JP7720105B2 - Gene therapy constructs for delivery of tissue-nonspecific alkaline phosphatase and methods of use thereof - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63/164,928, filed March 23, 2021, the contents of which are incorporated herein by reference in their entirety.
Reference to an electronically submitted sequence listing
The contents of the Sequence Listing submitted with this application, submitted electronically as an ASCII text file (designation 4627_002PC01_Seqlisting_ST25; size: 40,458 bytes; and creation date: March 22, 2022), are incorporated herein by reference in their entirety.

Hypophosphatasia (HPP) is a hereditary systemic bone disorder characterized by tissue-nonspecific alkaline phosphatase (TNALP) deficiency due to TNALP gene mutations, which result in abnormal bone and dental mineralization (Mornet, E (2007) Orphanet J Rare Dis 2: 40; Henthorn, PS, and Whyte, MP (1992) Clin Chem 38: 2501-2505; Millan, JL, and Whyte, MP (2016) Calcif Tissue Int 98: 398-416). Patients with infantile or perinatal HPP may appear normal at birth but gradually develop clinical rickets within 6 months of age. In many cases, subsequent craniosynostosis and nephrocalcinosis follow hypercalcemia and hypercalciuria (Albeggiani, A, and Cataldo, F (1982) Helv Paediatr Acta 37: 49-58). Additionally, pyridoxine-dependent seizures preceding skeletal changes are predictive of a fatal outcome (Whyte, MP (2016) Nat Rev Endocrinol 12: 233-246). Skeletal changes leading to thoracic deformities and rib fractures cause respiratory problems that are sometimes life-threatening in these HPP patients.

Adult patients with HPP have derive some benefit from several treatment approaches, including enzyme replacement therapy (ERT) to increase plasma alkaline phosphatase (ALP) levels and administration of parathyroid hormone (PTH) (Whyte, MP et al. (1984) J Pediatr 105: 926-933; Whyte, MP et al. (1986) J Pediatr 108: 82-88; Weninger, M, et al. (1989) Acta Paediatr Scand Suppl 360: 154-160; Camacho, PM, et al. (2008) Endocr Pract 14: 204-208; Whyte, MP, et al. (2007) J Clin Endocrinol Metab 92: 1203-1208). However, these therapies can result in the development of antibodies against the recombinant enzyme, and transport of the enzyme into the central nervous system can be difficult. Bone marrow transplantation has been reported to improve symptoms in patients with infantile HPP (Whyte, MP, et al. (2003) J Bone Miner Res 18: 624-636; Tadokoro, M et al. H (2009) J Pediatr 154: 924-930). Allogeneic mesenchymal stem cell transplantation was effective in prolonging survival and bone mineralization in patients with lethal perinatal HPP for 3 years. However, these therapies always involve chemotherapy, radiation therapy, and immunotherapy prior to cell therapy administration. Cell therapy can sometimes result in graft-versus-host disease after transplantation, which can be life-threatening for patients (Taketani, T, et al. (2015) Cell Transplant 24: 1931-1943).

Successful outcomes were observed in mice administered recombinant TNALP fused to decaaspartic acid (D10) for bone targeting (Millan, JL, et al. (2008) J Bone Miner Res 23: 777-787). These results led to the development of ERT using TNALP-D10 for human HPP patients (Bowden, SA, and Foster, BL (2018) Drug Des Devel Ther 12: 3147-3161). However, ERT requires frequent subcutaneous injections (2-3 times per week), which places a heavy burden on patients in terms of administration frequency, pain, and cost. The most common adverse effect of ERT is injection site reactions associated with frequent subcutaneous injections (Kishnani, PS, et al. (2017) Mol Genet Metab 122: 4-17).

Studies involving the treatment of TNALP knockout (Akp2 ^−/− ) mice, a rodent model of HPP, have shown that a single intravenous injection of a lentiviral or adeno-associated viral vector encoding TNALP-D10 resulted in sustained expression of TNALP in Akp2 ^−/− mice (Matsumoto, T, et al. (2011) Hum Gene Ther. 22: 1355-1364; Yamamoto, S, et al. (2011) J Bone Miner Res 26: 135-142). However, intravenous injection resulted in systemic distribution of the vector, and high ALP activity was observed in some organs, which could potentially lead to transduction of cancer or germ cells. Other studies involving the treatment of Akp2 ^−/− mice via intramuscular (IM) injection of a vector encoding TNALP with the muscle-specific creatine kinase (MCK) promoter demonstrated extended survival in Akp2 ^−/− mice (Nakamura-Takahashi, A, et al. (2016) Mol Ther Methods Clin Dev 3: 15059; Ikeue, R, T, et al. (2018) Mol Ther Methods Clin Dev 10: 361-370). However, intramuscular expression was not sufficient to mediate bone maturation in Akp2 ^−/− mice equivalent to that in Akp ^+/+ wild-type (WT) mice. To achieve sufficient plasma ALP activity using the MCK promoter, injection of as much as 15 μL of vector (5.0 × 10 ¹² vg/animal) into both quadriceps muscles of newborn mice was required. However, bone mineralization remained poor.

Mornet, E (2007) Orphanet J Rare Dis 2: 40 Henthorn, PS, and Whyte, MP (1992) Clin Chem 38: 2501-2505 Millan, JL, and Whyte, MP (2016) Calcif Tissue Int 98: 398-416 Albeggiani, A, and Cataldo, F (1982) Helv Paediatr Acta 37: 49-58 Whyte, MP (2016) Nat Rev Endocrinol 12: 233-246 Whyte, MP et al. (1984) J Pediatr 105: 926-933 Whyte, MP et al. (1986) J Pediatr 108: 82-88 Weninger, M, et al. (1989) Acta Paediatr Scand Suppl 360: 154-160 Camacho, PM, et al. (2008) Endocr Pract 14: 204-208 Whyte, MP, et al. (2007) J Clin Endocrinol Metab 92: 1203-1208 Whyte, MP, et al. (2003) J Bone Miner Res 18: 624-636 Tadokoro, M et al. H (2009) J Pediatr 154: 924-930 Taketani, T, et al. (2015) Cell Transplant 24: 1931-1943 Millan, JL, et al. (2008) J Bone Miner Res 23: 777-787 Bowden, SA, and Foster, BL (2018) Drug Des Devel Ther 12: 3147-3161 Kishnani, PS, et al. (2017) Mol Genet Metab 122: 4-17 Matsumoto, T, et al. (2011) Hum Gene Ther. 22: 1355-1364 Yamamoto, S, et al. (2011) J Bone Miner Res 26: 135-142 Nakamura-Takahashi, A, et al. (2016) Mol Ther Methods Clin Dev 3: 15059 Ikeue, R, T, et al. (2018) Mol Ther Methods Clin Dev 10: 361-370

Therefore, there remains a need to develop practical and safe gene therapies for HPP.

Certain aspects of the present disclosure are directed to a polynucleotide comprising a promoter (e.g., a CAG promoter) operably linked to a nucleic acid encoding a fusion protein comprising tissue-nonspecific alkaline phosphatase (TNALP) and a decaaspartic acid (D10) amino acid sequence, wherein the polynucleotide comprises a nucleic acid sequence having at least 85% identity to SEQ ID NO:1.

Certain aspects of the present disclosure are directed to a vector comprising a polynucleotide comprising a promoter (e.g., a CAG promoter) operably linked to a nucleic acid encoding a fusion protein comprising tissue-nonspecific alkaline phosphatase (TNALP) and a decaaspartic acid (D10) amino acid sequence, wherein the polynucleotide comprises a nucleic acid sequence having at least 85% identity to SEQ ID NO:1.

In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 1. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 1.

In some embodiments, the promoter is a CAG promoter. In some embodiments, the polynucleotide further comprises an enhancer, e.g., a CMV enhancer, upstream of the CAG promoter. In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3 or 21. In some embodiments, the CAG promoter comprises the nucleic acid sequence of SEQ ID NO: 21. In some embodiments, the CAG promoter and the CMV enhancer comprise the nucleic acid sequence of SEQ ID NO: 3.

In some embodiments, the nucleic acid encoding the fusion protein comprises a nucleic acid sequence having at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 2 or 5. In some embodiments, the nucleic acid encoding the fusion protein comprises the nucleic acid sequence of SEQ ID NO: 2 or 5.

In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 6. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 6.

In some embodiments, the fusion protein comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 9. In some embodiments, the polynucleotide encodes a fusion protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 9.

In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV is an AAV1 serotype, an AAV2 serotype, an AAV4 serotype, an AAV5 serotype, an AAV6 serotype, an AAV7 serotype, an AAV8 serotype, or an AAV9 serotype. In some embodiments, the AAV is an AAV7 serotype, an AAV8 serotype, or an AAV9 serotype. In some embodiments, the AAV is serotype 8 (AAV8). In some embodiments, the vector is a rhesus adeno-associated virus (AAVrh) vector. In some embodiments, the AAVrh is an AAVrh.74 serotype. In some embodiments, the AAVrh is an AAVrh.10 serotype.

In some embodiments, the AAV vector is suitable for intramuscular administration.

Certain aspects of the present disclosure are directed to pharmaceutical compositions comprising a vector of the present disclosure and a pharmaceutically acceptable carrier.

Certain aspects of the present disclosure are directed to pharmaceutical compositions comprising an adeno-associated virus (AAV) vector and a pharmaceutically acceptable carrier, wherein the AAV vector encapsulates a polynucleotide comprising a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:1, wherein the polynucleotide encodes a fusion protein comprising tissue-nonspecific alkaline phosphatase (TNALP) and decaaspartic acid (D10), and wherein the AAV serotype is AAV8.

Certain aspects of the present disclosure are directed to methods of increasing plasma alkaline phosphate (ALP) activity in a subject in need thereof, comprising administering to the subject in need thereof a polynucleotide of the present disclosure, a vector of the present disclosure, or a pharmaceutical composition of the present disclosure. In some aspects, the administration is by intramuscular injection. In some aspects, the administration is by single dose. In some aspects, the single dose is administered by single or multiple injections. In some aspects, the administration is by intramuscular injection at multiple sites.

Certain aspects of the present disclosure are directed to methods of increasing expression of tissue non-specific alkaline phosphatase (TNALP) in a subject in need thereof, comprising administering to the subject in need thereof a polynucleotide of the present disclosure, a vector of the present disclosure, or a pharmaceutical composition of the present disclosure. In some aspects, the administration is by intramuscular injection. In some aspects, the administration is by single dose. In some aspects, the single dose is administered by single or multiple injections. In some aspects, the administration is by intramuscular injection at multiple sites.

Certain aspects of the present disclosure are directed to methods of treating hypophosphatasia (HPP) in a subject in need thereof, comprising administering to the subject in need thereof a polynucleotide of the present disclosure, a vector of the present disclosure, or a pharmaceutical composition of the present disclosure. In some aspects, the administration is by intramuscular injection. In some aspects, the administration is by single dose. In some aspects, the single dose is administered by single or multiple injections. In some aspects, the administration is by intramuscular injection at multiple sites.

In some embodiments, the HPP is perinatal, infantile, pediatric, or adult HPP. In some embodiments, the HPP is a severe infantile form of HPP. In some embodiments, the subject is an infant or a child. In some embodiments, the subject is an adult.

In some embodiments, the subject's plasma alkaline phosphatase (ALP) level is about 5 U/mL to about 100 U/mL, about 15 U/mL to about 100 U/mL, about 25 U/mL to about 100 U/mL, about 50 U/mL to about 100 U/mL, about 75 U/mL to about 100 U/mL, about 5 U/mL to about 75 U/mL, about 5 U/mL to about 50 U/mL, about 5 U/mL to about 25 U/mL, or about 5 U/mL to about 15 U/mL two weeks after administration. In some embodiments, administration is by intramuscular injection. In some embodiments, administration is by single dose. In some embodiments, the single dose is administered by single or multiple injections. In some embodiments, administration is by intramuscular injection at multiple sites.

