y/+ mice. Arrows point to loss of the coronal suture in C57BL/6 FGFR2^C342Y/+ (Cz) mice.

Fig 2. Body weight and length of BALB/c and C57BL/6 mice.

Body weight and length of vehicle vs. TNAP treated mice are shown. FGFR2^C342Y/+ mice are lighter and smaller than FGFR2^+/+ mice, regardless of strain. Treatment does not alter body weight or body length. *p<0.01 between genotypes.

Fig 3. Isosurface micro CT images of control and TNAP treated FGFR2^C342Y/+ mice.

Micro CT isosurface axial images of representative day 28 BALB/c and day 21 C57BL/6 mouse skulls are shown. Denser bone tissues are lighter in color. Control indicates no lentiviral delivery of TNAP. Tx indicates lentiviral delivery of TNAP. Comparison of BALB/c FGFR2^+/+ (WT) mice (A), BALB/c FGFR2^C342Y/+ (CZ) vehicle treated mice (C), and BALB/c FGFR2^C342Y/+ (CZ) TNAP treated mice (E) indicates morphologic differences between genotypes but not treatment groups. Comparison of C57BL/6 FGFR2^+/+ (WT) mice (B), C57BL/6 /c FGFR2^C342Y/+ (CZ) control mice (D), and C57BL/6 FGFR2^C342Y/+ (CZ) TNAP treated mice (F) indicates morphologic differences between genotypes but not treatment groups. (A-F) Isosurface images taken at a bone threshold. Skull images of control (D) and TNAP treated (F) C57BL/6 FGFR2^C342Y/+ mice show both cranial vault and underlying cranial base due translucent poorly mineralized cranial bones in these mice. (G-I) Isosurface images taken at a threshold that includes both bone and soft tissue are provided for C57BL/6 FGFR2^+/+ (WT) mice (G), C57BL/6 FGFR2^C342Y/+ control mice (H), and C57BL/6 FGFR2^C342Y/+ TNAP treated mice (I) are provided for improved visualization of these skulls.

Fig 4. Coronal suture fusion in vehicle and TNAP treated FGFR2^C342Y/+ mice.

Percentage of mice with fusion of the coronal suture are shown. Fusion was scored in the following categories: 0) normal open suture, 1) diminished suture width with no fusion, 2) diminished suture width with point fusions across the suture, and 3) obliteration of the suture. Results show diminished suture obliteration in C57BL/6 FGFR2^C342Y/+ (Cz) mice with no changes noted in BALB/c FGFR2^C342Y/+ (Cz) mice. *p<0.03 between treatment groups.

Fig 5. Incidence of malocclusion in vehicle and TNAP treated FGFR2^C342Y/+ mice.

Percentage of FGFR2^C342Y/+ (Cz) mice with a class III malocclusion are shown. Treatment with TNAP lentivirus significantly diminished malocclusion in C57BL/6 FGFR2^C342Y/+ (Cz) but not BALB/c FGFR2^C342Y/+ (Cz) mice. *p<0.03.

Fig 6. Liver toxicity enzyme tests in vehicle and TNAP treated FGFR2^C342Y/+ mice.

Alanine amino transferase (ALT) and aspartate amino transferase (AST) liver enzymes were measured in serum of vehicle and treated C57BL/6 mice. When compared by groups, no differences were seen between FGFR2^+/+ and FGFR2^C342Y/+ vehicle treated mice. Treatment decreased serum ALT (A) and increased serum AST (B) in FGFR2^C342Y/+ mice. Linear regression analyses showed a significant reverse correlation (r = -.73; 95% CI of -.898 to -.361, p < .005) between AP and ALT enzyme levels in FGFR2^C342Y/+ mice (C). No correlation was found between AP and AST enzyme levels in FGFR2^C342Y/+ mice (D). No correlations were found between ALT (E) or AST (F) enzyme levels and body weight.