Topically administered TNAP to promote periodontal health

Abstract

Methods for treating periodontal disease, peri-implantitis, or preserving alveolar space in a subject are disclosed. Such methods include selecting a subject with periodontal disease, peri-implantitis, or in need of alveolar preservation, and locally administering to the periodontal tissue of the subject a therapeutically effective amount of a tissue-nonspecific alkaline phosphatase (TNAP) polypeptide or a nucleic acid molecule encoding a recombinant TNAP polypeptide. Also disclosed are methods for: i) promoting alveolar bone regeneration in a subject; ii) increasing the attachment of the periodontal ligament to the root surface of a tooth in a subject; iii) increasing cementum formation; and / or iv) increasing mineralization in a tooth in a subject. Pharmaceutical compositions for use in these methods are also disclosed.

Description

[Technical Field]

[0001] This invention was made with government support under DK121326 awarded by the National Institutes of Health. The government has certain rights in this invention. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 957,051, filed January 3, 2020, which is incorporated herein by reference in its entirety. Field of Disclosure The present application relates to the field of periodontal disease, and in particular to the use of tissue non-specific alkaline phosphatase (TNAP) polypeptides or nucleic acids encoding recombinant TNAP polypeptides to treat periodontal disease, treat peri-implantitis, or preserve the dental alveolus. [Background technology]

[0002] background Periodontal disease causes destruction of the cementum, periodontal ligament (PDL), and surrounding alveolar bone. Approximately 45% of adults in the United States suffer from periodontal disease, which, if left untreated, can lead to tooth loss (Eke et al. 2016, J Dent Res. 95(5):515-522). The goals of periodontal therapy are to eliminate pathogenic microorganisms, resolve inflammation, and restore periodontal structure and function, but current treatments for promoting tissue repair and regeneration are often suboptimal (Bosshardt 2005, J Dent Res. 84(5):390-406; Foster et al. 2007, Bone. 78:150-164; Sallum et al. 2019, Periodontol 2000. 79(1):22-55). This is especially true for cementum, a tissue characterized by limited reparative capacity. There remains a need for methods to treat periodontal disease. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Eke et al. 2016, J Dent Res. 95(5):515-522 [Non-patent document 2] Bosshardt 2005, J Dent Res. 84(5):390-406 [Non-patent document 3] Foster et al. 2007, Bone. 78:150-164 [Non-patent document 4] Sallum et al. 2019, Periodontol 2000. 79(1):22-55 Summary of the Invention [Means for solving the problem]

[0004] Disclosure Overview In some embodiments, a method for treating periodontal disease, peri-implantitis, or preserving a dental alveolus in a subject is disclosed. Such a method can include selecting a subject with periodontal disease, peri-implantitis, or in need of dental alveolar preservation, and locally administering to the periodontal tissue of the subject a therapeutically effective amount of a recombinant tissue non-specific alkaline phosphatase (TNAP) polypeptide comprising SEQ ID NO: 1, a variant thereof, or a nucleic acid molecule encoding the recombinant TNAP polypeptide.

[0005] In another embodiment, a method for treating a subject is disclosed, comprising locally administering to the periodontal tissue of the subject a therapeutically effective amount of a recombinant TNAP polypeptide comprising SEQ ID NO: 1, a variant thereof, or a nucleic acid molecule encoding the recombinant TNAP polypeptide, to i) promote alveolar bone regeneration in the subject, ii) increase periodontal ligament attachment to root surfaces of teeth in the subject, iii) increase cementum formation, and / or iv) increase mineralization in teeth in the subject.

[0006] In a further embodiment, a pharmaceutical composition is disclosed comprising a therapeutically effective amount of a recombinant TNAP polypeptide comprising SEQ ID NO: 1, a variant thereof, or a nucleic acid molecule encoding the recombinant TNAP polypeptide for use in treating periodontal disease, peri-implantitis, or in preserving the tooth socket, treating alveolar bone disease, or promoting dental implant retention.

[0007] The disclosed methods and compositions are used in subjects with periodontal disease, including subjects with hypophosphatasia (HPP) and subjects without HPP.

[0008] The foregoing and other features of the present disclosure will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0009] [Figure 1A-1B]Figures 1A-1G. Systemic delivery of TNAP-D10 restores cementum formation in Ibsp- / - mice. Mice were injected intramuscularly with TNAP-D10 lentiviral vector or PBS vehicle at 5 dpn and evaluated at 60 dpn. (A) TNAP-D10 increased plasma alkaline phosphatase (ALP) levels fivefold compared with PBS vehicle at 30 dpn, with no sustained difference at 60 dpn (n = 3 mice / group; *p < 0.05 by one-way ANOVA). (B) Compared with PBS, TNAP-D10 treatment significantly increased the thickness of both the buccal and lingual acellular cementum (AC) in Ibsp- / - mice at 60 dpn (3-fold and 2-fold, respectively), but cementum thickness remained less than that in WT mice (n = 3 mice / group; *p < 0.05, **p < 0.01 by t-test). (C) H&E staining reveals the absence of AC in the root dentin (D) and detachment of the periodontal ligament (PDL) from the root surface (*) in PBS-treated Ibsp- / - mice. TNAP-D10 reestablishes AC and PDL attachment (arrowheads) in Ibsp- / - mice. (D) Unlike the robust OPN localization in the AC of untreated control WT mice, PBS-treated Ibsp- / - mice lack OPN localization at the root surface. TNAP-D10 increases OPN at the root surface in Ibsp- / - mice. (E) In contrast to minimal DMP1 localization in the AC of WT and PBS-treated Ibsp- / - mice, TNAP-D10 increases DMP1 localization along the root surface. (F) Compared to WT, Ibsp- / - mice show increased localization of ASPN in the root-associated PDL. TNAP-D10 treatment reduces ASPN in the PDL of Ibsp- / - mice. (G) Picrosirius red staining revealed that TNAP-D10 induces PDL attachment in Ibsp- / - mice that is comparable to that in untreated control WT mice. Representative images from n = 3 mice. Scale bar: 20 μm. [Figures 1C-1G]Figures 1A-1G. Systemic delivery of TNAP-D10 restores cementum formation in Ibsp- / - mice. Mice were injected intramuscularly with TNAP-D10 lentiviral vector or PBS vehicle at 5 dpn and evaluated at 60 dpn. (A) TNAP-D10 increased plasma alkaline phosphatase (ALP) levels fivefold compared with PBS vehicle at 30 dpn, with no sustained difference at 60 dpn (n = 3 mice / group; *p < 0.05 by one-way ANOVA). (B) Compared with PBS, TNAP-D10 treatment significantly increased the thickness of both the buccal and lingual acellular cementum (AC) in Ibsp- / - mice at 60 dpn (3-fold and 2-fold, respectively), but cementum thickness remained less than that in WT mice (n = 3 mice / group; *p < 0.05, **p < 0.01 by t-test). (C) H&E staining reveals the absence of AC in the root dentin (D) and detachment of the periodontal ligament (PDL) from the root surface (*) in PBS-treated Ibsp- / - mice. TNAP-D10 reestablishes AC and PDL attachment (arrowheads) in Ibsp- / - mice. (D) Unlike the robust OPN localization in the AC of untreated control WT mice, PBS-treated Ibsp- / - mice lack OPN localization at the root surface. TNAP-D10 increases OPN at the root surface in Ibsp- / - mice. (E) In contrast to minimal DMP1 localization in the AC of WT and PBS-treated Ibsp- / - mice, TNAP-D10 increases DMP1 localization along the root surface. (F) Compared to WT, Ibsp- / - mice show increased localization of ASPN in the root-associated PDL. TNAP-D10 treatment reduces ASPN in the PDL of Ibsp- / - mice. (G) Picrosirius red staining revealed that TNAP-D10 induces PDL attachment in Ibsp- / - mice that is comparable to that in untreated control WT mice. Representative images from n = 3 mice. Scale bar: 20 μm.

[0010] [Figures 2A-2C]Figures 2A-2F. Systemic delivery of TNAP-D10 increases alveolar bone volume in Ibsp- / - mice. Mice were intramuscularly injected with TNAP-D10 lentiviral vector or PBS vehicle at 5 dpn and evaluated at 60 dpn. (A) 3D microCT rendering of the distal root of the first mandibular molar (B) and the mesial root of the second molar (C) and the surrounding alveolar bone (A). Scale bar: 500 μm. (B) TNAP-D10 significantly increases alveolar bone volume by 20% in Ibsp- / - mice, whereas (C) alveolar bone density remained unchanged (n = 3 mice; *p < 0.05 by t-test). (D) H&E staining reveals improved AB formation in TNAP-D10 vs. untreated Ibsp- / - mice. (E) IHC indicates increased OPN in the AB of Ibsp- / - vs. WT mice, with a further increase associated with TNAP-D10 treatment. (F) TNAP-D10 restores normal distribution of DMP1 in the AB of Ibsp- / - mice. Scale bar: 25 μm. [Figures 2D-2F] Figures 2A-2F. Systemic delivery of TNAP-D10 increases alveolar bone volume in Ibsp- / - mice. Mice were intramuscularly injected with TNAP-D10 lentiviral vector or PBS vehicle at 5 dpn and evaluated at 60 dpn. (A) 3D microCT rendering of the distal root of the first mandibular molar (B) and the mesial root of the second molar (C) and the surrounding alveolar bone (A). Scale bar: 500 μm. (B) TNAP-D10 significantly increases alveolar bone volume by 20% in Ibsp- / - mice, whereas (C) alveolar bone density remained unchanged (n = 3 mice; *p < 0.05 by t-test). (D) H&E staining reveals improved AB formation in TNAP-D10 vs. untreated Ibsp- / - mice. (E) IHC indicates increased OPN in the AB of Ibsp- / - vs. WT mice, with a further increase associated with TNAP-D10 treatment. (F) TNAP-D10 restores normal distribution of DMP1 in the AB of Ibsp- / - mice. Scale bar: 25 μm.

[0011] [Figure 3A-3B]Figures 3A-3G. Local delivery of rhTNAP increases the thickness of regenerated cementum. Fenestrated defects were created in 5-week-old WT and Ibsp- / - mice, and healing was analyzed on postoperative day (POD) 45. (A) Plasma ALP levels were unchanged by local delivery of rhTNAP in either WT or Ibsp- / - mice on POD 45 (n = 6 / group; p > 0.05 by independent samples t-test for pairwise comparisons within genotype). PBS: phosphate-buffered saline vehicle. (B) Compared with PBS, local delivery of rhTNAP significantly increased cementum thickness (21-fold and 42-fold, respectively) in both WT and Ibsp- / - mice on POD 45 (n = 6 mice / group; **p < 0.01, ***p < 0.001 by t-test). (C) H&E staining reveals the absence of acellular cementum (AC) in root dentin (D) and detachment of the periodontal ligament (PDL) from the root surface (*) in PBS-treated Ibsp- / - mice. rhTNAP promotes AC formation and PDL attachment (arrowheads) in Ibsp- / - mice. Thick AC formation (arrowheads) is observed in rhTNAP-treated WT mice. IHC reveals that rhTNAP increases the localization of (D) OPN and (E) DMP1 regenerated in Ibsp- / - mice, with no obvious difference between treated and untreated WT mice. (F) rhTNAP reduces ASPN localization within the periodontal ligament (PDL) of WT and Ibsp- / - mice. (G) Local delivery of rhTNAP induced PDL attachment in Ibsp- / - mice, as confirmed by picrosirius red staining. Scale bar: 200 μm. [Figure 3C-3G]Figures 3A-3G. Local delivery of rhTNAP increases the thickness of regenerated cementum. Fenestrated defects were created in 5-week-old WT and Ibsp- / - mice, and healing was analyzed on postoperative day (POD) 45. (A) Plasma ALP levels were unchanged by local delivery of rhTNAP in either WT or Ibsp- / - mice on POD 45 (n = 6 / group; p > 0.05 by independent samples t-test for pairwise comparisons within genotype). PBS: phosphate-buffered saline vehicle. (B) Compared with PBS, local delivery of rhTNAP significantly increased cementum thickness (21-fold and 42-fold, respectively) in both WT and Ibsp- / - mice on POD 45 (n = 6 mice / group; **p < 0.01, ***p < 0.001 by t-test). (C) H&E staining reveals the absence of acellular cementum (AC) in root dentin (D) and detachment of the periodontal ligament (PDL) from the root surface (*) in PBS-treated Ibsp- / - mice. rhTNAP promotes AC formation and PDL attachment (arrowheads) in Ibsp- / - mice. Thick AC formation (arrowheads) is observed in rhTNAP-treated WT mice. IHC reveals that rhTNAP increases the localization of (D) OPN and (E) DMP1 regenerated in Ibsp- / - mice, with no obvious difference between treated and untreated WT mice. (F) rhTNAP reduces ASPN localization within the periodontal ligament (PDL) of WT and Ibsp- / - mice. (G) Local delivery of rhTNAP induced PDL attachment in Ibsp- / - mice, as confirmed by picrosirius red staining. Scale bar: 200 μm.

[0012] [Figure 4A]Figures 4A-4E. Local delivery of rhTNAP increases alveolar bone regeneration. Fenestrated defects were created in 5-week-old WT and Ibsp- / - mice, and healing was analyzed on postoperative day (POD) 45. (A) 3D microCT rendering of the distal (B) and mesial (C) roots of the first mandibular molar and the surrounding alveolar bone (A). Scale bar: 500 µm. (B) rhTNAP increases regenerated alveolar bone volume by 37% and (C) bone mineral density by 5% in Ibsp- / - mice (n = 6 mice / group; *p < 0.05; **p < 0.01; ***p < 0.001 by t-test). Compared to PBS treatment, rhTNAP increases alveolar bone volume and bone mineral density by 17% and 5%, respectively, in WT mice. (D, E) IHC reveals that rhTNAP increases OPN and DMP1 in the regenerated alveolar bone of both WT and Ibsp- / - mice. Scale bar: 200 μm. [Figure 4B-4C] Figures 4A-4E. Local delivery of rhTNAP increases alveolar bone regeneration. Fenestrated defects were created in 5-week-old WT and Ibsp- / - mice, and healing was analyzed on postoperative day (POD) 45. (A) 3D microCT rendering of the distal (B) and mesial (C) roots of the first mandibular molar and the surrounding alveolar bone (A). Scale bar: 500 µm. (B) rhTNAP increases regenerated alveolar bone volume by 37% and (C) bone mineral density by 5% in Ibsp- / - mice (n = 6 mice / group; *p < 0.05; **p < 0.01; ***p < 0.001 by t-test). Compared to PBS treatment, rhTNAP increases alveolar bone volume and bone mineral density by 17% and 5%, respectively, in WT mice. (D, E) IHC reveals that rhTNAP increases OPN and DMP1 in the regenerated alveolar bone of both WT and Ibsp- / - mice. Scale bar: 200 μm. [Figure 4D-4E]Figures 4A-4E. Local delivery of rhTNAP increases alveolar bone regeneration. Fenestrated defects were created in 5-week-old WT and Ibsp- / - mice, and healing was analyzed on postoperative day (POD) 45. (A) 3D microCT rendering of the distal (B) and mesial (C) roots of the first mandibular molar and the surrounding alveolar bone (A). Scale bar: 500 µm. (B) rhTNAP increases regenerated alveolar bone volume by 37% and (C) bone mineral density by 5% in Ibsp- / - mice (n = 6 mice / group; *p < 0.05; **p < 0.01; ***p < 0.001 by t-test). Compared to PBS treatment, rhTNAP increases alveolar bone volume and bone mineral density by 17% and 5%, respectively, in WT mice. (D, E) IHC reveals that rhTNAP increases OPN and DMP1 in the regenerated alveolar bone of both WT and Ibsp- / - mice. Scale bar: 200 μm.

[0013] [Figures 5A-5D]Figures 5A-5H. bIAP increases mineralization in cementoblasts. WT and Ibsp- / - OCCM.30 immortalized mouse cementoblasts were used to examine the effect of bovine intestinal alkaline phosphatase IAP (bIAP) on cell expression and mineralization in vitro. (A) Alpl expression is reduced by 80% in Ibsp- / - versus WT cells on day 1 (**p<0.01). (B) Compared to medium from untreated WT controls, untreated Ibsp- / - cells exhibit a 40% reduction in Pi levels. Addition of 100 μg / ml bIAP increases Pi levels in both WT and Ibsp- / - cell medium (different letters, p<0.01). (C) Compared to WT cells, Ibsp- / - cells exhibit less mineralization. Addition of bIAP increases mineralization in both WT and Ibsp- / - cells. Mineral deposited by bIAP-treated Ibsp- / - cells lags behind that of untreated WT cells by day 6 (different letters p<0.01). (D) Addition of 0.1 mM phosphonoformic acid (PFA) inhibits the bIAP-induced increase in mineral nodule formation in both WT and Ibsp- / - cells, but the effect of PFA in Ibsp- / - cells is minimal, and mineral deposition by WT and Ibsp- / - cells treated with bIAP and PFA is comparable. (E) bIAP increases Spp1 expression in both control and Ibsp- / - cells. PFA inhibits Spp1 expression by 55% in both WT and Ibsp- / - cells (different letters p<0.001). (F) bIAP increases Dmp1 expression in both WT and Ibsp- / - cells versus untreated cells. Phosphate-induced Dmp1 expression in WT and Ibsp- / - cells is partially inhibited by PFA (different letters indicate p<0.001). (G) Aspn expression is increased in Ibsp- / - cells versus WT cells. bIAP reduces Aspn expression in both WT and Ibsp- / - cells (different letters indicate p<0.01). Pi-induced downregulation of Aspn expression is not affected by PFA. (H) Alpl expression in Ibsp- / - cells was significantly reduced by 37% by bIAP (p<0.0001), consistent with the trend in WT cells.In WT and Ibsp- / - cells, PFA tended to increase Alpl expression. [Figures 5E-5H]Figures 5A-5H. bIAP increases mineralization in cementoblasts. WT and Ibsp- / - OCCM.30 immortalized mouse cementoblasts were used to examine the effect of bovine intestinal alkaline phosphatase IAP (bIAP) on cell expression and mineralization in vitro. (A) Alpl expression is reduced by 80% in Ibsp- / - versus WT cells on day 1 (**p<0.01). (B) Compared to medium from untreated WT controls, untreated Ibsp- / - cells exhibit a 40% reduction in Pi levels. Addition of 100 μg / ml bIAP increases Pi levels in both WT and Ibsp- / - cell medium (different letters, p<0.01). (C) Compared to WT cells, Ibsp- / - cells exhibit less mineralization. Addition of bIAP increases mineralization in both WT and Ibsp- / - cells. Mineral deposited by bIAP-treated Ibsp- / - cells lags behind that of untreated WT cells by day 6 (different letters p<0.01). (D) Addition of 0.1 mM phosphonoformic acid (PFA) inhibits the bIAP-induced increase in mineral nodule formation in both WT and Ibsp- / - cells, but the effect of PFA in Ibsp- / - cells is minimal, and mineral deposition by WT and Ibsp- / - cells treated with bIAP and PFA is comparable. (E) bIAP increases Spp1 expression in both control and Ibsp- / - cells. PFA inhibits Spp1 expression by 55% in both WT and Ibsp- / - cells (different letters p<0.001). (F) bIAP increases Dmp1 expression in both WT and Ibsp- / - cells versus untreated cells. Phosphate-induced Dmp1 expression in WT and Ibsp- / - cells is partially inhibited by PFA (different letters indicate p<0.001). (G) Aspn expression is increased in Ibsp- / - cells versus WT cells. bIAP reduces Aspn expression in both WT and Ibsp- / - cells (different letters indicate p<0.01). Pi-induced downregulation of Aspn expression is not affected by PFA. (H) Alpl expression in Ibsp- / - cells was significantly reduced by 37% by bIAP (p<0.0001), consistent with the trend in WT cells.In WT and Ibsp- / - cells, PFA tended to increase Alpl expression.