In some embodiments, the subject has a plasma ALP level of at least 5 U/mL after two weeks of administration. In some embodiments, the subject's plasma ALP level is at least 10 U/mL after two weeks of administration. In some embodiments, the subject's plasma ALP level is at least 25 U/mL after two weeks of administration. In some embodiments, the subject's plasma ALP level is at least 50 U/mL after two weeks of administration. In some embodiments, the subject's plasma ALP level is at least 75 U/mL after two weeks of administration. In some embodiments, the subject's plasma ALP level is at least 100 U/mL after two weeks of administration.

In some embodiments, the subject's plasma ALP level is at least 5 U/mL, at least 6 U/mL, at least 7 U/mL, at least 8 U/mL, at least 9 U/mL, at least 10 U/mL, at least 25 U/mL, at least 50 U/mL, at least 75 U/mL, or at least 100 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, the subject's plasma ALP level is about 5 U/mL to about 100 U/mL, about 15 U/mL to about 100 U/mL, about 25 U/mL to about 100 U/mL, about 50 U/mL to about 100 U/mL, about 75 U/mL to about 100 U/mL, about 5 U/mL to about 75 U/mL, about 5 U/mL to about 50 U/mL, about 5 U/mL to about 25 U/mL, or about 5 U/mL to about 10 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, administration is by intramuscular injection. In some embodiments, administration is by single dose. In some embodiments, the single dose is administered by single or multiple injections. In some embodiments, administration is by intramuscular injection at multiple sites.

In some embodiments, the subject does not develop ectopic calcification or abnormal calcium metabolism within 3 months, 4 months, 5 months, or 6 months after administration.

In some embodiments, the vector is not detectable in the subject's liver, heart, bone, testes, ovaries, or any combination thereof within 3 months, 4 months, 5 months, or 6 months after administration. In some embodiments, administration is by intramuscular injection. In some embodiments, administration is by single dose. In some embodiments, the single dose is administered by single or multiple injections. In some embodiments, administration is by intramuscular injection at multiple sites.

In some embodiments, administration of a polynucleotide of the present disclosure, a vector of the present disclosure, or a pharmaceutical composition of the present disclosure does not cause liver or kidney dysfunction in a subject within 3 months, 4 months, 5 months, or 6 months after administration. In some embodiments, administration is by intramuscular injection. In some embodiments, administration is by single dose. In some embodiments, the single dose is administered by single or multiple injections. In some embodiments, administration is by intramuscular injection at multiple sites.

In some embodiments, administration of a polynucleotide of the present disclosure, a vector of the present disclosure, or a pharmaceutical composition of the present disclosure has no oncogenic effect in a subject within 3 months, 4 months, 5 months, or 6 months after administration. In some embodiments, administration is by intramuscular injection. In some embodiments, administration is by a single dose. In some embodiments, the single dose is administered by single or multiple injections. In some embodiments, administration is by intramuscular injection at multiple sites.

Certain aspects of the present disclosure include:
(a) administering to a subject a first dose of a vector comprising a polynucleotide, wherein the polynucleotide comprises a promoter (e.g., a CAG promoter) operably linked to a nucleic acid encoding a fusion protein comprising tissue-nonspecific alkaline phosphatase (TNALP) and a decaaspartic acid (D10) amino acid sequence, and the polynucleotide comprises a nucleic acid sequence having at least 85% identity to SEQ ID NO:1;
(b) measuring the level of plasma ALP in the subject after administering the first dose of the vector; and
(c) if the level of plasma ALP measured in step (b) is less than the therapeutically effective level of plasma ALP, administering to the subject a second dose of the vector.

In some embodiments, administration is by intramuscular injection.

In some embodiments, the therapeutically effective level of plasma ALP is at least about 5 U/mL, at least about 6 U/mL, at least about 7 U/mL, at least about 8 U/mL, at least about 9 U/mL, at least about 10 U/mL, at least about 25 U/mL, at least about 50 U/mL, at least about 75 U/mL, or at least about 100 U/mL.

In some embodiments, the therapeutically effective level of plasma ALP is about 5 U/mL to about 100 U/mL, about 15 U/mL to about 100 U/mL, about 25 U/mL to about 100 U/mL, about 50 U/mL to about 100 U/mL, about 75 U/mL to about 100 U/mL, about 5 U/mL to about 75 U/mL, about 5 U/mL to about 50 U/mL, about 5 U/mL to about 25 U/mL, or about 5 U/mL to about 10 U/mL. In some embodiments, the therapeutically effective level of plasma ALP is at least 5 U/mL. In some embodiments, the therapeutically effective level of plasma ALP is at least 10 U/mL. In some embodiments, the therapeutically effective level of plasma ALP is at least 25 U/mL. In some embodiments, the therapeutically effective level of plasma ALP is at least 50 U/mL. In some embodiments, the therapeutically effective level of plasma ALP is at least 75 U/mL.

In some embodiments, the subject's plasma ALP level is measured in (b) at least one week after administering the first dose of the vector.

In some embodiments, the subject's plasma ALP level is measured in (b) at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 18 months, or at least 2 years after administering the first dose of the vector.

In some embodiments, the method further includes (d) measuring the level of plasma ALP in the subject after administering the second dose of the vector; and (e) administering a third dose of the vector to the subject if the level of plasma ALP measured in (d) is less than the effective level of plasma ALP.

In some embodiments, the plasma ALP level is measured in (d) at least one week after administering the first dose of the vector.

In some embodiments, the subject's plasma ALP level is measured in (d) at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 18 months, or at least 2 years after administering the first dose of the vector.

In some embodiments, the method includes administering a vector (e.g., an AAV vector) containing a polynucleotide comprising a nucleic acid sequence having at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:1. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:1.

In some embodiments, the promoter is a CAG promoter. In some embodiments, the polynucleotide further comprises an enhancer, e.g., a CMV enhancer, upstream of the CAG promoter. In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3 or 21. In some embodiments, the CAG promoter comprises the nucleic acid sequence of SEQ ID NO: 21. In some embodiments, the CAG promoter and the CMV enhancer comprise the nucleic acid sequence of SEQ ID NO: 3.

In some embodiments, the method includes administering a vector (e.g., an AAV vector) containing a nucleic acid encoding a fusion protein comprising a nucleic acid sequence having at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 2 or 5. In some embodiments, the nucleic acid encoding the fusion protein comprises the nucleic acid sequence of SEQ ID NO: 2 or 5.

In some embodiments, the method includes administering a vector (e.g., an AAV vector) containing a polynucleotide comprising a nucleic acid sequence having at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:6. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:6.

In some embodiments, the method includes administering a vector (e.g., an AAV vector) comprising a polynucleotide encoding a fusion protein comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 9. In some embodiments, the polynucleotide encodes a fusion protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 9.

In some embodiments, the method includes administering an adeno-associated virus (AAV) vector. In some embodiments, the AAV is an AAV1 serotype, an AAV2 serotype, an AAV4 serotype, an AAV5 serotype, an AAV6 serotype, an AAV7 serotype, an AAV8 serotype, or an AAV9 serotype. In some embodiments, the AAV is an AAV7 serotype, an AAV8 serotype, or an AAV9 serotype. In some embodiments, the AAV is serotype 8 (AAV8).

In some embodiments, the vector is a rhesus adeno-associated virus (AAVrh) vector. In some embodiments, the AAVrh is the AAVrh.74 serotype. In some embodiments, the AAVrh is the AAVrh.10 serotype.

Figure 1 shows plasma ALP concentrations and lifespan of Akp2 ^-/- mice treated with the AAV8-TNALP-D10 vector. Akp2 ^-/- mice were injected with AAV8-TNALP-D10 at doses of 1.0 x ¹⁰¹² vector genomes (vg)/mouse, 1.0 x ¹⁰¹¹ vg/mouse, or 3.0 x ¹⁰¹¹ vg/mouse. Untreated Akp2 ^-/- mice and wild-type (WT) mice served as controls. Figure 1A shows a graph showing plasma ALP activity in newborn Akp2 ^-/- mice injected with AAV8-TNALP-D10 in the right quadriceps muscle. Plasma ALP activity was measured for up to 18 months after treatment. For plasma ALP activity, one unit (U) was defined as the amount of enzyme required to catalyze the production of 1 μmol of p-nitrophenol per minute (min). ALP activity in plasma was calculated as U/mL. Figure 1B shows plasma ALP concentrations and lifespan of Akp2 ^-/- mice treated with the AAV8-TNALP-D10 vector. Akp2 ^-/- mice were injected with AAV8-TNALP-D10 at doses of 1.0 x ¹⁰¹² vector genomes (vg)/mouse, 1.0 x ¹⁰¹¹ vg/mouse, or 3.0 x ¹⁰¹¹ vg/mouse. Untreated Akp2 ^-/- mice and wild-type (WT) mice served as controls. Figure 1B is a graph showing the lifespan of newborn Akp2 ^-/- mice injected with AAV8-TNALP-D10 in the right quadriceps muscle. Lifespan up to 18 months after treatment is shown. 1.0×10 ¹¹ vg/individual, n=5; 3.0×10 ¹¹ vg/individual, n=7; 1.0×10 ¹² vg/individual, n=7; wild-type (WT), n=5, untreated Akp2 ^−/− mice, n=4. Figure 2 shows bone mineralization and bone maturation in AAV8-TNALP-D10-treated Akp2 ^−/− mice. Figure 2A compares the morphology and gross size of Akp2 ^−/− mice treated with 1 × 10 ¹² vg/animal of AAV-TNALP-D10 and untreated wild-type (WT) mice using X-ray images obtained at 2 months of age. X-ray images were taken at 100 mA and 40 kV for 30 seconds using μFX-1000 film. Figure 2B shows bone mineralization and bone maturation in AAV8-TNALP-D10-treated Akp2 ^−/− mice. Figure 2B is a graph comparing body weight development in 1×10 ¹² vg/individual AAV-TNALP-D10-treated Akp2 ^−/− mice (n=5) and untreated WT mice (n=3) at 2 months of age. Figure 2C shows bone mineralization and bone maturation in AAV8-TNALP-D10-treated Akp2 ^−/− mice. Figure 2C shows representative X-ray images obtained by X-ray analysis of secondary ossification in untreated WT mice (left panel), untreated Akp2 ^−/− mice (middle panel), and Akp2 ^−/− mice treated with 1 × 10 ¹² vg/individual AAV-TNALP-D10 (right panel) at postnatal day 10. Secondary ossification centers were detected by X-ray analysis in all WT mice (left panel), but not in the majority of Akp2 ^−/− mice (middle panel). In contrast, secondary ossification centers were detected by X-ray analysis in Akp2 ^−/− mice treated with 1 × 10 ¹² vg/individual AAV-TNALP-D10 at postnatal day 10 (right panel). Figure 2D shows bone mineralization and bone maturation in AAV8-TNALP-D10-treated Akp2 ^−/− mice. Figure 2D shows representative X-ray images obtained from X-ray analysis of the knee joints of WT mice (left panel) and 1×10 ¹² vg/individual AAV-TNALP-D10-treated Akp2 ^−/− mice (right panel) at 2 months (56 days). Upon reaching adulthood on day 56, X-ray analysis of the knee joints showed similar mature bone in 1.0×10 ¹² vg/individual AAV8-TNALP-D10-treated Akp2 ^−/− mice as in WT mice. Figure 2E shows bone mineralization and bone maturation in AAV8-TNALP-D10-treated Akp2 ^−/− mice. Figure 2E is a graph comparing bone mineral density (BMD) at 18 months of age in 1×10 ¹² vg/individual AAV-TNALP-D10-treated Akp2 ^−/− mice (n=3) and WT mice injected with a GFP-expressing vector (n=3). BMD was determined by computed tomography (CT). Data represent the mean ± SD. Figure 1 shows histochemical (fast blue) staining of ALP ^activity in the tibiae of WT mice (left panel), AAV8-TNALP-D10-treated Akp2 ^−/− mice (middle panel), and AAV8-TNALP-D10-treated Akp2 ^−/− mice (right panel) at ² months of age. Original magnification, ×100. Figure 4A shows that mice with extremely high plasma ALP activity developed calculi on the mouse limbs. Figure 4A is a graph showing the time course of plasma ALP activity in mice after AAV8-TNALP-D10 injection. AAV8-TNALP-D10 was injected into the right quadriceps of 6-week-old male C57BL/6 mice at a dose of 2.8 × 10 ¹² , 1.1 × 10 ¹³ , or 2.8 × 10 ¹³ vg/individual in 50 μL of PBS, or into both quadriceps at a dose of 5.5 × 10 ¹³ vg/individual in 100 μL of PBS (n = 3 in each group). Untreated WT C57BL/6 mice served as controls. 4B shows that mice with extremely high plasma ALP activity developed stones on the mouse paws. 4B is an image showing stones in the forelimbs of 6-week-old C57BL/6 mice treated with 2.8 × 10 ¹³ vg/individual AAV8-TNALP-D10 (left panel) and 5.5 × 10 ¹³ vg/individual AAV8-TNALP-D10 (right panel). 4C shows that mice with extremely high plasma ALP activity developed stones in the mouse paws. 4D shows images of stones in the hind paws of 6-week-old C57BL/6 mice treated with 2.8 × 10 ¹³ vg/individual AAV8-TNALP-D10 (left panel) and 5.5 × 10 ¹³ vg/individual AAV8-TNALP-D10 (right panel). 1 is a graph showing TNALP levels in wild-type rats following administration of 1×10 ¹¹ , 1×10 ¹² , and 1×10 ¹³ vg/animal of AAV8-TNALP-D10 by intramuscular injection.

