Methods and compositions for treating hypophosphatasia

Abstract

Described herein are compositions and methods useful for treating cartilage disorders or treating hypophosphatase, comprising administering to a subject a viral vector comprising a mineral-targeted alkaline phosphatase under the control of a tissue-nonspecific promoter by intramuscular injection into a muscle, wherein administering the viral vector treats the cartilage disorder. The compositions disclosed herein are suitable for administration to a subject.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 193,366, filed May 26, 2021, the entirety of which is incorporated herein by reference.

[0002] Federally sponsored research statement This invention was made with Government support under Grant No. DE012889 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] Hypophosphatasia (HPP) is an inborn error of metabolism caused by loss-of-function mutations in the ALPL gene, which encodes tissue-nonspecific alkaline phosphatase (TNAP). TNAP is expressed in bones, teeth, liver, and kidneys. Deficiency of TNAP results in a decrease in the production of extracellular inorganic pyrophosphate (PP ), which is one of the main substrates for TNAP and a potent inhibitor of hydroxyapatite crystal formation and increase. i ) accumulation leads to impaired mineralization. Mouse studies have demonstrated that TNAP is expressed by mineralizing skeletal and dental cells, including osteoblasts, chondrocytes, ameloblasts, odontoblasts, and cementoblasts. Summary of the Invention

[0004] In certain aspects, disclosed herein is a method of treating a subject having a cartilage disorder, comprising administering to the subject a viral vector comprising a mineral-targeted alkaline phosphatase under the control of a tissue-non-specific promoter by intramuscular injection into a muscle, wherein administering the viral vector treats the cartilage disorder. In some embodiments, the tissue-non-specific promoter comprises a CAG promoter. In some embodiments, the viral vector comprises an adeno-associated vector. In some embodiments, the viral vector comprises an adeno-associated virus type 8 (AAV8) vector. In some embodiments, the mineral-targeted alkaline phosphatase comprises tissue-non-specific alkaline phosphatase (TNAP). In some embodiments, the mineral-targeted alkaline phosphatase comprises tissue-non-specific alkaline phosphatase (TNAP) and a bone-targeting sequence linked to the C-terminus of TNAP. In some embodiments, the bone-targeting sequence is decaaspartate (D 10 ) sequence. In some embodiments, the cartilage disease is hypophosphatasia (HPP). In some embodiments, the cartilage disease is juvenile hypophosphatasia or infantile hypophosphatasia (HPP). In some embodiments, the cartilage disease is late-onset hypophosphatasia (HPP). In some embodiments, the cartilage disease is caused by a deficiency in PHOSPHO1. In some embodiments, the subject is a human. In some embodiments, the subject is a mouse. In some embodiments, the mouse comprises a TNAP knockout mouse. In some embodiments, administration of the viral vector results in an increase in plasma alkaline phosphatase (ALP) activity for at least two months. In some embodiments, following administration of the viral vector, the viral vector is not detectable in the brain. In some embodiments, following administration of the viral vector, the viral vector is not detectable in the gonads. In some embodiments, following administration of the viral vector, the viral vector does not cause oncogenic effects in the subject. In some embodiments, the viral vector does not spread from muscle.

[0005] In certain aspects, compositions are disclosed herein that include a viral vector that includes a mineral-targeted alkaline phosphatase under the control of a tissue-nonspecific promoter. In certain aspects, compositions are disclosed herein for treating cartilage disease, the compositions include a viral vector that includes a mineral-targeted alkaline phosphatase under the control of a tissue-nonspecific promoter, the compositions treat the cartilage disease, and result in a higher plasma ALP activity than a subject not receiving the composition for at least 18 months. In certain aspects, compositions are disclosed herein for treating cartilage disease, the compositions include a viral vector that includes a mineral-targeted alkaline phosphatase under the control of a tissue-nonspecific promoter, the compositions treat the cartilage disease, and the viral vector does not spread from the target location. In some embodiments, the tissue-nonspecific promoter includes a CAG promoter. In some embodiments, the viral vector includes an adenovirus-associated virus. In some embodiments, the adenovirus-associated virus includes an adeno-associated virus type 8 (AAV8) vector. In some embodiments, the mineral-targeted alkaline phosphatase includes tissue-nonspecific alkaline phosphatase (TNAP). In some embodiments, the mineral-targeted alkaline phosphatase comprises a bone-targeting sequence linked to the C-terminus of TNAP. In some embodiments, the bone-targeting sequence is decaaspartate (D 10 ) sequence. In some embodiments, the cartilage disease is hypophosphatasia (HPP). In some embodiments, the hypophosphatasia is juvenile hypophosphatasia or infantile hypophosphatasia. In some embodiments, the hypophosphatasia is late-onset hypophosphatasia. In some embodiments, the cartilage disease is caused by a deficiency in PHOSPHO1. In some embodiments, disclosed herein is a method of treating a cartilage disease, the method comprising administering to a subject a composition described herein by intramuscular injection into a muscle.

[0006] In certain aspects, disclosed herein are methods of treating a subject having a dental disorder, comprising administering to the subject via intramuscular injection a viral vector comprising a mineral-targeted alkaline phosphatase under the control of a tissue-nonspecific promoter, wherein administering the viral vector treats the dental disorder. In some embodiments, the tissue-nonspecific promoter comprises a CAG promoter. In some embodiments, the viral vector comprises an adenovirus-associated virus. In some embodiments, the viral vector comprises an adeno-associated virus type 8 (AAV8) vector. In some embodiments, the mineral-targeted alkaline phosphatase comprises tissue-nonspecific alkaline phosphatase (TNAP). In some embodiments, the mineral-targeted alkaline phosphatase further comprises a bone-targeting sequence linked to the C-terminus of TNAP. In some embodiments, the bone-targeting sequence is decaaspartate (D 10 ) sequence. In some embodiments, the dental disorder comprises at least one of alveolar disease, dental hypomineralization, and periodontal disease.

[0007] Citation by reference All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications, patents, or patent applications incorporated by reference conflict with disclosure contained in this specification, the present specification is intended to supersede and / or take precedence over such conflicting material. [Brief description of the drawings]

[0008] This application for patent contains at least one color drawing. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0009] The novel features of the invention are set forth with particularity in the appended claims. To better understand the nature and advantages of the present invention, reference should be made to the following detailed description that sets forth illustrative embodiments in which the principles of the invention are utilized and the accompanying drawings.