[0014] [Figure 6] Figure 6. Periodontal fenestration defects (approximately 2 x 1.5 x 0.5 mm) were created by removal of alveolar bone at the buccal aspect of the distal root of the first mandibular molar and the mesial root of the second mandibular molar. Local delivery included 0.1 μg (in 1 μl) recombinant human tissue nonspecific alkaline phosphatase (rhTNAP) or a collagen scaffold with 1 μl phosphate-buffered saline (PBS) vehicle.

[0015] [Figure 7] Figure 7. Picrosirius red staining of intact mandibular periodontal tissue (distal root of first molar) in a WT mouse. PDL attachment was clearly observed in the intact periodontal tissue of a WT mouse (11 weeks of age, the same age as the surgical model). Scale bar: 200 μm.

[0016] [Figure 8] Figure 8. PFA did not affect bIAP-induced medium Pi concentrations in WT or Ibsp- / - cementoblasts. Pi levels were examined on day 1. Medium from untreated Ibsp- / - cells exhibited 80% lower Pi levels than untreated WT cells. Addition of 100 μg / ml bovine intestinal alkaline phosphatase (bIAP) increased Pi levels in both WT and Ibsp- / - cell medium (14-fold and 43-fold, respectively), as noted in Figure 5A. The increase in Pi levels by bIAP was not affected by the addition of 0.1 mM phosphonoformic acid (PFA; indicated by a P in the graph) in both WT and Ibsp- / - cells (n = 4 wells / treatment). Experimental groups marked with different upper / lowercase letters are significantly different (p < 0.0001 by one-way ANOVA), whereas groups sharing the same upper / lowercase letter are not different (p > 0.05). The results were reproduced in three independent experiments. DETAILED DESCRIPTION OF THE INVENTION

[0017] Sequence Listing The nucleic acid and amino acid sequences listed in the accompanying Sequence Listing are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids, as defined in Title 37 of the Code of Federal Regulations, Section 1.822 (37 C.FR 1.822). Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by reference to either of the displayed strands. The Sequence Listing is submitted as an ASCII text file [Sequence_Listing, December 31, 2020, 25.7 KB], which is incorporated herein by reference. In the accompanying Sequence Listing, the following are shown: SEQ ID NO: 1 is the amino acid sequence of a recombinant TNAP polypeptide, which is amino acids 18 to 504 of the human TNAP protein. SEQ ID NO: 2 is the amino acid sequence of the signal peptide. SEQ ID NO: 3 is the amino acid sequence of the histidine tag. SEQ ID NO:4 is the amino acid sequence of a recombinant TNAP polypeptide having a mineral binding domain. SEQ ID NO:5 is the amino acid sequence of a recombinant TNAP polypeptide without the mineral binding domain. SEQ ID NOs: 1 and 7 are the amino acid sequences of the signal peptides. SEQ ID NOs: 8 to 17 are primer sequences. SEQ ID NOs: 18 and 19 are alternative recombinant TNAP polypeptides. SEQ ID NO: 20 is the amino acid sequence of the signal peptide.

[0018] Detailed Description of Some Embodiments Recombinant TNAP polypeptides and methods for treating periodontal disease using nucleic acids encoding such TNAP polypeptides are disclosed. Such methods are useful in subjects with periodontal disease, including those with or without hypophosphatasia (HPP).

[0019] Delivery of recombinant TNAP polypeptide inhibits the growth of pyrophosphate (PP), a potent inhibitor of hydroxyapatite crystal growth.i ), and inorganic phosphate (P), a promoter of hydroxyapatite (HA) formation. i It has been disclosed that a pharmacological approach to increase TNAP can correct cementum defects and promote cementum regeneration in a mouse model of periodontal disease. In some embodiments, a lentiviral construct expressing mineral-targeted TNAP was used for systemic delivery (Yamamoto et al. 2011, J Bone Miner Res. 26(1):135-142). However, other viral vectors, such as AAV, can also be used. In addition, recombinant human TNAP was used for local delivery to treat periodontal fenestration defects in a mouse model (Rodrigues et al. 2011, J Periodontol. 82(12):1757-1766). A cementoblast cell line was also used to demonstrate the effects of bovine intestinal ALP (bIAP) on gene expression and mineralization in vitro.

[0020] Terminology Overview Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology can be found in Krebs et al. (eds.), Lewin's genes XII, published by Jones & Bartlett Learning, 2017. As used herein, unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" refer to both the singular and the plural. For example, the term "a polypeptide" includes singular or plural antigens and can be considered equivalent to the phrase "at least one polypeptide." As used herein, the term "comprises" means "includes."

[0021] It should be further understood that unless otherwise indicated, any and all base sizes or amino acid sizes and any molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for illustrative purposes. Although many methods and materials similar or equivalent to those described herein can be used, certain suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will prevail. Additionally, the materials, methods, and examples are merely illustrative and are not intended to be limiting. To facilitate review of various embodiments, the following explanations of terms are provided:

[0022] The term "about" denotes a range of + / -10% of a given value. For example, "about 10" indicates a range of 90% to 110% of 10, i.e., 9 to 11.

[0023] The term "administration" refers to the introduction of a composition into a subject by a selected route. Administration can be local or systemic. For example, if the selected route is intravenous, the composition is administered by introducing the composition into the subject's vein. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraosseous, intraperitoneal, and intravenous), sublingual, buccal, transmucosal, transdermal (e.g., topical), intranasal, and inhalation routes. In some embodiments, the composition is administered locally to the periodontal tissue of the subject.

[0024] The term "alkaline phosphate (ALP)" or "tissue non-specific alkaline phosphatase (TNAP, TNALP)" as used herein refers to an enzyme capable of dephosphorylating compounds. Also known as "basic phosphatase," ALP is a homodimeric protein enzyme optimally active in alkaline pH environments. This enzyme is found across a wide range of organisms, from prokaryotes such as bacteria to eukaryotes such as animal cells. An exemplary human TNAP protein sequence is provided in NCBI Reference Sequence NP_001356734.1, available on December 12, 2020, which is incorporated herein by reference, and the corresponding coding sequence is provided in NM_001369805.2. An exemplary mouse TNAP protein sequence is provided in NCBI Reference Sequence NP_031457.2, available on November 17, 2020, which is incorporated herein by reference.

[0025] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that have been modified later, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an α-carbon bonded to a hydrogen atom, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. "Amino acid mimetics" refer to chemical compounds that have a structure different from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids.

[0026] A variety of methods allow for the incorporation of unnatural amino acid derivatives or analogs into polypeptide chains in a site-specific manner, see, for example, WO02 / 086075.

[0027] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0028] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to nucleic acids that encode identical or essentially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also represents every possible silent variation of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0029] With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences that alter, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants," in that the alteration results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles.

[0030] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine ​​(C), Methionine (M) (See, e.g., Creighton, Proteins, W.H. Freeman and Co., NY (1984)).

[0031] In this application, amino acid residues are numbered according to their relative position in the unmodified (eg, wild-type) polypeptide sequence, starting from the left-most residue, which is numbered 1.

[0032] The term "control" refers to a reference standard, such as a biological sample from a subject that is untreated (e.g., not treated with a recombinant TNAP polypeptide or nucleic acid coding sequence provided herein) or that is treated with a substance known to be inert, such as a carrier.

[0033] The difference between the test sample and the control can be an increase or a decrease.The difference can be a qualitative difference or a quantitative difference, for example, a statistically significant difference.In some examples, the difference is an increase or decrease of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400% or at least about 500%, compared to the control.

[0034] The terms "effective amount" or "therapeutically effective amount," as used herein, refer to an amount sufficient to produce the intended effect for which the substance is administered. The effect can include a desired change in a biological process (e.g., improved regeneration of the dental root [cementum] or alveolar bone, or improved attachment of new periodontal ligament to the surrounding alveolar bone and / or dental root), as well as prevention, correction, or inhibition, to any detectable extent, of the progression of symptoms of the disease / condition and associated complications. The precise amount "effective" to achieve the desired effect depends on the nature of the therapeutic agent, the mode of administration, and the purpose of treatment, and can be ascertained by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms [vols. 1-3, 1992]; Lloyd, The Art, Science and Technology of Pharmaceutical Compounding

[1999] ; and Pickar, Dosage Calculations

[1999] ).

[0035] The term "expression" refers to the transcription or translation of a nucleic acid sequence, such as a TNAP sequence. For example, a coding nucleic acid sequence (such as a gene) can be expressed when its DNA is transcribed into RNA or an RNA fragment, which in some cases is processed into mRNA. A coding nucleic acid sequence (such as a gene) can also be expressed when its mRNA is translated into an amino acid sequence, such as a protein or protein fragment. In certain examples, a heterologous gene is expressed when it is transcribed into RNA. In other examples, a heterologous gene is expressed when its RNA is translated into an amino acid sequence. Regulation of expression can include control over transcription, translation, RNA transport and processing, degradation of intermediate molecules such as mRNA, or by activation, inactivation, compartmentalization, or degradation of a specific protein molecule after it is produced.

[0036] The term "expression control sequence" refers to a nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence (such as a TNAP sequence) to which it is operably linked. An expression control sequence is operably linked to a nucleic acid sequence if it controls and regulates the transcription and, if appropriate, translation of the nucleic acid sequence. Thus, an expression control sequence can include an appropriate promoter, enhancer, transcription terminator, a start codon (ATG) in front of a protein-coding gene, splice signals for introns (maintaining the correct reading frame of the gene to allow proper translation of mRNA), and a stop codon. The term "control sequence" includes, at a minimum, components whose presence can affect expression, and can also include additional components, such as leader sequences and fusion partner sequences. In one example, an expression control sequence is a promoter.

[0037] An "expression cassette" is a recombinantly or synthetically produced nucleic acid construct that has a series of specified polynucleotide elements that allow for transcription of a particular polynucleotide sequence (such as a TNAP sequence) in a host cell. An expression cassette can be part of a plasmid, a viral genome, or a nucleic acid fragment. Typically, an expression cassette contains the polynucleotide to be transcribed operably linked to a promoter.

[0038] The term "gene" refers to a segment of DNA involved in producing a polypeptide chain (e.g., a modified alkaline phosphatase disclosed herein). In some cases, this can include regions preceding and following the coding region (leader and trailer), as well as intervening sequences (introns) between individual coding segments (exons).

[0039] The term "heterologous," when used in the context of describing the relative positions of two elements, refers to two elements, such as polynucleotide sequences (e.g., promoters or protein / polypeptide coding sequences) or polypeptide sequences (e.g., recombinant TNAP polypeptides described herein), that are not naturally found in the same relative positions. Thus, a "heterologous promoter" of a gene refers to a promoter that is not operably linked to that gene in nature. Similarly, a "heterologous polypeptide" or "heterologous polynucleotide" with respect to a first polypeptide or polynucleotide is a polypeptide or polynucleotide that (1) is derived from a source different from that of the first polypeptide or polynucleotide, or (2) is derived from the same source but is not naturally connected to the first polypeptide or polynucleotide in the same manner that, when fused with the first polypeptide or polynucleotide, produces a longer, naturally occurring polypeptide or polynucleotide sequence.

[0040] "Gingiva" is a tissue that is the masticatory mucosa lining the alveolar bone surrounding the tooth neck. Gingival tissue provides a seal around the tooth with junctional epithelium and epithelial attachments to resist mechanical trauma and protect against microorganisms.

[0041] By "host cell" is meant a cell that contains an expression vector and supports the replication or expression of the expression vector, e.g., to produce a recombinant polypeptide (such as a TNAP polypeptide). Host cells can be prokaryotic cells such as E. coli, such as CHO, HeLa, etc., including cultured cells, explants, and cells in vivo, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells.

[0042] A "label," "detectable label," or "detectable moiety" is a composition detectable by radiological, spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include: 32 These include radioisotopes such as P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in ELISA), biotin, digoxigenin, or haptens and proteins that can be made detectable, for example, by incorporating a radioactive component into a polypeptide or can be used to detect antibodies specifically reactive with a polypeptide. Typically, a detectable label is a heterologous moiety attached to a probe or molecule (e.g., a protein or nucleic acid) with defined binding characteristics (e.g., a polypeptide or polynucleotide with known binding specificity) such that the presence of the probe / molecule (and thus its binding target) can be readily detected. The heterologous nature of the label ensures that the probe / molecule attached to the detectable label has an origin different from that of the probe or molecule it labels, such that it does not constitute a naturally occurring composition.

[0043] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof, in either single-stranded or double-stranded form. Unless otherwise specified, this term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, along with the sequence explicitly indicated. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA encoded by a gene, and mRNA.

[0044] The term "operably linked" refers to a first nucleic acid sequence being operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter, such as a CMV promoter, is operably linked to a coding sequence (such as a TNAP sequence) if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0045] The "pharmaceutically acceptable carrier(s)" used are conventional. Remington: The Science and Practice of Pharmacy, 22 nded., London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed agents, such as TNAP polypeptides or coding sequences.

[0046] Generally, the nature of the carrier will depend on the particular mode of administration being used. For example, parenteral formulations usually contain an injectable fluid containing pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solution, dextrose in water, glycerol, or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, added preservatives (such as non-natural preservatives), and pH buffering agents, e.g., sodium acetate or sorbitan monolaurate. In certain examples, the pharmaceutically acceptable carrier is sterile and suitable for parenteral administration to a subject, for example, by injection. In some embodiments, the active agent and pharmaceutically acceptable carrier are provided in unit dosage form, such as a pill, or in a selected amount in a vial. A unit dosage form can contain a single dose or multiple doses (eg, in a vial from which a metered dose of agent can be selectively dispensed).

[0047] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acid, as well as naturally occurring and non-naturally occurring amino acid polymers. As used herein, this term encompasses amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0048] A "promoter" is defined as a set of nucleic acid control sequences that direct the transcription of a polynucleotide sequence. As used herein, a promoter includes necessary polynucleotide sequences near the start site of transcription, such as a TATA element in the case of a polymerase II type promoter. A promoter also includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription, as needed. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to a functional linkage between a polynucleotide expression control sequence (such as a promoter or a set of transcription factor binding sites) and a second polynucleotide sequence, where the expression control sequence directs the transcription of the polynucleotide sequence corresponding to the second sequence.

[0049] The term "peri-implantitis" refers to a destructive inflammatory process that affects the soft and hard tissues around a dental implant. The soft tissue becomes inflamed, while the alveolar bone (hard tissue) that surrounds the implant for retention purposes is lost over time. Peri-implantitis is characterized by the absence of erythema, bleeding on probing, swelling, and suppuration.

[0050] "Periodontitis" refers to an acute or chronic inflammatory oral disease that progressively destroys the structures that support the teeth. This usually manifests as worsening gingivitis, followed by loosening and loss of teeth if left untreated. Systemic conditions such as heart disease, respiratory disease, and uncontrolled diabetes are associated with periodontitis. Severe periodontitis, including necrotizing periodontal disease, is commonly observed in immunocompromised patients, such as those with HIV. Diagnosis is based on physical examination, periodontal probing, and x-rays.

[0051] Periodontitis usually develops when gingivitis, usually caused by a large amount of plaque and calculus (calculus composed of bacteria, food debris, saliva, and mucus, as well as calcium and phosphate salts) below the gum line, is not properly treated. In periodontitis, deep pockets form in the periodontal tissues and can harbor anaerobic organisms that cause more damage than those typically present in simple gingivitis. Colonizing organisms include, but are not limited to, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Eikenella corrodens, and many of the bacteria that form the oral microbiome and biofilm.

[0052] These organisms induce the chronic release of inflammatory mediators, including cytokines, prostaglandins, and enzymes, from neutrophils and monocytes. The resulting inflammation affects the periodontal ligament, gingiva, cementum, and alveolar bone. The periodontal ligament progressively loses its attachment to the teeth, bone loss begins, and periodontal pockets deepen. With progressive bone loss, teeth can loosen and the gums recede. Tooth movement is common in later stages, and tooth loss can occur.

[0053] The term "promoting periodontal health" or any variation thereof encompasses any detectable improvement or enhancement in the regeneration of tooth roots (cementum), regeneration of alveolar bone, or attachment of new PDL to the surrounding alveolar bone and / or to the tooth roots (i.e., cementum). Such improvement or enhancement can occur in preventative as well as therapeutic contexts, for example, in regular (e.g., daily, weekly, or monthly, etc.) use by a person at risk of later developing periodontal disease, or in the process of preparing for or undergoing a dental implant.

[0054] The terms "purified" or "isolated" do not require absolute purity; rather, the terms are intended as relative terms. Thus, for example, a purified peptide preparation is one in which a peptide or protein (such as a TNAP polypeptide) is enriched relative to the peptide or protein in its natural environment within a cell. In one embodiment, a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation. A biological component (a nucleic acid, peptide, protein, or protein complex, such as a TNAP polypeptide) is isolated when it is substantially separated from, produced separately from, or purified away from other biological components, i.e., other chromosomal and extrachromosomal DNA and RNA and proteins, in the cells of an organism in which it naturally occurs. Thus, isolated nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids and proteins. An isolated nucleic acid, peptide or protein, e.g., a TNAP polypeptide, can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% pure.

[0055] The term "recombinant," when used with reference to, for example, a cell or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or by alteration of the native nucleic acid or protein, such as by truncation or substitution. Thus, for example, a recombinant cell expresses genes not found in the native (non-recombinant) form of the cell, or expresses native genes that are otherwise aberrantly expressed, under-expressed, or not expressed at all. Recombinant TNAP polypeptides are disclosed herein.

[0056] The term "sequence identity" refers to the identity between two or more nucleic acid sequences or two or more amino acid sequences, and is expressed in terms of the identity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the higher the sequence identity. Homologs and variants of TNAP polypeptides are typically characterized by possessing at least about 75% sequence identity, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, counted over the entire length of the amino acid sequence of interest.