Certain embodiments of the present disclosure relate to polynucleotides comprising a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:1. In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:1. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:1.

In some embodiments, the polynucleotide comprises a nucleic acid sequence comprising a promoter. In some embodiments, the promoter is a CAG promoter. In some embodiments, the polynucleotide comprises a nucleic acid sequence comprising an enhancer. In some embodiments, the enhancer is a CMV enhancer.

In some embodiments, the CAG promoter comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 21. In some embodiments, the CAG promoter comprises the nucleic acid sequence of SEQ ID NO: 21.

In some embodiments, the nucleic acid sequence comprises a CMV enhancer. In some embodiments, the nucleic acid sequence comprising the CMV enhancer has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:22. In some embodiments, the CMV enhancer comprises the sequence of SEQ ID NO:22.

In some embodiments, the nucleic acid sequence comprises a CMV enhancer upstream of the CAG promoter. In some embodiments, the nucleic acid sequence comprising a CMV enhancer upstream of the CAG promoter comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:3. In some embodiments, the nucleic acid sequence comprising a CMV enhancer upstream of the CAG promoter comprises the nucleic acid sequence of SEQ ID NO:3.

In some embodiments, the nucleic acid encoding the fusion protein comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2 or 5. In some embodiments, the nucleic acid encoding the fusion protein comprises the nucleic acid sequence of SEQ ID NO: 2 or 5.

In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:6. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:6.

In some embodiments, the fusion protein comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:9. In some embodiments, a polynucleotide encodes a fusion protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:9. Certain embodiments of the present disclosure relate to vectors (e.g., viral vectors) comprising a nucleic acid encoding a fusion protein, wherein the fusion protein comprises tissue-nonspecific alkaline phosphatase (TNALP) and a deca-aspartic acid (D10) amino acid sequence (TNALP-D10). In some embodiments, the vector comprises a polynucleotide comprising a promoter (e.g., a CAG promoter) operably linked to a nucleic acid encoding the TNALP fusion protein. In some embodiments, the polynucleotide comprises a CMV enhancer upstream of the CAG promoter. In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 1. In some embodiments, the vector comprises a polynucleotide encoding a fusion protein comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 9. In some embodiments, the polynucleotide encodes a fusion protein having 100% identity to SEQ ID NO: 9.

Certain embodiments of the present disclosure relate to adeno-associated virus (AAV) vectors or AAV capsids that contain or encapsulate a polynucleotide, where the polynucleotide comprises a promoter (e.g., a CAG promoter) operably linked to a nucleic acid encoding a TNALP-D10 fusion protein. In some embodiments, the polynucleotide comprises a CAG promoter and a CMV enhancer upstream of the CAG promoter. In some embodiments, the AAV vector or AAV capsid contains or encapsulates a polynucleotide comprising a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:1. In some embodiments, the AAV vector or AAV capsid comprises or encapsulates a polynucleotide comprising a nucleic acid sequence having at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 1. In some embodiments, the AAV vector or AAV capsid comprises or encapsulates a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 1.

In some embodiments, the AAV vector or AAV capsid contains or encapsulates a polynucleotide comprising a nucleic acid encoding a fusion protein comprising a nucleic acid sequence having at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO: 2 or 5. In some embodiments, the nucleic acid encoding the fusion protein comprises the nucleic acid sequence of SEQ ID NO: 2 or 5.

In some embodiments, the AAV vector or AAV capsid comprises or encapsulates a polynucleotide comprising a nucleic acid sequence having at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:6. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:6.

In some embodiments, the AAV vector or AAV capsid comprises or encapsulates a polynucleotide encoding a fusion protein comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 9. In some embodiments, the polynucleotide encodes a fusion protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 9.

In some embodiments, the AAV vector or capsid is an AAV1 serotype, AAV2 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, or AAV9 serotype vector or capsid. In some embodiments, the vector is a rhesus adeno-associated virus (AAVrh) vector. In some embodiments, the AAVrh is an AAVrh.74 serotype. In some embodiments, the AAVrh is an AAVrh.10 serotype.

Certain aspects of the present disclosure relate to pharmaceutical compositions comprising a vector (e.g., an AAV vector) disclosed herein and a pharmaceutically acceptable carrier.

Certain embodiments of the present disclosure relate to pharmaceutical compositions comprising an AAV vector or AAV capsid and a pharmaceutically acceptable carrier, wherein the AAV vector or capsid encapsulates a polynucleotide comprising a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, or at least 99% identity to SEQ ID NO:1, wherein the nucleic acid sequence encodes a TNALP-D10 fusion protein, and wherein the AAV serotype is AAV8. In some embodiments, the pharmaceutical composition comprises an AAV vector or AAV capsid and a polynucleotide comprising the nucleic acid of SEQ ID NO:1.

Certain aspects of the present disclosure relate to methods of treating a bone disease or disorder in a subject in need thereof, comprising administering to the subject in need thereof a vector or pharmaceutical composition disclosed herein. In some aspects, the bone disease or disorder is hypophosphatasia (HPP), rickets, hypercalcemia, nephrocalcinosis, odontophyophosphatasia, osteogenesis imperfecta, congenital dwarfism, or osteomalacia. In some aspects, the bone disease or disorder is HPP. In some aspects, the HPP is perinatal, infantile, pediatric, or adult HPP.

Certain aspects of the present disclosure relate to methods of increasing alkaline phosphatase (ALP) activity in a subject in need thereof, comprising administering to the subject in need thereof a vector or pharmaceutical composition disclosed herein. In some aspects, the vector or pharmaceutical composition disclosed herein can be administered to increase expression of tissue non-specific alkaline phosphatase (TNALP) in a subject in need thereof. In some aspects, the vector or pharmaceutical composition disclosed herein can be administered to increase plasma ALP in a subject in need thereof.

Certain aspects of the present disclosure are directed to methods for achieving therapeutically effective levels of plasma ALP in a subject in need thereof. In some aspects, the method includes: (a) administering to the subject a first dose of a vector comprising a polynucleotide, wherein the polynucleotide comprises a promoter (e.g., a CAG promoter) operably linked to a nucleic acid encoding a fusion protein comprising tissue-nonspecific alkaline phosphatase (TNALP) and a decaaspartic acid (D10) amino acid sequence, and the polynucleotide comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or 100% identity to SEQ ID NO:1; (b) measuring the level of plasma ALP in the subject after administering the first dose of the vector; and (c) administering to the subject a second dose of the vector if the level of plasma ALP measured in step (b) is less than a therapeutically effective level of plasma ALP. In some embodiments, the therapeutically effective level of plasma ALP is at least about 5 U/mL, at least about 10 U/mL, at least about 25 U/mL, at least about 50 U/mL, at least about 75 U/mL, or at least 100 U/mL. In some embodiments, the therapeutically effective level of plasma ALP is about 5 U/mL to about 100 U/mL, about 15 U/mL to about 100 U/mL, about 25 U/mL to about 100 U/mL, about 50 U/mL to about 100 U/mL, about 75 U/mL to about 100 U/mL, about 5 U/mL to about 75 U/mL, about 5 U/mL to about 50 U/mL, about 5 U/mL to about 25 U/mL, or about 5 U/mL to about 15 U/mL.

Non-limiting examples of various embodiments are provided in this disclosure.

I. Definitions In order that this disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed disclosure.

It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleic acid sequence" is understood to refer to one or more nucleic acid sequences unless otherwise specified. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

Furthermore, when used herein, "and/or" should be construed as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and/or" used herein in phrases such as "A and/or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and/or" used in phrases such as "A, B and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

Wherever embodiments are described herein with the word "comprising," it is understood that similar embodiments are also provided except that they are described with the words "consisting of" and/or "consisting essentially of."

The term "about" is used herein to mean approximately, roughly, around, or in the vicinity of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. Generally, the term "about" can modify a numerical value above and below the stated value by, for example, a variance of 10 percent above or below (higher or lower).

The term "at least" before a number or series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that could logically be included, if clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the recited property. When "at least" appears before a series of numbers or range, it is understood that "at least" can modify each number in the series or range. "At least" is also not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, and 5.18%, without considering the number of significant digits).

As used herein, "no more than" or "less than" is understood to refer to the value adjacent to the term and, if logical from the context, the next logically lower value or integer, down to zero. When "no more than" precedes a series of numbers or a range, it is understood that "no more than" can modify each of the numbers in the series or range.

As used herein, the term "vector" refers to any vehicle for cloning a nucleic acid and/or transferring a nucleic acid into a host cell, such as a plasmid, expression vector, expression cassette, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, capsid, etc. A vector can be a replicon to which another nucleic acid segment can be ligated so as to bring about replication of the ligated segment. A "replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replication under its own control. The term "vector" includes both viral and non-viral vehicles for introducing a nucleic acid into a cell in vitro, ex vivo, or in vivo. In some embodiments, the vector is selected from the group consisting of a viral vector (e.g., an AAV vector), a plasmid, a lipid, a protein particle, a bacterial vector, and a lysosome.

"Viral vector" refers to a vector that contains or is derived from components of a virus. Viral vectors can be used to deliver genetic material to cells. Viral vectors can be modified for specific uses. In some embodiments, a viral vector contains one or more polynucleotide regions that encode or contain a molecule of interest, such as a protein, peptide, and/or oligonucleotide. In some embodiments, the viral vector can be selected from the group consisting of an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, or a retroviral vector. In some embodiments, the viral vector is an AAV vector.

The term "adeno-associated viral vector" or "AAV vector," as used herein, refers to any vector that contains or is derived from adeno-associated vector components and is suitable for infecting mammalian cells, preferably human cells. The term AAV vector typically refers to an AAV-type viral particle or virion that contains a payload. AAV vectors can be derived from various serotypes, including combinations of serotypes (i.e., "pseudotyped" AAV), or from various genomes (e.g., single-stranded or self-complementary genomes). Additionally, AAV vectors can be replication-deficient and/or targeted. As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3, (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAVrh8, AAVrh10, AAVrh.74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, the AAV serotypes and clades disclosed by Gao et al. (J. Virol. 78:6381 (2004)) and Morris et al. (Virol. 33:375 (2004)), and any other AAV now known or later discovered. See, e.g., FIELDS et al. VIROLOGY, Vol. 2, Chapter 69 (4th ed., Lippincott-Raven Publishers). In some embodiments, "AAV vector" includes derivatives of known AAV vectors. In some embodiments, "AAV vector" includes modified or artificial AAV vectors. The terms "AAV genome" and "AAV vector" can be used interchangeably. In some embodiments, the AAV vector is modified relative to a wild-type AAV serotype sequence.

