Preclinical evaluation of the efficacy and safety of adeno-associated virus 8-tissue-nonspecific alkaline phosphatase-D10 in Alpl-/- and AlplPrx1/Prx1 mouse models for the treatment of early and late-onset hypophosphatasia

Abstract

We previously documented successful resolution of skeletal and dental disease in the infantile and late-onset murine models of hypophosphatasia (HPP) with a single injection of an adeno-associated serotype 8 vector encoding mineral-targeted TNAP (AAV8-TNAP-D10). Here, we conducted dosing studies in both HPP mouse models. A single escalating dose from 4 × 108 up to 4 × 1010 (vg/b) was intramuscularly injected into 4-day-old Alpl-/- mice (an infantile HPP model) and a single dose from 4 × 106 up to 4 × 109 (vg/b) was administered to 8-wk-old AlplPrx1/Prx1 mice (a late-onset HPP model). Wild-type littermates were used as controls. Serum alkaline phosphatase activity was increased, and PPi levels were decreased in a dose-dependent manner in both the Alpl-/- and AlplPrx1/Prx1 models. Radiographic and μCT analysis of long bones of female and male Alpl-/- mice showed full correction of skeletal phenotype at 4 × 1010 vg/b. We observed full correction of the bone phenotype at 4 × 108 and 4 × 109 in female AlplPrx1/Prx1 mice, but bones remained hypomineralized with the 4 × 106 and 4 × 107 (vg/b) doses after 70 d of treatment. We observed skeletal improvements using the 4 × 109 (vg/b) dose, but the phenotype was not fully corrected in male AlplPrx1/Prx1. Immunohistochemistry using anti-TNAP and anti-D10 antibodies showed high immunolocalization in the femurs of female AlplPrx1/Prx1 mice, while D10 immunolocalization was high in the liver of male AlplPrx1/Prx1 mice at a dose of 4 × 109 (vg/b). This sex-dependent difference was not seen in the infantile HPP model. A serum proteome analysis showed enhanced inflammatory pathways in treated AlplPrx1/Prx1 males compared to treated female mice. We also found a few areas of ectopic calcification in soft organs at the highest tested dose of 4 × 1010 (vg/b) in Alpl-/- or 4 × 109 (vg/b) in the AlplPrx1/Prx1 model. This pre-clinical study will inform the design of clinical trials to develop gene therapy in early-onset and late-onset HPP patients.

Keywords: AAV8-TNAP-D10skeletal mineralization; adult-HPP; gene therapy; hypophosphatasia; infantile-HPP; inflammation.

Graphical Abstract

Figure 1

Survival, biochemical profile, and vector distribution from female and male Alpl^-/- mice under AAV8-TNAP-D₁₀ (4 × 10⁷ to 4 × 10¹⁰ vg/b) treatment at 180 dpn. (A) Percent of survival rate. (B) Serum alkaline phosphatase (ALP) activity. (C) Plasma PP_i levels. (D) Vector distribution in bone, muscle, liver, kidney, spleen, heart, aorta, lung and brain of female and male WT and Alpl^-/- treated mice at 4 × 10¹⁰ vg/b. n = 3 for each group. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. ^*p < .05. ^**p < .01. ^***p < .001. ^****p < .0001.

Figure 2

μCT analysis of femur in Alpl^-/- and WT-treated mice. (A) 2D and 3D μCT images of the femur from females and males treated with AAV8-TNAP-D₁₀ after 70 dpn. (B) Quantification of trabecular and cortical bone parameters. Statistical analysis was performed by two-way ANOVA followed by Tukey’s multiple comparison test. Differences were statistically significant at ^*p < .05. Abbreviations: BV: Bone volume. TV, total volume; BV/TV, bone volume to total volume; Tb, trabecular; Tb.Th, trabecular thickness; Ct.Ar/Tt.Ar, cortical bone area versus the total bone area; Tt.Ar, total bone area.