[0057] Methods of alignment of sequences for comparison are well known. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2(4):482-489, 1981; Needleman and Wunsch, J. Mol. Biol. 48(3):443-453, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85(8):2444-2448, 1988; Higgins and Sharp, Gene, 73(1):237-244, 1988; Higgins and Sharp, Bioinformatics, 5(2):151-3, 1989; Corpet, Nucleic Acids Res. 16(22):10881-10890, 1988; Huang et al. Bioinformatics, 8(2):155-165, 1989. 1992; and Pearson, Methods Mol. Biol. 24:307-331, 1994. Altschul et al., J. Mol. Biol. 215(3):403-410, 1990, presents a detailed discussion of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215(3):403-410, 1990) is available from several sources, including the National Center for Biological Information and on the Internet, for use with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. blastn is used to compare nucleic acid sequences, while blastp is used to compare amino acid sequences. Further information can be found on the NCBI website.

[0058] Generally, when two sequences are aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue occurs in both sequences. The percent sequence identity between the two sequences is determined by dividing the number of matches by either the length of the sequence shown in the identified sequence or by the explicit length (such as 100 consecutive nucleotides or amino acid residues from the sequence shown in the identified sequence), and then multiplying the resulting value by 100.

[0059] A "subject" is a living multi-cellular vertebrate organism, a category that includes humans and non-human mammals. In some examples, the subject is a mammal, such as a human. In some examples, the mammal is a veterinary subject, such as a horse, pig, cat, or dog.

[0060] The "socket" is the hole in the bone where the tooth was removed. After the tooth is extracted, a blood clot forms in the socket to protect the bone and nerves underneath. The blood clot may become dislodged or dissolve 2-3 days after the tooth is extracted, which is called a "dry socket" or "alveolitis."

[0061] A "transformed" cell is a cell into which a nucleic acid molecule has been introduced by molecular biological techniques. As used herein, the term transformed and the like (e.g., transformation, transfection, transduction, etc.) encompasses any technique by which a nucleic acid molecule (such as a TNAP coding sequence) can be introduced into such a cell, including transduction with a viral vector, transformation with a plasmid vector, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.

[0062] A "vector" refers to an entity containing a nucleic acid molecule (such as a DNA or RNA molecule) operatively linked to a coding sequence for a protein of interest (such as the TNAP coding sequence) and having a promoter(s) capable of expressing the coding sequence. Non-limiting examples include naked or packaged (lipid and / or protein) DNA, naked or packaged RNA, a subcomponent of a virus or bacteria or other microorganism that may be replication-incompetent, or a virus or bacteria or other microorganism that may be replication-competent. A vector is sometimes referred to as a construct. A recombinant DNA vector is a vector that contains recombinant DNA. A vector can contain a nucleic acid sequence that enables it to replicate in a host cell, such as an origin of replication. A vector can also contain one or more selectable marker genes and other genetic elements known in the art. A viral vector is a recombinant nucleic acid vector that contains at least some nucleic acid sequences derived from one or more viruses. In one example, the viral vector is a lentiviral vector. In another example, the viral vector is an adeno-associated viral vector.

[0063] In this disclosure, the term "or" is generally used in its sense including "and / or" unless the content clearly dictates otherwise.

[0064] A. Recombinant TNAP Polypeptide In some embodiments, the recombinant TNAP polypeptide used in the disclosed methods comprises the amino acid sequence of SEQ ID NO:1: LVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANEGTVGVSAATERSRC NTTQGNEVTSILRWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKNKTDVEYESDEKARGTRLDGLDLVDTWKSFKP RYKHSHFIWNRTELLTLDPHNVDYLLGLFEPGDMQYELNRNNVTDPSLSEMVVVAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHEAVEMDRAIGQAGSLTSSEDTLTVVTADHSHVF TFGGYTPRGNSIFGLAPMLSDTDKKPFTAILYGNGPGYKVVGGERENVSMVDYAHNNYQAQSAVPLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHVMAYAACIGANLGHCAPASSAGS This sequence corresponds to amino acids 18 to 505 of the human TNAP protein as provided in the NCBI reference sequence NP_001356734.1, available on December 12, 2020, which is incorporated herein by reference.

[0065] In some embodiments, the recombinant TNAP polypeptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 1. Homologs and variants, such as polypeptides that are about 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 1, are also used. In some embodiments, a polypeptide that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1, is used in the methods disclosed herein, wherein the polypeptide i) promotes alveolar bone regeneration in a subject; ii) increases periodontal ligament attachment to the root surface of a tooth in a subject; iii) increases cementum formation in a subject; and / or iv) increases mineralization in a tooth in a subject. In further embodiments, the polypeptide comprises at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 conservative substitutions in SEQ ID NO: 1, and the polypeptide i) promotes alveolar bone regeneration in a subject; ii) increases periodontal ligament attachment to the root surface of a tooth in the subject; iii) increases cementum formation in the subject; and / or iv) increases mineralization in a tooth in the subject.

[0066] The locations of functional domains in the TNAP protein are disclosed, for example, in J Biol Chem. 2001 Aug 17;276(33):31171-8. doi: 10.1074 / jbc.M102788200, which provides the locations and functions of domains in the TNAP protein and is incorporated herein by reference. Five regions are known in human TNAP. The TNAP structure was investigated using mutations associated with hypophosphatasia (HPP) and the consequences of these mutations on the activity or structure of the enzyme. Mutations are clustered within five regions: the active site and its vicinity, the active site valley, the homodimer interface, the crown domain, and the metal-binding site. The crown domain and metal-binding domain are mammalian-specific. The crown domain contains the collagen-binding loop. Synchrotron radiation X-ray fluorescence studies confirmed that the metal in the metal-binding site is a calcium ion, which may be important for TNAP function (Mornet et al., J Biol Chem. 2001 Aug 17;276(33):31171-8. doi: 10.1074 / jbc.M102788200; Kozlenkov et al., J Biol Chem. 2002 Jun 21;277(25):22992-9. doi: 10.1074 / jbc.M202298200, incorporated herein by reference). In addition, residue 108 in TNAP determines the specificity of inhibition by L-homoarginine (L-hArg), and the conserved Tyr-371 is also required for L-hArg binding. Binding of levamisole to TNAP was largely dependent on His-434 and Tyr-371, but not on residues 108 or 109, and the major determinant of sensitivity to theophylline was His-434 (Kozlenkov et al., J Bone Miner Res. 2004 Nov;19(11):1862-72. doi: 10.1359 / JBMR.040608, incorporated herein by reference).See Mornet et al., J Biol Chem. 2001 Aug 17;276(33):31171-8. doi: 10.1074 / jbc.M102788200, incorporated herein by reference, which provides structural evidence of the functional role of human tissue-nonspecific alkaline phosphatase in bone mineralization. See also Kozlenkov et al., J Biol Chem. 2002 Jun 21;277(25):22992-9. doi: 10.1074 / jbc.M202298200, incorporated herein by reference, and Kozlenkov et al., J Bone Miner Res. 2004 Nov;19(11):1862-72. doi: 10.1359 / JBMR.040608, incorporated herein by reference, which identify residues that determine the binding specificity of uncompetitive inhibitors for tissue-nonspecific alkaline phosphatase. Thus, in some embodiments, one or more of these domains / residues are maintained in the variant. In further embodiments, substitutions are made outside the domain required for TNAP activity.

[0067] In some embodiments, the recombinant TNAP polypeptide further comprises 4 to 12 aspartic acid residues at the C-terminus or 4 to 12 glutamic acid residues at the C-terminus. In additional embodiments, the recombinant TNAP polypeptide further comprises 4 to 10 aspartic acid residues at the C-terminus or 4 to 10 glutamic acid residues at the C-terminus. In still other embodiments, the recombinant TNAP polypeptide further comprises 6 to 8 aspartic acid residues at the C-terminus or 6 to 8 glutamic acid residues at the C-terminus. In still other embodiments, the recombinant TNAP polypeptide comprises 4, 5, 6, 7, 8, 9, 10, 11, or 12 aspartic acid residues (e.g., 4, 5, 6, 7, 8, 9, 10, 11, or 12 aspartic acid residues added to the C-terminal amino acid of SEQ ID NO: 1). In additional embodiments, the recombinant TNAP polypeptide comprises 4, 5, 6, 7, 8, 9, 10, 11, or 12 glutamic acid residues (e.g., 4, 5, 6, 7, 8, 9, 10, 11, or 12 glutamic acid residues added to the C-terminal amino acid of SEQ ID NO: 1). In some non-limiting examples, the recombinant TNAP polypeptide further comprises 4-12 aspartic acid residues at the C-terminus. In other non-limiting examples, the recombinant TNAP polypeptide further comprises 4-10 aspartic acid residues at the C-terminus. In even further non-limiting examples, the recombinant TNAP polypeptide further comprises 6-8 aspartic acid residues at the C-terminus.

[0068] In additional embodiments, the recombinant TNAP polypeptide comprises a stretch of aspartic acid at the C-terminus, such as 4-10 aspartic acid residues at the C-terminus or 4-10 glutamic acid residues at the C-terminus. In yet other embodiments, the recombinant TNAP polypeptide further comprises 6-8 aspartic acid residues. Thus, the recombinant TNAP polypeptide can comprise 4, 5, 6, 7, 8, 9, 10, 11, or 12 aspartic acid residues.

[0069] In still other embodiments, the recombinant TNAP polypeptide does not contain more than one aspartic acid or glutamic acid residue at the C-terminus. In further embodiments, the recombinant TNAP polypeptide does not contain any aspartic acid or glutamic acid residues at the C-terminus. In these embodiments, the recombinant TNAP polypeptide does not contain repeated aspartic acid or glutamic acid residues at the C-terminus.

[0070] In some embodiments, the recombinant TNAP polypeptide includes a signal peptide at its N-terminus (e.g., appended to the N-terminal amino acid of SEQ ID NO: 1). The signal peptide sequence can include any suitable signal peptide sequence. In one embodiment, the signal peptide sequence is MRGPAVLLTVALATLLAPGAGA (SEQ ID NO: 2). However, other signal peptides are also used, such as a mouse immunoglobulin light chain kappa signal sequence, such as an amino acid sequence comprising or consisting of MDFQVQIFSFLLISASVIMSRG (SEQ ID NO: 6). Other signal sequences known in the art can be utilized. In another example, the signal peptide sequence is a human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor sequence, such as an amino acid sequence comprising or consisting of LLVTSLLLCELPHPAFLLIPDT (SEQ ID NO: 7). In a further example, the signal peptide sequence is an IL-2 signal peptide. In another example, the signal peptide is MTRLTVLALLAGLLASSRA (SEQ ID NO: 20).

[0071] Although a signal peptide sequence can facilitate expression of a recombinant TNAP polypeptide, the presence of a signal peptide sequence is not required for TNAP function. After expression of the TNAP polypeptide, the signal peptide sequence may be cleaved off from the recombinant TNAP polypeptide if it is produced synthetically. If the protein is secreted in vitro, the signal sequence may be cleaved off by the cell. Thus, in some embodiments, the TNAP polypeptide lacks a signal peptide sequence.

[0072] In some embodiments, the recombinant TNAP polypeptide comprises a histidine tag. In some embodiments, the histidine tag comprises at least 6 histidines. In other embodiments, the histidine tag comprises 6-12 histidines, such as 6, 7, 8, 9, 10, 11, or 12 histidines. The histidine tag can comprise 8-12, e.g., 9-11, e.g., 10 histidines. The histidine tag can be at the N-terminus or C-terminus of the TNAP polypeptide. Thus, in specific, non-limiting examples, a) the TNAP polypeptide comprises a histidine tag N-terminal to SEQ ID NO: 1; or b) the TNAP polypeptide comprises a histidine tag C-terminal to SEQ ID NO: 1. In additional embodiments, the histidine tag is [ka] In specific, non-limiting examples, a) the TNAP polypeptide comprises SEQ ID NO:3 so that it is N-terminal to SEQ ID NO:1; or b) the TNAP polypeptide comprises SEQ ID NO:3 so that it is C-terminal to SEQ ID NO:1.

[0073] In this embodiment, the bolded sequence is a TEV cleavage sequence. TEV protease can cleave this protein between Q (shown in bold) and G (shown in bold), such that the N-terminal portion is removed. In some embodiments, a histidine tag is not included. In some embodiments, a GM is retained at either the C- or N-terminus of a TNAP polypeptide, such as one comprising the amino acid sequence of SEQ ID NO: 1.

[0074] In some embodiments, the TNAP polypeptide comprises residues GM at either the N- or C-terminus. In some non-limiting examples, the TNAP polypeptide comprises GM C-terminally relative to SEQ ID NO: 1. In other non-limiting examples, the TNAP polypeptide comprises GM N-terminally relative to SEQ ID NO: 1.

[0075] In additional embodiments, the recombinant TNAP polypeptide comprises, in order from N- to C-terminus, a signal peptide, a histidine tag, and amino acids 18-504 of TNAP or a homolog or variant thereof. In other embodiments, a recombinant TNAP polypeptide comprising a signal peptide and a histidine tag is synthesized, but the signal peptide is then removed for use. In further embodiments, a recombinant TNAP polypeptide comprising a signal peptide and a histidine tag is synthesized, but at least a portion of the signal peptide and the histidine tag are removed for use.

[0076] In yet other embodiments, the recombinant TNAP polypeptide is [ka] In this sequence, the signal peptide is underlined, the histidine tag is shown in bold, and the C-terminal aspartic acid is shown in italics. Additional aspartic acid residues may be included at the C-terminus, such as 1 to 6 additional aspartic acid residues, if desired.

[0077] In a further embodiment, the recombinant TNAP polypeptide comprises the amino acid sequence: [ka] In this sequence, the signal peptide is underlined and the histidine tag is in bold.

[0078] In some embodiments, the recombinant TNAP polypeptide comprises or consists of the amino acid sequence set forth as SEQ ID NO:4 or SEQ ID NO:5. Homologs and variants are also used, such as polypeptides that are about 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, the recombinant TNAP polypeptide is at least 95% identical to the amino acid sequence of SEQ ID NO:4, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:4, and the polypeptide i) promotes alveolar bone regeneration in a subject; ii) increases periodontal ligament attachment to root surfaces of teeth in a subject; iii) increases cementum formation in a subject; and / or iv) increases mineralization in teeth in a subject. In some embodiments, the recombinant TNAP polypeptide is at least 95% identical to the amino acid sequence of SEQ ID NO:5, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:5, and the polypeptide i) promotes alveolar bone regeneration in a subject; ii) increases periodontal ligament attachment to the root surface of a tooth in a subject; iii) increases cementum formation in a subject; and / or iv) increases mineralization in a tooth in a subject.

[0079] In further embodiments, the recombinant TNAP polypeptide comprises at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 conservative substitutions in SEQ ID NO:4, and the polypeptide i) promotes alveolar bone regeneration in a subject; ii) increases periodontal ligament attachment to root surfaces of teeth in the subject; iii) increases cementum formation in the subject; and / or iv) increases mineralization in teeth in the subject. In additional embodiments, the recombinant TNAP polypeptide comprises at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 conservative substitutions in SEQ ID NO:5, and the polypeptide i) promotes alveolar bone regeneration in a subject; ii) increases periodontal ligament attachment to the root surface of a tooth in the subject; iii) increases cementum formation in the subject; and / or iv) increases mineralization in a tooth in the subject.

[0080] In some embodiments, the recombinant TNAP polypeptide comprises a polypeptide that is about 95%, 96%, 97%, 98%, or 99% identical to amino acids 1-40 of SEQ ID NO:4 and amino acids 41-528 of SEQ ID NO:4, wherein the polypeptide i) promotes alveolar bone regeneration in a subject; ii) increases periodontal ligament attachment to the root surface of a tooth in a subject; iii) increases cementum formation in a subject; and / or iv) increases mineralization in a tooth in a subject. In some embodiments, the recombinant TNAP polypeptide comprises a polypeptide that is about at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 1-40 of SEQ ID NO:4 and amino acids 41-528 of SEQ ID NO:4, wherein the polypeptide i) promotes alveolar bone regeneration in a subject; ii) increases periodontal ligament attachment to the root surface of a tooth in a subject; iii) increases cementum formation in a subject; and / or iv) increases mineralization in a tooth in a subject. In further embodiments, the recombinant TNAP polypeptide comprises amino acids 1-40 of SEQ ID NO:4 and has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 conservative substitutions in amino acids 41-528 of SEQ ID NO:4, wherein the polypeptide i) promotes alveolar bone regeneration in a subject; ii) increases periodontal ligament attachment to the root surface of a tooth in a subject; iii) increases cementum formation in a subject; and / or iv) increases mineralization in a tooth in a subject. Optionally, the polypeptide also comprises an additional aspartic acid at the C-terminus, such as 4 to 12 aspartic acid residues.

[0081] In some embodiments, the recombinant TNAP polypeptide comprises a polypeptide that is about 95%, 96%, 97%, 98%, or 99% identical to amino acids 1-40 of SEQ ID NO:5 and amino acids 41-528 of SEQ ID NO:5, wherein the polypeptide i) promotes alveolar bone regeneration in a subject; ii) increases periodontal ligament attachment to the root surface of a tooth in a subject; iii) increases cementum formation in a subject; and / or iv) increases mineralization in a tooth in a subject. In some embodiments, the recombinant TNAP polypeptide comprises a polypeptide that is about at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to amino acids 1-40 of SEQ ID NO:5 and amino acids 41-528 of SEQ ID NO:5, wherein the polypeptide i) promotes alveolar bone regeneration in a subject; ii) increases periodontal ligament attachment to the root surface of a tooth in a subject; iii) increases cementum formation in a subject; and / or iv) increases mineralization in a tooth in a subject. In further embodiments, the recombinant TNAP polypeptide comprises amino acids 1-40 of SEQ ID NO:5 and a polypeptide with at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 conservative substitutions in amino acids 41-528 of SEQ ID NO:5, wherein the polypeptide i) promotes alveolar bone regeneration in a subject; ii) increases periodontal ligament attachment to the root surface of a tooth in the subject; iii) increases cementum formation in the subject; and / or iv) increases mineralization in a tooth in the subject.

[0082] In other embodiments, the recombinant TNAP polypeptide is MHHHHHHENLYFQGMLVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANEGTVG VSAATERSRCNTTQGNEVTSILRWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKNKTDVEYESDEKARGTRLDGLDLVDTW KSFKPRYKHSHFIWNRTELLTLDPHNVDYLLGLFEPGDMQYELNRNNVTDPSLSEMVVVAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHEAVEMDRAIGQAGSLTSSEDTLTVVTADHSHVF TFGGYTPRGNSIFGLAPMLSDTDKKPFTAILYGNGPGYKVVGGERENVSMVDYAHNNYQAQSAVPLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHVMAYAACIGANLGHCAPASSAGSL-(D) n=4-12 (SEQ ID NO: 18).