As used herein, an "AAV capsid" or "AAV particle" is an AAV virus comprising an AAV vector having at least one payload region (e.g., a polynucleotide encoding a therapeutic protein or peptide, such as a TNALP-D10 fusion protein) and at least one inverted terminal repeat (ITR) region. In some embodiments, the terms "AAV vector of the present disclosure," "AAV vector," or "AAV capsid" refer to an AAV vector comprising, for example, a polynucleotide encoding a fusion protein, packaged within the AAV capsid.

In some embodiments, the AAV vector or AAV capsid is an AAV1 serotype, an AAV2 serotype, an AAV4 serotype, an AAV5 serotype, an AAV6 serotype, an AAV7 serotype, an AAV8 serotype, or an AAV9 serotype. In some embodiments, the AAV vector or AAV capsid is an AAV7 serotype, an AAV8 serotype, or an AAV9 serotype. In some embodiments, the AAV vector or AAV capsid is an AAV8 serotype. In some embodiments, the vector is a rhesus adeno-associated virus (AAVrh) vector. In some embodiments, the AAVrh is an AAVrh.74 serotype. In some embodiments, the AAVrh is an AAVrh.10 serotype.

As used herein, the term "promoter" refers to a DNA sequence recognized by cellular or introduced synthetic machinery required to initiate the specific transcription of a gene. The term "promoter" is also meant to encompass nucleic acid elements sufficient for promoter-dependent gene expression that is regulatable for cell-type-specific expression, tissue-specific expression, or inducible by external signals or agents; such elements can be located in the 5' or 3' regions of the native gene.

In some embodiments, the promoter is a constitutively active promoter, a cell type-specific promoter, or an inducible promoter. In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter comprises a CMV enhancer upstream of the CAG promoter.

The terms "operably linked," "operably inserted," "operably positioned," "under control," or "under transcriptional control" mean that a promoter is in the correct location and orientation relative to a gene or nucleic acid (e.g., a DNA sequence) of interest to control RNA polymerase initiation and expression of the gene or nucleic acid of interest. The term "operably linked" means that a gene or nucleic acid (e.g., a DNA sequence) of interest and a regulatory sequence(s) are linked in a manner that allows expression of the gene or nucleic acid of interest when the appropriate molecule (e.g., a transcriptional activator protein) binds to the regulatory sequence(s). The term "operably inserted" means that a gene or nucleic acid of interest introduced into a cell is positioned adjacent to a DNA sequence that directs the transcription and translation of the introduced gene or nucleic acid of interest (i.e., promotes production of a polypeptide encoded by the gene or nucleic acid of interest, for example).

The term "expression vector" or "expression construct" refers to any type of genetic construct containing a nucleic acid from which part or all of a nucleic acid coding sequence can be transcribed.

A "coding sequence," or a sequence "encoding" a particular molecule (e.g., a therapeutic protein or peptide), is a nucleic acid that is transcribed (in the case of DNA) or translated (in the case of mRNA) into a polypeptide, either in vitro or in vivo, when operably linked to appropriate regulatory sequences, such as a promoter. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Coding sequences include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence is typically located 3' to the coding sequence.

As used herein, the term "administration" or "administering" refers to the administration of a composition of the present disclosure (e.g., a vector, AAV vector, AAV capsid, or pharmaceutical composition disclosed herein) to a subject or system. Administration to an animal subject (e.g., to a human) can be by any suitable route, such as intramuscular injection.

"Nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably in this application. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA. The terms "nucleic acid," "polynucleotide," and "oligonucleotide," as used herein, are defined as a molecule comprising two or more covalently linked nucleosides, as commonly understood by those of skill in the art. Such covalently linked nucleosides may also be referred to as a nucleic acid molecule or oligomer. Polynucleotides can be produced recombinantly, enzymatically, or synthetically, for example, by solid-phase chemical synthesis followed by purification. Reference to the sequence of a polynucleotide or nucleic acid refers to the sequence or order of the nucleobase moieties of the covalently linked nucleotides or nucleosides, or modifications thereof.

As used herein, the term "polypeptide" is intended to encompass the singular form "polypeptide" as well as the plural form "polypeptides," and includes any chain or chains of two or more amino acids. Thus, as used herein, "peptide," "peptide subunit," "protein," "amino acid chain," "amino acid sequence," "fusion protein," or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide," even though each of these terms may have a more specific meaning. The term "polypeptide" can be used in place of or interchangeably with any of these terms. This term further includes polypeptides that have undergone post-translational or post-synthetic modifications, such as palmitoyl conjugation, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, or modification with non-natural amino acids. As used herein, the term "peptide" encompasses full-length peptides and fragments, variants, or derivatives thereof. The peptides disclosed herein can be part of a fusion polypeptide that includes an additional component, such as an aspartic acid peptide (e.g., a decaaspartic acid (D10) peptide). In some embodiments, the peptides can also be derivatized in a number of different ways. In some embodiments, the peptides can include modifications such as, for example, the conjugation of a palmitoyl group.

As used herein, the term "fusion protein" refers to a complex of two or more amino acid sequences that are linked or bound to each other.

A "TNALP-D10" fusion protein refers to a complex comprising a TNALP amino acid sequence and a deca-aspartic acid (D10) amino acid sequence. In some embodiments, the TNALP-10 fusion protein comprises amino acids having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:9. In some embodiments, the TNALP-D10 fusion protein has an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:9. In some embodiments, the TNALP-10 fusion is encoded by a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:5. In some embodiments, the sequence encoding the TNALP-D10 fusion has a nucleic acid sequence that is at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:5.

As used herein, the terms "effective amount," "therapeutically effective amount," "therapeutically effective level," and "sufficient amount," for example, of an AAV vector, AAV capsid, or pharmaceutical composition disclosed herein, refer to an amount sufficient to produce a beneficial or desired result, including a clinical result, when administered to a subject, including a human; therefore, "effective amount" or synonyms thereto will depend on the context in which it is applied. In some aspects, a therapeutically effective amount of an agent (e.g., a polynucleotide, vector, viral vector, AAV capsid, or pharmaceutical composition disclosed herein) is an amount that produces a beneficial or desired result in a subject when compared to a control.

In some embodiments, the amount of a given agent (e.g., a polynucleotide, vector, viral vector, AAV capsid, or pharmaceutical composition disclosed herein) corresponds to an amount that varies depending on various factors, such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, identity of the subject (e.g., age, sex, and/or weight) or host being treated, etc.

As used herein, the term "in vitro" refers to events that occur not within a living organism (e.g., an animal, plant, or microorganism), but rather in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a petri dish, etc.

As used herein, the term "in vivo" refers to events that occur within an organism (e.g., an animal, plant, or microorganism, or its cells or tissues).

"Percent (%) sequence identity" to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in the reference polynucleotide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be accomplished in a variety of ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. For example, percent sequence identity values can be generated using the sequence comparison computer program BLAST.

"Level" refers to the level or activity of a protein or the mRNA encoding the protein, optionally compared to a reference. The reference can be any useful reference as defined herein. A "decreased level" or "increased level" of a protein refers to a decrease or increase in protein level compared to a reference.

Protein levels can be expressed as mass/volume (e.g., g/dL, mg/mL, μg/mL, ng/mL) or as a percentage of total protein or mRNA in a sample. Protein activity can be expressed in U/mL. One unit (U) is the amount of enzyme required to catalyze the production of a particular substrate. In some embodiments, one unit (U) is the amount of enzyme required to catalyze the production of 1 μmol of p-nitrophenol per minute.

The term "pharmaceutical composition," as used herein, refers to a composition comprising a compound or molecule described herein, e.g., a polynucleotide, vector, viral vector, or AAV capsid disclosed herein, formulated with a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition may be manufactured or sold with the approval of a government regulatory agency as part of a therapeutic regimen for the treatment of a disease in a mammal.

As used herein, "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" means any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving an active compound) that has substantially non-toxic and non-inflammatory properties in a patient. In some embodiments, the pharmaceutically acceptable carrier is PBS, water, Ringer's solution, dextrose solution, and 5% human serum albumin, or any combination thereof.

As used herein, the term "subject" refers to any organism to which a composition disclosed herein, e.g., an AAV vector of the present disclosure, can be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). A subject can be a human or animal seeking or in need of treatment, requesting treatment, receiving treatment, or planning to receive future treatment, or receiving care by a trained professional for a particular disease or condition.

As used herein, the terms "treat," "treated," and "treating" refer to both therapeutic treatments and prophylactic or preventative measures whose goal is to prevent or delay (alleviate) an undesirable physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical result. In some aspects, treating reduces or alleviates symptoms associated with a disease or disorder. In some aspects, treating produces a beneficial or desired clinical result.

Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; a decrease in the severity of a condition, disorder, or disease; a stable (i.e., not worsening) state of a condition, disorder, or disease; a delay in the onset or slowing of the progression of a condition, disorder, or disease; an improvement or remission (whether partial or total) of a condition, disorder, or disease state, whether detectable or undetectable; an improvement in at least one measurable physical parameter that may not necessarily be discernible by the patient; or an enhancement or amelioration of a condition, disorder, or disease. In some embodiments, treatment involves eliciting a clinically significant response without excessive levels of side effects. In some embodiments, treatment involves prolonging survival compared to expected survival in the absence of treatment. As used herein, the term "improvement" or "ameliorating" refers to a reduction in the severity of at least one indicator of a condition or disease. As used herein, the term "prevent" or "prevention" refers to delaying or forestalling the onset, development, or progression of a condition or disease for a period of time, such as weeks, months, or years.

As used herein, the term "decaaspartic acid" or "D10" refers to a peptide containing 10 aspartic acid residues. In some embodiments, the D10 peptide targets the fusion protein to bone.

As used herein, the term "TNALP" or "TNSALP" refers to tissue-nonspecific alkaline phosphatase. In some embodiments, the TNALP is human TNALP. In some embodiments, the TNALP has an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:7. In some embodiments, the TNALP comprises an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:7. In some embodiments, the TNALP is encoded by a nucleic acid sequence comprising at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:2. In some embodiments, the sequence encoding the TNALP has a nucleic acid sequence that has at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:2.

II. Composition
II.A. Fusion Proteins Certain embodiments of the present disclosure are directed to polynucleotides for encoding fusion proteins. In some embodiments, the fusion protein comprises a tissue-nonspecific alkaline phosphatase (TNALP) and a series of aspartic acid (D) amino acids. In some embodiments, the aspartic acid amino acids comprise 8 aspartic acids (D8) (SEQ ID NOS: 13 and 17), 9 aspartic acids (D9) (SEQ ID NOS: 14 and 18), 10 aspartic acids (or decaaspartic acids (D10)) (SEQ ID NOS: 4 and 8), 11 aspartic acids (D11) (SEQ ID NOS: 15 and 19), or 12 aspartic acids (D12) (SEQ ID NOS: 16 and 20). In some embodiments, the aspartic acid amino acids comprise 8 to 12 aspartic acids. In some embodiments, the aspartic acid amino acids are directly linked to the TNALP amino acid sequence. In some embodiments, the aspartic acid amino acids are linked to the TNALP amino acid sequence by a linker.

The present disclosure provides a polynucleotide comprising a nucleic acid encoding a fusion protein. In some aspects, the nucleic acid encodes a fusion protein (TNALP-D10) comprising tissue-nonspecific alkaline phosphatase (TNALP) and a decaaspartic acid (D10) amino acid sequence.

The present disclosure provides a vector (e.g., an AAV vector or an AAV capsid) comprising a nucleic acid encoding a fusion protein. In some embodiments, the fusion protein comprises tissue-nonspecific alkaline phosphatase (TNALP) and a decaaspartic acid (D10) amino acid sequence (TNALP-D10).