[0010] [Figure 1A] Figures 1A-1G show that AAV8-TNAP-D10 improves survival and PPi metabolic conversion in AAV8-TNAP-D10-treated Alpl- / - mice. Figure 1A shows that the average body weight of AAV8-TNAP-D10-treated male Alpl- / - mice was similar to that of WT littermates, whereas female Alpl- / - mice weighed less than WT. [Figure 1B] Figure 1A-1G show that AAV8-TNAP-D10 improves survival rate and PPi metabolic conversion in AAV8-TNAP-D10-treated Alpl- / - mice. Figure 1B shows that serum ALP activity is significantly increased in AAV8-TNAP-D10-treated Alpl- / - mice and AAV8-TNAP-D10-treated Alpl- / -WT mice compared to untreated WT mice. [Figure 1C] Figure 1A-1G show that AAV8-TNAP-D10 improves survival and PPi metabolic conversion in AAV8-TNAP-D10-treated Alpl- / - mice. Figure 1C shows that plasma PPi levels in AAV8-TNAP-D10-treated Alpl- / - mice are significantly lower than those in WT controls. [Figure 1D] Figure 1A-1G show that AAV8-TNAP-D10 improves survival and PPi metabolic conversion in AAV8-TNAP-D10-treated Alpl- / - mice. Figure 1D shows that urinary PPi concentrations in AAV8-TNAP-D10-treated Alpl- / - mice remain significantly higher than those in both treated and untreated WT mice (70 dpn). [Figure 1E]Figure 1A-G shows that AAV8-TNAP-D10 improves survival and PPi metabolic conversion in AAV8-TNAP-D10-treated Alpl- / - mice. Figure 1E shows that urinary PPi concentrations in untreated Alpl- / - and heterozygous (Alpl+ / -) mice are higher than those in WT mice (10 dpn). [Figure 1F] Figure 1A-G shows that AAV8-TNAP-D10 improves survival and PPi metabolic conversion in AAV8-TNAP-D10-treated Alpl- / - mice. Figure 1F shows that qPCR of kidney RNA shows reduced Alpl expression in Alpl- / - mice, but no significant differences in other genes related to PPi metabolism (Ank, Enpp1, and Abcc6) or inflammation (Il6 and Tnf). [Figure 1G] Figure 1A-G shows that AAV8-TNAP-D10 improves survival rate and PPi metabolic conversion in AAV8-TNAP-D10-treated Alpl- / - mice. Figure 1G shows that histochemical staining of kidneys shows no ALP activity in proximal tubules of AAV8-TNAP-D10-treated Alpl- / - mice (upper panel). Light microscopy shows no obvious structural changes in glomeruli or tubules of AAV8-TNAP-D10-treated Alpl- / - mice stained with H&E (lower panel). [Figure 2A] Figures 2A-D show improved radiographic findings in AAV8-TNAP-D10-treated Alpl- / - mice. Figure 2A shows whole-skeletal radiographic images of female WT littermate controls and female Alpl- / - mice treated with AAV8-TNAP-D10. [Figure 2B] Figures 2A-2D show the improvement of radiographic findings in AAV8-TNAP-D10-treated Alpl- / - mice, and Figure 2B shows the absence of signs of rickets (e.g., curvature and metaphyseal expansion, and craniosynostosis) in AAV8-TNAP-D10-treated Alpl- / - mice. [Figure 2C]Figures 2A-2D show improved radiographic findings in AAV8-TNAP-D10-treated Alpl- / - mice, and Figure 2C shows that AAV8-TNAP-D10-treated female Alpl- / - mice have shorter limb lengths compared to their WT littermates. [Figure 2D] Figures 2A-D show improved radiographic findings in AAV8-TNAP-D10-treated Alpl- / - mice. Figure 2D shows that nose length, skull length, and skull width were not significantly different between groups. Unpaired t-tests with Welch correction were performed to compare differences between WT and Alpl- / - treated male mice. One-way ANOVA was performed followed by Turkey's multiple comparison test to compare differences between WT, treated Alpl- / -, and treated WT female mice. Significance was indicated on the graphs as *P<0.05, **P<0.01, ***P<0.001. [Figure 3A] Figures 3A-C show that bone microstructure is partially normalized in AAV8-TNAP-D10 treated Alpl- / - mice. Figure 3A shows 2D and 3D micro-CT images of the femur of treated Alpl- / - mice compared to WT controls. Treated Alpl- / - females show shorter femurs compared to WT controls. Red arrows point to the abnormal articular surfaces of the medial and distal femurs of treated Alpl- / - mice. [Figure 3B] Figures 3A-C show partial normalization of bone microarchitecture in AAV8-TNAP-D10-treated Alpl- / - mice, and Figure 3B shows quantification of trabecular parameters from 50 sections proximal to the growth plate of the distal femur. [Figure 3C] Figures 3A-C show partial normalization of bone microarchitecture in AAV8-TNAP-D10-treated Alpl- / - mice. Figure 3C shows quantification of cortical bone parameters from 50 mid-shaft femoral sections. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison test, *P<0.05, **P<0.01. [Figure 4A] Figures 4A-D show improved histological findings in AAV8-TNAP-D10-treated Alpl- / - mice. Figure 4A shows histological analysis of femurs from WT and AAV8-TNAP-D10-treated Alpl- / - mice. Von Kossa / van Gieson staining of femurs and lumbar vertebrae shows no measurable osteoid surface. [Figure 4B] Figure 4A-4D show the improvement of histological findings in AAV8-TNAP-D10-treated Alpl- / - mice. Figure 4B shows that the BV / TV values ​​of the lumbar spine in AAV8-TNAP-D10-treated Alpl- / - mice were significantly lower than those in WT mice (WT: n=5; male: n=2, female: n=3; KO: n=3; male: n=1, female: n=2). [Figure 4C] Figures 4A-4D show the histological improvement of AAV8-TNAP-D10-treated Alpl- / - mice. Figure 4C shows that H&E and Safranin O staining of demineralized tibiae reveals abnormal distribution of chondrocytes in the secondary ossification center of AAV8-TNAP-D10-treated Alpl- / - mice (black arrows). [Figure 4D] Figures 4A-4D show the histological improvement of AAV8-TNAP-D10-treated Alpl- / - mice. Figure 4D shows that femoral histochemical staining reveals strong ALP activity in the hypertrophic layer of the epiphyseal growth plate, metaphysis, and diaphysis of WT mice, but only slight ALP activity is observed in the growth plate and diaphysis of AAV8-TNAP-D10-treated Alpl- / - mice. [Figure 5A] Figures 5A-5I show that AAV8-TNAP-D10 prevents HPP-associated dentoalveolar defects in Alpl- / - mice. Figures 5A-5F show that 3D and 2D microCT renderings of first molars (M1) and incisors (INC) show normal dental structure in AAV8-TNAP-D10-treated Alpl- / - mice, similar to that of WT controls (70 dpn). [Figure 5B]Figures 5A-5I show that AAV8-TNAP-D10 prevents HPP-associated dentoalveolar defects in Alpl- / - mice. Figures 5A-5F show that 3D and 2D microCT renderings of first molars (M1) and incisors (INC) show normal dental structure in AAV8-TNAP-D10-treated Alpl- / - mice, similar to that of WT controls (70 dpn). [Figure 5C] Figures 5A-5I show that AAV8-TNAP-D10 prevents HPP-associated dentoalveolar defects in Alpl- / - mice. Figures 5A-5F show that 3D and 2D microCT renderings of first molars (M1) and incisors (INC) show normal dental structure in AAV8-TNAP-D10-treated Alpl- / - mice, similar to that of WT controls (70 dpn). [Figure 5D] Figures 5A-5I show that AAV8-TNAP-D10 prevents HPP-associated dentoalveolar defects in Alpl- / - mice. Figures 5A-5F show that 3D and 2D microCT renderings of first molars (M1) and incisors (INC) show normal dental structure in AAV8-TNAP-D10-treated Alpl- / - mice, similar to that of WT controls (70 dpn). [Figure 5E] Figures 5A-5I show that AAV8-TNAP-D10 prevents HPP-associated dentoalveolar defects in Alpl- / - mice. Figures 5A-5F show that 3D and 2D microCT renderings of first molars (M1) and incisors (INC) show normal dental structure in AAV8-TNAP-D10-treated Alpl- / - mice, similar to that of WT controls (70 dpn). [Figure 5F]Figures 5A-5I show that AAV8-TNAP-D10 prevents HPP-associated dentoalveolar defects in Alpl- / - mice. Figures 5A-5F show that 3D and 2D microCT renderings of first molars (M1) and incisors (INC) show normal dental structure in AAV8-TNAP-D10-treated Alpl- / - mice, similar to that of WT controls (70 dpn). [Figure 5G] Figure 5A-I show that AAV8-TNAP-D10 prevents HPP-associated dentoalveolar defects in Alpl- / - mice. Figure 5G shows that there are no significant differences in enamel density, dentin volume, or dentin density between groups in first molars. Dental enamel is reduced in volume in treated Alpl- / - molars compared to WT controls. [Figure 5H] Figures 5A-I show that AAV8-TNAP-D10 prevents HPP-associated dentoalveolar defects in Alpl- / - mice, and Figure 5H shows that continued expression in the incisors did not result in significant defects in either enamel or dentin volume or density between groups. [Figure 5I] Figure 5A-I show that AAV8-TNAP-D10 prevents HPP-associated alveolar defects in Alpl- / - mice. Figure 5I shows that AAV8-TNAP-D10 significantly improves alveolar bone volume in treated Alpl- / - mice versus WT mice, but reduces alveolar bone mineral density by 4% in treated Alpl- / - mice versus WT mice. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. EN=enamel; DE=dentin; AB=alveolar bone. [Figure 6A]Figures 6A-G show improved cementum and PDL attachment in AAV8-treated Alpl- / - mice. Figure 6A shows that H&E staining reveals no obvious tooth loss in treated Alpl- / - mice compared to WT controls. Boxed areas are shown at higher magnification (left). [Figure 6B] Figures 6A-G show improved cementum and PDL attachment in AAV8-treated Alpl- / - mice. Figure 6B shows in situ hybridization with an Alpl probe (left) confirms the absence of Alpl expression in treated Alpl- / - mice. TNAP IHC (right) shows weak staining (brown) around the treated Alpl- / - alveolar bone (AB) and darker staining in WT mice. [Figure 6C] Figures 6A-G show improved cementum and PDL attachment in AAV8-treated Alpl- / - mice. Figure 6C shows BSP IHC (left) shows pronounced staining (brown) in acellular cementum (AC, white arrows) and alveolar bone (AB) in treated Alpl- / - and WT mice. OPN IHC (right) shows comparable staining (brown) in AC and AB in treated Alpl- / - mice versus WT mice. [Figure 6D] Figures 6A-G show improved cementum and PDL attachment in AAV8-treated Alpl- / - mice. Figure 6D shows ImageJ color maps (left) and (right) showing improved cell density in the PDL space (yellow symbols) and AC width (yellow line) in treated Alpl- / - mice versus WT mice. [Figure 6E]Figures 6A-6G show the improvement of cementum and PDL attachment in AAV8-treated Alpl- / - mice. Figures 6E-6G show the quantification of acellular cementum thickness, mantle dentin (MD), and cell density in the PDL space. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison test, *P<0.05 and **P<0.01, ns: no specificity. [Figure 6F] Figures 6A-6G show the improvement of cementum and PDL attachment in AAV8-treated Alpl- / - mice. Figures 6E-6G show the quantification of acellular cementum thickness, mantle dentin (MD), and cell density in the PDL space. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison test, *P<0.05 and **P<0.01, ns: no specificity. [Figure 6G] Figures 6A-6G show the improvement of cementum and PDL attachment in AAV8-treated Alpl- / - mice. Figures 6E-6G show the quantification of acellular cementum thickness, mantle dentin (MD), and cell density in the PDL space. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison test, *P<0.05 and **P<0.01, ns: no specificity. [Figure 7A] Figures 7A-B show the size of Alpl- / - and WT pups. Figure 7A shows that untreated Alpl- / - mice were significantly smaller than WT mice at 10 dpn. [Figure 7B] Figures 7A-7B show the size of Alpl- / - and WT pups. Figure 7B shows that Alpl- / - mice injected with AAV8-TNAP-D10 within 5 dpn were not significantly smaller than WT mice at 15 dpn. [Figure 8] FIG. 8 shows that histochemical staining of the liver revealed that ALP activity (black arrows), which was evident in WT mice, was not seen in the hepatic artery branches of AAV8-TNAP-D10-treated Alpl− / − mice. [Figure 9]FIG. 9. Von Kossa staining shows no ectopic calcification in the aorta, coronary arteries, brain, or kidney in WT and AAV8-TNAP-D10-treated Alpl− / − mice at 70 dpn. [Figure 10] Figure 10 shows Safranin O staining of demineralized tibiae reveals abnormal distribution of chondrocytes in the secondary ossification center in AAV8-TNAP-D10-treated Alpl- / - mice (black arrows). Sections were scanned with the Aperio AT2 system to capture images of the entire tibia. The images of WT and Alpl- / - shown in the upper panel were the same as those shown in Figure 4C (observed under a microscope). [Figure 11A] Figures 11A-C show hypomineralization of teeth and periodontal tissue damage in untreated Alpl- / - mice compared to WT mice. Figure 11A shows hypomineralization of the molar root by H&E staining of the first mandibular molar (upper panel, red arrow). The lower panel shows hypoplasia of acellular cementum (*) and loss of periodontal attachment. [Figure 11B] Figures 11A-C show hypomineralization of teeth and periodontal tissue damage in untreated Alpl- / - mice compared to WT mice. Figure 11B shows ImageJ color map of H&E stained images revealing abnormal PDL cells in Alpl- / - mice compared to WT mice. [Figure 11C] Figures 11A-11C show tooth hypomineralization and periodontal tissue damage in untreated Alpl- / - compared to WT mice. Figure 11C shows a bar graph of cell counts in the boxed area, revealing that there are less PDL cells in Alpl- / - mice compared to WT mice, although not significantly so. Statistical analysis was performed by Student's t-test, ns: no specificity. [Figure 12A] Figures 12A-12L show biochemical analysis of serum / plasma collected from adult HPP mice and WT littermates under AAV8-TNAP-D10 treatment or AAV8-GFP control 60 days after injection. Figures 12A-12B show body weight. [Figure 12B]Figures 12A-12L show biochemical analysis of serum / plasma collected from adult HPP mice and WT littermates under AAV8-TNAP-D10 treatment or AAV8-GFP control 60 days after injection. Figures 12A-12B show body weight. [Figure 12C] Figures 12A-12L show biochemical analysis of serum / plasma collected from adult HPP mice and WT littermates under AAV8-TNAP-D10 treatment or AAV8-GFP control 60 days after injection. Figures 12C-12D show serum alkaline phosphatase activity. [Figure 12D] Figures 12A-12L show biochemical analysis of serum / plasma collected from adult HPP mice and WT littermates under AAV8-TNAP-D10 treatment or AAV8-GFP control 60 days after injection. Figures 12C-12D show serum alkaline phosphatase activity. [Figure 12E] Figures 12A-12L show biochemical analysis of serum / plasma collected from adult HPP mice and WT littermates under AAV8-TNAP-D10 treatment or AAV8-GFP control 60 days after injection. Figures 12E-12F show plasma PPi levels. [Figure 12F] Figures 12A-12L show biochemical analysis of serum / plasma collected from adult HPP mice and WT littermates under AAV8-TNAP-D10 treatment or AAV8-GFP control 60 days after injection. Figures 12E-12F show plasma PPi levels. [Figure 12G] Figures 12A-12L show biochemical analysis of serum / plasma collected from adult HPP mice and WT littermates under AAV8-TNAP-D10 treatment or AAV8-GFP control 60 days after injection. Figures 12G-12H show serum calcium concentrations. [Figure 12H] Figures 12A-12L show biochemical analysis of serum / plasma collected from adult HPP mice and WT littermates under AAV8-TNAP-D10 treatment or AAV8-GFP control 60 days after injection. Figures 12G-12H show serum calcium concentrations. [Figure 12I]Figures 12A-12L show biochemical analysis of serum / plasma collected from adult HPP mice and WT littermates under AAV8-TNAP-D10 treatment or AAV8-GFP control 60 days after injection. Figures 121-12J show serum phosphorus concentrations. [Figure 12J] Figures 12A-12L show biochemical analysis of serum / plasma collected from adult HPP mice and WT littermates under AAV8-TNAP-D10 treatment or AAV8-GFP control 60 days after injection. Figures 121-12J show serum phosphorus concentrations. [Figure 12K] Figures 12A-12L show biochemical analysis of serum / plasma collected from adult HPP mice and WT littermates under AAV8-TNAP-D10 treatment or AAV8-GFP control 60 days after injection. Figures 12K-12L show blood urea nitrogen (BUN) levels in serum. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 12L] Figures 12A-12L show biochemical analysis of serum / plasma collected from adult HPP mice and WT littermates under AAV8-TNAP-D10 treatment or AAV8-GFP control 60 days after injection. Figures 12K-12L show blood urea nitrogen (BUN) levels in serum. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 13A] Figures 13A-J show biochemical analysis of serum / plasma collected from 2-month-old adult female and male HPP mice and WT siblings prior to injection. Figures 13A-B show serum alkaline phosphatase activity. [Figure 13B] Figures 13A-J show biochemical analysis of serum / plasma collected from 2-month-old adult female and male HPP mice and WT siblings prior to injection. Figures 13A-B show serum alkaline phosphatase activity. [Figure 13C]Figures 13A-J show biochemical analysis of serum / plasma collected from 2-month-old adult female and male HPP mice and WT siblings prior to injection, and Figures 13C-D show plasma PPi levels. [Figure 13D] Figures 13A-J show biochemical analysis of serum / plasma collected from 2-month-old adult female and male HPP mice and WT siblings prior to injection, and Figures 13C-D show plasma PPi levels. [Figure 13E] Figures 13A-J represent biochemical analyses of serum / plasma collected from 2-month-old adult female and male HPP mice and WT siblings prior to injection, and Figures 13E-F represent serum calcium assays. [Figure 13F] Figures 13A-J represent biochemical analyses of serum / plasma collected from 2-month-old adult female and male HPP mice and WT siblings prior to injection, and Figures 13E-F represent serum calcium assays. [Figure 13G] Figures 13A-J show biochemical analysis of serum / plasma collected from 2-month-old adult female and male HPP mice and WT siblings prior to injection. Figures 13G-H show serum phosphorus concentrations. [Figure 13H] Figures 13A-J show biochemical analysis of serum / plasma collected from 2-month-old adult female and male HPP mice and WT siblings prior to injection. Figures 13G-H show serum phosphorus concentrations. [Figure 13I] Figures 13A-J show biochemical analysis of serum / plasma collected from 2-month-old female and male adult HPP mice and WT siblings before injection. Figures 13I-J show blood urea nitrogen (BUN) levels in serum. Statistical analysis was performed by unpaired t-test. *P<0.05. **P<0.01. ****P<0.0001. [Figure 13J] Figures 13A-J show biochemical analysis of serum / plasma collected from 2-month-old female and male adult HPP mice and WT siblings before injection. Figures 13I-J show blood urea nitrogen (BUN) levels in serum. Statistical analysis was performed by unpaired t-test. *P<0.05. **P<0.01. ****P<0.0001. [Figure 14A] Figures 14A-J show biochemical analysis of serum / plasma collected from Phospho1 KO mice under AAV8-TNAP-D10 treatment or AAV8-GFP treatment as a control 45 and 90 days after injection. WT littermates were treated with AAV8-TNAP-D10 vector. Figures 14A-B show serum alkaline phosphatase activity. [Figure 14B] Figures 14A-J show biochemical analysis of serum / plasma collected from Phospho1 KO mice under AAV8-TNAP-D10 treatment or AAV8-GFP treatment as a control 45 and 90 days after injection. WT littermates were treated with AAV8-TNAP-D10 vector. Figures 14A-B show serum alkaline phosphatase activity. [Figure 14C] Figures 14A-J show biochemical analysis of serum / plasma collected from Phospho1 KO mice under AAV8-TNAP-D10 treatment or AAV8-GFP treatment as a control 45 and 90 days after injection. WT littermates were treated with AAV8-TNAP-D10 vector. Figures 14C-D show plasma PPi levels. [Figure 14D] Figures 14A-J show biochemical analysis of serum / plasma collected from Phospho1 KO mice under AAV8-TNAP-D10 treatment or AAV8-GFP treatment as a control 45 and 90 days after injection. WT littermates were treated with AAV8-TNAP-D10 vector. Figures 14C-D show plasma PPi levels. [Figure 14E] Figures 14A-J show biochemical analysis of serum / plasma collected from Phospho1 KO mice under AAV8-TNAP-D10 treatment or AAV8-GFP treatment as a control 45 and 90 days after injection. WT littermates were treated with AAV8-TNAP-D10 vector. Figures 14E-F show serum calcium concentration. [Figure 14F]Figures 14A-J show biochemical analysis of serum / plasma collected from Phospho1 KO mice under AAV8-TNAP-D10 treatment or AAV8-GFP treatment as a control 45 and 90 days after injection. WT littermates were treated with AAV8-TNAP-D10 vector. Figures 14E-F show serum calcium concentration. [Figure 14G] Figures 14A-J show biochemical analysis of serum / plasma collected from Phospho1 KO mice under AAV8-TNAP-D10 treatment or AAV8-GFP treatment as a control 45 and 90 days after injection. WT littermates were treated with AAV8-TNAP-D10 vector. Figures 14G-H show serum phosphorus concentration. [Figure 14H] Figures 14A-J show biochemical analysis of serum / plasma collected from Phospho1 KO mice under AAV8-TNAP-D10 treatment or AAV8-GFP treatment as a control 45 and 90 days after injection. WT littermates were treated with AAV8-TNAP-D10 vector. Figures 14G-H show serum phosphorus concentration. [Figure 14I] Figures 14A-J show biochemical analysis of serum / plasma collected from Phospho1 KO mice under AAV8-TNAP-D10 treatment or AAV8-GFP treatment as a control 45 and 90 days after injection. WT littermates were treated with AAV8-TNAP-D10 vector. Figures 14I-J show blood urea nitrogen (BUN) levels in serum. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison test. *P<0.05. **P<0.01. ***P<0.001. ****P<0.0001. [Figure 14J]Figures 14A-J show biochemical analysis of serum / plasma collected from Phospho1 KO mice under AAV8-TNAP-D10 treatment or AAV8-GFP treatment as a control 45 and 90 days after injection. WT littermates were treated with AAV8-TNAP-D10 vector. Figures 14I-J show blood urea nitrogen (BUN) levels in serum. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparison test. *P<0.05. **P<0.01. ***P<0.001. ****P<0.0001. [Figure 15A] Figures 15A-D show radiographic findings of the bone phenotype of adult female HPP mice. Radiographic images of whole skeletal tissues, including spine (2x), vertebrae (2x), and mandible (3x), as well as skull at higher magnification. Figures 15A-15B show female WT treated with control AAV8-GFP and female WT treated with AAV8-TNAP-D10. [Figure 15B] Figures 15A-D show radiographic findings of the bone phenotype of adult female HPP mice. Radiographic images of whole skeletal tissues, including spine (2x), vertebrae (2x), and mandible (3x), as well as skull at higher magnification. Figures 15A-15B show female WT treated with control AAV8-GFP and female WT treated with AAV8-TNAP-D10. [Figure 15C] Figures 15A-D show radiographic findings of the bone phenotype of adult female HPP mice. Radiographic images of whole skeletal tissues, including spine (2x), vertebrae (2x), and mandible (3x), as well as skull at higher magnification. Figures 15C-15D show AAV8-GFP or AAV8-TNAP-D10 treated adult HPP mice 60 days after injection. [Figure 15D] Figures 15A-D show radiographic findings of the bone phenotype of adult female HPP mice. Radiographic images of whole skeletal tissues, including spine (2x), vertebrae (2x), and mandible (3x), as well as skull at higher magnification. Figures 15C-15D show AAV8-GFP or AAV8-TNAP-D10 treated adult HPP mice 60 days after injection. [Figure 16A]Figures 16A-D show radiographic findings of the bone phenotype of male adult HPP mice. Radiographic images of whole skeletal tissues, including spine (2x), vertebrae (2x), and mandible (3x), as well as skull at higher magnification. Figures 16A-16B show male WT treated with vehicle AAV8-GFP and male WT treated with AAV8-TNAP-D10. [Figure 16B] Figures 16A-D show radiographic findings of the bone phenotype of male adult HPP mice. Radiographic images of whole skeletal tissues, including spine (2x), vertebrae (2x), and mandible (3x), as well as skull at higher magnification. Figures 16A-16B show male WT treated with vehicle AAV8-GFP and male WT treated with AAV8-TNAP-D10. [Figure 16C] Figures 16A-D show radiographic findings of the bone phenotype of male adult HPP mice. Radiographic images of whole skeletal tissues, including spine (2x), vertebrae (2x), and mandible (3x), as well as skull at higher magnification. Figures 16C-16D show AAV8-GFP or AAV8-TNAP-D10 treated male adult HPP mice 60 days after injection. [Figure 16D] Figures 16A-D show radiographic findings of the bone phenotype of male adult HPP mice. Radiographic images of whole skeletal tissues, including spine (2x), vertebrae (2x), and mandible (3x), as well as skull at higher magnification. Figures 16C-16D show AAV8-GFP or AAV8-TNAP-D10 treated male adult HPP mice 60 days after injection. [Figure 17A] Figures 17A-17B represent radiographic findings and H&E staining of long bones of adult HPP mice bone phenotype. Figure 17A represents radiographic images of female and male HPP mice treated with AAV8-TNAP-D10 or AAV8-GFP and WT littermate controls 60 days after injection. Long bone defects in HPP mice include defects in the proximal and distal femur and proximal tibia in addition to the patellar articular surface. TNAP treatment partially restored epiphyseal and metaphyseal bone regions (highlighted in red). [Figure 17B]Figures 17A-17B show radiographic findings and H&E staining of long bones of adult HPP mice bone phenotype. Figure 17B shows H&E staining revealing disorganized growth plate in AAV8-GFP-treated HPP mice and substantial improvement in bone morphology parameters in AAV8-TNAP-D10-treated HPP mice. [Figure 18A] Figures 18A-B show microCT analysis of femurs from adult HPP mice and WT littermates. Figure 18A shows 2D and 3D microCT images of female and male femurs 60 days after injection, treated with AAV8-TNAP-D10 or AAV8-GFP. [Figure 18B] Figures 18A-B show micro-CT analysis of femurs from adult HPP mice and WT littermates, and Figure 18B shows micro-CT analysis of bone parameters in femurs. [Figure 19A] Figures 19A-B show radiographic findings of the bone phenotype of female Phospho1 KO mice. Radiographic images of whole skeletal tissues, including spine (2x), head (4x), mandible (3x), vertebrae (2x), and long bones (2x), as well as skull at higher magnification. Female Phospho1 KO mice treated with control AAV8-GFP or AAV8-TNAP-D10, 90 days after injection. [Figure 19B] Figures 19A-B show radiographic findings of the bone phenotype of female Phospho1 KO mice. Radiographic images of whole skeletal tissues, including spine (2x), head (4x), mandible (3x), vertebrae (2x), and long bones (2x), as well as skull at higher magnification. Female Phospho1 KO mice treated with control AAV8-GFP or AAV8-TNAP-D10, 90 days after injection. [Figure 20A]Figures 20A-B show radiographic findings of the bone phenotype of female Phospho1 KO mice. Radiographic images of whole skeletal tissues, including spine (2x), head (4x), mandible (3x), vertebrae (2x), and long bones (2x), as well as skull at higher magnification. Male Phospho1 KO mice treated with control AAV8-GFP or AAV8-TNAP-D10, 90 days after injection. [Figure 20B] Figures 20A-B show radiographic findings of the bone phenotype of female Phospho1 KO mice. Radiographic images of whole skeletal tissues, including spine (2x), head (4x), mandible (3x), vertebrae (2x), and long bones (2x), as well as skull at higher magnification. Male Phospho1 KO mice treated with control AAV8-GFP or AAV8-TNAP-D10, 90 days after injection. [Figure 21A] Figure 21A shows representative radiographic images of the spine of 90-day-old female and male Phospho1 KO mice. Three-day-old mice were intramuscularly administered a single dose of AAV8-TNAP-D10 or AAV8-GFP (3x1011vg / body) as a control. Scoliosis was observed in the control AAV8-GFP-treated Phospho1 KO mice. Gene therapy using the AAV8-TNAP-D10 vector led to the correction of the scoliotic deformity of the spine. [Figure 21B] FIG. 21B shows that H&E staining reveals differential organization of vertebrae in mice treated with AAV8-GFP or TNAP-encoding vectors, revealing increased trabecular bone and decreased bone marrow area in AAV8-TNAP-D10-treated mice. [Figure 22A] Figures 22A-22C show micro-CT analysis of tibiae from Phospho1 KO mice and WT littermates. Figure 22A shows 2D and 3D micro-CT images of female and male tibiae 90 days after injection, treated with AAV8-TNAP-D10 or AAV8-GFP. [Figure 22B]Figures 22A-22C show micro-CT analysis of tibiae from Phospho1 KO mice and WT littermates, and Figures 22B-22C show quantification of bone parameters. [Figure 22C] Figures 22A-22C show micro-CT analysis of tibiae from Phospho1 KO mice and WT littermates, and Figures 22B-22C show quantification of bone parameters. [Figure 23A] 23A-23B show alizarin red staining of soft organs of adult HPP and WT mice. Females and males were treated with AAV8-GFP or AAV8-TNAP-D10. 60 days after injection of the TNAP-encoding vector, no evidence of ectopic calcification was found. Upper panel: kidney. Middle panel: heart. Lower panel: aorta (20xmag). [Figure 23B] 23A-23B show alizarin red staining of soft organs of adult HPP and WT mice. Females and males were treated with AAV8-GFP or AAV8-TNAP-D10. 60 days after injection of the TNAP-encoding vector, no evidence of ectopic calcification was found. Upper panel: kidney. Middle panel: heart. Lower panel: aorta (20xmag). [Figure 24A] Figures 24A-24B show ectopic calcification in soft organs of adult female HPP and WT mice under CKD diet. Figure 24A shows that Alizarin Red staining was performed and ectopic calcification was observed in soft organs and vasculature in late-onset HPP mouse model and WT littermates as controls. [Figure 24B]Figures 24A-24B show ectopic calcification in soft organs of female adult HPP and WT mice under CKD diet. Figure 23B represents histological sections of kidney, heart, and aorta for the following experimental groups: WT mice treated with control AAV8-GFP or AAV8-TNAP-D10, and adult HPP mice injected with AAV8-GFP or AAV8-TNAP-D10. Kidney: upper panel - 996 μM, lower panel - 100 μM mag. Heart: upper panel - 996 μM, lower panel - 50 μM mag. Aorta: upper panel - 100 μM, lower panel - 50 μM mag. Figure 24B represents quantification of Alizarin Red S. [Figure 25A] Figures 25A-C show microCT analyses of the dentoalveolar complex of adult HPP mice and WT littermates. Figure 25A shows 2D and 3D microCT images of the first molar and incisor of AAV8-TNAP-D10 or AAV8-GFP treated mice 60 days after injection. [Figure 25B] Figures 25A-C show micro-CT analysis of the dento-alveolar complex of adult HPP mice and WT littermates, and Figure 25B shows quantification of enamel, dentin, alveolar bone, and dental pulp parameters. [Figure 25C] Figures 25A-C show micro-CT analysis of the dentoalveolar complex of adult HPP mice and WT littermates. Figure 25C shows H&E staining of dental organization. There are no obvious differences in organization, periodontal attachment, or acellular cementum between AAV8-TNAP-D10 or AAV8-GFP treated mice. M1: first molar. EN: enamel. DE: dentin. AB: alveolar bone. [Figure 26A] Figures 26A-D show micro-CT analysis of the dentoalveolar complex of Phospho1 KO and WT littermate mice, and Figures 26A-C show 2D and 3D micro-CT images of the first molars and incisors of AAV8-TNAP-D10 or AAV8-GFP treated mice 90 days after injection. [Figure 26B]Figures 26A-D show micro-CT analysis of the dentoalveolar complex of Phospho1 KO and WT littermate mice, and Figures 26A-C show 2D and 3D micro-CT images of the first molars and incisors of AAV8-TNAP-D10 or AAV8-GFP treated mice 90 days after injection. [Figure 26C] Figures 26A-D show micro-CT analysis of the dentoalveolar complex of Phospho1 KO and WT littermate mice, and Figures 26A-C show 2D and 3D micro-CT images of the first molars and incisors of AAV8-TNAP-D10 or AAV8-GFP treated mice 90 days after injection. [Figure 26D] Figures 26A-D show micro-CT analysis of the dentoalveolar complex of Phospho1 KO and WT littermate mice. Figure 26D shows quantification of enamel, dentin, alveolar bone, and cellular cementum parameters. There are no obvious differences in organization, periodontal attachment, except for higher dentin and acellular cementum volumes in AAV8-TNAP-D10-treated WT mice compared to AAV8-TNAP-D10 and AAV8-GFP-treated Phospho1 KO mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hypophosphatasia (HPP) is caused by loss-of-function mutations in the ALPL gene, which encodes tissue-nonspecific alkaline phosphatase (TNAP). Deficiency of TNAP results in the secretion of extracellular inorganic pyrophosphate (PP i HPP is characterized by skeletal and dental hypomineralization, and disease severity ranges from life-threatening perinatal or infantile forms to milder forms that manifest in adulthood or affect only the dentition.