[0083] In a further embodiment, the recombinant TNAP polypeptide is MHHHHHHENLYFQGMLVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANEGTVG VSAATERSRCNTTQGNEVTSILRWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKNKTDVEYESDEKARGTRLDGLDLVDTW KSFKPRYKHSHFIWNRTELLTLDPHNVDYLLGLFEPGDMQYELNRNNVTDPSLSEMVVVAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHEAVEMDRAIGQAGSLTSSEDTLTVVTADHSHVF TFGGYTPRGNSIFGLAPMLSDTDKKPFTAILYGNGPGYKVVGGERENVSMVDYAHNNYQAQSAVPLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHVMAYAACIGANLGHCAPASSAGSL-(E) n=4-12 (SEQ ID NO: 19). These polypeptides contain the elements: (1) the amino acid sequence set forth in SEQ ID NO: 1; (2) 6×His; and (3) 4 to 12×Asp or 4 to 12×Glu.

[0084] In some embodiments, the recombinant TNAP polypeptide has 6xHis positioned at its N-terminus, immediately following Met as the first amino acid of the polypeptide. In further embodiments, the polypeptide has 6xHis positioned at its C-terminus.

[0085] In some embodiments, the recombinant TNAP polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18 or 19. In these embodiments, 4-12×Asp or 4-12×Glu includes any number of Asps or Glus, such as 4, 5, 6, 7, 8, 9, 10, 11, up to 12, e.g., 4, 6, 8, 10, 12×Asp or 4, 6, 8, 10, or 12×Glu.

[0086] In further embodiments, a recombinant TNAP polypeptide is provided that includes (1) the amino acid sequence set forth in SEQ ID NO: 1; (2) 6xHis; and (3) 4-12xAsp or 4-12xGlu elements. In some embodiments, the 6xHis is positioned N-terminally after Met as the first amino acid of the polypeptide. In other embodiments, the 6xHis is positioned C-terminally of the polypeptide. In further embodiments, the histidine tag comprises a TEV cleavage sequence. TEV protease can cleave this protein between Q (shown in bold) and G (shown in bold) such that the N-terminal portion is removed. In some embodiments, the histidine tag is not included in the polypeptide administered to a subject.

[0087] B. Chemical Synthesis and Supports The amino acid sequences disclosed herein can also be chemically synthesized using conventional peptide synthesis or other known protocols. Polypeptides can be synthesized by solid-phase peptide synthesis using procedures similar to those described by Merrifield et al., J. Am. Chem. Soc., 85:2149-2156 (1963); Barany and Merrifield, Solid-Phase Peptide Synthesis, in The Peptides: Analysis, Synthesis, Biology Gross and Meienhofer (eds.), Academic Press, NY, vol. 2, pp. 3-284 (1980); and Stewart et al., Solid Phase Peptide Synthesis 2nd ed., Pierce Chem. Co., Rockford, Ill. (1984). During synthesis, N-α-protected amino acids with protected side chains are added stepwise to a growing polypeptide chain that is linked by its C-terminus to a solid support, i.e., polystyrene beads. Peptides are synthesized by linking the amino group of an N-α-deprotected amino acid to the α-carboxy group of an N-α-protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide. Attachment of the free amino group to the activated carboxyl results in peptide bond formation. The most commonly used N-α-protecting groups include the acid-labile Boc and base-labile Fmoc.

[0088] The recombinant TNAP polypeptide may be contained on a solid support. Materials suitable for use as solid supports are well known to those skilled in the art and include, but are not limited to, halomethyl resins such as chloromethyl or bromomethyl resins; hydroxymethyl resins; phenolic resins such as 4-(α-[2,4-dimethoxyphenyl]-Fmoc-aminomethyl)phenoxy resin; tert-alkyloxycarbonyl-hydrazide resins, and the like. Such resins are commercially available, and methods for their preparation are known to those skilled in the art.

[0089] Briefly, a C-terminal N-α-protected amino acid is first attached to a solid support. The N-α-protecting group is then removed. The deprotected α-amino group is attached to the activated α-carboxylic acid group of the next N-α-protected amino acid. This process is repeated until the desired peptide is synthesized. The resulting peptide is then cleaved from the insoluble polymer support, and the amino acid side chains are deprotected. Longer peptides can be obtained by condensation of protected peptide fragments. Details of appropriate chemistries, resins, protecting groups, protected amino acids, and reagents are well known in the art and will not be described in detail herein (see Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press (1989) and Bodanszky, Peptide Chemistry, A Practical Textbook, 2nd Ed., Springer-Verlag (1993)).

[0090] Biocompatible scaffolds can be used. The recombinant TNAP polypeptide can be included in a suitable matrix implant, including, but not limited to, a biopolymer or synthetic polymer or polymer matrix (to encapsulate the recombinant TNAP polypeptide prior to introduction into the subject's body, such as for slow release). Examples of biopolymers include, but are not limited to, fibronectin, fibrin, fibrinogen, thrombin, collagen, and proteoglycans, which may be chemically modified or shaped. In some embodiments, the polymer allows for controlled release. In one example, the recombinant TNAP polypeptide is part of or attached to a hydrogel or microsphere.

[0091] The three-dimensional gel is used with the recombinant TNAP polypeptide contained within the gaps of the three-dimensional gel. Numerous biocompatible scaffolds are known and available. An example of a biocompatible scaffold that can be used is hydroxyapatite / tricalcium phosphate. PLGA or other types of scaffolds can be used. The biocompatible scaffold can be a collagen sponge or gel.

[0092] C. Cloning and Subcloning of the Coding Sequence of Recombinant TNAP Polypeptide A polynucleotide sequence encoding a TNAP polypeptide can be determined based on its amino acid sequence and available information on various known TNAPs, and can be isolated from a vector containing the known coding sequence or from a genomic library containing one or more TNAP genes, or can be synthesized by a commercial supplier before further modification.

[0093] A nucleic acid sequence encoding naturally occurring TNAP can be isolated from a cDNA or genomic DNA library using standard cloning techniques, such as the polymerase chain reaction (PCR). The most commonly used techniques for this purpose are described in standard textbooks, such as Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Ausubel et al., eds., Current Protocols in Molecular Biology (1994).

[0094] Suitable cDNA libraries for obtaining coding sequences are commercially available or can be constructed. General methods for isolating mRNA, generating cDNA by reverse transcription, ligating the cDNA into a recombinant vector, transfecting it into a recombinant host for propagation, screening, and cloning are well known (see, e.g., Gubler and Hoffman, Gene, 25: 263-269 (1983); Ausubel et al., supra). After obtaining an amplified segment of a nucleotide sequence by PCR, the segment can be further used as a probe to isolate a longer polynucleotide sequence encoding TNAP from a cDNA library. An overview of suitable procedures can be found in Sambrook and Russell, supra.

[0095] Based on sequence homology, degenerate oligonucleotides can be designed as primer sets, and PCR can be performed under appropriate conditions (see, e.g., White et al., PCR Protocols: Current Methods and Applications, 1993; Griffin and Griffin, PCR Technology, CRC Press Inc. 1994) to amplify segments of nucleotide sequences from cDNA or genomic libraries. The amplified segments can be used as probes to obtain longer lengths of nucleic acid encoding recombinant TNAP polypeptides, such as SEQ ID NO:1.

[0096] After obtaining a nucleic acid sequence encoding TNAP, the coding sequence can be modified as appropriate (e.g., by deletion, addition, and / or substitution of one or more amino acid residues, or fusion with one or more coding sequences of heterologous origin, including the addition of a coding sequence for an affinity tag, e.g., a 6xHis tag or a GST tag, SEQ ID NO: 3, etc.), and then subcloned into a vector, e.g., an expression vector, such that recombinant TNAP polypeptide can be produced from the resulting construct after transfection and culturing host cells under conditions that allow recombinant protein expression directed by a promoter operably linked to the coding sequence.

[0097] D. Modifying Nucleic Acids for Preferred Codon Usage in the Host Organism Polynucleotide sequences encoding the recombinant TNAP polypeptides disclosed herein can be further modified to conform to the preferred codon usage of a particular host. For example, the preferred codon usage of a strain of bacterial cells can be used to obtain a polynucleotide encoding a recombinant TNAP polypeptide and containing codons favored by that strain. The frequency of preferred codon usage exhibited by a host cell can be calculated by averaging the frequency of preferred codon usage in a large number of genes expressed by that host cell (e.g., calculation services are available from the website of the Kazusa DNA Research Institute, Japan). This analysis is preferably limited to genes that are highly expressed by the host cell.

[0098] Upon completion of modification, the coding sequence can be verified by sequencing and then subcloned into an appropriate expression vector for recombinant production of the recombinant TNAP polypeptide.

[0099] E. Expression and Purification of Recombinant TNAP Polypeptide Using the coding sequences, recombinant TNAP polypeptides can be produced using recombinant genetics relying on the polynucleotide sequences encoding the polypeptides disclosed herein.

[0100] 1. Expression system To obtain high-level expression of a nucleic acid encoding a recombinant TNAP polypeptide, the polynucleotide encoding the polypeptide is typically subcloned into an expression vector containing a strong promoter (typically heterologous) to direct transcription, a transcription / translation terminator, and a ribosome binding site for translation initiation. Suitable bacterial promoters include, for example, those described in Sambrook and Russell, supra, and Ausubel et al., supra. Bacterial expression systems for expressing recombinant polypeptides are available for, for example, E. coli, Bacillus sp., Salmonella, and Caulobacter. Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known and commercially available. In one embodiment, the eukaryotic expression vector is an adenoviral vector, an adeno-associated vector (AAV), or a retroviral vector.

[0101] The promoter used to direct the expression of heterologous nucleic acid can depend on specific application.If necessary, promoter is positioned at a distance from the heterologous transcription start site that is approximately the same as the distance from the transcription start site in its natural context.However, some variations in this distance can be accommodated without losing promoter function.

[0102] In addition to a promoter, an expression vector typically contains a transcription unit or expression cassette that contains all of the additional elements required for expression of a recombinant TNAP polypeptide in a host cell. Thus, a typical expression cassette contains a promoter operably linked to the coding sequence, as well as signals required for efficient polyadenylation of the transcript, a ribosome binding site, and translation termination. The nucleic acid sequence encoding the recombinant TNAP polypeptide is typically linked to a cleavable signal peptide sequence to facilitate secretion of the recombinant polypeptide by transformed cells. Such signal peptides include, among others, signal peptides derived from tissue plasminogen activator, insulin, and neuron growth factor, and Heliothis virescens juvenile hormone esterase. Additional elements of the cassette can include enhancers and, when genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.

[0103] In addition to a promoter sequence, the expression cassette may also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from a different gene.

[0104] The particular expression vector used to transport the genetic information into the cell is not critical. Any conventional vector used for expression in eukaryotic or prokaryotic cells can be used. Standard bacterial expression vectors include plasmids based on pBR322, pSKF, pET23d, pcDNA™, 3.4 TOPO®, and other plasmids, as well as fusion expression systems such as GST and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient isolation methods, such as c-myc.

[0105] Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, such as SV40 vectors, papillomavirus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009 / A. + , pMTO10 / A + These vectors include pMAMneo-5, baculovirus pDSVE, and any other vector that allows expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown to be effective for expression in eukaryotic cells. Lentiviral vectors are also used.

[0106] Some expression systems have markers that result in gene amplification, such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. Alternatively, high-yield expression systems that do not involve gene amplification are also suitable, such as baculovirus vectors in insect cells with a polynucleotide sequence encoding a recombinant TNAP polypeptide under the direction of the polyhedrin promoter or other strong baculovirus promoter.

[0107] Elements typically included in expression vectors include a replicon that functions in E. coli, a gene encoding antibiotic resistance to allow selection of bacteria carrying the recombinant plasmid, and a unique restriction site in a non-essential region of the plasmid to allow insertion of eukaryotic sequences. The particular antibiotic resistance gene selected is not essential, and any of the many resistance genes known in the art are suitable. The prokaryotic sequence is selected as needed so as not to interfere with DNA replication in eukaryotic cells, if necessary. Similar to antibiotic resistance selection markers, metabolic selection markers based on known metabolic pathways can also be used as a means to select transformed host cells.

[0108] If periplasmic expression of a recombinant protein (e.g., a recombinant TNAP polypeptide) is desired, the expression vector further comprises a sequence encoding a secretion signal, such as the E. coli OppA (periplasmic oligopeptide-binding protein) secretion signal or a modified version thereof, connected directly 5' to the coding sequence of the protein to be expressed. This signal sequence directs recombinant proteins produced in the cytoplasm through the cell membrane into the periplasmic space. The expression vector may further comprise a coding sequence for signal peptidase 1, which can enzymatically cleave the signal sequence when the recombinant protein enters the periplasmic space. Further details on periplasmic production of recombinant proteins can be found, for example, in Gray et al., Gene 39: 247-254 (1985), U.S. Patent Nos. 6,160,089 and 6,436,674.

[0109] 2. Transfection Method Standard transfection methods are used to generate bacterial, mammalian, yeast, insect, or plant cell lines that express large amounts of the recombinant polypeptide, which is then purified using standard techniques (see, e.g., Colley et al., J. Biol. Chem. 264: 17619-17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells is carried out according to standard techniques (see, e.g., Morrison, J. Bact. 132: 349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101: 347-362 (Wu et al., eds, 1983)).

[0110] Any known procedure for introducing foreign nucleotide sequences into host cells can be used. Such procedures include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors, and any other well-known method for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into host cells (see, e.g., Sambrook and Russell, supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing a recombinant polypeptide. Exemplary host cells include, but are not limited to, Chinese hamster ovary cells, such as ExpiCHO cells.

[0111] 3. Detection of Recombinant Expression of Recombinant TNAP Polypeptide in Host Cells After the expression vector is introduced into a suitable host cell, the transfected cells are cultured under conditions favorable for expression of the recombinant TNAP polypeptide. The cells are then screened for expression of the recombinant polypeptide, which is then recovered from the culture using standard techniques (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Patent No. 4,673,641; Ausubel et al., supra; and Sambrook and Russell, supra).

[0112] Several general methods for screening gene expression are known. First, gene expression can be detected at the nucleic acid level. Various methods of specific DNA and RNA measurement using nucleic acid hybridization techniques are commonly used (see, e.g., Sambrook and Russell, supra). Some methods involve electrophoretic separation (e.g., Southern blot for detecting DNA and Northern blot for detecting RNA), although DNA or RNA detection can also be performed without electrophoresis (e.g., by dot blot). The presence of nucleic acid encoding a recombinant TNAP polypeptide in transfected cells can also be detected by PCR or RT-PCR using sequence-specific primers.

[0113] Second, gene expression can be detected at the polypeptide level. Various immunological assays are routinely used by those skilled in the art, particularly to measure gene product levels using polyclonal or monoclonal antibodies specifically reactive with recombinant TNAP polypeptides (e.g., Harlow and Lane, Antibodies, A Laboratory Manual, Chapter 14, Cold Spring Harbor, 1988; Kohler and Milstein, Nature, 256: 495-497 (1975)). Such techniques require antibody preparation by selecting antibodies with high specificity for recombinant TNAP polypeptides. Methods for producing polyclonal and monoclonal antibodies are well established; see, e.g., Harlow and Lane, supra; Kohler and Milstein, Eur. J. Immunol., 6: 511-519 (1976). A more detailed description of preparing antibodies against recombinant TNAP polypeptides and performing immunological assays to detect antigen-containing polypeptides is provided in later sections.

[0114] 4. Purification of Recombinantly Produced Recombinant TNAP Polypeptide Once expression of the recombinant TNAP polypeptide in the transfected host cells has been confirmed, the host cells are cultured at an appropriate scale for purposes of purifying the recombinant polypeptide.

[0115] a. Purification of recombinantly produced polypeptides from bacteria When recombinant TNAP polypeptides are recombinantly produced in large quantities by transformed bacteria, typically after promoter induction (although expression can be constitutive), the polypeptides may form insoluble aggregates. Several protocols exist for the purification of protein inclusion bodies. For example, purification of aggregated proteins (hereafter referred to as inclusion bodies) typically involves extraction, isolation, and / or purification of inclusion bodies by disruption of bacterial cells, e.g., by incubation in a buffer containing approximately 100-150 μg / ml lysozyme and 0.1% Nonidet P40 (a non-ionic detergent). Cell suspensions can be ground using a Polytron grinder (Brinkman Instruments, Westbury, NY). Alternatively, cells can be sonicated on ice. Alternative methods for lysing bacteria are described in Ausubel et al. and Sambrook and Russell, both referenced above, and will be apparent to those skilled in the art.

[0116] The cell suspension is typically centrifuged, and the pellet containing the inclusion bodies is resuspended in a buffer that does not dissolve the inclusion bodies but washes them, such as 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 150 mM NaCl, and 2% Triton®-X 100 (a non-ionic detergent). Repeated washing steps may be necessary to remove as much cell debris as possible. The remaining inclusion body pellet can be resuspended in a suitable buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl). Other suitable buffers will be apparent to those skilled in the art.

[0117] After the washing step, the inclusion bodies are solubilized by the addition of a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor (or a combination of solvents each possessing one of these properties). The proteins that formed the inclusion bodies can then be renatured by dilution with a compatible buffer or by dialysis. Suitable solvents include, but are not limited to, urea (about 4M to about 8M), formamide (at least about 80%, volume / volume basis), and guanidine hydrochloride (about 4M to about 8M). Some solvents that can solubilize aggregate-forming proteins, such as SDS (sodium dodecyl sulfate) and 70% formic acid, may be unsuitable for use in this procedure due to the potential for irreversible denaturation of the protein, resulting in immunogenicity and / or loss of activity. While guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible; renaturation can occur after removal of the denaturant (e.g., by dialysis) or dilution, allowing for the re-formation of an immunologically and / or biologically active protein of interest. After solubilization, the protein can be separated from other bacterial proteins by standard separation techniques. For a further description of the purification of recombinant polypeptides from bacterial inclusion bodies, see, e.g., Patra et al., Protein Expression and Purification 18: 182-190 (2000).

[0118] Alternatively, recombinant polypeptides, such as recombinant TNAP polypeptides, can be purified from bacterial periplasm. If recombinant proteins are transported into the bacterial periplasm, the bacterial periplasmic fraction can be isolated by low-temperature osmotic shock, in addition to other methods known to those skilled in the art (see, for example, Ausubel et al., supra). To isolate recombinant proteins from the periplasm, bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% ​​sucrose. To lyse the cells, the bacteria are centrifuged, and the pellet is resuspended in ice-cold 5 mM MgSO4 and kept in an ice bath for approximately 10 minutes. The cell suspension is centrifuged, and the supernatant is decanted and saved. The recombinant protein present in the supernatant can be separated from host proteins by standard separation techniques well known to those skilled in the art.

[0119] b. Standard protein isolation techniques for purification When a recombinant polypeptide, such as a recombinant TNAP polypeptide, is expressed in a soluble form in a host cell, its purification can follow standard protein purification procedures described below. A histidine tag can be used for purification. Several standard purification procedures are also suitable for purifying recombinant TNAP polypeptides obtained from chemical synthesis.