In some embodiments, the nucleic acid encoding the fusion protein comprises a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:2 (the nucleic acid sequence of TNALP). In some embodiments, the nucleic acid encoding the fusion protein comprises a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:4 (the nucleic acid sequence of D10). In some embodiments, the nucleic acid encoding the fusion protein comprises a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 5 (the nucleic acid sequence of TNALP-D10).

In some embodiments, the vector comprises a polynucleotide, wherein the polynucleotide comprises a promoter (e.g., a CAG promoter) operably linked to a nucleic acid encoding the TNALP-D10 fusion protein. In some embodiments, the CAG promoter has a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:21. In some embodiments, the CAG promoter comprises the nucleic acid sequence of SEQ ID NO:21.

In some embodiments, the vector comprises a polynucleotide, wherein the polynucleotide comprises a CMV enhancer. In some embodiments, the CMV enhancer has a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:22. In some embodiments, the CMV enhancer comprises the nucleic acid sequence of SEQ ID NO:22.

In some embodiments, the vector comprises a polynucleotide, wherein the polynucleotide comprises a CMV enhancer upstream of a CAG promoter operably linked to a nucleic acid encoding a TNALP-D10 fusion protein. In some embodiments, the nucleic acid sequence comprising the CMV enhancer upstream of the CAG promoter has a nucleic acid sequence that has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:3. In some embodiments, a polynucleotide comprising a CMV enhancer upstream of a CAG promoter operably linked to a nucleic acid sequence encoding a fusion protein has a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:6.

In some embodiments, the TNALP of the fusion protein comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 7. In some embodiments, the D10 of the fusion protein comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 8. In some embodiments, the fusion protein (TNALP-D10) comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:9.

In some embodiments, the vector comprises a nucleic acid sequence in Table 1. In some embodiments, the vector comprises a nucleic acid sequence encoding an amino acid sequence in Table 2. In some embodiments, the fusion protein comprises an amino acid sequence in Table 2.

In some embodiments, a polynucleotide comprising a CAG promoter operably linked to a nucleic acid encoding a TNALP-D10 fusion protein is inserted into a viral vector (e.g., an AAV vector) disclosed herein. In some embodiments, the polynucleotide comprises a CMV enhancer upstream of the CAG promoter.

In some embodiments, the vector is a viral vector (e.g., an AAV vector). In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV is an AAV1 serotype, an AAV2 serotype, an AAV3 serotype, an AAV4 serotype, an AAV5 serotype, an AAV6 serotype, an AAV7 serotype, an AAV8 serotype, an AAV9 serotype, or an AAV10 serotype. In some embodiments, the AAV is an AAV7 serotype, an AAV8 serotype, or an AAV9 serotype. In some embodiments, the vector is an AAV serotype 8 (AAV8) vector.

In some embodiments, the vector is a rhesus adeno-associated virus (AAVrh) vector. In some embodiments, the AAVrh is the AAVrh.74 serotype. In some embodiments, the AAVrh is the AAVrh.10 serotype.

In some embodiments, a polynucleotide comprising a CAG promoter operably linked to a nucleic acid encoding a TNALP-D10 fusion protein is located between two inverted terminal repeats (ITRs). In some embodiments, the ITRs are AAV1 ITRs, AAV2 ITRs, AAV3 ITRs, AAV4 ITRs, AAV5 ITRs, AAV6 ITRs, AAV7 ITRs, AAV8 ITRs, AAV9 ITRs, AAV10 ITRs, or any combination thereof. In some embodiments, the ITRs are AAV2 ITRs. In some embodiments, the polynucleotide comprises a CMV enhancer upstream of the CAG promoter.

In some embodiments, the polynucleotide has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1. In some embodiments, the polynucleotide has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 10.

In some embodiments, the vector is an AAV serotype 8 (AAV8) vector comprising a polynucleotide, wherein the polynucleotide comprises a CAG promoter operably linked to a nucleic acid encoding a TNALP-D10 fusion protein, wherein the polynucleotide has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to SEQ ID NO:1. In some embodiments, the polynucleotide comprises a CMV enhancer upstream of the CAG promoter.

In some embodiments, the vectors disclosed herein are suitable for treating a disease or disorder (e.g., a bone disease or disorder). In some embodiments, the bone disease or disorder is hypophosphatasia (HPP), rickets, hypercalcemia, nephrocalcinosis, odontophyophosphatasia, osteogenesis imperfecta, congenital dwarfism, or osteomalacia. In some embodiments, the bone disease or disorder is HPP. In some embodiments, the HPP is perinatal, infantile, pediatric, or adult HPP. In some embodiments, the vector is administered to treat HPP in a subject in need thereof. In some embodiments, the HPP is a severe infantile form of HPP.

In some aspects, the vectors disclosed herein can be administered to increase plasma alkaline phosphatase (ALP) activity in a subject in need thereof. In some aspects, the vectors disclosed herein can be administered to increase expression of tissue non-specific alkaline phosphatase (TNALP) in a subject in need thereof. In some aspects, the vectors disclosed herein can be administered to increase plasma alkaline phosphatase (TNALP) in a subject in need thereof.

In some embodiments, the subject in need thereof is an infant, child, or adult. In some embodiments, the subject in need thereof is an infant or child. In some embodiments, the subject in need thereof is an adult. In some embodiments, the subject in need thereof is a non-human primate. In some embodiments, the subject in need thereof is a human.

In some aspects, the vectors disclosed herein can be administered into a muscle. In some aspects, the vectors disclosed herein can be administered via intramuscular injection. In some aspects, the vectors disclosed herein can be administered via a single dose. In some aspects, the vectors can be administered at multiple sites via intramuscular injection.

In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity from about 1 U/mL to about 20 U/mL to about 5 U/mL to about 15 U/mL, about 5 U/mL to about 10 U/mL, about 5 U/mL to about 25 U/mL, about 5 U/mL to about 50 U/mL, about 5 U/mL to about 75 U/mL, about 5 U/mL to about 100 U/mL, about 15 U/mL to about 100 U/mL, about 25 U/mL to about 100 U/mL, about 50 U/mL to about 100 U/mL, or about 75 U/mL to about 100 U/mL after two weeks of administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity from about 5 U/mL to about 15 U/mL after two weeks of administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 5 U/mL after two weeks of administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 10 U/mL after two weeks of administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 25 U/mL after two weeks of administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 50 U/mL after two weeks of administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 75 U/mL after two weeks of administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 100 U/mL after two weeks of administration.

In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to about 5 U/mL to about 10 U/mL, about 5 U/mL to about 25 U/mL, about 5 U/mL to about 50 U/mL, about 5 U/mL to about 75 U/mL, about 5 U/mL to about 100 U/mL, about 15 U/mL to about 100 U/mL, about 25 U/mL to about 100 U/mL, about 50 U/mL to about 100 U/mL, or about 75 U/mL to about 100 U/mL at 2 weeks, 1 month, 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 5 U/mL at 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 10 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 25 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 50 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 75 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, plasma ALP levels are about 5 U/mL to about 10 U/mL 2 weeks, 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, plasma ALP levels are about 5 U/mL 2 weeks, 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, plasma ALP levels are about 10 U/mL 2 weeks, 2 months, 6 months, 10 months, or 18 months after administration.

In some embodiments, the vectors disclosed herein can be administered to increase the expression of tissue non-specific alkaline phosphatase (TNALP) with enhanced safety in a subject in need thereof. In some embodiments, administration of the vectors disclosed herein does not cause the subject to develop ectopic calcification or abnormal calcium metabolism for at least six months after administration. In some embodiments, administration of the vectors disclosed herein does not cause liver or kidney dysfunction in the subject. In some embodiments, administration of the vectors disclosed herein does not have a carcinogenic effect in the subject.

II.B. Vectors In some embodiments, the vector is a plasmid, expression vector, expression cassette, virus, or capsid. In some embodiments, the vector is a viral vector, a non-viral vector, a plasmid, a lipid, or a lysosome. In some embodiments, the vector is a viral vector (e.g., an AAV vector). In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV is an AAV1 serotype, an AAV2 serotype, an AAV3 serotype, an AAV4 serotype, an AAV5 serotype, an AAV6 serotype, an AAV7 serotype, an AAV8 serotype, an AAV9 serotype, or an AAV10 serotype. In some embodiments, the AAV is an AAV7 serotype, an AAV8 serotype, or an AAV9 serotype. In some embodiments, the vector is an AAV serotype 8 (AAV8) vector. In some embodiments, the vector is a rhesus adeno-associated virus (AAVrh) vector. In some embodiments, the AAVrh is the AAVrh.74 serotype. In some embodiments, the AAVrh is the AAVrh.10 serotype.

In some embodiments, the vector comprises a polynucleotide. In some embodiments, the polynucleotide comprises a nucleic acid encoding a fusion protein. In some embodiments, the fusion protein comprises tissue-nonspecific alkaline phosphatase (TNALP) and a decaaspartic acid (D10) amino acid sequence (TNALP-D10).

In some embodiments, the nucleic acid encoding the TNALP has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2. In some embodiments, the deca-aspartic acid comprises a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 4. In some embodiments, the nucleic acid encoding TNALP-D10 has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:5.

In some embodiments, the vector comprises a polynucleotide, wherein the polynucleotide comprises a CAG promoter operably linked to a nucleic acid encoding a TNALP-D10 fusion protein. In some embodiments, the CAG promoter has a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:21. In some embodiments, the CAG promoter has the nucleic acid sequence of SEQ ID NO:21.

In some embodiments, the vector comprises a polynucleotide, wherein the polynucleotide comprises a CMV enhancer. In some embodiments, the CMV enhancer has a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:22. In some embodiments, the CMV enhancer has the nucleic acid sequence of SEQ ID NO:22.

In some embodiments, the vector comprises a polynucleotide, wherein the polynucleotide comprises a CAG promoter operably linked to a nucleic acid encoding TNALP-D10. In some embodiments, the vector comprises a polynucleotide, wherein the polynucleotide comprises a CMV enhancer upstream of the CAG promoter. In some embodiments, the nucleic acid sequence comprising the CMV enhancer upstream of the CAG promoter has a nucleic acid sequence that has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:3. In some embodiments, a nucleic acid sequence comprising a CMV enhancer upstream of a CAG promoter operably linked to a nucleic acid sequence encoding TNALP-D10 has a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:6.

In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a TNALP having an amino acid sequence at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding D10 having an amino acid sequence at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding TNALP-D10 having an amino acid sequence that has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:9.

In some embodiments, the polynucleotide has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1. In some embodiments, the polynucleotide has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 10.

In some embodiments, the polynucleotide comprising the nucleic acid encoding TNALP-D10 is located between two inverted terminal repeats (ITRs).

In some embodiments, the AAV serotype 8 (AAV8) vector comprises a polynucleotide comprising a CAG promoter operably linked to a nucleic acid encoding TNALP-D10, wherein the polynucleotide has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to SEQ ID NO:1. In some embodiments, the polynucleotide comprises a CMV enhancer upstream of the CAG promoter.

In some embodiments, the vector comprises a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1. In some embodiments, the vector comprises a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 10.

In some embodiments, the vectors disclosed herein are suitable for treating a disease or disorder (e.g., a bone disease or disorder). In some embodiments, the bone disease or disorder is hypophosphatasia (HPP), rickets, hypercalcemia, nephrocalcinosis, odontophyophosphatasia, osteogenesis imperfecta, congenital dwarfism, or osteomalacia. In some embodiments, the bone disease or disorder is HPP. In some embodiments, the HPP is perinatal, infantile, pediatric, or adult HPP. In some embodiments, the vector is administered to treat HPP in a subject in need thereof. In some embodiments, the HPP is a severe infantile form of HPP.

In some aspects, the vectors disclosed herein can be administered to increase plasma alkaline phosphatase (ALP) activity in a subject in need thereof. In some aspects, the vectors disclosed herein can be administered to increase expression of tissue non-specific alkaline phosphatase (TNALP) in a subject in need thereof. In some aspects, the vectors disclosed herein can be administered to increase plasma alkaline phosphatase (TNALP) in a subject in need thereof.