[0012] Asfotase alfa contains the TNAP ectodomain, a human IgG1 Fc domain for one-step purification, and a terminal decaaspartate (D) that targets mineral. 10Infantile HPP, TNAP knockout (Akp2 - / - Or Alpl - / - In a mouse model, daily subcutaneous injections of asfotase alfa preserved life span, improved the skeletal phenotype, and prevented epileptic seizures and tooth loss. In humans, subcutaneous injections of asfotase alfa 3-7 times per week have demonstrated substantial and sustained efficacy in children and adults with HPP while maintaining a good safety profile. Asfotase alfa has saved the lives of subjects with severe neonatal and infantile HPP and improved bone mineralization, motor function, and quality of life in subjects with adult HPP. At the same time, the patient burden of multiple weekly injections to maintain efficacy of asfotase alfa and the associated healthcare costs have spurred preclinical research into alternative strategies to treat HPP.

[0013] Human chimeric recombinant alkaline phosphatase, ChimAP, and TNAP-D 10 Several forms of viral vectors expressing Alpl - / - Mice have longer life spans, are protected from seizures, and have improved skeletal phenotypes. 10 A single intravenous injection of lentivirus or adeno-associated virus type 8 (AAV8) vectors encoding Alpl - / - Although correction of the skeletal phenotype in mice was maintained, the resulting widespread distribution of the vector genome throughout the body raised concerns about the potential for germ cell transduction.

[0014] Previous approaches to correct HPP-associated mineralized defects have met with mixed success or have inherent limitations or drawbacks. - / -It has been shown to be highly effective in correcting skeletal and dental alveolar defects in mice, and these translational studies led to its approval in 2015 for the treatment of patients with perinatal / infantile and adolescent-onset HPP. As an exception, asfotase alfa is approved for all ages in Japan. However, asfotase alfa ERT has a half-life of 2.28 days, requires multiple injections per week, is associated with injection site reactions, and is expensive, prompting further preclinical research into alternative strategies to treat HPP. Daily subcutaneous injections of soluble non-mineral-targeted recombinant chimeric alkaline phosphatase (ChimAP) have been shown to enhance the expression of alkaline phosphatase in patients with HPP. - / - Seizures were prevented and survival rates increased in mice, but skeletal and dental alveolar phenotypes were only partially ameliorated. Studies using several different types of viral vectors with different modes of administration have been reported. 10 (HIV-TNAP-D 10 ) or AAV8-TNAP-D 10 A single intravenous injection of either the lentiviral vector containing Alpl - / - Although this resulted in a sustained increase in circulating TNAP and phenotypic correction in mice, viral sequences were detected in soft tissues, including the liver, lungs, and heart, raising safety concerns about the oncogenic potential of the integrated lentiviral vector in these tissues. 10 Intramuscular injection of AAV8, consisting of a self-complementary AAV type 8 (scAAV8) vector and muscle creatine kinase (MCK) promoter, was performed to restrict viral distribution and enhance Alpl - / - The skeletal phenotype in mice was improved, but long bones showed limited elongation and remained hypomineralized. 10 Our approach using a single intramuscular injection of combines a more practical gene therapy with a potentially improved safety profile due to limited tissue distribution, yet remains highly effective in correcting mineralization defects.

[0015] Treatments for HPP other than asfotase alfa have been tried in clinical settings but have not been subjected to rigorous preclinical or translational studies. A cross-sectional study enrolling 51 patients with childhood- or adult-onset HPP showed clinical and radiological improvement in two of four patients treated with teriparatide (parathyroid hormone 1–34). In a phase IIA open-label study of eight adult patients with HPP, treatment with anti-sclerostin monoclonal antibody (BPS804) resulted in increases in bone formation markers and bone mineral density. However, these anabolic agents are not approved in HPP, and effective medical therapies for adult-onset HPP patients are needed. At the same time, novel treatments with fewer injections may further benefit patients with childhood-onset HPP.

[0016] AAV8-TNAP-D in human patients 10 Concerns regarding administration of PP i These include potential effects on metabolism, as well as the development or worsening of ectopic calcification. 10 A single intramuscular injection of extracellular PP resulted in extremely high circulating ALP activity, 500-fold higher than that in WT mice. i TNAP-D is known to be a central regulator of biomineralization and is important for controlling inappropriate soft tissue mineralization in the body. 10 By PP i By hydrolyzing extracellular PP in soft tissues, i However, AAV8-TNAP-D may result in over-suppression of 10 Treated Alpl - / - In mice, plasma PP iConcentrations were suppressed and no soft tissue calcification developed during the 70 dpn observation window. This contrasts with previous results obtained from genetically modified mouse models targeting TNAP overexpression in the vasculature. Overexpression of TNAP in smooth muscle cells by using Tagln-Cre resulted in massive arterial calcification in the ascending and descending aorta, carotid arteries, and subclavian arteries. Overexpression of TNAP in endothelial cells by using Tie2-Cre resulted in partial calcification in cardiac, renal, mesenteric, pancreatic, and splenic arteries. These mouse models showed 20- to 30-fold higher circulating ALP activity but no plasma PP i The concentrations of circulating ALP and PP2 were similar to those of the WT. i The concentrations are all local PP i It can be inferred that the local PP i Although TNAP levels cannot be measured in vivo, it is believed that high TNAP expression leads to low levels in target tissues. 10 Treated Alpl - / - Urinary PP in mice i The increased concentration of PP in urine i Metabolism is largely dependent on TNAP, which is expressed on the luminal surface of renal proximal tubules, and serum ALP activity and circulating PP i qPCR analysis of kidneys from treated mice revealed that systemic PP1, including Ank, Enpp1, and Abcc6, were downregulated in the kidneys of treated mice. i It was suggested that genes of key regulators of metabolism were not affected by the treatment.

[0017] Herein, we present a mouse model of severe infantile HPP, the TNAP knockout (Alpl - / - ) TNAP-D to increase lifespan and improve skeletal and dental phenotypes in mice 10 The efficacy of a single intramuscular administration of adeno-associated virus type 8 (AAV8) encoding Alpl - / - Mice were transfected with AAV8-TNAP-D within 5 days after birth (dpn). 10 3×1011 The mice received 1000 vector genomes / body. 10 This increases serum ALP activity and plasma PP i The treatment suppressed Alpl - / - The life span of the mice was extended, and no ectopic calcification was observed in the kidney, aorta, coronary artery, or brain during the 70 dpn observation window. - / - Mice did not show signs of rickets, including bowing of long bones, enlargement of the epiphysis, or fractures. - / - The bone microstructure of the mice was similar to that of wild-type mice (WT), with some persistent small cortical and trabecular defects. - / - Although there was no measurable osteoid accumulation in the mice, bone volume fraction was reduced. - / - Mice featured normal molar and incisor alveolar tissues, except for slightly reduced molar enamel and alveolar bone density. Histology demonstrated the presence of cementum and normal periodontal ligament attachment. These results support gene therapy as a viable alternative to ERT for treating HPP.

[0018] method In some embodiments, disclosed herein is a method useful for treating a subject in need thereof, comprising administering a composition described herein. In some embodiments, the composition described herein comprises a viral vector described herein. In some embodiments, the disease is a cartilage disease, a dental disorder, or a combination thereof. In some embodiments, the method comprises administering to the subject a viral vector comprising a mineral-targeted alkaline phosphatase under the control of a tissue-nonspecific promoter by intramuscular injection into the muscle.

[0019] In certain aspects, described herein is a method of administering the compositions described herein to a subject in need thereof.In some embodiments, the method improves at least one of symptoms selected from the list consisting of lifespan, skeletal abnormalities, seizures, tooth loss, bone mineralization, motor function, and quality of life.In some embodiments, the symptoms improve compared to subjects who do not receive treatment.In some embodiments, the symptoms improve compared to subjects before receiving treatment.

[0020] In some embodiments, described herein are methods of increasing the lifespan of a subject in need, comprising administering to a subject a composition described herein. In some embodiments, described herein are methods of decreasing skeletal abnormalities in a subject in need, comprising administering to a subject a composition described herein. In some embodiments, described herein are methods of decreasing seizures in a subject in need, comprising administering to a subject a composition described herein. In some embodiments, described herein are methods of decreasing tooth loss in a subject in need, comprising administering to a subject a composition described herein. In some embodiments, described herein are methods of increasing bone mineralization in a subject in need, comprising administering to a subject a composition described herein. In some embodiments, described herein are methods of increasing motor function in a subject in need, comprising administering to a subject a composition described herein. In some embodiments, described herein are methods of increasing the quality of life of a subject in need, comprising administering to a subject a composition described herein. In some embodiments, the composition comprises a viral vector for delivery of alkaline phosphatase.

[0021] Diseases and Disorders Patients with HPP suffer from characteristic rickets and / or osteomalacia with a wide range of severity, as well as dental defects. There are seven main types of HPP, including the life-threatening perinatal and infantile type (OMIM#241500), the benign perinatal, mild and severe pediatric type (OMIM#241510), the adult type (OMIM#136300), and odonto-limited HPP (OMIM#146300). Patients with perinatal HPP, the most severe form of HPP, often die in utero or shortly after birth due to severe skeletal hypomineralization, respiratory failure due to hypoplasia of the thoracic cage and lungs, and elevated intracranial pressure due to craniosynostosis. A dentoalveolar phenotype, including premature avulsion of primary teeth, periodontal disease, and enamel alternation, is commonly observed in patients with all types of HPP.

[0022] In some embodiments, the subject has a cartilage disorder. In some embodiments, the cartilage disorder is hypophosphatasia (HPP). In some embodiments, the hypophosphatasia is juvenile hypophosphatasia or infantile hypophosphatasia. In some embodiments, the hypophosphatasia is late-onset hypophosphatasia.

[0023] In some embodiments, the disease comprises dental disorders. All forms of HPP include dental problems, with premature tooth loss being one of the most common symptoms. - / - Characteristic dentoalveolar abnormalities observed in mice include impaired formation of acellular cementum in the roots, detachment of the PDL, and mineralization defects in enamel and dentin. As described herein, qualitative and quantitative analyses demonstrated that teeth and associated periodontal tissues were transformed with AAV8-TNAP-D. 10 These results suggest that AAV8-TNAP-D significantly improved the disease. 10 Treated Alpl - / - Mice showed normal formation and mineralization of enamel and dentin in molars and incisors, as well as restoration of acellular cementum, PDL cell density, and PDL-cementum attachment.- / - Mice were shown to have well-developed alveolar bone with a small decrease in bone mineral density. In some embodiments, the dental disorder comprises at least one of alveolar disease, dental hypomineralization, and periodontal disease.

[0024] There are over 400 mutant alleles identified for ALPL. The inheritance pattern of perinatal and infantile HPP is often autosomal recessive, with most patients being compound heterozygous for pathogenic ALPL mutations resulting in nearly null ALP activity, but some patients are homozygous for recessive alleles, and most adult and odonto-limited HPP patients have a single dominant-negative ALPL allele. In some embodiments, the subject has a mutation in the ALPL gene. In some embodiments, the subject has a deficiency of PHOSPHO1.

[0025] Administration In some embodiments, the viral vector is administered at least once. In some embodiments, administering the viral vector results in an increase in plasma alkaline phosphatase (ALP) activity. In some embodiments, the ALP activity is increased for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, or 6 months. In some embodiments, the ALP activity is increased for at least 1 week. In some embodiments, the ALP activity is increased for at least 2 weeks. In some embodiments, the ALP activity is increased for at least 3 weeks. In some embodiments, the ALP activity is increased for at least 3 weeks. In some embodiments, the ALP activity is increased for at least 4 weeks. In some embodiments, the ALP activity is increased for at least 2 months. In some embodiments, the ALP activity is increased for at least 3 months. In some embodiments, the ALP activity is increased for at least 4 months. In some embodiments, the ALP activity is increased for at least 5 months. In some embodiments, the ALP activity is increased for at least 6 months.