[0120] i. Soluble fraction Often used as an initial step, especially when the protein mixture is complex, initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture medium) from the recombinant protein of interest. A preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate based on their solubility. The more hydrophobic a protein, the more likely it is to precipitate at lower ammonium sulfate concentrations. A typical protocol is to add saturated ammonium sulfate to the protein solution so that the resulting ammonium sulfate concentration is between 20 and 30%. This procedure precipitates the most hydrophobic proteins. The precipitate is discarded (unless the protein of interest is hydrophobic), and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer, and excess salt is removed, if necessary, by either dialysis or diafiltration. Other methods relying on protein solubility, such as cold ethanol precipitation, are well known to those skilled in the art and can be used to fractionate complex protein mixtures.

[0121] ii. Size-specific filtration Based on the calculated molecular weight, proteins of larger and smaller sizes can be isolated using ultrafiltration through membranes of different pore sizes (e.g., Amicon or Millipore membranes). As a first step, the protein mixture is ultrafiltered through a membrane with a pore size that has a molecular weight cutoff lower than the molecular weight of the protein of interest, such as a recombinant TNAP polypeptide. The retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cutoff higher than the molecular weight of the protein of interest. The recombinant protein passes through the membrane and becomes the filtrate. The filtrate can then be subjected to chromatography as described below.

[0122] iii. Column chromatography A protein of interest (such as a recombinant TNAP polypeptide) can also be separated from other proteins based on its size, histidine tag, net surface charge, hydrophobicity, or affinity for a ligand. In addition, antibodies raised against a recombinant TNAP polypeptide can be conjugated to a column matrix to immunopurify the recombinant TNAP polypeptide. All of these methods are well known in the art.

[0123] It will be apparent to one skilled in the art that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (eg, Pharmacia Biotech).

[0124] F. Method Provided herein are pharmaceutical compositions comprising a recombinant TNAP polypeptide or a polynucleotide encoding a recombinant TNAP polypeptide for use in promoting periodontal health in a patient. In some embodiments, a method is provided for treating periodontal disease in a subject. The method includes selecting a subject with periodontal disease and locally administering to the subject's periodontal tissues a therapeutically effective amount of a recombinant TNAP polypeptide or a nucleic acid molecule encoding a recombinant TNAP polypeptide as disclosed herein. In other embodiments, a method is provided for treating peri-implantitis in a subject. The method includes selecting a subject with peri-implantitis and locally administering to the subject's periodontal tissues a therapeutically effective amount of a recombinant TNAP polypeptide or a nucleic acid molecule encoding a recombinant TNAP polypeptide as disclosed herein. In additional embodiments, a method is provided for preserving a dental alveolus in a subject. The method includes selecting a subject with a dental alveolus, such as after tooth extraction, and locally administering to the subject's periodontal tissues a therapeutically effective amount of a recombinant TNAP polypeptide or a nucleic acid molecule encoding a recombinant TNAP polypeptide as disclosed herein. Local administration may be to the gums.

[0125] The pharmaceutical composition can be administered locally via various local routes, such as orally, by injection into the alveolar cavity and / or into periodontal tissues such as the gingiva. However, administration can also be systemic, such as subcutaneously, transdermally, nasally, intramuscularly, intravenously, or intraperitoneally. Routes for administering the pharmaceutical composition include local periodontal delivery to a subject at a daily dose of about 0.01 to 5000 mg, such as about 0.01 to 20 mg, including the weight of the scaffold, which can be about 10 to about 90% of either composition. In other embodiments, about 0.1 μg to about 10 mg or about 0.1 μg to about 20 mg is used per bone defect, depending on the size of the defect. In one non-limiting example, about 0.1 μg of protein is used for a bone defect measuring 1 mm x 2 mm x 0.5 mm (depth). The appropriate dose can be administered in a single daily dose or as divided doses given at appropriate intervals, e.g., two, three, four, or more partial doses per day. The dose can be administered as a single dose or can be repeated, such as daily for 1, 2, 3, 4, or 5 days. The dose can be adjusted based on the type and size of the defect.

[0126] Periodontal disease is characterized by gingivitis, destruction of alveolar bone and periodontal ligament, and apical migration of epithelial attachment, which leads to the formation of periodontal pockets. Many different tissues are involved, including epithelium, cartilage, and bone. The disclosed method promotes the healing and regeneration of gum tissue (epithelial tissue), periodontal ligament (cartilage), and jaw (bone). The pulp and dentin tissues in teeth eroded or attacked by periodontal disease can be regenerated using this method. In some embodiments, the method i) promotes alveolar bone regeneration in a subject; ii) increases the attachment of periodontal ligament to the root surface of the tooth in a subject; iii) increases cementum formation; and / or iv) increases mineralization in the tooth in a subject. In some examples, the method promotes alveolar bone regeneration in a subject by increasing alveolar bone regeneration in the subject by at least 20%, at least 25%, at least 40%, at least 50%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300% or more, compared to the absence of administration of a TNAP polypeptide or coding sequence. In some examples, the method increases periodontal ligament attachment to tooth root surfaces in a subject, e.g., by at least 20%, at least 25%, at least 40%, at least 50%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300% or more, compared to the absence of administration of a TNAP polypeptide or coding sequence. In some examples, the method increases cementum formation by at least 20%, at least 25%, at least 40%, at least 50%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300% or more, for example, compared to the absence of administration of the TNAP polypeptide or coding sequence.In some cases, the method increases the mineralization in the subject's teeth by at least 20%, at least 25%, at least 40%, at least 50%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300% or more, for example, compared to the absence of administration of a TNAP polypeptide or coding sequence.In some cases, a combination of these effects is achieved.The method can also include measuring: i) alveolar bone regeneration in the subject; ii) periodontal ligament attachment to the root surface of the subject's teeth; iii) cementum formation; and / or iv) mineralization in the subject's teeth.

[0127] In some embodiments, a composition containing an effective amount of recombinant TNAP polypeptide (or a TNAP coding sequence, e.g., as part of a viral vector) is drawn into a syringe and administered to a subject. This procedure can be used to administer multiple injections. The use of such a procedure can target the TNAP polypeptide to any predetermined site and is relatively non-traumatic.

[0128] In some embodiments, compositions containing recombinant TNAP polypeptides (or TNAP coding sequences, e.g., as part of a viral vector) are combined with a pharmaceutically acceptable carrier substance for topical administration. Examples of pharmaceutically acceptable carriers include, for example, commercially available inert gels or liquids supplemented with albumin, methylcellulose, or collagen matrices. Typical of such formulations are ointments, creams, and gels. These offer advantages, for example, they are non-invasive.

[0129] Viscous solutions such as ointments or creams can also be used for external application and / or injection. Ointments can also be prepared using an oily base containing fixed oils or hydrocarbons, such as white petrolatum or mineral oil, or an absorbent base consisting of absorbent anhydrous material(s), such as anhydrous lanolin. After the base is formed, the active ingredient is added at the desired concentration. Creams generally contain an oil phase (internal phase) typically containing fixed oils, hydrocarbons, etc., such as wax, petrolatum, mineral oil, etc., and an aqueous phase (continuous phase) containing water and any water-soluble material, such as added salts. The two phases are stabilized by the use of emulsifiers, e.g., surfactants such as sodium lauryl sulfate; hydrophilic colloids such as acacia colloidal clay, veegum, etc. After the emulsion is formed, the active ingredient is added at the desired concentration. Gels are composed of a base selected from oily bases, water, or emulsion suspension bases, as previously described. The gelling agent is added to the base, forming a matrix in the base, and increasing its viscosity to a semi-solid consistency.The examples of gelling agent are hydroxypropyl cellulose, acrylic acid polymer, etc.Before adding the gelling agent, the active ingredient is added to the formulation at a desired concentration.These can be applied to the gums, for example.

[0130] Biocompatible scaffolds can be used for treatment. The recombinant TNAP polypeptide can be contained in a suitable matrix implant, including, but not limited to, a biopolymer or synthetic polymer or polymer matrix (to encapsulate the recombinant TNAP polypeptide prior to introduction into the subject's body, such as for slow release). Examples of biopolymers include, but are not limited to, fibronectin, fibrin, fibrinogen, thrombin, collagen, and proteoglycans, which may be chemically modified or shaped. In some embodiments, the polymer allows for controlled release. Hydrogels or microspheres can also be used. These can be applied to periodontal tissues, such as the gums, or inserted into the dental socket.

[0131] The three-dimensional gel is used with the recombinant TNAP polypeptide contained within the gaps of the three-dimensional gel. Numerous biocompatible scaffolds are known and available in the art. An example of a biocompatible scaffold that can be used is hydroxyapatite / tricalcium phosphate. PLGA or other types of scaffolds can be used. The biocompatible scaffold can be a collagen sponge or gel. These can be applied to periodontal tissues such as the gingiva or inserted into the dental socket.

[0132] Useful matrix materials include, but are not limited to, synthetic homopolymers and copolymers of glycolic and lactic acid, hydroxyapatite, tricalcium phosphate and other calcium phosphates, and granular demineralized guanidine-extracted species-specific (allogeneic) bone. Matrices containing recombinant TNAP polypeptides can be applied to shapes that span bone or cartilage defects to serve as a "temporary scaffold" and substratum as a basis for the anchorage and proliferation of differentiated tissue cells.

[0133] Bone collagen matrix (see, e.g., U.S. Pat. No. 4,975,526, incorporated herein by reference) can be used as a carrier for bone and / or cartilage applications. The collagen matrix described in this patent is a biodegradable, biocompatible, mineral-free, insoluble type I bone collagen particle that has been depleted of non-collagenous proteins. The collagen matrix particles can have an average diameter of approximately 70 μm to 850 μm and an increased intra-particle surface area compared to the intact material. In this embodiment, recombinant TNAP polypeptide is first dissolved in a suitable solvent, such as buffered sterile saline, and then added to the collagen matrix. The mixture is vortexed, and the matrix is ​​lyophilized as desired, shaped, or implanted into areas of bone or cartilage by packing.

[0134] 1. Additional Pharmaceutical Compositions for Use in the Disclosed Methods When used to deliver an effective amount of a recombinant TNAP polypeptide (or a TNAP coding sequence, e.g., as part of a viral vector) to a recipient, a pharmaceutical composition can contain certain physiologically acceptable excipient(s) and, if necessary, be formulated for a particular preferred delivery method. Pharmaceutical compositions containing (1) an effective amount of a recombinant TNAP polypeptide (or a TNAP coding sequence, e.g., as part of a viral vector) and (2) at least one additional, possibly physiologically acceptable excipient or carrier are therefore useful in both prophylactic and therapeutic applications designed to treat or prevent various periodontal diseases and conditions involving reduced regeneration of cementum or alveolar bone or weakened PDL attachment to cementum and / or alveolar bone. A physiologically or pharmaceutically acceptable carrier is an inert agent that does not detectably alter or affect the functionality of an active ingredient (e.g., a recombinant TNAP polypeptide) present in the same composition in which the active ingredient is used. Pharmaceutical compositions can be used, for example, in the methods disclosed herein. Some examples include cryoprotectants; surfactants; bulking agents; osmotic agents; stabilizers; preservatives; and buffers. The composition is suitable for use in various drug delivery systems. Suitable formulations for use can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). For a review of methods for drug delivery, see Langer, Science 249: 1527-1533 (1990).

[0135] To prepare pharmaceutical compositions containing recombinant TNAP polypeptides (or TNAP coding sequences, e.g., as part of a viral vector), inert, pharmaceutically acceptable carriers can be used. Pharmaceutical carriers can be either solid or liquid. Solid form preparations include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents; it can also be an encapsulating material.

[0136] In dusts and powders, the carrier is generally a finely divided solid that is in admixture with the finely divided active ingredient, e.g., recombinant TNAP polypeptide. In tablets, the active ingredient (recombinant TNAP polypeptide) is mixed with a carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

[0137] For preparing pharmaceutical compositions in the form of suppositories, a low-melting wax such as a mixture of fatty acid glycerides and cocoa butter is first melted and the active ingredient is dispersed therein by, for example, stirring. The molten homogeneous mixture is then poured into convenient sized molds and allowed to cool and solidify.

[0138] The powders and tablets preferably contain about 5% to about 70% by weight of the active ingredient. Suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low-melting wax, cocoa butter, and the like.

[0139] Pharmaceutical compositions can include formulations of active compounds of recombinant TNAP polypeptide or TNAP coding sequences (e.g., as part of a viral vector) with an encapsulating material as a carrier, thereby providing a capsule in which the carrier surrounds the polypeptide (with or without other carriers) so that the carrier is associated with the compound. Cachets can also be included in a similar manner. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration.

[0140] Liquid pharmaceutical compositions include, for example, solutions suitable for oral or parenteral administration, suspensions and emulsions suitable for oral administration. A sterile aqueous solution of an active ingredient (e.g., a recombinant TNAP polypeptide) or a sterile solution of the active ingredient in a solvent including water, buffered water, saline, PBS, ethanol, or propylene glycol are examples of liquid compositions suitable for parenteral administration. The composition may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, osmotic pressure adjusting agents, wetting agents, detergents, etc.

[0141] Sterile solutions can be prepared by dissolving the active ingredient (e.g., recombinant TNAP polypeptide) in a desired solvent system and then sterilizing the resulting solution by passing it through a membrane filter, or by dissolving the sterile compound in a previously sterilized solvent under sterile conditions. The resulting aqueous solution can be packaged for immediate use or lyophilized, with the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparation is typically between 3 and 11, such as between 5 and 9 or between 7 and 8.

[0142] Pharmaceutical compositions containing recombinant TNAP polypeptides (or TNAP coding sequences, e.g., as part of a viral vector) can be administered for prophylactic and / or therapeutic treatments. In therapeutic applications, the compositions are administered to patients already suffering from periodontal disease in an amount sufficient to prevent, cure, reverse, or at least partially slow or halt the symptoms of the condition and its complications. The appropriate amount to achieve this depends on the severity of the disease or condition, the size of the bone defect, and the patient's general condition. Localized periodontal delivery can be applied to a subject at a daily dose of about 0.01 to 5000 mg, such as about 0.01 to 20 mg, including the weight of the scaffold, which can be about 10 to about 90% of either composition. In other embodiments, about 0.1 μg to about 10 mg or about 0.1 μg to about 20 mg is used per bone defect, depending on the size of the defect. In one non-limiting example, about 0.1 μg of protein is used for a bone defect measuring 1 mm x 2 mm x 0.5 mm (depth). The appropriate dose can be administered in a single daily dose or as divided doses given at appropriate intervals, for example, 2, 3, 4 or more partial doses per day.The dose can be administered as a single dose or can be repeated, such as every day for 1, 2, 3, 4 or 5 days.The dose can be adjusted based on the size and type of defect.

[0143] In prophylactic applications, pharmaceutical compositions containing recombinant TNAP polypeptide (or a TNAP coding sequence, e.g., as part of a viral vector) are administered to patients susceptible to or otherwise at risk of developing periodontal disease or periodontal disorders in an amount sufficient to delay or prevent the onset of symptoms. Such an amount is defined as a "prophylactically effective dose." Routes of administration of pharmaceutical compositions include localized periodontal delivery to a subject at a daily dose of about 0.01 to 5000 mg, such as about 0.01 to 20 mg, including about 10 to about 90% of the weight of the scaffold of either composition. In other embodiments, about 0.1 μg to about 10 mg or about 0.1 μg to about 20 mg is used per bone defect, depending on the size of the defect. In one non-limiting example, about 0.1 μg of protein is used for a bone defect measuring 1 mm x 2 mm x 0.5 mm (depth). The appropriate dose can be administered in a single daily dose or as divided doses given at appropriate intervals, for example, 2, 3, 4 or more partial doses per day. The dose can be administered as a single dose or can be repeated, such as daily for 1, 2, 3, 4 or 5 days. The dose can be adjusted based on the type and size of the defect.

[0144] Single or multiple administrations of the composition can be carried out with the dose level and pattern selected by the treating physician, dentist, or health care provided. In either case, the pharmaceutical formulation should provide a sufficient amount of recombinant TNAP polypeptide to effectively promote periodontal health in the patient, either therapeutically or prophylactically.

[0145] When a recombinant TNAP polypeptide (or a TNAP-encoding sequence, e.g., as part of a viral vector) is intended for use in promoting periodontal health, treating periodontal disease, peri-implantitis, preserving the alveolus, or alleviating symptoms, the recombinant TNAP polypeptide can be the only active agent in a pharmaceutical composition, or the composition can further include one or more other active agents suitable, for example, for inhibiting bacterial infection, promoting biomineralization of periodontal tissues (e.g., cementum and alveolar bone), promoting mineralization, or promoting or inhibiting dental caries. The importance of the extracellular matrix, including the small integrin-binding ligand N-linked glycoprotein (SIBLING family) of proteins, in skeletal mineralization and bone remodeling is disclosed, for example, in J Endocrinol. 2012 Sep;214(3):241-55. doi: 10.1530 / JOE-12-0143, incorporated herein by reference.

[0146] The compositions can be administered by any suitable route, although in some embodiments, the compositions can be formulated for topical administration, which can be in the form of an ointment, lotion, cream, paste, gel, drops, spray, liquid, or powder. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable. The compositions can be in the form of, including but not limited to, tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, or aerosols (as solids). In some cases, the compositions can contain, for example, up to 1%, 2%, 5%, 10%, or more of the active agent (e.g., recombinant TNAP) by weight.

[0147] In certain embodiments, the composition is in the form of dental care products, including but not limited to toothpaste, tooth powder, mouthwash / mouth rinse, dental floss, liquid dentifrice, dental tablets, external gel, lozenges, chewing gum, toothpaste, gum massage cream, mouthwash, lozenges and food.In addition, the composition can be formulated for injection, particularly for local injection, and delivered directly into patient's mouth, such as oral epithelial tissue or periodontal tissue.An example of delivery method is injection into gum or into the recess in cementum or alveolar bone, for example, immediately before, during or immediately after dental implantation process.

[0148] 2. Use of Nucleic Acid Molecules in the Disclosed Methods Therapeutic approaches involving the introduction of a nucleic acid encoding recombinant TNAP such that expression of the polypeptide results in a reduction or elimination of the causes and symptoms of periodontal disease in the recipient can treat or alleviate periodontal disease and peri-implantitis, and preserve the dental alveolus. Compositions and methods for nucleic acid-based treatment schemes are provided herein.

[0149] a. Vectors for nucleic acid delivery For delivery to cells or organisms, the nucleic acid encoding the recombinant TNAP polypeptide can be incorporated into a vector. Examples of vectors used for such purposes include expression plasmids that can direct the expression of the recombinant TNAP polypeptide in target cells. In another example, the vector is a viral vector system in which a polynucleotide coding sequence is incorporated into the viral genome, which can transfect target cells. In some embodiments, the coding sequence can be operably linked to expression and control sequences that can direct the transcription of the sequence in the desired target tissue. Thus, improved periodontal health can be achieved in target tissues, such as periodontal tissues.