In some embodiments, the subject in need thereof is an infant, child, or adult. In some embodiments, the subject in need thereof is an infant or child. In some embodiments, the subject in need thereof is an adult. In some embodiments, the subject in need thereof is a non-human primate. In some embodiments, the subject in need thereof is a human.

In some aspects, the vectors disclosed herein can be administered intramuscularly. In some aspects, the vectors disclosed herein can be administered via intramuscular injection. In some aspects, the vectors disclosed herein can be administered via a single dose. In some aspects, the single dose is administered via single or multiple injections. In some aspects, the vectors can be administered at multiple sites via intramuscular injection.

In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity from about 1 U/mL to about 20 U/mL to about 5 U/mL to about 15 U/mL, about 5 U/mL to about 10 U/mL, about 5 U/mL to about 25 U/mL, about 5 U/mL to about 50 U/mL, about 5 U/mL to about 75 U/mL, about 5 U/mL to about 100 U/mL, about 15 U/mL to about 100 U/mL, about 25 U/mL to about 100 U/mL, about 50 U/mL to about 100 U/mL, or about 75 U/mL to about 100 U/mL after two weeks of administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity from about 5 U/mL to about 15 U/mL after two weeks of administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 5 U/mL after two weeks of administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 10 U/mL after two weeks of administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 25 U/mL after two weeks of administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 50 U/mL after two weeks of administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 75 U/mL after two weeks of administration.

In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to about 5 U/mL to about 10 U/mL, about 5 U/mL to about 25 U/mL, about 5 U/mL to about 50 U/mL, about 5 U/mL to about 75 U/mL, about 5 U/mL to about 100 U/mL, about 15 U/mL to about 100 U/mL, about 25 U/mL to about 100 U/mL, about 50 U/mL to about 100 U/mL, or about 75 U/mL to about 100 U/mL at 2 weeks, 1 month, 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 5 U/mL at 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 10 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 25 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 50 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, the vectors disclosed herein can be administered to increase a subject's plasma ALP activity to at least 75 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, plasma ALP levels are about 5 U/mL to about 10 U/mL 2 weeks, 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, plasma ALP levels are about 5 U/mL 2 weeks, 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, plasma ALP levels are about 10 U/mL 2 weeks, 2 months, 6 months, 10 months, or 18 months after administration.

In some embodiments, the vectors disclosed herein can be administered to increase the expression of tissue non-specific alkaline phosphatase (TNALP) with enhanced safety in a subject in need thereof. In some embodiments, administration of the vectors disclosed herein does not cause the subject to develop ectopic calcification or abnormal calcium metabolism for at least six months after administration. In some embodiments, administration of the vectors disclosed herein does not cause liver or kidney dysfunction in the subject. In some embodiments, administration of the vectors disclosed herein does not have a carcinogenic effect in the subject.

In some embodiments, the vector is a viral capsid that encapsulates a polynucleotide. In some embodiments, the polynucleotide comprises a nucleic acid encoding a fusion protein. In some embodiments, the fusion protein comprises tissue-nonspecific alkaline phosphatase (TNALP) and a decaaspartic acid (D10) amino acid sequence (TNALP-D10).

In some embodiments, the capsid is an adeno-associated virus (AAV) capsid. In some embodiments, the AAV is an AAV1 serotype, an AAV2 serotype, an AAV3 serotype, an AAV4 serotype, an AAV5 serotype, an AAV6 serotype, an AAV7 serotype, an AAV8 serotype, an AAV9 serotype, or an AAV10 serotype. In some embodiments, the AAV is an AAV7 serotype, an AAV8 serotype, or an AAV9 serotype. In some embodiments, the capsid is an AAV serotype 8 (AAV8). In some embodiments, the vector is a rhesus adeno-associated virus (AAVrh) vector. In some embodiments, the AAVrh is an AAVrh.74 serotype. In some embodiments, the AAVrh is an AAVrh.10 serotype.

In some embodiments, the capsid comprises a polynucleotide. In some embodiments, the polynucleotide comprises a nucleic acid encoding a fusion protein. In some embodiments, the fusion protein comprises tissue-nonspecific alkaline phosphatase (TNALP) and a decaaspartic acid (D10) amino acid sequence (TNALP-D10).

In some embodiments, the nucleic acid encoding the TNALP has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2. In some embodiments, the deca-aspartic acid comprises a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 4. In some embodiments, the nucleic acid encoding TNALP-D10 has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:5.

In some embodiments, the capsid comprises a polynucleotide, wherein the polynucleotide comprises a CAG promoter operably linked to a nucleic acid encoding a TNALP-D10 fusion protein. In some embodiments, the CAG promoter has a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:21. In some embodiments, the CAG promoter comprises the nucleic acid sequence of SEQ ID NO:21.

In some embodiments, the capsid comprises a polynucleotide, wherein the polynucleotide comprises a CMV enhancer. In some embodiments, the CMV enhancer has a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:22. In some embodiments, the CMV enhancer comprises the nucleic acid sequence of SEQ ID NO:22.

In some embodiments, the capsid comprises a polynucleotide, wherein the polynucleotide comprises a CAG promoter operably linked to a nucleic acid encoding TNALP-D10. In some embodiments, the capsid comprises a polynucleotide, wherein the polynucleotide comprises a CMV enhancer upstream of the CAG promoter. In some embodiments, the nucleic acid comprising a CMV enhancer upstream of the CAG promoter has a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:3. In some embodiments, the nucleic acid sequence comprising a CMV enhancer upstream of a CAG promoter operably linked to TNALP-D10 has a nucleic acid sequence that has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:6.

In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a TNALP having an amino acid sequence at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding D10 having an amino acid sequence at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding TNALP-D10 having an amino acid sequence that has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:9.

In some embodiments, the polynucleotide has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1. In some embodiments, the polynucleotide has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 10.

In some embodiments, the polynucleotide comprising the nucleic acid encoding TNALP-D10 is located between two inverted terminal repeats (ITRs).

In some embodiments, an AAV serotype 8 (AAV8) capsid encapsulates a polynucleotide comprising a nucleic acid encoding TNALP-D10, wherein the polynucleotide has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to SEQ ID NO:1.

In some embodiments, the capsid comprises a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1. In some embodiments, the capsid comprises a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 10.

In some embodiments, the capsids disclosed herein are suitable for treating a disease or disorder (e.g., a bone disease or disorder). In some embodiments, the bone disease or disorder is hypophosphatasia (HPP), rickets, hypercalcemia, nephrocalcinosis, odontophyophosphatasia, osteogenesis imperfecta, congenital dwarfism, or osteomalacia. In some embodiments, the bone disease or disorder is HPP. In some embodiments, the HPP is perinatal, infantile, pediatric, or adult HPP. In some embodiments, the capsids are administered to treat HPP in a subject in need thereof. In some embodiments, the HPP is a severe infantile form of HPP.

In some aspects, the capsids disclosed herein can be administered to increase plasma alkaline phosphatase (ALP) activity in a subject in need thereof. In some aspects, the capsids disclosed herein can be administered to increase expression of tissue-nonspecific alkaline phosphatase (TNALP) in a subject in need thereof. In some aspects, the capsids disclosed herein can be administered to increase plasma alkaline phosphatase (TNALP) in a subject in need thereof.

In some embodiments, the subject in need thereof is an infant, child, or adult. In some embodiments, the subject in need thereof is an infant or child. In some embodiments, the subject in need thereof is an adult. In some embodiments, the subject in need thereof is a non-human primate. In some embodiments, the subject in need thereof is a human.

In some embodiments, the capsids disclosed herein can be administered into muscle. In some embodiments, the capsids as disclosed herein can be administered via intramuscular injection. In some embodiments, the capsids disclosed herein can be administered via a single dose or a single intramuscular injection. In some embodiments, a single dose is administered via single or multiple injections. In some embodiments, the capsids are administered at multiple sites via intramuscular injection. In some embodiments, a single dose includes multiple intramuscular injections within 24 hours, 12 hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour.

In some embodiments, the capsids disclosed herein can be administered to increase a subject's plasma ALP activity from about 1 U/mL to about 20 U/mL to about 5 U/mL to about 15 U/mL, about 5 U/mL to about 10 U/mL, about 5 U/mL to about 25 U/mL, about 5 U/mL to about 50 U/mL, about 5 U/mL to about 75 U/mL, about 5 U/mL to about 100 U/mL, about 15 U/mL to about 100 U/mL, about 25 U/mL to about 100 U/mL, about 50 U/mL to about 100 U/mL, or about 75 U/mL to about 100 U/mL after two weeks of administration. In some embodiments, the capsids disclosed herein can be administered to increase a subject's plasma ALP activity from about 5 U/mL to about 15 U/mL after two weeks of administration. In some embodiments, the capsids disclosed herein can be administered to increase a subject's plasma ALP activity to at least 5 U/mL after two weeks of administration. In some embodiments, the capsids disclosed herein can be administered to increase a subject's plasma ALP activity to at least 10 U/mL after two weeks of administration. In some embodiments, the capsids disclosed herein can be administered to increase a subject's plasma ALP activity to at least 25 U/mL after two weeks of administration. In some embodiments, the capsids disclosed herein can be administered to increase a subject's plasma ALP activity to at least 50 U/mL after two weeks of administration. In some embodiments, the capsids disclosed herein can be administered to increase a subject's plasma ALP activity to at least 75 U/mL after two weeks of administration.

In some embodiments, the capsids disclosed herein can be administered to increase a subject's plasma ALP activity to about 5 U/mL to about 10 U/mL, about 5 U/mL to about 25 U/mL, about 5 U/mL to about 50 U/mL, about 5 U/mL to about 75 U/mL, about 5 U/mL to about 100 U/mL, about 15 U/mL to about 100 U/mL, about 25 U/mL to about 100 U/mL, about 50 U/mL to about 100 U/mL, or about 75 U/mL to about 100 U/mL 2 weeks, 1 month, 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, the capsids disclosed herein can be administered to increase a subject's plasma ALP activity to at least 5 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, the capsids disclosed herein can be administered to increase a subject's plasma ALP activity to at least 10 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, the capsids disclosed herein can be administered to increase a subject's plasma ALP activity to at least 25 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, the capsids disclosed herein can be administered to increase a subject's plasma ALP activity to at least 50 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, the capsids disclosed herein can be administered to increase a subject's plasma ALP activity to at least 75 U/mL 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, the plasma ALP level is about 5 U/mL to about 10 U/mL 2 weeks, 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, the plasma ALP level is about 5 U/mL 2 weeks, 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, the plasma ALP level is about 10 U/mL 2 weeks, 2 months, 6 months, 10 months, or 18 months after administration.

In some aspects, the capsids disclosed herein can be administered to increase the expression of tissue-nonspecific alkaline phosphatase (TNALP) with enhanced safety in a subject in need thereof.

Upregulation of TNALP has been reported to play an important role in medial vascular calcification. Sheen, CR, et al. (2014) Journal of Bone and Mineral Research. Although hyperphosphatemia is usually associated with disease states such as malignant tumors or metabolic bone disorders such as osteomalacia, rickets, Paget's disease, or osteoporosis, elevated plasma ALP levels are not usually the cause of severe disease. Transient hyperphosphatasia can be observed in infants and young children. These children exhibit plasma phosphatase levels approximately five times higher than the normal range. This is usually referred to as "benign" hyperphosphatasia. Nevertheless, no young patients receiving enzyme replacement therapy using the bone-targeted recombinant human TNALP asfotase alfa have experienced ectopic or vascular calcification associated with elevated plasma ALP activity of approximately 24 U/mL within 4 weeks of ERT initiation or 3-6 U/mL 5 years after ERT initiation. Kitaoka, T, et al. (2017). Clin Endocrinol (Oxf) 87: 10-19; Whyte, MP, et al. (2016). JCI Insight 1: e85971.