[0026] In some embodiments, the viral vector is administered intramuscularly. In some embodiments, the viral vector does not spread away from the injection site. In some embodiments, the viral vector does not spread away from the muscle. In some embodiments, after administration of the viral vector, the viral vector is not detected in the brain, gonads, or a combination thereof. In some embodiments, after administration of the viral vector, the viral vector is not detected in the brain. In some embodiments, after administration of the viral vector, the viral vector is not detected in the gonads.

[0027] In patients with HPP, both with and without treatment, several types of ectopic calcifications are commonly observed, such as ocular calcification, nephrocalcinosis, and painful periarthritis. In some embodiments, after administration of the viral vector, the subject does not develop ectopic calcification. In some embodiments, the ectopic calcification is selected from the list consisting of ocular calcification, nephrocalcinosis, painful periarthritis, or kidney stones. In some embodiments, the subject does not develop hypercalcemia. In some embodiments, the subject does not develop hypercalciuria. In some embodiments, the subject does not develop ectopic calcification in the subject's tissue. In some embodiments, the tissue is selected from the list consisting of kidney, aorta, coronary artery, and brain. In some embodiments, after administration of the viral vector, the viral vector does not cause canceration in the subject.

[0028] composition In certain aspects, compositions are described herein for delivering a therapeutic peptide to a subject in need thereof. In some embodiments, viral vectors are described herein for delivering alkaline phosphatase to a subject in need thereof. In some embodiments, the viral vector comprises a sequence of a therapeutic peptide. In some embodiments, the therapeutic peptide comprises alkaline phosphatase. In some embodiments, the therapeutic peptide comprises mineral-targeted alkaline phosphatase. In some embodiments, the mineral-targeted alkaline phosphatase comprises tissue non-specific alkaline phosphatase (TNAP).

[0029] In some embodiments, the alkaline phosphatase comprises an alkaline phosphatase comprising the sequence of SEQ ID NO: 15. In some embodiments, the alkaline phosphatase comprises an alkaline phosphatase having at least 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100% sequence identity to SEQ ID NO: 15.

[0030] In some embodiments, the mineral-targeted alkaline phosphatase comprises a bone-targeting sequence. The bone-targeting sequence may be linked to the C-terminus of the mineral-targeted alkaline phosphatase. The bone-targeting sequence may be linked to the N-terminus of the mineral-targeted alkaline phosphatase. The bone-targeting sequence may comprise at least one aspartate. The bone-targeting sequence may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more aspartates. In some embodiments, the bone-targeting sequence comprises decaaspartate (D 10) sequence. In some embodiments, the bone targeting sequence comprises the sequence DDDDDDDDDD (SEQ ID NO: 17). In some embodiments, the bone targeting peptide comprises a repeat sequence of aspartate-serine-serine (DSS, SEQ ID NO: 18). In some embodiments, the bone targeting sequence comprises at least 1, 2, 3, 4, 5, 6, 7, 8, or more repeats of aspartate-serine-serine. In some embodiments, the bone targeting sequence comprises the sequence KRRTPVRE (SEQ ID NO: 19). In some embodiments, the bone targeting sequence comprises the sequence KNFQSRSH (SEQ ID NO: 20). In some embodiments, the bone targeting sequence comprises the sequence KTYASMQW (SEQ ID NO: 21). In some embodiments, the bone targeting sequence comprises at least one sequence selected from the list consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0031] The peptides described herein can be encoded by nucleic acids. A nucleic acid is a type of polynucleotide that contains two or more nucleotide bases. In certain embodiments, a nucleic acid is a component of a vector that can be used to transcribe a polynucleotide that encodes a polypeptide into a cell. As used herein, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid to which it is linked. One type of vector is a genomic integration vector or "integration vector" that can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, e.g., a nucleic acid that can replicate extrachromosomally. A vector that can direct the expression of a gene to which it is operably linked is referred to herein as an "expression vector." Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. In expression vectors, the regulatory elements used to control transcription, such as promoters, enhancers, polyadenylation signals, and the like, can be derived from mammalian, bacterial, viral, or insect genes.

[0032] In some embodiments, the viral vector comprises a tissue-non-specific promoter. In some embodiments, the tissue-non-specific promoter comprises a CAG promoter. In some embodiments, the viral vector comprises a promoter having a sequence of SEQ ID NO: 16. In some embodiments, the viral vector comprises a tissue-non-specific promoter having at least 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100% sequence identity to SEQ ID NO: 16.

[0033] Usually, the replication ability in the host, which is conferred by a replication origin, and a selection gene for facilitating the recognition of transformants can be further incorporated. A vector derived from a virus, such as a lentivirus, a retrovirus, an adenovirus, or an adeno-associated virus, can be used. In some embodiments, the vector comprises an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 vector. In some embodiments, the vector is an AAV8 vector.

[0034] The plasmid vector can be linearized for integration into a chromosomal location. The vector may contain sequences that direct site-specific integration (e.g., AttP-AttB recombination) into a defined location or set of restricted sites within the genome. Additionally, the vector may contain sequences derived from transposable elements for integration.

[0035] Pharmaceutical Compositions The compositions disclosed herein may be formulated in any suitable manner for administration. Any suitable technique, carrier, and / or excipient is contemplated for use with the compositions disclosed herein. Non-limiting examples of cosmetically, dermatologically or pharma- ceutically acceptable carriers and excipients suitable for the formulation can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; Pharmaceutical Dosage Forms and Drug Delivery Systems, Eighth Ed. (Lippincott Williams & Wilkins 2004); and Muller, RH, et al., Advanced Drug Delivery Reviews 59 (2007)522-530, each of which is incorporated by reference in its entirety.

[0036] In some embodiments, the pharma- ceutically acceptable carriers or excipients disclosed herein include, but are not limited to, one or more of pH adjusting agents (e.g., buffering agents), stabilizers, thickening agents, coloring agents, preservatives, emulsifiers, solubilizers, antioxidants, or any combination thereof. Other suitable compounds contemplated herein and within the knowledge of an experienced practitioner in the relevant art are found in Handbook of Pharmaceutical Excipients, 4th Ed. (2003), the entirety of which is incorporated herein by reference.

[0037] In some embodiments, the compositions disclosed herein comprise one or more preservatives.When used, the preservative is in an amount sufficient to extend the shelf life or storage stability, or both, of the topical formulations disclosed herein.Exemplary preservatives include, but are not limited to, tetrasodium ethylenediaminetetraacetate (EDTA), methyl, ethyl, butyl, and propyl paraben, benzophenone-4, methylchloroisothiazolinone, methylisothiazolinone, sodium benzoate, paraoxybenzoic acid ester, chlorobutanol, benzyl alcohol, phenylethyl alcohol, dehydroacetic acid, sorbic acid, benzalkonium chloride (BKC), benzethonium chloride, phenol, phenylmercuric nitrate, and thimerosal.

[0038] Specific Terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are merely exemplary and illustrative, and are not intended to limit the claimed subject matter. In this application, the use of the singular includes the plural unless expressly stated otherwise. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Furthermore, the use of the term "including," as well as other forms such as "include," "includes," and "included," is not limited to the described term. The section headings used herein are for organizational purposes only and should not be construed as limiting the described subject matter.

[0039] As used herein, the term "effective amount" or "therapeutically effective amount" generally refers to an amount of an agent or compound sufficient to alleviate to some extent one or more symptoms of the disease or disorder being treated, or to reduce the risk of onset. The result may include reduction and / or alleviation of the signs, symptoms, or causes of the disease, or any other desired change in a biological system. The term "effective amount" or "therapeutically effective amount" typically includes, for example, a prophylactically effective amount. For example, a "prophylactically effective amount" is the amount of a composition described herein required to reduce the risk of pathogen attachment, infection, or function in an individual, or the risk of pathogen infection to another individual.

[0040] The terms "subject", "individual", or "patient" are often used interchangeably herein. A "subject" may be a biological entity containing expressed genetic material. The biological entity may be, for example, a plant, an animal, or a microorganism, including bacteria, viruses, fungi, and protozoa. A subject may be tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro. A subject may be a mammal. A mammal may be a human. A subject may be a mouse. A subject may be a TNAP knockout mouse. A subject may be diagnosed or suspected to be at high risk for a disease. In some cases, a subject is not necessarily diagnosed or suspected to be at high risk for a disease.

[0041] As used herein, the term "treatment" or "treating" is used in reference to a pharmaceutical or other intervention regimen to obtain a beneficial or desired result in a recipient. Beneficial or desired results include, but are not limited to, therapeutic benefit and / or prophylactic benefit. Therapeutic benefit may refer to the eradication or amelioration of the condition or underlying disease being treated. Therapeutic benefit may also be achieved by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disease, whereby an improvement is observed in the subject, even though the subject still suffers from the underlying disease. Prophylactic benefit includes delaying, preventing, or eliminating the appearance of a disease or illness, delaying or eliminating the onset of symptoms of a disease or illness, slowing, halting, or reversing the progression of a disease or illness, or any combination thereof. For prophylactic benefit, subjects at risk of developing a particular disease, or who report one or more physiological symptoms of the disease, even if the disease has not been diagnosed, may receive treatment.

[0042] As used herein, the term "about" or "approximately" generally means within an acceptable error range for a particular value as determined by a person skilled in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation according to the practice in the art. Alternatively, "about" may mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude of a value, within a range of 5-fold, more preferably within a range of 2-fold. EXAMPLES

[0043] The following examples are illustrative, but non-limiting, to the scope of the compositions, methods, and formulations described herein.

[0044] Example 1 Mouse model of infantile HPP TNAP knockout (Alpl - / - (Narisawa S, Frohlander N, Millan JL. Inactivation of two mouse alkaline phosphatase genes and establishment of a model of infantile hypophosphatasia. Dev Dyn. Mar 1997; 208(3):432-46.)Alpl - / - The mice phenocopy human infantile HPP and appear normal at birth, making them indistinguishable from their siblings. The mice show little circulating ALP activity, develop epileptic seizures, become cachectic, and die by 10-12 days postnatal (dpn) without further supportive care. - / - Mice were maintained on a 12.5% ​​C57B1 / 6 and 87.5% 129J background and genotyped by PCR using genomic DNA extracted from nail samples within 5 days after birth. All animals in this study (breeders, foster dams, pups, and weaners) were fed ad libitum a normal diet with standard levels of vitamin B6 (2018 Teklad global 18% protein extruded rodent diets or 2019 Teklad global 19% protein rodent diets, Envigo, Indianapolis, IN, USA). Elevated levels of vitamin B6 increased the risk of Alpl - / - It was reported that the life span of mice was improved. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC).

[0045] Human TNAP-D 10 Viral vectors encoding cDNA TNAP-D 10We report here that we have synthesized recombinant human soluble TNAP (sALP) and a C-terminal decaaspartate (DAS) that allows TNAP to target mineralized tissues such as bone and teeth. 10 ) sequence. The human IgG1 Fc domain present in asfotase alpha, which allows for one-step purification, is not present in the product of this vector, since purification is not required for in vivo expression mediated by viral vectors. As previously reported, TNAP-D, placed under the control of the CAG promoter, was expressed by a triple transfection method using the HEK293 cell line. 10 A recombinant AAV8 vector encoding TNAP-D 10 ) were generated, purified, and then titrated. As a control, a recombinant AAV8 vector encoding GFP (AAV8-GFP) was used. Within 5 dpn, 3 × 10 11 AAV8-TNAP-D at dose of vector / genome (vg) / body 10 In this study, a single injection of Alpl - / - Eleven mice (male n = 6, female n = 5) and 14 wild-type (WT) controls (male n = 7, female n = 7) were included. After genotyping, Alpl - / - 3 × 10 11 Dose of vector / genome (vg) / body of AAV8-TNAP-D 10 Three control WT mice were injected with the same dose of AAV8-TNAP-D 10 If endogenous TNAP activity is present, PP i Its effects on metabolism and soft tissue mineralization were evaluated. Seven WT mice were administered the same amount of control AAV8-GFP vector, and four WT mice were untreated, and because there was no substantial difference between them, these data were combined and analyzed together as WT. After intraperitoneal administration of Avertin, mice were euthanized by exsanguination at 70 dpn. In mice, 70 dpn corresponds to the end of puberty, which corresponds to 20 years of human age, and root tip formation and cellular cementum formation are complete. Untreated Alpl - / - , 17 WT, 7 heterozygotes (Alpl+ / - ), and Alpl - / - Ten pups were harvested at 10 dpn and euthanized by exsanguination after intraperitoneal administration of Avertin.

[0046] Sample collection and biochemical analysis Mice were weighed at 35 and 70 dpn. Blood was collected from the orbital sinus of isoflurane-anesthetized mice using a Pasteur pipette every 4 weeks after injection. Spot urine samples were collected at the same time.