[0150] b. Gene delivery system A gene delivery system is any means for delivering a nucleic acid to a target cell. Viral vector systems useful for introducing and expressing a recombinant TNAP polypeptide include, for example, naturally occurring or recombinant viral vector systems. Depending on the specific application, suitable viral vectors include replication-competent, replication-deficient, and conditionally replicating viral vectors. For example, viral vectors can be derived from the genomes of human or bovine adenoviruses, vaccinia viruses, herpesviruses, adeno-associated viruses, minute virus of mice (MVM), lentiviruses such as HIV, Sindbis viruses, and retroviruses (including, but not limited to, Rous sarcoma virus and lentiviruses), and MoMLV. Typically, the coding sequence for a recombinant TNAP polypeptide is inserted into such a vector, typically allowing packaging of the gene construct with accompanying viral DNA, followed by infection of susceptible host cells and expression of the polypeptide.

[0151] Similarly, viral envelopes used to package genetic constructs containing coding sequences for recombinant TNAP polypeptides can be modified by the addition of receptor ligands or antibodies specific for receptors that allow receptor-mediated endocytosis into specific cells (see, e.g., WO93 / 20221, WO93 / 14188, and WO94 / 06923).

[0152] Retroviral vectors can also be useful for introducing recombinant TNAP polypeptides into target cells or organisms. Retroviral vectors are produced by genetically manipulating retroviruses. The viral genome of retroviruses is RNA. After infection, this genomic RNA is reverse transcribed into a DNA copy, which is integrated into the chromosomal DNA of transduced cells with high stability and efficiency. The integrated DNA copy is called a provirus and is inherited by daughter cells like any other gene. Wild-type retroviral genomes and proviral DNA contain three genes: gag, pol, and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes internal structural (nucleocapsid) proteins; the pol gene encodes an RNA-directed DNA polymerase (reverse transcriptase); and the env gene encodes viral envelope glycoproteins. The 5' and 3' LTRs act to promote transcription and polyadenylation of virion RNA. The 5'LTR is flanked by sequences necessary for reverse transcription of the genome (tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (Psi site) (see Mulligan, In: Experimental Manipulation of Gene Expression, Inouye (ed), 155-173 (1983); Mann et al., Cell 33:153-159 (1983); Cone and Mulligan, Proceedings of the National Academy of Sciences, USA, 81:6349-6353 (1984)).

[0153] The design of retroviral vectors is well known to those skilled in the art. Briefly, when sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the result is a cis-acting defect that prevents encapsidation of the genomic RNA. However, the resulting mutant is still capable of directing the synthesis of all virion proteins. Retroviral genomes deleted of these sequences, as well as cell lines containing mutant genomes stably integrated into chromosomes, are well known in the art and are used to construct retroviral vectors. The preparation and use of retroviral vectors is described in many publications, including, for example, European Patent Application EPA 0178220; U.S. Patent No. 4,405,712, Gilboa Biotechniques 4:504-512 (1986); Mann et al., Cell 33:153-159 (1983); Cone and Mulligan Proc. Natl. Acad. Sci. USA 81:6349-6353 (1984); Eglitis et al. Biotechniques 6:608-614 (1988); Miller et al. Biotechniques 7:981-990 (1989); Miller (1992), supra; Mulligan (1993), supra; and WO 92 / 07943.

[0154] Retroviral vector particles are prepared by recombinantly inserting a desired coding sequence into a retroviral vector and packaging the vector with retroviral capsid proteins using a packaging cell line. The resulting retroviral vector particles cannot replicate in host cells, but can be integrated into the host cell genome as a proviral sequence containing the desired nucleotide sequence. As a result, patients can produce recombinant TNAP polypeptide, thereby reducing or eliminating the underlying causes of various periodontal diseases.

[0155] Packaging cell lines used to prepare retroviral vector particles are typically recombinant mammalian tissue culture cell lines that produce the necessary viral structural proteins required for packaging but are incapable of producing infectious virions. Defective retroviral vectors, on the other hand, lack such structural genes but encode the remaining proteins necessary for packaging. To prepare packaging cell lines, an infectious clone of the desired retrovirus can be constructed in which the packaging site has been deleted. Cells containing this construct express all structural viral proteins but are unable to package the introduced DNA. Alternatively, packaging cell lines can be generated by transforming a cell line with one or more expression plasmids encoding the appropriate core and envelope proteins. In such cells, the gag, pol, and env genes can be derived from the same or different retroviruses.

[0156] Several packaging cell lines are also available. Examples of such cell lines include Crip, GPE86, PA317, and PG13 (see Miller et al., J. Virol. 65:2220-2224 (1991)). Other exemplary packaging cell lines are described in Cone and Mulligan Proceedings of the National Academy of Sciences, USA, 81:6349-6353 (1984); Danos and Mulligan Proceedings of the National Academy of Sciences, USA, 85:6460-6464 (1988); Eglitis et al. (1988), supra; and Miller (1990), supra.

[0157] C. Pharmaceutical formulations of nucleic acid molecules When used for pharmaceutical purposes, nucleic acids encoding recombinant TNAP polypeptides are generally formulated in a suitable buffer, which can be any pharmaceutically acceptable buffer, such as phosphate-buffered saline or sodium phosphate / sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to those skilled in the art, such as those described by Good et al. Biochemistry 5:467 (1966).

[0158] The composition may further comprise stabilizers, enhancers, and / or other pharmaceutically acceptable carriers or vehicles. Pharmaceutically acceptable carriers may contain, for example, physiologically acceptable compounds that act to stabilize the nucleic acid and any associated vectors. Physiologically acceptable compounds may include, for example, carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low-molecular-weight proteins, or other stabilizers or excipients. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents, or preservatives that are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. Examples of carriers, stabilizers, or adjuvants can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985).

[0159] d. Administration of formulations containing nucleic acid molecules The formulation containing the nucleic acid encoding recombinant TNAP polypeptide can be delivered to any tissue or organ using any delivery method known to those skilled in the art.In some embodiments, the nucleic acid is formulated in mucosal, topical and / or buccal formulations, particularly mucoadhesive gel and topical gel formulations.Exemplary permeation enhancing compositions, polymer matrices and mucoadhesive gel preparations for transdermal delivery are disclosed in U.S. Patent No. 5,346,701.

[0160] Formulations containing the encoding nucleic acid are typically administered to a patient systemically or locally, such as directly to the periodontal tissue. In some embodiments, the encoding nucleic acid is introduced into the periodontal tissue by methods such as microinjection, calcium phosphate precipitation, liposome fusion, ultrasound, electroporation, or biolistics. In further embodiments, the nucleic acid is taken up directly by the tissue of interest (e.g., the periodontal tissue).

[0161] In some embodiments, the encoding nucleic acid is administered ex vivo to cells or tissues explanted from the patient and then returned to the patient. Examples of ex vivo administration of therapeutic gene constructs include Nolta et al., Proc Natl. Acad. Sci. USA 93(6):2414-9 (1996); Koc et al., Seminars in Oncology 23(1):46-65 (1996); Raper et al., Annals of Surgery 223(2):116-26 (1996); Dalesandro et al., J. Thorac. Cardi. Surg., 11(2):416-22 (1996); and Makarov et al., Proc. Natl. Acad. Sci. USA 93(1):402-6 (1996).

[0162] The effective dosage of a formulation varies depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, and other drugs administered. Thus, treatment dosages must be titrated to optimize safety and efficacy. In determining the effective amount of vector to be administered, the physician should evaluate the specific nucleic acid used, the diagnosed disease state, the patient's age, weight, and general condition, circulating plasma levels, vector toxicity, disease progression, and the production of anti-vector antibodies. The size of the dose is also determined by the existence, nature, and extent of any adverse side effects associated with the administration of a particular vector.

[0163] In some embodiments, doses ranging from about 10 ng to 1 g, 100 ng to 100 mg, about 1 μg to 10 mg, or about 30 to 300 μg of encoding nucleic acid per patient are used. Doses generally range from about 0.01 to about 50 mg per kilogram of body weight, preferably about 0.1 to about 5 mg / kg body weight, or about 10 μg per injection. 8 ~10 10 or 10 12 Generally, the equivalent dose of naked nucleic acid from a vector is about 1 μg to 100 μg for a typical 70 kg patient, and the dose of vector containing retroviral particles is calculated to obtain an equivalent amount of nucleic acid encoding a recombinant TNAP polypeptide.

[0164] G.Kit Kits are provided for promoting periodontal health, treating periodontal disease, peri-implantitis, or preserving dental alveoli by administering a recombinant TNAP polypeptide or a nucleic acid encoding the polypeptide according to the methods disclosed herein. The kits typically include a first container (such as a glass or plastic vial) containing a pharmaceutical composition having an effective amount of a recombinant TNAP polypeptide (or a TNAP coding sequence, e.g., as part of a viral vector), and, optionally, a second container (such as a glass or plastic vial) containing one or more agents active in promoting dental and periodontal health, such as compounds that provide fluoride, calcium, or phosphate ions, antibacterial agents for inhibiting bacterial infection, biomineralization promoters for controlling or promoting tissue biomineralization in periodontal tissues, or agents capable of inhibiting dental caries. For example, U.S. Patent Nos. 6,811,769; 8,999,298; 9,211,240; 9,290,555; and 10,314,776 disclose therapeutically active agents and formulations that can be used in combination with the disclosed recombinant TNAP polypeptides.

[0165] Pharmaceutical compositions containing a recombinant TNAP polypeptide (or a TNAP coding sequence, e.g., as part of a viral vector) and, optionally, another active agent capable of promoting dental / periodontal health, can be in any suitable form, including, but not limited to, tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solids or in liquid media), e.g., ointments containing up to 10% by weight of the active compound, soft and hard gelatin capsules, sterile injectable solutions, and sterile packaged powders.

[0166] In some embodiments, the pharmaceutical composition is in the form of a dental care product, including, but not limited to, toothpaste, tooth powder, mouthwash, dental floss, liquid dentifrice, dental tablets, topical gels, lozenges, chewing gum, tooth powder, gum massage cream, mouthwash, lozenges, and foods. In other embodiments, the composition can be formulated for injection to deliver the active ingredient(s), such as a recombinant TNAP polypeptide, or another active agent directly into the periodontal tissue, e.g., the gingiva, or a depression in the cementum or alveolar bone, or into the oral epithelial tissue, near the site where a periodontal condition may exist or may later develop.

[0167] In some embodiments, the kit also includes informational material containing instructions on how to dispense the pharmaceutical composition, including a description of the types of patients who may be treated (e.g., those who have periodontal disease or who are at increased risk of later developing periodontal disease), the schedule (e.g., dose and frequency) and route of administration, etc.

[0168] The present disclosure is illustrated by the following non-limiting examples. [Example]

[0169] Identification of novel therapeutic approaches inspired by the developmental biology of periodontal tissues can lead to improved treatment outcomes. Research has identified inorganic phosphate (P), a stimulator of hydroxyapatite (HA) formation. i), and pyrophosphate (PP), a potent inhibitor of hydroxyapatite crystal growth. i ) is crucial for proper mineralization, and acellular cementum is particularly sensitive to this regulatory mechanism (Ao et al. 2017, Bone. 105:134-147; Chu et al. 2020, Bone. 136:115-329; Foster et al. 2012, Bone. 78:150-164; Thumbigere-Math et al. 2018, J Dent Res. 97(4):432-441; Nagasaki et al. 2020, J Dent Res. In press). i / PP i Regulatory factors work in concert to induce mineralization of periodontal tissues. Tissue-nonspecific alkaline phosphatase (gene: Alpl in mouse, ALPL in human; protein: TNAP) regulates PP i is hydrolyzed to give P i TNAP promotes mineralization by producing PP (Millan 2006, Purinergic Signal. 2(2):335-341). TNAP is expressed by mineralizing cells, including cementoblasts and osteoblasts, and loss-of-function mutations result in increased PP. i This leads to a genetic hypomineralization disorder, hypophosphatasia (HPP; OMIM#241500, 241510, 146300), which is characterized by tooth loss due to cementum defects (Bowden and Foster 2019, Adv Exp Med Biol. 1148:279-322). Progressive ankylosis proteins (Ank / ANK in mice, ANKH / ANK in humans) mediate the transport of PP into the extracellular space. i Ectonucleotide pyrophosphatase phosphodiesterase 1 (Enpp1 / ENPP1) is a transmembrane regulator of nucleotide transport (Ho et al. 2000, Science. 289(5477):265-270; Szeri et al. 2020, PLoS Genet. 16(7):e1008884). iBoth ANK and ENPP1 are ectoenzymes that cleave extracellularly to form PP1 (Rutsch et al. 2003, Nat Genet. 34(4):379-381). i Loss of function of either gene increases PP levels. i Loss of ANK or ENPP1 leads to dramatically thicker acellular cementum (Ao et al. 2017, Bone. 105:134-147; Chu et al. 2020, Bone. 136:115-329; Foster et al. 2012, Bone. 78:150-164; Thumbigere-Math et al. 2018, J Dent Res. 97(4):432-441). - / - Alpl - / - PP by crossing with mice i Genetic reduction of P corrected the cementum development defect. Furthermore, loss of ANK or ENPP1 promoted cementum regeneration in a periodontal fenestration model (Rodrigues et al. 2011, J Periodontol. 82(12):1757-1766; Nagasaki et al. 2020, J Dent Res. In press), suggesting that P i / PP i Implications for metabolic modifiers as strategies to promote periodontal regeneration.

[0170] Mice lacking the extracellular matrix (ECM) protein bone sialoprotein (BSP / Ibsp) exhibit severe periodontal damage, including reduced cementum, PDL detachment and tissue breakdown, and alveolar bone destruction (Foster et al. 2015; Foster et al. 2013, J Dent Res. 92(2):166-172). - / - Ibsp - / - PP by crossing with mice i The genetic decline of Ibsp - / -This resulted in correction of cementum defects noted in mice (Ao et al. 2017, Bone. 105:134-147). As disclosed herein, delivery of TNAP enhances PP i Decrease P i Pharmacological approaches to increase levels of Ibsp in periodontal disease - / - It was determined that Ibsp can correct cementum defects and promote cementum regeneration in a model. Using a lentiviral construct expressing mineral-targeted TNAP (Yamamoto et al. 2011, J Bone Miner Res. 26(1):135-142) for systemic delivery, Ibsp can be transfected into the graft. - / - Recombinant human TNAP (rhTNAP) was used for local delivery to treat developmental defects in the adult Ibsp model. - / - Periodontal fenestration defects in mice were treated (Rodrigues et al. 2011, J Periodontol. 82(12):1757-1766). - / - A cementoblast cell line was used to determine the effect of bovine intestinal ALP (bIAP) on gene expression and mineralization in vitro.

[0171] Example 1 Materials and Methods Animals: Ibsp on a 129P3 / CD1 background - / - Mice were previously described (Ao et al. 2017, Bone. 105:134-147). Male wild-type (WT) control and Ibsp - / - Mice were used to minimize potential gender variations. At weaning, mice were fed a soft diet (Diet Gel 31M, ClearH2O, Portland, ME) in addition to their regular chow (NIH-31) and maintained at Ibsp - / - Reduced incisor malocclusion in mice.

[0172] Systemic TNAP lentiviral vector delivery: For systemic delivery, high levels of alkaline phosphatase (ALP / Alpl) are produced, and Alpl - / -Mineral-targeted TNAP with a decaaspartic acid tail (TNAP-D), previously shown to correct skeletal defects in mice, 10 A lentiviral vector expressing TNAP-D in phosphate-buffered saline (PBS) was used (Cellomics Technology, Halethorpe, MD) (Yamamoto et al. 2011, J Bone Miner Res. 26(1):135-142). 10 Lentivirus (5.0 × 10 7 Transducing units (TU) were mixed with Evans blue dye (Sigma-Aldrich Inc, St. Louis, MO) and monitored by intramuscular injection using a Hamilton syringe and a 34-gauge Hamilton needle (Hamilton Company, Reno, NV). Five-day-old (dpn) bone sialoproteinase (Ibsp) knockout mice were injected intramuscularly. - / - ) Mice were injected into the quadriceps muscle (n=3). - / - and wild-type (WT) mice were injected with PBS vehicle (n = 3 / genotype). Blood was collected from the retro-orbital sinus at 30 dpn and by cardiac puncture at 60 dpn for measurement of plasma ALP levels. Mice were euthanized by cervical dislocation at 60 dpn.

[0173] Local recombinant human TNAP delivery: A mouse fenestration defect model, modified from a previous report, was used to deliver rhTNAP (0.1 μg in 1 μl) (R&D Systems, Minneapolis, MN) to 5-week-old WT and Ibsp mice. - / -This study was performed in mice (n = 6 / genotype) (Figure 6) (King et al. 1997, J Dent Res. 76(8):1460-1470; Rodrigues et al. 2011, J Periodontol. 82(12):1757-1766). PBS (1 μl) was used as a negative control (n = 6 / group). Prior to surgery, mice were anesthetized with 100 mg / kg ketamine and 7 mg / kg xylazine intraperitoneally. A skin incision was made around the buccal aspect of the left mandible. After securing visualization of the surgical site using an operating microscope, periodontal fenestration bone defects (approximately 2 × 1.5 × 0.5 mm) were created in the buccal aspect of the mandible around the distal root of the first molar and the mesial root of the second molar. Type I collagen scaffolds (Zimmer Collagen Patch Absorbable Wound Dressing, Collagen Matrix Inc., Oakland, NJ) were used to deliver rhTNAP or PBS. Tissues were repositioned and sutured using 4-0 Coated VICRYL (Polyglactin 910; Ethicon Inc., Somerville, NJ). Buprenorphine (0.05 mg / 20 g) was administered immediately before or after surgery for pain management. Mice were euthanized on postoperative day (POD) 45.

[0174] Blood biochemistry: To measure plasma alkaline phosphatase activity (ALP), whole-body TNAP-D 10 Blood was collected at 30 and 60 days after administration (n=3) for the experiment, and on day 45 of administration (n=6) for the local delivery experiment of rhTNAP. From the mice enrolled in the systemic delivery experiment, blood was collected from the retro-orbital sinus at 30 days after administration. The mice were euthanized by cervical dislocation, and then blood was collected by cardiac puncture in lithium heparin gel tubes (Becton, Dickinson and Company, Franklin Lakes, NJ). Plasma samples were kept at room temperature for 30 minutes and then centrifuged at 10,000 × g for 30 minutes at 4 ° C. Plasma samples were stored at -80 ° C until ALP measurement was performed.