Discontinuing clinical use of ERT or asfotase alfa in patients with hypophosphatasia can be fatal, and lifelong treatment can be a burden to patients. Hofmann, CE, et al. (2019). J Clin Endocrinol Metab 104: 2735-2747; Rockman-Greenberg, C (2019) J Clin Endocrinol Metab 104: 3146-3147.

In some embodiments, administration of the capsids disclosed herein does not cause the subject to develop ectopic calcification or abnormal calcium metabolism for at least six months after administration. In some embodiments, administration of the capsids disclosed herein does not cause liver or kidney dysfunction in the subject. In some embodiments, administration of the capsids disclosed herein does not have a carcinogenic effect in the subject.

II.C. Pharmaceutical Compositions Some embodiments of the present disclosure relate to pharmaceutical compositions comprising a vector (e.g., an AAV vector or an AAV capsid) disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is PBS, water, Ringer's solution, dextrose solution, 5% human serum albumin, or any combination thereof. In some embodiments, the pharmaceutical composition is suitable for delivery to a muscle. In some embodiments, the pharmaceutical composition can be administered via intramuscular injection. In some embodiments, the pharmaceutical composition can be administered via a single dose. In some embodiments, the single dose is administered via single or multiple injections. In some embodiments, the pharmaceutical composition is administered at multiple sites via intramuscular injection. In some embodiments, the pharmaceutical composition comprises an AAV serotype 8 (AAV8) capsid encapsulating a polynucleotide comprising a nucleic acid encoding TNALP-D10, wherein the polynucleotide has at least 85%, at least 90%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or at least 100% identity to SEQ ID NO: 1. In some embodiments, the polynucleotide has 100% identity to SEQ ID NO: 1.

In some aspects, the pharmaceutical compositions disclosed herein can be administered to increase plasma alkaline phosphatase (ALP) activity in a subject in need thereof. In some aspects, the pharmaceutical compositions disclosed herein can be administered to increase expression of tissue non-specific alkaline phosphatase (TNALP) in a subject in need thereof. In some aspects, the pharmaceutical compositions disclosed herein can be administered to increase plasma alkaline phosphatase (TNALP) in a subject in need thereof.

In some embodiments, the subject in need thereof is an infant, child, or adult. In some embodiments, the subject in need thereof is an infant or child. In some embodiments, the subject in need thereof is an adult. In some embodiments, the subject in need thereof is a non-human primate. In some embodiments, the subject in need thereof is a human.

In some embodiments, the pharmaceutical compositions disclosed herein can be administered intramuscularly. In some embodiments, the pharmaceutical compositions disclosed herein can be administered via intramuscular injection. In some embodiments, the pharmaceutical compositions disclosed herein can be administered via a single dose or a single intramuscular injection. In some embodiments, a single dose is administered via single or multiple injections. In some embodiments, the pharmaceutical compositions are administered at multiple sites via intramuscular injection. In some embodiments, a single dose comprises multiple intramuscular injections within 24 hours, within 12 hours, within 6 hours, within 4 hours, within 3 hours, within 2 hours, or within 1 hour.

In some embodiments, the pharmaceutical compositions disclosed herein can be administered to increase a subject's plasma ALP activity from about 1 U/mL to about 20 U/mL to about 5 U/mL to about 15 U/mL, or about 5 U/mL to about 10 U/mL, about 5 U/mL to about 25 U/mL, about 5 U/mL to about 50 U/mL, about 5 U/mL to about 75 U/mL, about 5 U/mL to about 100 U/mL, about 15 U/mL to about 100 U/mL, about 25 U/mL to about 100 U/mL, about 50 U/mL to about 100 U/mL, or about 75 U/mL to about 100 U/mL after two weeks of administration. In some embodiments, the pharmaceutical compositions disclosed herein can be administered to increase a subject's plasma ALP activity from about 5 U/mL to about 15 U/mL after two weeks of administration. In some embodiments, the pharmaceutical compositions disclosed herein can be administered to increase a subject's plasma ALP activity to at least 5 U/mL after two weeks of administration. In some embodiments, the pharmaceutical compositions disclosed herein can be administered to increase a subject's plasma ALP activity to at least 10 U/mL after two weeks of administration. In some embodiments, the pharmaceutical compositions disclosed herein can be administered to increase a subject's plasma ALP activity to at least 25 U/mL after two weeks of administration. In some embodiments, the pharmaceutical compositions disclosed herein can be administered to increase a subject's plasma ALP activity to at least 50 U/mL after two weeks of administration. In some embodiments, the pharmaceutical compositions disclosed herein can be administered to increase a subject's plasma ALP activity to at least 75 U/mL after two weeks of administration.

In some embodiments, the pharmaceutical compositions disclosed herein can be administered to increase a subject's plasma ALP activity to about 5 U/mL to about 10 U/mL, about 5 U/mL to about 25 U/mL, about 5 U/mL to about 50 U/mL, about 5 U/mL to about 75 U/mL, about 5 U/mL to about 100 U/mL, about 15 U/mL to about 100 U/mL, about 25 U/mL to about 100 U/mL, about 50 U/mL to about 100 U/mL, or about 75 U/mL to about 100 U/mL at 2 weeks, 1 month, 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, the pharmaceutical compositions disclosed herein can be administered to increase a subject's plasma ALP activity to at least 5 U/mL at 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, the pharmaceutical compositions disclosed herein can be administered to increase a subject's plasma ALP activity to at least 10 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, the pharmaceutical compositions disclosed herein can be administered to increase a subject's plasma ALP activity to at least 25 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, the pharmaceutical compositions disclosed herein can be administered to increase a subject's plasma ALP activity to at least 50 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, the pharmaceutical compositions disclosed herein can be administered to increase a subject's plasma ALP activity to at least 75 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, plasma ALP levels are about 5 U/mL to about 10 U/mL 2 weeks, 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, plasma ALP levels are about 5 U/mL 2 weeks, 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, plasma ALP levels are about 10 U/mL 2 weeks, 2 months, 6 months, 10 months, or 18 months after administration.

In some embodiments, the pharmaceutical compositions disclosed herein can be administered to increase the expression of tissue non-specific alkaline phosphatase (TNALP) with enhanced safety in a subject in need thereof. In some embodiments, administration of the pharmaceutical compositions disclosed herein does not cause the subject to develop ectopic calcification or abnormal calcium metabolism for at least six months after administration. In some embodiments, administration of the pharmaceutical compositions disclosed herein does not cause liver or kidney dysfunction in the subject. In some embodiments, administration of the pharmaceutical compositions disclosed herein does not have a carcinogenic effect in the subject.

III. Methods of Treatment and Use Some embodiments of the present disclosure are directed to methods for treating a disease or disorder (e.g., a bone disease or disorder) in a subject in need thereof, comprising administering to the subject a vector or pharmaceutical composition disclosed herein. In some embodiments, the bone disease or disorder is hypophosphatasia (HPP), rickets, hypercalcemia, nephrocalcinosis, odontophyophosphatasia, osteogenesis imperfecta, congenital dwarfism, or osteomalacia. In some embodiments, the bone disease or disorder is HPP. In some embodiments, the HPP is perinatal, infantile, pediatric, or adult HPP. In some embodiments, a vector is administered to treat HPP in a subject in need thereof. In some embodiments, the HPP is a severe infantile form of HPP.

Some aspects of the present disclosure are directed to methods of increasing plasma alkaline phosphatase (ALP) activity in a subject in need thereof, comprising administering to the subject in need thereof a vector or pharmaceutical composition disclosed herein. In some aspects, a vector or pharmaceutical composition disclosed herein is administered to increase expression of tissue non-specific alkaline phosphatase (TNALP) in a subject in need thereof. In some aspects, a vector or pharmaceutical composition disclosed herein is administered to increase plasma alkaline phosphatase (TNALP) in a subject in need thereof.

In some embodiments, a vector or pharmaceutical composition disclosed herein is administered to increase a subject's plasma ALP activity from about 1 U/mL to 20 U/mL to about 5 U/mL to about 15 U/mL, or about 5 U/mL to about 10 U/mL, about 5 U/mL to about 25 U/mL, about 5 U/mL to about 50 U/mL, about 5 U/mL to about 75 U/mL, about 5 U/mL to about 100 U/mL, about 15 U/mL to about 100 U/mL, about 25 U/mL to about 100 U/mL, about 50 U/mL to about 100 U/mL, or about 75 U/mL to about 100 U/mL after two weeks of administration. In some embodiments, a vector or pharmaceutical composition disclosed herein is administered to increase a subject's plasma ALP activity from about 5 U/mL to about 15 U/mL after two weeks of administration. In some embodiments, a vector or pharmaceutical composition disclosed herein is administered to increase a subject's plasma ALP activity to at least 5 U/mL after two weeks of administration. In some embodiments, a vector or pharmaceutical composition disclosed herein is administered to increase a subject's plasma ALP activity to at least 10 U/mL after two weeks of administration. In some embodiments, a vector or pharmaceutical composition disclosed herein is administered to increase a subject's plasma ALP activity to at least 25 U/mL after two weeks of administration. In some embodiments, a vector or pharmaceutical composition disclosed herein is administered to increase a subject's plasma ALP activity to at least 50 U/mL after two weeks of administration. In some embodiments, a vector or pharmaceutical composition disclosed herein is administered to increase a subject's plasma ALP activity to at least 75 U/mL after two weeks of administration.

In some embodiments, a vector or pharmaceutical composition disclosed herein is administered to increase a subject's plasma ALP activity to about 5 U/mL to about 10 U/mL, about 5 U/mL to about 25 U/mL, about 5 U/mL to about 50 U/mL, about 5 U/mL to about 75 U/mL, about 5 U/mL to about 100 U/mL, about 15 U/mL to about 100 U/mL, about 25 U/mL to about 100 U/mL, about 50 U/mL to about 100 U/mL, or about 75 U/mL to about 100 U/mL at 2 weeks, 1 month, 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, a vector or pharmaceutical composition disclosed herein is administered to increase a subject's plasma ALP activity to at least 5 U/mL at 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, a vector or pharmaceutical composition disclosed herein is administered to increase a subject's plasma ALP activity to at least 10 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, a vector or pharmaceutical composition disclosed herein is administered to increase a subject's plasma ALP activity to at least 25 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, a vector or pharmaceutical composition disclosed herein is administered to increase a subject's plasma ALP activity to at least 50 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, a vector or pharmaceutical composition disclosed herein is administered to increase a subject's plasma ALP activity to at least 75 U/mL after 2 months, 6 months, 10 months, or 18 months of administration. In some embodiments, plasma ALP levels are about 5 U/mL to about 10 U/mL 2 weeks, 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, plasma ALP levels are about 5 U/mL 2 weeks, 2 months, 6 months, 10 months, or 18 months after administration. In some embodiments, plasma ALP levels are about 10 U/mL 2 weeks, 2 months, 6 months, 10 months, or 18 months after administration.

Some aspects of the present disclosure are directed to methods for increasing expression of tissue non-specific alkaline phosphatase (TNALP) with enhanced safety in a subject in need thereof, comprising administering to the subject a vector or pharmaceutical composition disclosed herein. In some aspects, administration of a vector or pharmaceutical composition disclosed herein does not cause the subject to develop ectopic calcification or abnormal calcium metabolism for at least six months after administration. In some aspects, administration of a vector or pharmaceutical composition disclosed herein does not cause liver or kidney dysfunction in the subject. In some aspects, administration of a vector or pharmaceutical composition disclosed herein does not have a carcinogenic effect in the subject.

In some embodiments, the subject in need thereof is an infant, child, or adult. In some embodiments, the subject in need thereof is an infant or child. In some embodiments, the subject in need thereof is an adult. In some embodiments, the subject in need thereof is a non-human primate. In some embodiments, the subject in need thereof is a human.