[0047] Blood was collected into two BD Microtinas (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) coated with either clot activator or lithium heparin. Blood and urine samples for plasma collection were placed on ice. The Microtinas were then centrifuged at 7,000g for 10 min. 20 pL of heparinized plasma was deproteinized using a Microcon-10kDa Centrifugal Filter Unit with an Ultracel-10 membrane (MilliporeSigma, Merck KGaA, Darmstadt, Germany) and centrifuged at 14,000g for 20 min. Urine was diluted 1:3 with 10 mM HEPES. Samples were stored at -80°C for further analysis.

[0048] Plasma PP was prepared according to the previously described protocol. i The concentrations of PP were measured in a range of 0.125 μM to 20 μM in 5 μL of deproteinized plasma samples. iStandards (sodium pyrophosphate decahydrate, Sigma-Aldrich, St. Louis, MO, USA) were added to 45 μL of assay mixture containing 0.9 U / mL recombinant yeast ATP sulfurylase / MET3 (R&D Systems, Inc., Minneapolis, MN, USA) along with 90 μM adenosine 5' phosphosulfate sodium salt (APS) (Sigma-Aldrich), 22.5 μM MgCl2, 11.25 mM HEPES. The mixture was incubated at 37°C for 30 min and heat inactivated at 90°C for 10 min. 10 μL of each sample was then transferred to a white-bottom 96-well plate and mixed with 50 μl of BacTiter-Glo Microbial Cell Viability Assay (Promega Corporation, Madison, WI, USA). Luminescence was measured with FilterMax F5 Multimode Microplate Readers (Molecular Devices, LLC., San Jose, CA, USA).

[0049] Serum ALP activity was measured using an enzymatic assay. Five microliters of serum and recombinant human ALP standard (0.099–216.0 ng / mL) were mixed with 95 μL of 10 mM pNPP in diethanolamine (DEA) buffer (pH 9.8) containing 1.0 mM MgCl2 and 20 μM ZnCl2. The increment in A405 nm was measured using an OptiMax Microplate Absorbance Reader (Molecular Devices, LLC., San Jose, CA, USA) for 15 min. Serum and urinary calcium, urinary creatinine, and serum urea concentrations were measured using QuantiChrom Calcium Assay Kit, QuantiChrom Creatinine Assay Kit, and QuantiChrom Urea Assay Kit (BioAssay Systems, Hayward, CA, USA), respectively. Serum and urinary phosphorus concentrations were measured using Stanbio Phosphorus Liqui-UV (EKF Diagnostics-Stanbio Laboratory, Boerne, TX, USA).

[0050] Quantitative polymerase chain reaction (qPCR) Total RNA was extracted from kidneys using the RNAeasy Plus Kit (Qiagen LLC, Germantown, MD, USA) and reverse transcribed using PrimeScript RT Master Mix (Takara Bio USA, Inc., Mountain View, CA, USA). Real-time qPCR was performed in 384-well plates with an Applied Biosystems 7900HT Fast Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) using cDNA equivalent to 25ng total RNA and a DyNAmo Flash SYBR Green qPCR Kit (Thermo Fisher Scientific, Waltham, MA, USA). The reaction was run for 40 cycles with an initial temperature of 95°C for 7 min, followed by 95°C for 10 s, followed by 60°C for 15 s. Ct values ​​were determined by the software, and amplification of the target genes was normalized to 18S ribosomal RNA (Rn18s).The sequences of the primer pairs used for PCR are as follows: Alpl (NM_007431.3) F-CTGCCACTGCCTACTTGTGT (SEQ ID NO: 1) and R-GATGGATGTGACCTCATTGC (SEQ ID NO: 2); Ank (NM_020332.4) F-CTGCTGCTACAGAGGCAGTG (SEQ ID NO: 3) and R-GACAAAACAGAGCGTCAGCGA (SEQ ID NO: 4); Enpp1 (NM_001308327.1) F-TCACGCCACCGAGACTAAATA (SEQ ID NO: 5) and R-TGCAGTAGGGTGTCATGAAGG (SEQ ID NO: 6), Abcc6 (NM_018795.2) F-CATCTTGCCAGGAATCAACACT (SEQ ID NO: 7) and R-ACCAGGGACAAGCACAGGTA (SEQ ID NO: 8); Il6 (Interleukin 6) (NM_031168.2) F-CAAAGCCAGAGTCCTTCAGAGAG (SEQ ID NO: 9) and R-TTAGCCACTCCTTCTGTGACTCC (SEQ ID NO: 10); Tnf (TNF-alpha) (NM_013693.3) F-CAGCCTCTTCTCATTCCTGCT (SEQ ID NO: 11) and R-GCCATTTGGGAACTTCTCATC (SEQ ID NO: 12); Rn18s (NR_003278.3) F-TTGATTAAGTCCCTGCCCTTTGT (SEQ ID NO: 13) and R-CGATCCGAGGGCCTCACTA (SEQ ID NO: 14).

[0051] Radiography and micro-computed tomography (μCT) Radiographic images of the entire skeleton, as well as the forelimbs, hindlimb, and skull, were obtained with a Faxitron MX-20DC4 (Chicago, IL, USA) at an energy of 20 kV. Femoral, tibia, humerus, and radius lengths were measured using ImageJ (Rasband, WS, ImageJ, National Institutes of Health, Bethesda, MD, USA, https: / / imagej.nih.gov / ij / , 1997–2018). Head measurements were performed using the following landmarks: (Liu J, Nam HK, Wang E, Hatch NE. Further analysis of the Crouzon mouse: effects of the FGFR2(C342Y) mutation are cranial bone-dependent. Calcif Tissue Int. 2013 May;92(5):451-66.) Snout length, i.e., the length from the rostral intersection of the nasal bones to the caudal intersection of the nasal bones; skull length, i.e., the length from the intersection of the anastomoses of the nasal bones to the midpoint (mid) point of the posterior margin of the foramen magnum; skull width, i.e., the length from the right junction of the squamosal body to the zygomatic process of the temporal bone to its left counterpart.

[0052] Mandibles and femurs were fixed in 4% paraformaldehyde / PBS solution and then scanned with a μCT 50 scanner (Scanco Medical, Bassersdorf, Switzerland) at 70 kV, 76 μA, 0.5 A1 filter, integration time 900 ms, and voxel dimensions of 6 or 10 μm for mandibles and femurs, respectively. Reconstructed images were calibrated to five known densities of hydroxyapatite and analyzed using AnalyzePro (version 1.0; AnalyzeDirect, Overland Park, KS). For femurs, trabecular and cortical bone were calibrated at 350 and 650 mg HA / cm, respectively. 3 The bone was segmented into bone volume (BV), total volume (TV), bone volume fraction (Tb.BV / TV), trabecular number (Tb.N), thickness (Tb.Th), spacing (Tb.Sp), and connectivity density (1 / mm 3Fifty sections (total 0.5 mm) proximal to the growth plate of the distal femur were used to trace the trabecular bone to quantify the cortical bone volume fraction (Ct.BV / TV), cortical thickness (Ct.Th), porosity, and bone mineral density (Ct.BMD). For cortical bone, fifty sections from the mid-femoral portion of each bone were used to quantify the cortical bone volume fraction (Ct.BV / TV), cortical thickness (Ct.Th), porosity, and bone mineral density (Ct.BMD).

[0053] The first mandibular molar and its associated alveolar bone were quantitatively analyzed as previously described. The alveolar bone region of interest (ROI) included the area between 240 μm mesial to the most proximal point of the first molar root and 240 μm distal to the most distal point of the distal root. Enamel was prepared using a concentration of 1,600 mg HA / cm. 3 The dentin / cementum and alveolar bone were segmented at 550–1,600 mg HA / cm 3 was segmented.

[0054] Tissue collection and histological examination Skeletal and soft tissues were fixed in 4% paraformaldehyde / PBS solution and processed for histological analysis. Fixed non-demineralized bone samples were placed in 30% sucrose / PBS solution and then cryo-embedded in Optimal Cutting Temperature (OCT) compound (Tissue-Tek, Torrance, CA, USA) in a hexane-dry ice bath and sectioned by Kawamoto's method. (Kawamoto T. Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants. Arch Histol Cytol. May 2003;66(2):123-43) Tibias and femurs were decalcified in 0.125M EDTA / 10% formalin (pH 7.3) solution for 7 days and then embedded in paraffin. Soft organs were either embedded in paraffin or embedded in OCT in an ethanol-dry ice bath. Hematoxylin and eosin (H&E), von Kossa, von Kossa / van Gieson, and Safranin O staining were performed according to standard methods. Tissue ALP activity was assayed by incubating OCT-embedded sections in freshly mixed substrate solution (pH 8.9) consisting of 1 volume of 0.2 mg naphthol AS-MX phosphate disodium salt per mL water and 1 volume of 1.2 mg fast violet B salt per mL 0.2 M Tris-HCl at room temperature for 60 min and counterstaining in methyl green solution. Sections were viewed under an IX81 Olympus Microscope (Olympus Corporation, Center Valley, PA, USA) or scanned with an Aperio AT2 system (Leica Biosystems of Leica Microsystems Inc., Buffalo Grove, IL, USA). Bone volume fraction (BV / TV) was measured using ImageJ.

[0055] The left mandible was fixed overnight in Bouin's solution, decalcified in acetic acid / formalin / sodium chloride solution, processed, embedded in paraffin, and sectioned at 5 μm thickness in the coronal plane. Paraffin sections were stained with H&E to evaluate the teeth and their associated periodontal tissues. Immunohistochemical staining (IHC) procedures were performed as previously described. (Foster BL, Ao M, Salmon CR, Chavez MB, Kolli TN, Tran AB, et al. Osteopontin regulates dentin and alveolar bone development and mineralization. Bone. 2018 February;107:196-207.) Primary antibodies included monoclonal rat anti-human alkaline phosphatase IgG (TNAP) (R&D systems, Minneapolis, MN, USA), polyclonal rabbit anti-mouse bone sialoprotein (BSP) IgG, and polyclonal LF-175 rabbit anti-mouse osteopontin (OPN) IgG. For mouse Alpl, in situ hybridization was performed as previously described (RNAscope 2.5 HD Detection reagent kit-RED assay, Advanced Cell Diagnostics) according to the manufacturer's instructions. (Zhang H, Chavez MB, Kolli TN, Tan MH, Fong H, Chu EY, et al. Dentoalveolar Defects in the Hyp Mouse Model of X-linked Hypophosphatemia. J Dent Res. 2020 Apr;99(4):419-28) To perform acellular cementum / PDL analysis, H&E stained images taken with the same acquisition parameters were segmented using the color map function (5Ramps) in ImageJ. This method pseudocolorizes the images to make the differences between pixel values ​​more obvious and to visualize tissue refinement. Acellular cementum and mantle dentin thickness values ​​represent the average of three linear measurements taken at 90 μm, 100 μm, and 110 μm from the cement-enamel junction (CEJ) using the ImageJ linear function.The outer layer of the mantle dentin is the dentin adjacent to the acellular cementum, which is less mineralized. For the assessment of cell density, a 5.5 mm area of ​​100 μm from the apical side toward the CEJ was used to count cells in the PDL space. 2 The area was defined as follows.

[0056] statistics All statistical analyses were performed using GraphPad Prism version 9.0.0 (GraphPad Software, San Diego, CA, USA). In the charts, data are presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was performed followed by multiple comparison tests by Turkey's method to compare the differences between control WT, treated Alpl - / - The differences were compared between WT mice and treated Alpl mice. - / - To compare the expression of each gene in mouse kidney, an unpaired t-test with Welch correction was performed. Significance was determined at P < 0.05 and is shown in the chart. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P<0.0001.

[0057] Example 2 AAV8-TNAP-D 10 Treated Alpl - / - Improved survival and plasma PP in mice i Correction of Untreated Alpl - / - The pups died within 10–12 days after birth. 10 Alpl treated - / - All mice survived to the end of the study at 70 dpn without developing epileptic seizures. - / - The pups did not feed well and were significantly smaller than their WT littermates at 10 dpn (Figure 7). In contrast, within 5 dpn, AAV8-TNAP-D 10 Injected Alpl - / -The mice showed catch-up growth, and at 15 dpn the weight of the mice was not significantly different from that of WT littermates (Figure 7). 10 Treated male Alpl - / - Mice were similar in weight to their WT littermates, but were significantly outweighted compared to treated female Alpl mice. - / - Mice weighed less than their WT littermates at both 35 and 70 dpn ( Fig. 1A ).