[0175] Micro-computed tomography (micro-CT): To measure the volume and density of regenerated bone, micro-CT analysis was performed using a modification of a previously described approach (Ao et al. 2017). Formalin-fixed unilateral mandibles were scanned in 70% ethanol in a μCT 50 (Scanco Medical, Bassersdorf, Switzerland) at 70 kVp, 85 μA, a 0.5 mm Al filter, a 900 ms integration time, and a 6 μm voxel dimension. DICOM files were created from the scans and uploaded to AnalyzePro 1.0 (AnalyzeDirect, Overland Park, KS). Density (mg HA / cm) of known bone was measured. 3 A standard curve was determined using five hydroxyapatite (HA) standards. To measure regenerated bone, a region of interest was created starting from the center between the mesial and distal roots of the first molar and extending 200 slices (1,200 μm) mesial and distal from the root apex and 175 slices (1,050 μm) coronally.

[0176] Histology: Tissues were decalcified in acetic acid / formalin / sodium chloride (AFS) solution and embedded in paraffin for 5 μm sections (Ao et al. 2017). Hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), picrosirius red staining, and histomorphometry were performed. Mandibles were fixed in 10% neutral buffered formalin for 48 hours at room temperature and then placed in 70% ethanol for storage. After microCT scanning, specimens were demineralized in AFS (acetic acid, formaldehyde, sodium chloride) for 4 weeks as previously described (Ao et al. 2017, Bone. 105:134-147). After decalcification, specimens were washed twice in 70% ethanol and embedded in paraffin for 5 μm serial sectioning. TNAP-D 10In the development experiment, the mandible was sectioned in the coronal direction. In the local delivery experiment of rhTNAP, the mandible was sectioned in the axial direction. Due to the limited size of the defects for local delivery of rhTNAP, approximately 20 serial sections were prepared from each specimen. For standardization, slides at the same axial level were used for each staining. H&E staining was performed for morphological analysis. TNAP-D was visualized using NDP.view2 Viewing software (Hamamatsu Photonics KK, Hamamatsu, Shizuoka, Japan). 10 In the development experiment, acellular cementum thickness was measured apically at 100 μm from the cementoenamel junction (CEJ) on the buccal and lingual sides, and in the local delivery experiment of rhTNAP, cementum thickness was measured at the same axial level. Immunohistochemistry (IHC) was performed using an avidin-biotinylated peroxidase (ABC)-based kit (Vectastain Elite, Vector Labs, Burlingame, CA) and 3-amino-9-ethylcarbazole (AEC) substrate, which produces a reddish-brown product, as previously described (Ao et al. 2017). Primary antibodies included rabbit polyclonal LF-175 rabbit anti-mouse osteopontin (OPN; Kerafast, Inc., Boston, MA) diluted 1:200, rabbit polyclonal M176 anti-dentin matrix protein 1 (DMP1; TakaRa Bio Inc., Kusatsu, Shiga, Japan) diluted 1:600, and goat polyclonal ab31303 anti-asporin (ASPN; Abcam Inc., Cambridge, MA) diluted 1:50. Negative controls included slides processed by the same protocol except for the absence of primary antibody addition. To analyze the periodontal ligament (PDL), picrosirius red staining (Polysciences, Inc., Warrington, PA) was performed according to the manufacturer's instructions.

[0177] Cell culture: Using CRISPR / Cas9 to transfect Ibsp - / -Immortalized mouse cementoblasts (OCCM.30) were engineered (Ao et al. 2017, Bone. 105:134-147). WT parental cells served as a normal control. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Life Technologies Corporation, Grand Island, NY) containing 10% fetal bovine serum (FBS; Hyclone Laboratories, South Logan, UT), 2 mM L-glutamine (Life Technologies Corporation), 100 U / ml penicillin (Life Technologies Corporation), and 100 μg / ml streptomycin (Life Technologies Corporation). - / - For all in vitro experiments, 4 × 10 cells were maintained in growth medium at 2.5 ml / well in 6-well cell culture dishes or 0.5 ml / well in 24-well cell culture dishes. 4 Cells were seeded at 100 cells / ml. After 24 hours, the medium was replaced with calcification medium consisting of DMEM supplemented with 2% FBS, 50 μg / ml ascorbic acid (AA; Sigma-Aldrich Inc.), and 10 mM β-glycerophosphate (BGP; Sigma-Aldrich Inc.) (Osathanon et al. 2009), 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin.

[0178] To increase ALP levels in vitro, we used bovine intestinal alkaline phosphatase (bIAP) (Sigma-Aldrich Inc) (Osathanon et al. 2009, Biomaterials. 30(27):4513-4521). bIAP is a TNAP analog encoded by the gene AlpI, and has a different substrate specificity from TNAP, but it also inhibits PP. ibIAP has been shown to hydrolyze ATP and BGP (Manes et al. 1998, J Biol Chem. 273(36):23353-23360; Millan 2006, Purinergic Signal. 2(2):335-341; Weissig et al. 1993, Biochem J. 290 ( Pt 2):503-508). bIAP was not used in cell culture experiments because large amounts of the enzyme are required, making cost a factor in experimental design. bIAP (100 μg / ml) was added when maintenance medium was replaced with calcification medium.

[0179] Phosphonoformic acid (PFA; Sigma-Aldrich Inc.) was used to inhibit sodium-phosphate cotransport (Foster et al. 2012, PLoS One. 7(6):e38393). Type II sodium-phosphate cotransporter i PFA, a nonspecific, low-affinity competitive inhibitor of the cotransporter, was used to i We investigated how transfection affects signal transduction and mineralization in vitro (Addison et al. 2007, J Biol Chem. 282(21):15872-15883; Foster et al. 2006, Calcif Tissue Int. 78(2):103-112). A concentration of 0.1 mM PFA was used to reduce any potential direct effects on mineral nodule formation described (Villa-Bellosta et al. 2009, Am J Physiol Renal Physiol. 296(4):F691-699). 0.1 mM was tested and determined to be nontoxic to OCCM.30 cells. 24 hours after plating, cells were pretreated or untreated with PFA for 2 hours and then treated with BGP + / - bIAP for 48 hours.

[0180] Medium P according to the manufacturer's instructions (Sigma-Aldrich Inc.) iLevels were analyzed. Calcification was measured by Alizarin Red staining (EMS, Hatfield, PA) as previously described (Foster et al. 2012, PLoS One. 7(6):e38393).

[0181] Inorganic phosphate (P i ) Measurement: P in cell culture medium i To measure the concentration of P, cells were cultured as mentioned above and the medium was collected after 24 hours. The cell culture medium at confluence was analyzed for P using a phosphate assay kit according to the manufacturer's instructions (Sigma-Aldrich Inc.). i The P in the medium was analyzed using a plate reader at 620 nm. i Levels were measured and calculated using a standard curve included with the test kit.

[0182] Alizarin Red Staining: To analyze in vitro mineral nodule formation, cells exposed to mineralization medium for 2, 4, or 6 days were assayed as previously described (Foster et al. 2012, PLoS One. 7(6):e38393). Briefly, alizarin red stain (Electron Microscopy Sciences, Hatfield, PA) bound to mineral deposits was eluted with cetylpyridinium chloride and measured using a plate reader set at a wavelength of 490 nm.

[0183] Quantitative polymerase chain reaction (qPCR): Total RNA was extracted from cells using the MagMAX mirVava Total RNA Isolation Kit (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) and quantified using standard spectrophotometric methods. Complementary DNA (cDNA) was synthesized from 250 μg of total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Indianapolis, IN). Gapdh was used as an internal control for normalization. Primer sequences for qPCR are listed in Table 1. Amplification with the SYBR Green I Master Kit for qPCR (Roche Diagnostics, Indianapolis, IN) was performed in a LightCycler 480 thermal cycler.

[0184] [Table 1]

[0185] Statistical analysis: Results are reported as mean ± standard deviation. Independent sample t-test (p < 0.05) * ), p<0.01( ** ) or p<0.001( *** ) indicates significance) or one-way ANOVA with post hoc Tukey's test (experimental groups marked by different letters are significantly different (p<0.05); groups sharing the same letter are not significant (p>0.05)) and data were analyzed (Prism 7; GraphPad Software, La Jolla, CA).

[0186] Example 2 Whole body TNAP-D 10 Delivery is by Ibsp - / - Improves periodontal development in mice Systemic delivery of TNAP is - / -To determine whether the developmental cementum defect in mice can be overcome, we administered mineral-targeted TNAP-D to 5-day-old mice (5 dpn; before molar root formation). 10 The mice were intramuscularly injected with a lentivirus encoding TNAP-D. 10 Treatment consisted of PBS-treated WT and Ibsp at 30 dpn. - / - mice, resulting in an almost five-fold increase in plasma ALP, which returned to normal levels by 60 dpn (Fig. 1A).

[0187] Cementum and PDL were evaluated by histology. 10 PBS treatment Ibsp - / - Although PBS-treated Ibsp mice increased cementum thickness on both the buccal and lingual sides (3-fold and 2-fold, respectively), the cementum thickness remained reduced by 20-40% compared to WT mice (Figure 1B). - / - Mice exhibited a detached and disorganized PDL, whereas TNAP-D 10 The treatment reestablished PDL attachment and organization comparable to that of untreated control WT mice (Figure 1C). The restored cementum was characterized by immunostaining for mineralization markers, osteopontin (OPN) and dentin matrix protein-1 (DMP1). PBS-treated Ibsp - / - Unlike WT controls, TNAP-D mice lacked obvious OPN localization, indicating acellular cementum at the root surface (Figure 1D). 10 Ibsp - / - PBS treatment increased OPN deposition on the root surface of mice. - / - and TNAP-D in contrast to minimal DMP1 expression in the cementum of untreated control WT mice. 10Increased DMP1 expression along the root surface (Figure 1E). Asporin (Aspn / ASPN), a negative regulator of mineralization (Kajikawa et al. 2014, J Dent Res. 93(4):400-405; Yamada et al. 2007, J Biol Chem. 282(32):23070-23080), was upregulated in Ibsp1 compared with untreated control WT mice, as previously demonstrated by qPCR (Ao et al. 2017, Bone. 105:134-147). - / - TNAP-D was increased in the PDL of the aged (Fig. 1F). 10 Treatment is continued until the Ibsp - / - Picrosirius red staining showed that TNAP-D decreased ASPN in mice. 10 but comparable to untreated control WT mice, Ibsp - / - We found that it induced PDL adhesion in mice (Figure 1G).

[0188] Alveolar bone was assessed by micro-CT and histology. Alveolar bone volume was significantly increased by TNAP-D. 10 Treated vs. untreated Ibsp - / - The volume increased by 20% in the untreated mice, but remained reduced relative to untreated control WT levels (Figures 2A-C). - / - It did not significantly alter mouse alveolar bone density, which remained reduced relative to WT controls. - / - Vs. untreated control WT alveolar bone revealed increased OPN staining, whereas TNAP-D 10 Ibsp - / - TNAP-D further increased OPN deposition in mice (Figure 2E). 10 Ibsp - / - It also increased DMP1 expression in the alveolar bone of mice, restoring levels to those comparable to those in WT mice ( Fig. 2F ).

[0189] Example 3 Local delivery of rhTNAP was performed using Ibsp - / - Promotes cementum and alveolar bone regeneration in mice TNAP-D 10 Systemic delivery of Ibsp - / - Restores cementum formation and periodontal function in mice, and P i / PP i Having confirmed that pharmacological modulation of rhTNAP can overcome the inherent mineralization defect, we determined the efficacy of local rhTNAP delivery; this is a more translational approach closer to the clinical scenario for the treatment of periodontal disease. Fenestrated defects were created in 5-week-old mice. The postoperative day 45 (POD45) recovery period was uneventful, with no overt signs of inflammation or infection. Local delivery of rhTNAP did not affect plasma ALP levels (Figure 3A).

[0190] Cementum and PDL were evaluated by histology. rhTNAP treatment increased cementum thickness and Ibsp in WT mice. - / - rhTNAP promoted the formation of a cementum-like layer on the root surface of the mice (21-fold and 42-fold, respectively), and the mineralized matrix extended into the PDL space, but no ankylosis of the tooth-bone joint was observed (Fig. 3B, Fig. 3C). - / - Improved PDL attachment and organization compared to mice (Figure 3C). - / - Although both OPN and DMP1 localization increased in response to rhTNAP treatment in mice, treatment differences were not evident in WT mice, and tissues with compromised cementum, e.g., Ibsp - / - This suggests more active cementum formation in the PDL of mice (Fig. 3D, Fig. 3E). ASPN localization in the PDL was significantly higher in Ibsp mice treated with rhTNAP than in the vehicle control. - / - The intensity of TNAP-D appeared to be lower in mice (Fig. 3F). 10 Similar to the above, local delivery of rhTNAP was confirmed by picrosirius red staining. - / - PDL attachment in PBS- and rhTNAP-treated WT mice was induced (Figure 3G). However, PDL attachment / insertion into the cementum in PBS- and rhTNAP-treated WT mice was less evident than in untreated WT mice (Figure 7), suggesting that PDL regeneration occurs more slowly than cementum regeneration.

[0191] Alveolar bone was evaluated by micro-CT and histology. rhTNAP significantly reduced the alveolar bone mass in WT (17% and 5%, respectively) and Ibsp. - / - Local rhTNAP delivery significantly increased alveolar bone volume and density in both WT and Ibsp mice (37% and 5%, respectively) (Figure 4A-4C). - / - Increased expression of OPN and DMP1 in the regenerated alveolar bone of both mice (Figures 4D and 4E).

[0192] Example 4 bIAP increases mineralization in cementoblasts To further define the mechanistic aspects of TNAP-mediated periodontal regeneration, Ibsp, which exhibits reduced mineral formation, - / - Immortalized mouse cementoblasts (OCCM.30) were used versus WT cells (Ao et al. 2017, Bone. 105:134-147). Alpl expression was measured by Ibsp expression on day 1. - / - 80% reduction in Ibsp cells compared to medium from WT controls (p<0.01; Fig. 5A). - / - The cells showed a 40% reduction in P i The levels were shown. Addition of 100 μg / ml bIAP significantly reduced the levels of WT and Ibsp - / - P in both cell culture media i The levels of Ibsp were increased approximately 2-3 fold (p<0.01) (Figure 5B). - / - The cells showed less mineralization. Addition of bIAPs significantly reduced the mineralization of WT and Ibsp - / - bIAP treatment increased mineralization in both Ibsp and Ibsp cells. - / - Mineral deposition by α-glucan lagged behind treated WT cells by day 6 (p<0.01) (Fig. 5C).

[0193] In addition to its contribution to HA crystal growth, P ibIAP treatment increased the expression of IL-1, which acts as a signaling factor for many cells, including cementoblasts (Beck et al. 2000, Proc Natl Acad Sci U S A. 97(15):8352-8357; Chaudhary et al. 2016, Matrix Biol. 52-54:284-300; Rutherford et al. 2006, J Dent Res. 85(6):505-509). i Based on the concentration, P i We examined whether inhibiting transport would impair the positive effects of type II sodium P on mineralization. i We used PFA, a nonspecific, low-affinity competitive inhibitor of the cotransporter (Addison et al. 2007, J Biol Chem. 282(21):15872-15883; Foster et al. 2006, Calcif Tissue Int. 78(2):103-112). As previously described (Villa-Bellosta et al. 2009, Am J Physiol Renal Physiol. 296(4):F691-699), we used a relatively low concentration of 0.1 mM PFA to investigate the altered P activity in mineral nodule formation. i The addition of PFA reduced the potential direct effect of WT and Ibsp. - / - inhibited the bIAP-induced increase in mineral nodule formation in both cells, but Ibsp - / - The effect of PFA on cells was minimal, with bIAP and PFA-treated WT and Ibsp - / - Mineral deposition by cells was equivalent (Figure 5D). In the presence of bIAP, control and Ibsp - / - Spp1 expression in both WT and Ibsp cells was increased by 29- and 26-fold, respectively (p<0.0001). - / - bIAP inhibited Spp1 expression in both WT and Ibsp1 cells by 55% (p<0.001) (Figure 5E). - / - It increased Dmp1 expression by 42-fold and 10-fold in both WT and Ibsp cells (p<0.0001).- / - Phosphate-induced Dmp1 expression in cells was inhibited by PFA by 60% and 49%, respectively (p<0.001) (Fig. 5F). Aspn expression was upregulated by Ibsp - / - bIAPs were increased 7-fold in WT vs. WT cells (p<0.001). - / - Both α-glucan and α-glucan reduced Aspn expression in α-glucan-containing cells by 97% and 57% (p<0.01), but the P of Aspn expression i The induced downregulation was not affected by PFA (Figure 5G). - / - Alpl expression in cells was significantly reduced by 37% by bIAP (p<0.0001), consistent with the trend in WT cells. - / - There was a trend for increased Alpl expression by PFA in cells (Fig. 5H), but PFA did not significantly increase the expression of WT and Ibsp - / - Both bIAP-inducing medium P i These results suggest that bIAP-induced calcification by WT cells was more potent than that by WT cells. i While dependent on import (and potentially signaling), Ibsp - / - bIAP-induced calcification in cells i This suggests that it may act both through import and through a direct effect on the mineralization process.

[0194] Periodontal tissues, especially cementum, are i / PP i It is sensitive to metabolic regulators (Chu et al. 2020, Bone. 136:115329; Thumbigere-Math et al. 2018, J Dent Res. 97(4):432-441; Zweifler et al. 2015, Int J Oral Sci. 7(1):27-41; Nagasaki et al. 2020, J Dent Res. In press). Novel Ibsp of periodontal decay - / - Using a mouse model, we investigated the P i / PP iIt was determined whether pharmacological modulation of TNAP-D could promote cementum formation. 10 Systemic delivery of increases circulating plasma ALP levels and Ibsp - / - Restores cementum formation and periodontal function in mice, and P i / PP i Pharmacological approaches to modulate Ibsp - / - We confirmed that local delivery of rhTNAP in a periodontal fenestration defect model can overcome the inherent periodontal defect in mice. - / - We confirmed that increased TNAP promotes periodontal regeneration by promoting increased cementum and bone regeneration and restoring periodontal attachment in mice. Importantly, rhTNAP also increased the thickness of regenerated cementum in WT mice, supporting this as a generalizable approach for periodontal regeneration. Increased ALP by delivery of bIAPs was associated with increased Ibsp activity in vitro. - / - It partially rescued the mineralization defect in cementoblasts, implicating a direct effect on mineralization and ECM protein expression.

[0195] Dental defects, including acellular cementum hypoplasia, periodontal disease, and premature loss of primary and secondary teeth, are common among individuals with HPP. These periodontal defects are phenocopied in mouse models of HPP (Foster et al. 2017, J Dent Res. 96(1):81-91; McKee et al. 2011, J Dent Res. 90(4):470-476; Zweifler et al. 2015, Int J Oral Sci. 7(1):27-41). The role of P in the mineralization process i Further evidence of the important role played by PHEX is highlighted by the inherited hypophosphatemic disorders X-linked hypophosphatemia (XLH; OMIM #307800) is caused by inactivating mutations in PHEX and results in reduced serum P. iThis results in impaired skeletal and dental mineralization, including low levels of bone, thin cementum, poorly mineralized alveolar bone, and a high prevalence of periodontal disease (Biosse Duplan et al. 2017, J Dent Res. 96(4):388-395).