Figure 3

μCT analysis of dentoalveolar complex in infantile-HPP Alpl^-/- and WT littermates treated mice. (A) 2D and 3D μCT images of first molar (M1) and incisor (INC) from AAV8-TNAP-D₁₀ at 4 × 10⁸ to 4 × 10¹⁰ vg/b treated mice after 70 dpn. (B) Quantification of molar and incisor enamel, dentin, alveolar bone and pulp parameters. Statistical analysis was done by two-way ANOVA followed by Tukey’s multiple comparison test. Differences were statistically significant at ^*p < .05. Abbreviations: EN, enamel; DE, dentin; AB, alveolar bone.

Figure 4

Biochemical analysis and vector distribution from female and male Alpl^Prx1/Prx1 mice under AAV8-TNAP-D₁₀ (4 × 10⁶ to 4 × 10⁹ vg/b) treatment at 70 post-injection. (A) Serum alkaline phosphatase (ALP) activity in females. (B) ALP activity in males. (C) Plasma PP_i levels. (D) Vector distribution in bone, muscle, liver, kidney, spleen, heart, aorta, lung, and brain of female and male WT and Alpl^Prx1/Prx1 treated mice at 4 × 10⁹ vg/b. n = 3 for each group. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. ^*p < .05. ^**p < .01. ^***p < .001. ^****p < .0001.

Figure 5

Radiographic findings of long bones in adult-HPP Alpl^Prx1/Prx1 treated mice. (A) Representative X-ray images from female (top) and male (bottom) Alpl^Prx1/Prx1 mice treated with AAV8-TNAP-D₁₀ at 4 × 10⁶ up to 4 × 10⁹ vg/b at 70 post-treatment. (B) Femur and tibia length measurement.

Figure 6

μCT analysis of femur in adult-HPP Alpl^Prx1/Prx1 treated mice after 70 d post-treatment. (A) 2D and 3D μCT images of the femur from females and males treated with AAV8-TNAP-D₁₀. (B) Trabecular and cortical bone parameters quantification. Statistical analysis was performed by two-way ANOVA followed by Tukey’s multiple comparison test. Differences were statistically significant at ^*p < .05. Abbreviations: BV, bone volume; TV, total volume; BV/TV, bone volume to total volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; Ct.Th, cortical thickness; Ct.BMD, cortical bone mineral density; Ct.Ar/Tt.Ar, cortical bone area versus the total bone area; Tt.Ar, total bone area.

Figure 7

Serum proteomic analysis from Alpl^Prx1/Prx1 mice treated with AAV8-TNAP-D₁₀ at 4 × 10⁹ vg/b at 70 post-treatment. (A) Ingenuity pathway analysis (QIAGEN IPA) identification of 66 pathways according to z-score in the comparison of male vs female treated mice. (B) Heatmap of genes identified from IPA analysis, upregulated in males (in red), and downregulated in females (in blue), and related to the immune system. (C) Heatmap of genes identified from IPA analysis, upregulated in males (in red), and downregulated in females (in blue), and related to the inflammatory pathways. (D) Heatmap of genes identified from IPA analysis, upregulated in males (in red), and downregulated in females (in blue), and related to the MAPK signaling. (E) Heatmap of genes identified from IPA analysis, upregulated in males (in red), and downregulated in females (in blue), and related to bone metabolism. (F) Volcano plot of selected genes upregulated (in red) or downregulated (in blue) in treated male Alpl^Prx1/Prx1 mice.

Figure 8

Gene expression in liver from Alpl^Prx1/Prx1 mice treated with AAV8-TNAP-D₁₀ at 4 × 10⁹ vg/b at 70 post-treatment. (A) IL-1b, (B) IL-6, (C) Tnfa, (D) Notch1, (E) Egfr; (F) Rap1a, (G) Rap1b, (H) Serpina1e, (I) Anxa2, (J) Pdia3, (K) LgalS3, and (L) Ywhab. The data presented are results of the mean ± SD of 3 independent experiments (n = 3/group/experiment). Statistical analysis was performed by the unpaired Student’s t-test (^*p < .05. ^**p < .01. ^***p < .001). Abbreviation: ns, not significant.