[0058] Serum ALP activity was significantly increased by AAV8-TNAP-D 10 Injected Alpl - / - The serum ALP activity was significantly higher in the WT and IFN-γ mice and was approximately 500-800-fold higher than that in the control WT mice (Fig. 1B, Table 1). There were no significant differences in serum calcium, serum phosphorus, serum urea, urinary calcium, or urinary phosphorus concentrations between the experimental groups (Table 1). i The concentration of AAV8-TNAP-D 10 Alpl treated - / - TNAP-D mice and WT mice were nearly undetectable (Figure 1C, Table 1). 10 Urinary PP in AlpP-mice treated with i The concentration remained significantly higher than in WT mice at 70 dpn (Fig. 1D, Table 1). - / - Mouse and Alpl + / - In the study, urinary PP i Increased concentrations of Alpl were also observed (Figure IE). - / - Alpl gene expression was not observed in mice, and PP i No significant differences were observed in other genes related to metabolism (Ank, Enpp1, and Abcc6) or in genes related to inflammation (Il6 and Tnf) (Figure 1F, Table 2). Histochemical staining of the kidney and liver showed that AAV8-TNAP-D 10 Treated Alpl - / -No ALP activity was observed in the renal proximal tubules or hepatic arterial branches of mice (Figure 1G and Figure 8). Light microscopy did not reveal any obvious structural changes in H&E-stained glomeruli or renal tubules (Figure 1G). No ectopic calcification was observed in the aorta, coronary arteries, brain, or kidney by 70 dpn (Figure 9).

[0059] [Table 1]

[0060] [Table 2]

[0061] AAV8-TNAP-D 10 Treated Alpl - / - Improving bone microarchitecture in mice Previous studies have shown that untreated Alpl - / - Mice showed severe skeletal abnormalities, including decreased bone mineral density and bone fractures in tissues. 10 Treated Alpl - / - Radiographic images of the mice revealed grossly normal-appearing skeletal development comparable to that of WT littermates (Figure 2A). - / - Mice did not show any characteristic features of rickets or osteomalacia, such as bowing of long bones, enlargement of the epiphyses, or fractures (Figure 2B). - / - The length of the long bones of treated female Alpl mice was not significantly different from that of WT mice, whereas the length of the long bones of treated female Alpl mice was not significantly different from that of WT mice. - / - Mice were significantly smaller than their WT littermates (Fig. 2C). - / - No craniosynostosis was observed in mice (Figure 2B). Nose length, cranial length, and cranial width did not differ significantly among the treatment groups, but nose length and cranial length varied significantly among individuals, especially after AAV8-TNAP-D. 10 Treated Alpl - / - Variability occurred in mice (Figure 2D, Table 3).

[0062] [Table 3]

[0063] AAV8-TNAP-D 10 Three-dimensional μCT rendering of the treated femur showed similar bone morphology to that of the WT femur, whereas the treated Alpl - / - The femoral bones of treated Alpl mice were approximately 7–9% shorter compared to WT controls (Figure 2C, Figure 3A). - / - The femoral joints of mice had abnormal articular surfaces compared to those of WT controls. Quantitative μCT analysis revealed that treated male and female Alpl - / - Similar trabecular and cortical parameters were seen in the femurs of treated Alpl compared to WT controls. The distal femoral metaphysis showed no significant differences in total volume (TV), bone volume (BV), or trabecular bone volume fraction (Tb.BV / TV) between the groups (Figure 3B). However, the treated Alpl - / - In females, there was an increase in Tb.BV / TV, which was associated with improved trabecular connectivity compared to treated WT mice (Figure 3B). No significant differences were detected in trabecular number (Tb.N), thickness (Tb.Th), spacing (Tb.Sp), or bone mineral density (Tb.BMD) between groups. However, AAV8-TNAP-D 10 Treated Alpl - / - In females, there was a decrease in the cortical BV / TV ratio associated with a decrease in cortical thickness. - / - In males, increased cortical porosity was observed, but Ct.BV / TV was not significantly affected. 10 However, cortical bone mineral density (Ct.BMD) was not completely restored by the treatment (Fig. 3C), suggesting that restoration of the cortical bone defect was incomplete.

[0064] Von Kossa / van Gieson staining of femurs and lumbar vertebrae revealed no measurable osteoid surface (Fig. 4A ), whereas AAV8-TNAP-D 10 Treated Alpl- / - The BV / TV ratio of the lumbar spine of the mice was significantly lower than that of WT mice (Fig. 4B; WT, n = 5; male, n = 2, female, n = 3; KO, n = 3; male, n = 1, female, n = 2). H&E and Safranin O staining of the tibia after decalcification showed no elongation of the growth plate of the tibial epiphysis, but AAV8-TNAP-D 10 Treated Alpl - / - Abnormal distribution of chondrocytes was observed in the secondary ossification center in the WT mice (Fig. 4C, Fig. S10). Histochemical staining of femurs revealed strong ALP activity in the hypertrophic layer of the epiphyseal growth plate, metaphysis, and diaphysis of the WT mice, but not in the AAV8-TNAP-D mice. 10 Treated Alpl - / - Only slight ALP activity was observed in the mouse growth plate and diaphysis (Fig. 4D).

[0065] AAV8-TNAP-D 10 By Alpl - / - Prevents HPP-associated alveolar defects in mice Data from male and female dental alveolar tissues were combined together because of the substantial lack of sex-related differences in previous reports of HPP mouse models and because these data showed no sex-related trends. 10 Treated Alpl - / - The first mandibular molars and surrounding alveolar bone of mice appeared macroscopically normal compared to untreated and treated WT controls (Figure 5A-F). Enamel volume was significantly increased in AAV8-TNAP-D mice compared to the WT group. 10 Treated Alpl - / - The volume or density of the dentin in the molars was decreased by 12–14% in the AAV8-TNAP-D mice, whereas no significant difference was observed in the enamel bone mineral density (Figure 5G). 10 Treated Alpl - / - No difference was observed between the Alpl and WT groups (Figure 5G). - / -Incisors that continued to erupt in mice showed no difference in enamel or dentin volume or density compared to the WT group (Figure 5H). 10 Treated Alpl - / - Although no difference in volume was found between the mice and WT controls, they showed approximately 4% lower bone mineral density than the WT group ( Fig. 5I ).

[0066] AAV8-TNAP-D 10 Treated Alpl - / - Histology of the mouse mandibles revealed normal tooth structure largely indistinguishable from WT controls, with similar morphology, organization, and presence of acellular cementum on the root surfaces and periodontal attachment (Figure 6A). 10 Treated Alpl - / - Although endogenous Alpl expression was not observed in mouse tissues, IHC detected low levels of TNAP localized to the PDL (Figure 6B), suggesting that AAV8-mediated TNAP contributed to the improvement of the defect. 10 Treated Alpl - / - In mice, BSP and OPN immunostaining was comparable to that in WT controls, confirming the presence of acellular cementum (Figure 6C). 10 Acellular cementum thickening was observed in the treated WT teeth, whereas the treated Alpl - / - No significant differences in the thickness of acellular cementum were observed in the molars of mice with HPP versus WT mice (Figures 6D and 6E). The outermost mantle dentin was enlarged and hypomineralized in humans with HPP, whereas Alpl - / - In mice, calcification was delayed, and the treated Alpl - / - The IL-1 expression level was decreased in both untreated Alpl mice and WT mice (Figures 6D and 6F). - / - In mice, there was a loss of PDL cells associated with acellular cementum and loss of the PDL attachment (Figure 11), but treatment restored normal cell density (Figures 6D, 6G) and the periodontal architecture appeared intact.

[0067] Example 3: AAV8-TNAP-D 10 Analysis of mice under treatment method Human TNAP-D 10 Viral vectors encoding cDNA The HEK293 cell line was used to transfect the mineral-targeted TNAP-encoding recombinant adeno-associated viral vector serotype 8 (AAV8) (AAV8-TNAP-D 10 As a control, a recombinant AAV8 vector encoding GFP (AAV8-GFP) was used. Prxl / Prxl Mice and WT sibling mice, as well as Phospho1 - / - and 3 × 10 11 AAV8-TNAP-D at a dose of vector genome (vg) / body 10 Or, as a control, a single intramuscular injection of AAV8-GFP vector was administered.

[0068] Induction of uremia 8-week-old Alpl Prxl / Prxl Mice and WT sibling mice were fed a diet containing adenine (0.2%) for 4 weeks, followed by an adenine + high phosphorus diet (0.2% adenine and 1.8% phosphorus) for another 4 weeks, using a protocol adapted from Tani et al., Inhibition of tissue-nonspecific alkaline phosphatase protects against medial arterial calcification and improves survival probability in the CKD-MBD mouse model. J Pathol. 01 2020; 250(1):30-41, to induce a uremic state recapitulating CKD, which leads to vascular calcification. Animals were weighed and monitored weekly for posture and clinical signs, including gait and feeding difficulties. On the same day as induction of uremia, animals were administered a single dose of AAV8-TNAP-D 10After eight weeks, the mice were sacrificed and soft organs, including the kidneys, aorta, and heart, were harvested from each animal to assess the status of calcification and whether the treatments might have exacerbated ectopic calcification.

[0069] biochemical analysis Mice were first weighed and then anesthetized with isoflurane to collect blood (plasma / serum) from the orbital sinus of the mice using a Pasteur pipette before treatment and 60 days after injection for late-onset HPP mice and before treatment and 45 and 90 days after treatment for sham HPP mice. Blood samples were centrifuged at 7,500 rpm for 10 min. PP i For analysis, 20 μL of heparinized plasma was deproteinized using a Microcon-10 kDa Centrifugal Filter Unit with an Ultracel-10 membrane (MilliporeSigma, Merck KGaA, Darmstadt, Germany) and centrifuged at 14,000 g for 20 min. Samples were stored at −80°C for further analysis.

[0070] plasma PP i The concentrations of PP were measured in a range of 0.125 μM to 20 μM in 5 μL of deproteinized plasma samples. iStandards (sodium pyrophosphate decahydrate, Sigma-Aldrich, St. Louis, MO, USA) were added to 45 μL of assay mixture containing 0.9 U / mL recombinant yeast ATP sulfurylase / MET3 (R&D Systems, Inc., Minneapolis, MN, USA) along with 90 μM adenosine 5' phosphosulfate sodium salt (APS) (Sigma-Aldrich), 22.5 μM MgCl2, 11.25 mM HEPES. The mixture was incubated at 37°C for 30 min and heat inactivated at 90°C for 10 min. 10 μL of each sample was then transferred to a white-bottom 96-well plate and mixed with 50 μL of BacTiter-Glo Microbial Cell Viability Assay (Promega Corporation, Madison, WI, USA). Luminescence was measured with FilterMax F5 Multimode Microplate Readers (VWR International, LLC., Radnor, PA, USA). Serum ALP activity was measured using an enzymatic assay. 5 μL of serum and recombinant human ALP standards (0.099–216.0 ng / mL) were mixed with 95 μL of 10 mM pNPP in diethanolamine (DEA) buffer (pH 9.8) containing 1.0 mM MgCl2 and 20 μM ZnCl2. Kinetic assay absorbance was measured at 405 nm for 15 min using an OptiMax Microplate Absorbance Reader (Molecular Devices, LLC., San Jose, CA, USA). Serum calcium and serum urea concentrations were measured using QuantiChrom Calcium Assay Kit and QuantiChrom Urea Assay Kit (BioAssay Systems, Hayward, CA, USA), respectively. Serum phosphorus concentrations were measured using Stanbio Phosphorus Liqui-UV (EKF Diagnostics-Stanbio Laboratory, Boerne, TX, USA).

[0071] Radiography and micro-computed tomography (micro-CT) Radiographic images of the whole skeleton and single bones, such as the skull, vertebrae, long bones, and mandible, were obtained with a Faxitron MX-20DC4 radiographic imaging system (Chicago, IL, USA) at an energy of 20 kV.

[0072] Prior to micro-CT analysis, the long bones and right mandible were fixed in 4% paraformaldehyde / PBS solution. The femur was scanned using a SkyScan 1172 scanner (Bruker Micro-CT, Kontig, Belgium) at 55 kV, 181 μA, 0.5 mm A1 filter, integration time 280 ms, and voxel dimension 10 μm. Micro-CT images were reconstructed with NRecon software, and reconstructed images were calibrated to three known densities of hydroxyapatite and analyzed using AnalyzePro (version 1.0; AnalyzeDirect, Overland Park, KS). Trabecular and cortical bone of the femur were analyzed using 400 and 550 mg HA / cm, respectively. 3 The femur was segmented by CT. Micro-CT analysis of the femur was performed. Briefly, bone volume (BV), total volume (TV), bone volume fraction (Tb.BV / TV), trabecular number (Tb.N), thickness (Tb.Th), spacing (Tb.Sp), and connectivity density (1 / mm 3 Fifty slices (total 0.5 mm) proximal to the growth plate of the distal femur were used to trace the trabecular bone to quantify the cortical bone volume fraction (Ct.BV / TV), cortical thickness (Ct.Th), medullary cavity area, and bone mineral density (Ct.BMD). For cortical bone, fifty slices from the mid-femoral portion of each bone were used to quantify the cortical bone volume fraction (Ct.BV / TV), cortical thickness (Ct.Th), medullary cavity area, and bone mineral density (Ct.BMD).