[0196] Conversely, loss-of-function mutations in ENPP1 or ANKH, respectively, result in reduced extracellular PP in the disorder. i This leads to high levels of ectopic calcium deposition, generalized arterial calcium deposition in infancy (GACI; OMIM#208000), and craniometaphyseal dysplasia (CMD; OMIM#123000). In mouse models of these disorders, acellular cementum increases dramatically (Dutra et al. 2013, J Dent Res. 92(2):173-179; Foster et al. 2012, PLoS One. 7(6):e38393; Zweifler et al. 2015, Int J Oral Sci. 7(1):27-41), and similarly expanded acellular cementum is attributed to GACI in humans (Thumbigere-Math et al. 2018, J Dent Res. 97(4):432-441). i Confirming that concentration is the primary driver of these outcomes, Alpl - / - and Ank - / - Crossbreeding of the mice resulted in correction of cementum formation and restoration of periodontal attachment and function (Chu et al. 2020, Bone. 136:115329). Collectively, these data support the role of P as an evolutionarily conserved mechanism regulating periodontal tissues. i / PP i Supports adjustment. P i Addition of P i / PP iCorrection of the ratio improved mineralization in primary PDL and dental pulp cells obtained from subjects with HPP (Rodrigues et al. 2012, J Periodontol. 83(5):653-663; Rodrigues et al., 2012, J Endod. 38(7):907-912). The FDA-approved mineral-targeting TNAP-D, designed to deliver enzymatic activity to the mineralization front, 10 The molecule was remarkably effective in correcting skeletal effects, promoting dental development, and extending lifespan in severe HPP (Bowden and Foster 2019, Adv Exp Med Biol. 1148:279-322; Foster et al. 2012, PLoS One. 7(6):e38393; McKee et al. 2011, J Dent Res. 90(4):470-476; Millan 2006, Purinergic Signal. 2(2):335-341; Whyte 2016, Nat Rev Endocrinol. 12(4):233-246).

[0197] The data presented herein demonstrate that PP i showed that pharmacological reduction of levels was effective in promoting cementum formation. - / - TNAP-D has been shown to increase survival time and improve skeletal mineralization in mice 10 Systemic delivery experiments using lentiviral constructs encoding TNAP-D 10 However, Ibsp in early postnatal age - / - We demonstrated that TNAP promoted cementum formation in mice, along with the maintenance of periodontal attachment and alveolar bone, demonstrating that pharmacological administration of TNAP can enhance cementum formation in the absence of BSP, which lacks mineralization.

[0198] The effect of local delivery of rhTNAP in the periodontal fenestration model is similar to the clinical scenario of periodontal disease. As disclosed herein, local delivery of rhTNAP is - / -In both WT and WT mice, ASPN significantly increased alveolar bone volume and bone mineral density, promoted cementum regeneration and PDL attachment at the root surface, and increased the ECM localization of mineralization markers, i.e., OPN and DMP1. Previous studies have shown that ASPN is an inhibitor of mineralization (Kajikawa et al. 2014, J Dent Res. 93(4):400-405; Yamada et al. 2007, J Biol Chem. 282(32):23070-23080), and Ibsp - / - The localization of ASPN was increased in the PDL of mice (Ao et al. 2017, Bone. 105:134-147). - / - The PDL of mice was reduced. Thus, local delivery of rhTNAP is a viable strategy for promoting periodontal regeneration. The benefits of local delivery of recombinant proteins include limited promineralization activity concentrated in local tissues and potentially fewer side effects than systemic delivery. Both TNAP and its intestinal analog IAP possess anti-inflammatory properties through detoxification of bacterial lipopolysaccharides, adding another potential additional therapeutic benefit.

[0199] Cell culture experiments have shown that bIAPs are - / - We confirmed in vivo results demonstrating partial rescue of mineralization deficiency in OCCM.30 cementoblasts, which was consistent with the in vivo data, by increasing P i and associated with altered gene expression. i Addition of PFA, a nonspecific, low-affinity competitive inhibitor of cotransporters, blocked the bIAP-induced increase in calcification. Thus, without being bound by theory, the positive effects of TNAP in vivo may be due, in part, to the P i This may be related to its ability to increase local levels of

[0200] Several in vivo and in vitro approaches disclosed herein demonstrate the efficacy of TNAP for P i / PPi We demonstrate that pharmacological modulation of metabolism can overcome periodontal destruction and achieve regeneration and restoration to functional periodontal tissues.

[0201] In view of the many possible embodiments to which the principles of Applicants' invention may be applied, it should be recognized that the described embodiments are merely exemplary of the invention and should not be considered limitations on the scope of the invention. Rather, the scope of the invention is defined by the following claims. Applicants therefore claim as their invention all that comes within the scope and spirit of such claims. In certain embodiments, for example, the following are provided: (Item 1) 1. A method of treating periodontal disease, peri-implantitis, or preserving a dental alveolus in a subject, comprising: selecting a subject with periodontal disease, peri-implantitis or in need of alveolar preservation; locally administering to the periodontal tissue of the subject an effective amount of a recombinant tissue non-specific alkaline phosphatase (TNAP) polypeptide comprising SEQ ID NO: 1, or a nucleic acid molecule encoding the recombinant TNAP polypeptide; thereby treating the periodontal disease, peri-implantitis, or preserving the dental alveolus in the subject. (Item 2) 2. The method of claim 1, wherein the subject does not have hypophosphatasia (HPP). (Item 3) 3. The method of claim 1, wherein the recombinant TNAP polypeptide has a C-terminus and an N-terminus, and the recombinant TNAP polypeptide further comprises 4 to 12 aspartic acid residues at the C-terminus or 4 to 12 glutamic acid residues at the C-terminus. (Item 4) 4. The method of claim 3, wherein the recombinant TNAP polypeptide comprises 6 to 8 aspartic acid residues at the C-terminus. (Item 5) 3. The method of claim 1 or 2, wherein the recombinant TNAP polypeptide does not contain repeats of aspartic acid residues at the C-terminus. (Item 6) 6. The method of any one of items 1 to 5, wherein the recombinant TNAP polypeptide further comprises a signal sequence at the N-terminus. (Item 7) 7. The method of item 6, wherein the signal sequence comprises SEQ ID NO: 2. (Item 8) 8. The method of any one of items 1 to 7, wherein the recombinant TNAP polypeptide further comprises a histidine tag polypeptide comprising at least six histidine residues. (Item 9) 9. The method of claim 8, wherein the histidine tag polypeptide comprises SEQ ID NO: 3. (Item 10) 10. The method of any one of items 1 to 4 or 6 to 9, wherein the recombinant TNAP polypeptide comprises, in order from the amino (N) to carboxy (C) terminus, a signal sequence, a tag polypeptide, the amino acid sequence set forth as SEQ ID NO: 1, and 4 to 8 aspartic acid residues. (Item 11) 11. The method of claim 10, wherein the recombinant TNAP polypeptide comprises 6 to 8 aspartic acid residues. (Item 12) 12. The method of claim 11, wherein the recombinant TNAP polypeptide comprises SEQ ID NO:4. (Item 13) 10. The method of any one of items 1-2 or 6-9, wherein the recombinant TNAP polypeptide comprises SEQ ID NO:5. (Item 14) 14. The method of any one of items 1 to 13, wherein the locally administering step comprises injection into the periodontal tissue. (Item 15) 15. The method of any one of items 1 to 14, comprising locally administering the recombinant TNAP polypeptide to the subject using a biocompatible scaffold. (Item 16) 16. The method of any one of items 1 to 15, wherein i) promoting alveolar bone regeneration in said subject, ii) increasing the attachment of the periodontal ligament to the root surfaces of the teeth of said subject, iii) increasing cementum formation in said subject, and / or iv) increasing mineralization in the teeth of said subject. (Item 17) 1. A method of treating a subject, comprising: locally administering to the periodontal tissue of the subject an effective amount of a recombinant TNAP polypeptide comprising SEQ ID NO: 1, or a nucleic acid molecule encoding said recombinant TNAP polypeptide. i) promoting alveolar bone regeneration in said subject; ii) increasing the attachment of the periodontal ligament to the root surface of a tooth in said subject; iii) increasing cementum formation; and / or iv) increasing mineralization in a tooth in said subject. (Item 18) 18. The method of item 17, wherein the subject has alveolar bone loss caused by periodontal conditions such as periodontitis and tooth extraction, dental implants, peri-implantitis and / or sinus enlargement, and inherited hypomineralization disorders, hypophosphatasia (HPP), or other disorders characterized by tooth loss due to cementum defects. (Item 19) 19. The method of claim 17 or 18, wherein the subject does not have hypophosphatasia (HPP). (Item 20) 20. The method of any one of items 17 to 19, wherein the recombinant TNAP polypeptide has a C-terminus and an N-terminus, and the recombinant TNAP polypeptide further comprises 4 to 12 aspartic acid residues at the C-terminus or 4 to 12 glutamic acid residues at the C-terminus. (Item 21) 21. The method of claim 20, wherein the recombinant TNAP polypeptide comprises 6 to 8 aspartic acid residues at the C-terminus. (Item 22) 20. The method of any one of items 17 to 19, wherein the recombinant TNAP polypeptide has a C-terminus and an N-terminus, and the recombinant TNAP polypeptide does not contain aspartic acid repeats at the C-terminus. (Item 23) 23. The method of any one of items 17 to 22, wherein the recombinant TNAP polypeptide further comprises a signal sequence at the N-terminus. (Item 24) 24. The method of claim 23, wherein the signal sequence comprises SEQ ID NO: 2. (Item 25) 25. The method of any one of items 17 to 24, wherein the recombinant TNAP polypeptide further comprises a tag polypeptide comprising at least six histidine residues. (Item 26) 26. The method of item 25, wherein the tag polypeptide comprises SEQ ID NO: 3. (Item 27) 26. The method of any one of items 17 to 21 or 23 to 25, wherein the recombinant TNAP polypeptide comprises, in order from the amino (N) to the carboxy (C) terminus, a signal sequence, a tag polypeptide, the amino acid sequence set forth as SEQ ID NO: 1, and 4 to 8 aspartic acid residues. (Item 28) 28. The method of claim 27, wherein the recombinant TNAP polypeptide comprises 6 to 8 aspartic acid residues. (Item 29) 29. The method of claim 28, wherein the recombinant TNAP polypeptide comprises SEQ ID NO:4. (Item 30) 26. The method of any one of items 22 to 25, wherein the recombinant TNAP polypeptide comprises the amino acid sequence of SEQ ID NO: 5. (Item 31) 31. The method of any one of items 17 to 30, wherein the locally administering step comprises injection into the periodontal tissue. (Item 32) 31. The method of any one of items 17 to 30, wherein the step of locally administering the TNAP polypeptide comprises administration using a biocompatible scaffold. (Item 33) 1. A pharmaceutical composition comprising an effective amount of a tissue non-specific alkaline phosphatase (TNAP) polypeptide comprising SEQ ID NO: 1, or a nucleic acid molecule encoding said recombinant TNAP polypeptide, for use in treating periodontal disease according to the method of any one of items 1 to 16, treating alveolar bone disease according to the method of any one of items 18 to 32, or promoting dental implant retention and alveolar preservation according to the method of any one of items 18 to 32. (Item 34) A recombinant polypeptide comprising, in order from N to C terminus, a signal sequence, a histidine tag and the amino acid sequence of SEQ ID NO:1. (Item 35) 35. The recombinant polypeptide of item 34, wherein the signal sequence comprises the amino acid sequence of SEQ ID NO:2. (Item 36) 36. The recombinant polypeptide of claim 34 or 35, wherein the histidine tag comprises the amino acid sequence of SEQ ID NO: 3. (Item 37) 37. The recombinant polypeptide according to any one of items 34 to 36, further comprising 4 to 12 aspartic acid residues at the C-terminus. (Item 38) 38. The recombinant polypeptide according to any one of items 34 to 37, comprising 6 to 8 aspartic acid residues at the C-terminus. (Item 39) 36. The recombinant polypeptide according to any one of items 34 to 35, comprising the amino acid sequence of SEQ ID NO: 5. (Item 40) 40. The method of any one of items 1 to 32, or the recombinant polypeptide of any one of items 34 to 39, wherein the recombinant TNAP polypeptide is present on or in a collagen (sponge / gel) scaffold or another biocompatible scaffold. (Item 41) 34. A biocompatible scaffold comprising the pharmaceutical composition according to item 33. (Item 42) 40. A nucleic acid molecule encoding the recombinant polypeptide according to any one of items 34 to 39. (Item 43) 43. A recombinant vector comprising the nucleic acid molecule of item 42.

Claims

1. 1. A composition for use in a method of: i) treating periodontal disease affecting alveolar bone or cementum, or periodontal disease involving weakened periodontal ligament attachment to cementum and / or alveolar bone; ii) treating peri-implantitis; or iii) preserving a dental alveolus in a subject, said composition comprising a recombinant tissue non-specific alkaline phosphatase (TNAP) polypeptide comprising SEQ ID NO: 1; said method comprising: selecting a subject having said periodontal disease or said peri-implantitis or in need of alveolar preservation; locally administering the recombinant TNAP to the periodontal tissue of the subject; thereby treating the periodontal disease or the peri-implantitis or preserving the dental alveolus in the subject.

2. The composition of claim 1 , wherein the subject does not have hypophosphatasia (HPP).

3. 3. The composition of claim 1 or claim 2, wherein the recombinant TNAP polypeptide has a C-terminus and an N-terminus, and the recombinant TNAP polypeptide further comprises 4 to 12 aspartic acid residues at the C-terminus or 4 to 12 glutamic acid residues at the C-terminus.

4. The composition of claim 3, wherein the recombinant TNAP polypeptide comprises 6 to 8 aspartic acid residues at the C-terminus.

5. The composition described in claim 1 or claim 2, wherein the recombinant TNAP polypeptide has a C-terminus and an N-terminus, and the recombinant TNAP polypeptide does not contain repeated aspartic acid residues at the C-terminus.

6. The composition of any one of claims 3 to 5, wherein the recombinant TNAP polypeptide further comprises a signal sequence at the N-terminus.

7. The composition of claim 6 , wherein the signal sequence comprises SEQ ID NO:

2.

8. The composition of any one of claims 1 to 7, wherein the recombinant TNAP polypeptide further comprises a histidine tag polypeptide comprising at least six histidine residues.

9. The composition of claim 8 , wherein the histidine tag polypeptide comprises SEQ ID NO:

3.

10. 10. The composition of any one of claims 1 to 4 or 6 to 9, wherein the recombinant TNAP polypeptide comprises, in order from amino (N) to carboxy (C) terminus, a signal sequence, a tag polypeptide, the amino acid sequence set forth as SEQ ID NO: 1, and 4 to 8 aspartic acid residues.

11. The composition of claim 10, wherein the recombinant TNAP polypeptide comprises 6 to 8 aspartic acid residues.

12. The composition of claim 11 , wherein the recombinant TNAP polypeptide comprises SEQ ID NO:

4.

13. The composition of any one of claims 1-2 or 6-9, wherein the recombinant TNAP polypeptide comprises SEQ ID NO:

5.

14. The composition of any one of claims 1 to 13, wherein the locally administering step comprises injection into the periodontal tissue.

15. The composition of any one of claims 1 to 14, wherein the method comprises locally administering the recombinant TNAP polypeptide to the subject using a biocompatible scaffold.

16. 16. The composition of any one of claims 1 to 15, wherein the method i) promotes alveolar bone regeneration in the subject, ii) increases the attachment of the periodontal ligament to the root surfaces of teeth in the subject affected by cementum and / or alveolar bone, iii) increases cementum formation in the subject, and / or iv) increases mineralization in teeth in the subject.

17. 1. A composition for use in a method of treating a subject, said composition comprising a recombinant TNAP polypeptide comprising SEQ ID NO:1, said method comprising: locally administering said recombinant TNAP polypeptide to the periodontal tissue of said subject. wherein the method comprises: i) promoting alveolar bone regeneration in the subject; ii) increasing the attachment of the periodontal ligament to the root surface of teeth in the subject having affected cementum and / or alveolar bone; iii) increasing cementum formation; and / or iv) increasing mineralization in teeth in the subject.

18. 18. The composition of claim 17, wherein the subject has alveolar bone loss caused by periodontal conditions such as periodontitis and tooth extraction, dental implants, peri-implantitis and / or sinus augmentation, and inherited hypomineralization disorders, hypophosphatasia (HPP), or other disorders characterized by tooth loss due to cementum defects.

19. 19. The composition of claim 17 or claim 18, wherein the subject does not have hypophosphatasia (HPP).

20. 20. The composition of any one of claims 17-19, wherein the recombinant TNAP polypeptide has a C-terminus and an N-terminus, and the recombinant TNAP polypeptide further comprises 4 to 12 aspartic acid residues at the C-terminus or 4 to 12 glutamic acid residues at the C-terminus.

21. 21. The composition of claim 20, wherein the recombinant TNAP polypeptide comprises 6 to 8 aspartic acid residues at the C-terminus.

22. 20. The composition of any one of claims 17 to 19, wherein the recombinant TNAP polypeptide has a C-terminus and an N-terminus, and wherein the recombinant TNAP polypeptide does not contain aspartic acid repeats at the C-terminus.

23. The composition of any one of claims 17 to 22, wherein the recombinant TNAP polypeptide has a C-terminus and an N-terminus, and the recombinant TNAP polypeptide further comprises a signal sequence at the N-terminus.

24. 24. The composition of claim 23, wherein the signal sequence comprises SEQ ID NO:

2.

25. The composition of any one of claims 17 to 24, wherein the recombinant TNAP polypeptide further comprises a tag polypeptide comprising at least six histidine residues.

26. The composition of claim 25 , wherein the tag polypeptide comprises SEQ ID NO:

3.

27. 26. The composition of any one of claims 17-21 or 23-25, wherein the recombinant TNAP polypeptide comprises, in order from amino (N) to carboxy (C) terminus, a signal sequence, a tag polypeptide, the amino acid sequence set forth as SEQ ID NO:1, and 4 to 8 aspartic acid residues.

28. 28. The composition of claim 27, wherein the recombinant TNAP polypeptide comprises 6 to 8 aspartic acid residues.

29. 29. The composition of claim 28, wherein the recombinant TNAP polypeptide comprises SEQ ID NO:

4.

30. The composition of any one of claims 22 to 25, wherein the recombinant TNAP polypeptide comprises the amino acid sequence of SEQ ID NO:

5.

31. The composition of any one of claims 17 to 30, wherein the step of locally administering comprises injection into the periodontal tissue.

32. The composition of any one of claims 17 to 30, wherein the step of locally administering the TNAP polypeptide comprises administration using a biocompatible scaffold.

33. 33. The composition of any one of claims 1 to 32, wherein the recombinant TNAP polypeptide is present on or in a collagen (sponge / gel) scaffold or another biocompatible scaffold.

34. 34. A biocompatible scaffold comprising the composition of claim 33.

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