[0073] Mandibles were scanned using a microCT50 scanner (Scanco Medical, Bassersdorf, Switzerland) at 70 kV, 76 μA, 0.5 A1 filter, integration time 900 ms, and voxel dimension 6 μm. Reconstructed images were calibrated to five known densities of hydroxyapatite and analyzed using AnalyzePro (version 1.0; AnalyzeDirect, Overland Park, KS). Dental microCT analysis was performed using the first mandibular molar and its associated alveolar bone. The alveolar bone region of interest was defined to include 240 μm mesial to the most proximal point of the first molar mesial root and 240 μm distal to the most distal point of the distal root. Alveolar bone and dentin / cementum were measured at 650–1,600 mg / cm2. 3 Segmented with HA, enamel is 1,600 mg / cm 3 Segmented beyond the HA.

[0074] Tissue collection and processing for histological examination Treated Alpl Prxl / Prxl and Phospho1 - / - Mice were euthanized by exsanguination 60 and 90 days after intraperitoneal administration of Avertin, respectively, and skeletal / dental tissues were harvested, fixed in 4% paraformaldehyde / PBS or Bouin's solution, and processed for histological analysis. Long bones and vertebrae were decalcified in 0.125 M EDTA / 10% formalin (pH 7.3) solution for 7 days, then embedded in paraffin and sectioned at 5 μm thickness. Mandibles were fixed in Bouin's solution, decalcified in acetic acid / formalin / sodium chloride solution, processed, embedded in paraffin, and sectioned at 5 μm thickness. Hematoxylin and eosin (H&E) and Alizarin Red staining were performed according to standard methods. Sections were scanned with an Aperio AT2 system (Leica Biosystems of Leica Microsystems Inc., Buffalo Grove, IL, USA).

[0075] statistical analysis All statistical analyses were performed using GraphPad Prism version 9.0.0 (GraphPad Software, San Diego, CA, USA) using one-way ANOVA followed by Tukey's multiple comparison test for comparisons of three or more groups, or unpaired t-test for comparisons of two groups. Values ​​are expressed as mean ± SD. Differences were * P<0.05 was statistically significant.

[0076] result AAV8-TNAP-D 10 Biochemical analysis of mice under treatment Pretreatment and AAV8-TNAP-D 10 Sixty days after injection, adult HPP and WT mice were monitored for body weight and biological markers in serum and / or plasma. Before injection, the body weight of female adult HPP mice was slightly lower than that of WT littermates. After 60 days of treatment, there was an increase in body weight, but no difference was observed in males. In male adult HPP mice, AAV8-TNAP-D was significantly higher than that of HPP AAV8-GFP injected mice compared to HPP AAV8-GFP injected mice. 10 After treatment, an increase in body weight was observed (Figures 12A to 12B). The results of serum ALP activity were significantly lower in female and male adult HPP mice than in WT mice before treatment, and PP i were significantly higher (Figures 13A-D, Table 4). Except for female HPP, there were no differences in serum calcium, blood urea nitrogen (BUN), and phosphorus levels in HPP and WT mice before treatment, but lower levels of phosphorus were observed in female HPP compared to WT mice before injection (Figures 13E-J, Table 4).

[0077] [Table 4]

[0078] AAV8-TNAP-D 10Sixty days after administration, serum ALP activity was significantly higher in adult female and male HPP mice and WT mice compared to the vehicle (AAV8-GFP) group (Figures 12C-D, Table 5). i Plasma PP concentrations were higher in both female and male adult HPP mice when compared to WT in control AAV8-GFP-treated mice. i Levels were significantly increased in AAV8-TNAP-D compared to AAV8-GFP-treated adult HPP mice. 10 The AAV8-TNAP-D expression level was significantly lower in treated adult male HPP mice. 10 It was not significantly lower in treated female mice (Figures 12E-F, Table 5). 10 Treatment increased calcium levels in WT females but not in males. Calcium levels in adult HPP mice were not affected by TNAP treatment (Figures 12G-H, Table 5). 10 High phosphorus levels were observed in adult HPP mice treated with AAV8-TNAP-D (Figures 12I-J, Table 5). 10 and there was no significant difference in BUN obtained from serum samples of HPP and WT mice treated with AAV8-GFP (Figures 12K-12L, Table 5).

[0079] [Table 5]

[0080] AAV8-TNAP-D 10 At 45 and 90 days after treatment, Phospho1 - / - Biochemical markers were evaluated in mice. 10 Serum ALP activity and plasma PP were significantly higher in treated mice compared to AAV8-GFP-treated mice. i The levels were low (Figures 14A to 14D, Table 6). 10AAV8-TNAP-D compared to treated WT mice 10 90 days after injection, female Phospho1 - / - Calcium levels increased in mice, whereas in males Phospho1 - / - The phosphorus or BUN levels did not increase in mice treated with AAV8-TNAP-D (Figures 14E to 14F). 10 No significant differences were observed after treatment with .

[0081] [Table 6]

[0082] No evidence of ectopic calcification in soft organs, AAV8-TNAP-D 10 Improvement of the skeleton through treatment Radiographic images of adult female and male HPP mice revealed normal skeletal development compared to WT littermates (Figures 15A-D, 16A-D). However, the long bones of adult female and male HPP mice exhibited defects in the epiphyseal and diaphyseal regions of the femur and tibia, as well as the patellar articular surface, when compared to WT mice (Figure 17A). 10 Long bones in treated adult HPP mice were partially restored, as evidenced by improved proximal tibia and distal femur regions, when compared to AAV8-GFP-treated adult HPP mice (Figure 17B). 10 There was no change in bone phenotype in treated WT mice compared to AAV8-GFP treated WT mice (Figure 17A). Histological sections of femoral bones stained with H&E from adult female and male HPP mice confirmed the radiographically visualized damaged areas compared to WT mice. Disorganized growth plates with modified chondrocytes in columnar organization were observed in adult HPP mice but not in WT mice (Figure 17B). AAV8-TNAP-D 10The femurs of treated adult HPP mice displayed preserved growth plate regions, as shown by bone morphology associated with AAV8-GFP-treated HPP mice (Figure 17B). 10 No histological changes in the bone phenotype of treated WT mice were observed when compared with AAV8-GFP-treated WT mice (FIG. 17B).

[0083] Micro-CT analysis of bone parameters demonstrated that untreated and AAV8-TNAP-D significantly improved bone function in untreated HPP mice. 10 A statistically significant decrease in cortical bone volume fraction (Ct.BV / TV) was demonstrated compared to treated WT littermates. The decrease in cortical bone BV / TV in untreated HPP mice was associated with a significant decrease in cortical thickness (Ct.Th) and an increase in bone marrow area (Ma.Ar) compared to untreated and treated WT littermates. However, cortical bone mineral density (Ct.BMD) in untreated HPP mice was not statistically significantly different from treated and untreated WT littermates. AAV8-TNAP-D 10 Gene therapy using the vector significantly improved the cortical bone of HPP mice, including bone fraction (Ct.BV / TV), thickness, and bone marrow area. Notably, no statistically significant differences were observed between treated and untreated WT mice, and AAV8-TNAP-D over 60 days was significantly improved in HPP mice. 10 It was demonstrated that vector-mediated gene therapy had no adverse effects (Figures 18A-B).

[0084] Female and male Phospho1 - / - Radiographic images of mice showed no changes in the spinal cord, i.e., no changes were observed in the untreated Phospho1 - / - The mice were shown to have developed a normal-looking skeleton, with moderate and severe scoliosis in the vertebrae (Figures 19A-B, 20A-B). Interestingly, a single dose of AAV8-TNAP-D 10 The scoliosis curve was improved by 90 days after injection in both female and male Phospho1 mice. - / -This congenital condition was corrected in mice (Figure 21A). H&E staining of vertebrae revealed that AAV8-TNAP-D was significantly associated with a higher phenotype compared to AAV8-GFP controls. 10 Treated mice showed increased trabecular bone mass and decreased bone marrow area (Figure 21B). 10 Phospho1 - / - MicroCT analysis of the tibiae of treated mice revealed that AAV8-GFP Phospho1 - / - Treated mice and AAV8-TNAP-D 10 The trabecular bone volume (Tb.BV) and trabecular connectivity density (Tb.Conn.D) were shown to be increased, respectively, when compared to treated WT mice (FIGS. 22A-C).

[0085] ★Furthermore, AAV8-TNAP-D 10 Vector treatment did not promote ectopic calcification in the kidney, heart, or aorta of female and male adult HPP mouse models during this short treatment window (60 days of treatment) (Figures 23A-B), and did not increase soft organ calcification in adult HPP when chronic kidney disease was superimposed as a comorbidity after 60 days of treatment (Figures 24A-M).

[0086] Characterization of the dentoalveolar complex in both osteomalacia models Micro-CT analysis of the first mandibular molar and associated alveolar bone revealed that untreated WT or AAV8-TNAP-D 10 Compared with treated WT and HPP mice, adult HPP mice did not have obvious defects in enamel, dentin / cementum, bone volume, and bone mineral density. However, AAV8-TNAP-D 10There was a statistically significant difference in enamel volume between treated WT and untreated HPP. Similarly, microCT analysis of consecutively erupted incisors showed no significant differences in enamel and dentin volume and density. Collectively, these results indicate that Alpl ablation in Prx1-expressing dental mesenchymal cells may be essential for alveolar formation and mineralization. Furthermore, since no differences were observed between treated and untreated WT teeth, the 60-day AAV8-TNAP-D 10 AAV8-mediated gene therapy was shown to have no adverse effects. Histological analysis of untreated HPP teeth revealed no morphological differences compared to untreated WT or to both AAV8-treated HPP and WT littermates. There were no obvious differences in organization, periodontal attachment, or acellular cementum between treated and untreated HPP (Figures 25A-C). - / - Similar results were seen in the mouse dentoalveolar complex, which showed a more mild mineralization defect. - / - AAV8-TNAP-D compared to treated and untreated mice 10 In treated WT mice, there was no obvious phenotypic change except for an increase in cellular cementum volume (FIG. 26).

[0087] [Table 7]

[0088] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used in implementing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims, and their equivalents, be covered thereby.

Claims

A pharmaceutical composition comprising a viral vector for use in treating a cartilage disease in a human subject, wherein the viral vector comprises a sequence encoding a mineral-targeted alkaline phosphatase under the control of a tissue-nonspecific promoter, the mineral-targeted alkaline phosphatase comprises tissue-nonspecific alkaline phosphatase (TNAP), the viral vector is administered to the muscle of the human subject in intramuscular injection, and wherein the cartilage disease is caused by (1) a mutation in the ALPL gene or (2) a mutation in the PHOSPHO1 gene.

2. The pharmaceutical composition according to claim 1, wherein the tissue-nonspecific promoter comprises (1) a CAG promoter, (2) a promoter having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 16, or (3) a combination of (1) or (2).

3. The pharmaceutical composition according to claim 1, wherein the viral vector comprises an adeno-associated vector.

4. The pharmaceutical composition according to claim 3, wherein the adeno-associated viral vector comprises an adeno-associated virus type 8 (AAV8) vector.

5. The pharmaceutical composition according to claim 1, wherein the mineral-targeted alkaline phosphatase further comprises a bone-targeting sequence.

6. The pharmaceutical composition according to claim 5, wherein the bone-targeting sequence is linked to the C-terminus of the TNAP.

7. The pharmaceutical composition according to claim 5, wherein the bone-targeting sequence comprises the amino acid sequence set forth in any one of SEQ ID NOs: 17 to 21.

8. The pharmaceutical composition according to claim 5, wherein the bone-targeting sequence is a decaaspartate (D10) sequence.

9. The pharmaceutical composition according to claim 1, wherein the cartilage disease is caused by a mutation in the ALPL gene.

10. The pharmaceutical composition according to claim 1, wherein the cartilage disease is caused by a mutation in the PHOSPHO1 gene.

11. The pharmaceutical composition according to claim 10, wherein the cartilage disease is caused by a deficiency of PHOSPHO1.

12. The pharmaceutical composition according to claim 1, wherein the cartilage disease is hypophosphatasia (HPP) or pseudo-HPP.

13. The pharmaceutical composition according to claim 12, wherein the cartilage disorder is said HPP.

14. The pharmaceutical composition according to claim 13, wherein the cartilage disorder is juvenile HPP or infantile HPP.

15. The pharmaceutical composition according to claim 13, wherein the cartilage disorder is late-onset HPP.

16. The pharmaceutical composition according to claim 12, wherein the cartilage disorder is pseudo-HPP.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein after administration of the viral vector, the plasma alkaline phosphatase (ALP) activity in the human subject increases for at least two months.

18. The pharmaceutical composition according to any one of claims 1 to 16, wherein after administration of the viral vector, the viral vector is not detected in the brain and / or gonads of the human subject.

19. The pharmaceutical composition according to any one of claims 1 to 16, wherein after administration of the viral vector, the viral vector or alkaline phosphatase (ALP) activity is not detected in the liver or soft tissue of the human subject for at least two months.

20. The pharmaceutical composition according to any one of claims 1 to 16, wherein after administration of the viral vector, the viral vector does not cause carcinogenesis in the human subject.

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