Some embodiments of the present disclosure are directed to methods of achieving a therapeutically effective level of plasma ALP in a subject in need thereof. In some embodiments, the method includes: (a) administering to the subject a first dose of a vector comprising a polynucleotide, wherein the polynucleotide comprises a CAG promoter operably linked to a nucleic acid encoding a fusion protein comprising tissue-nonspecific alkaline phosphatase (TNALP) and a decaaspartic acid (D10) amino acid sequence, and the polynucleotide comprises a nucleic acid sequence having at least 85% identity to SEQ ID NO: 1; (b) measuring the level of plasma ALP in the subject after administering the first dose of the vector; and (c) administering to the subject a second dose of the vector if the plasma ALP level measured in step (b) is less than the therapeutically effective level of plasma ALP. In some embodiments, the polynucleotide comprises a CMV enhancer upstream of the CAG promoter.

In some embodiments, the therapeutically effective level of plasma ALP is at least about 5 U/mL. In some embodiments, the therapeutically effective level of plasma ALP is at least about 10 U/mL. In some embodiments, the therapeutically effective level of plasma ALP is between about 5 U/mL and about 10 U/mL. In some embodiments, the therapeutically effective level of plasma ALP is between about 5 U/mL and about 25 U/mL. In some embodiments, the therapeutically effective level of plasma ALP is between about 5 U/mL and about 50 U/mL. In some embodiments, the therapeutically effective level of plasma ALP is between about 5 U/mL and about 75 U/mL. In some embodiments, the therapeutically effective level of plasma ALP is between about 5 U/mL and about 100 U/mL. In some embodiments, the therapeutically effective level of plasma ALP is at least 5 U/mL. In some embodiments, the therapeutically effective level of plasma ALP is at least 10 U/mL. In some embodiments, the therapeutically effective level of plasma ALP is at least 25 U/mL. In some embodiments, the therapeutically effective level of plasma ALP is at least 50 U/mL. In some embodiments, the therapeutically effective level of plasma ALP is at least 75 U/mL.

In some embodiments, the subject's plasma ALP level is measured in (b) at least one week after administering the first dose of the vector. In some embodiments, the subject's plasma ALP level is measured in (b) at least two weeks, at least three weeks, at least four weeks, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least 11 months, at least one year, at least 18 months, or at least two years after administering the first dose of the vector.

In some embodiments, the method further includes (d) measuring the level of plasma ALP in the subject after administering the second dose of the vector; and (e) administering a third dose of the vector to the subject if the level of plasma ALP measured in (d) is less than the effective level of plasma ALP. In some embodiments, the plasma ALP level is measured in (d) at least one week after administering the first dose of the vector.

In some embodiments, the subject's plasma ALP level is measured in (d) at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 18 months, or at least 2 years after administering the first dose of the vector.

In some embodiments, the nucleic acid encoding the TNALP has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2. In some embodiments, the deca-aspartic acid comprises a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 4. In some embodiments, the nucleic acid encoding TNALP-D10 has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:5.

In some embodiments, the nucleic acid encoding TNALP-D10 is operably linked to a promoter. In some embodiments, the promoter is a CAG promoter. In some embodiments, the CAG promoter has a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:21.

In some embodiments, the nucleic acid comprises a CMV enhancer. In some embodiments, the CMV enhancer has a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 22. In some embodiments, the CMV enhancer is upstream of the CAG promoter.

In some embodiments, the nucleic acid encoding TNALP-D10 is operably linked to a promoter. In some embodiments, the promoter is a CAG promoter. In some embodiments, the nucleic acid encoding TNALP-D10 comprises a CMV enhancer upstream of the CAG promoter. In some embodiments, the nucleic acid sequence comprising a CMV enhancer upstream of the CAG promoter has a nucleic acid sequence that has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:3. In some embodiments, the nucleic acid sequence comprising a CMV enhancer upstream of a CAG promoter operably linked to TNALP-D10 has a nucleic acid sequence that has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:6.

In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a TNALP having an amino acid sequence at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding D10 having an amino acid sequence at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding TNALP-D10 having an amino acid sequence that has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:9.

In some embodiments, the polynucleotide has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1. In some embodiments, the polynucleotide has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 10.

In some embodiments, the polynucleotide comprising the nucleic acid encoding TNALP-D10 is located between two inverted terminal repeats (ITRs).

In some embodiments, administration is via intramuscular injection. In some embodiments, administration includes a single dose or multiple doses (e.g., 2, 3, or 4). In some embodiments, administration is via a single dose (e.g., a single intramuscular injection or multiple intramuscular injections (e.g., 2, 3, 4, 5, or 6 injections)). In some embodiments, a single dose is administered by single or multiple injections. In some embodiments, administration is by intramuscular injection at multiple sites. In some embodiments, a single dose includes multiple intramuscular injections within 24 hours, 12 hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour. In some embodiments, a subject is administered a single dose of about 1× ¹⁰ to about 1× ¹⁰ , about 1× ¹⁰ to about 1× ¹⁰ , about 1× ¹⁰ to about 5× ¹⁰ , or about 1× ¹⁰ to about 1× ¹⁰ vg/kg. In some embodiments, a subject is administered a single dose of 1× ¹⁰ to 1× ¹⁰ , 1× ¹⁰ to 1× ¹⁰ , 1× ¹⁰ to 5× ¹⁰ , or 1× ¹⁰ to 1× ¹⁰ vg/kg. In some embodiments, the single dose is administered in a volume of about 50 μL to about 150 μL (e.g., 50 μL to 150 μL). In some embodiments, a single dose is administered in a volume of about 75 μL to about 125 μL (e.g., 75 μL to 125 μL). In some embodiments, a single dose is administered in a volume of about 75 μL, about 80 μL, about 85 μL, about 90 μL, about 95 μL, or about 100 μL. In some embodiments, a single dose is administered by single or multiple injections.

IV. Administration The vectors and pharmaceutical compositions (e.g., AAV vectors or AAV capsids) disclosed herein can be administered to a subject by any route that results in a therapeutically effective outcome. In some embodiments, a vector or pharmaceutical composition of the present disclosure is administered to a subject by intramuscular (IM), intravenous (IV), or intradermal injection. In some embodiments, a vector or pharmaceutical composition of the present disclosure is administered to a subject by intramuscular injection. In some embodiments, a vector or pharmaceutical composition is administered to a subject as a single dose or a single intramuscular injection. In some embodiments, a single dose is administered by single or multiple injections. In some embodiments, a vector or pharmaceutical composition is administered at multiple sites by intramuscular injection. In some embodiments, a single dose comprises multiple intramuscular injections within 24 hours, 12 hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour.

In some embodiments, a subject is administered a single dose of about 1× ¹⁰ to about 1× ¹⁰ , about 1× ¹⁰ to about 1× ¹⁰ , about 1× ¹⁰ to about 5× ¹⁰ , or about 1× ¹⁰ to about 1× ¹⁰ vg/kg. In some embodiments, a subject is administered a single dose of 1× ¹⁰ to 1× ¹⁰ , 1× ¹⁰ to 1× ¹⁰ , 1× ¹⁰ to 5× ¹⁰ , or 1× ¹⁰ to 1× ¹⁰ vg/kg. In some embodiments, the single dose is administered in a volume of about 50 μL to about 150 μL (e.g., 50 μL to 150 μL). In some embodiments, a single dose is administered in a volume of about 75 μL to about 125 μL (e.g., 75 μL to 125 μL). In some embodiments, a single dose is administered in a volume of about 75 μL, about 80 μL, about 85 μL, about 90 μL, about 95 μL, or about 100 μL.

In some embodiments, the subject is an infant, child, or adult. In some embodiments, the subject is a human or a non-human primate. In some embodiments, the subject is a human suffering from a bone disease or disorder. In some embodiments, the bone disease or disorder is hypophosphatasia (HPP), rickets, hypercalcemia, nephrocalcinosis, odontophyophosphatasia, osteogenesis imperfecta, congenital dwarfism, or osteomalacia. In some embodiments, the bone disease or disorder is HPP. In some embodiments, the HPP is perinatal, infantile, pediatric, or adult HPP. In some embodiments, the HPP is a severe infantile form of HPP.

Example 1: Generation of AAV8-TNALP-D10 An AAV vector encoding TNALP-D10 driven by a tissue-nonspecific CAG promoter was generated.

An AAV vector plasmid containing cDNA for expression of the human TNALP gene driven by the CAG promoter was constructed. TNALP-D10 was produced using a previously described method. Yamamoto, S, et al. (2011) J Bone Miner Res 26: 135-142.

AAV8-TNALP-D10 was produced using the HEK293 cell line by triple transfection and purified as previously described. Salvetti, A, et al. (1998). Hum Gene Ther 9: 695-706; Hermens, WT, et al. (1999). Hum Gene Ther 10: 1885-1891; Kurai, T, et al. (2007) Mol Ther 15: 38-43. A recombinant AAV type 8 vector encoding GFP (AAV8-GFP) was used as a control for evaluation of bone maturation over an 18-month period. AAV vector titers were determined using real-time PCR (7500 Fast, Applied Biosystems, Tokyo, Japan) as previously described. Salvetti, A, et al. (1998) Hum Gene Ther 9: 695-706; Hermens, WT, et al. (1999) Hum Gene Ther 10: 1885-1891; Kurai, T, et al. (2007) Mol Ther 15: 38-43; Noro, T, et al. (2004) Cancer Res 64: 7486-7490.

The nucleic acid sequence of the AAV8-TNALP-D10 vector is provided in Table 3. The TNALP-D10 amino acid sequence is provided in Table 2.

Example 2: Survival time and persistent TNALP expression in mice intramuscularly injected with >3.0 x 10 ¹¹ vg/animal of AAV8-TNALP-D10
The exogenous plasma ALP activity required for survival of Akp2 ^−/− mice was calculated to be approximately 10 U/mL, which is 10- to 100-fold higher than the endogenous activity in WT mice. Yamamoto, S, et al. (2011) J Bone Miner Res 26: 135-142; Matsumoto, T, et al. (2011) Hum Gene Ther 22: 1355-1364. To determine the amount of vector required to achieve a titer capable of supporting Akp2 ^−/− mouse survival, newborn mice were first intramuscularly injected with AAV8-TNALP-D10 (1.0 × 10 ¹² vector genomes (vg)/animal).

The generation and characterization of Akp2 ^−/− mice have been previously described. Narisawa, S, et al. (1997) Dev Dyn 208: 432-446. To select ^{Akp2 −/−} mice, all newborn TNALP/129/B16 mice were genotyped using PCR as previously described. Matsumoto, T, et al. (2011) Hum Gene Ther 22: 1355-1364. The primers used were 5'-AGTCCGTGGGCATTGTGACTA-3' (SEQ ID NO: 11) and 5'-TGCTGCTCCACTCACGTCGAT-3' (SEQ ID NO: 12). Akp2 ^−/− mice closely mimic infantile HPP and appear healthy at birth. Growth retardation becomes evident within one week, and the majority of Akp2 ^−/− mice die within two to three weeks. Approximately half of these mice have severe symptoms and experience seizures before death. Narisawa, S, et al. (1997) Dev Dyn 208: 432-446. For this study, newborn (1, 2, or 3 days after birth) Akp2 ^-/- mice were injected with AAV8-TNALP-D10 (1.0 x 10 ¹¹ , 3.0 x 10 ¹¹ , or 1.0 x 10 ¹² vector genomes (vg) in 2 μL of PBS). TNALP/129/Bl6 Akp2 ^+/+ wild-type (WT) mice served as controls for treatment experiments. As a control, WT mice were injected with the AAV8-GFP vector (1.0 x 10 ¹² vg/animal in 2 μL of PBS) for 18-month bone analysis.

Injections were performed into the right quadriceps muscle using a 22-gauge Hamilton syringe. Blood samples were collected from the orbital sinus of anesthetized animals using a heparin-coated capillary, and plasma alkaline phosphatase activity was assessed at 1, 2, 3, 6, and 12 months (3.0 × 10 ¹¹ vg/animal in 2 μL of PBS) and at 1, 2, 3, 6, 12, and 18 months (1.0 × 10 ¹¹ and 1.0 × 10 ¹² vg/animal in 2 μL of PBS). Mice behavior was also observed. Mice were sacrificed under deep anesthesia by perfusion with PBS containing heparin (10 U/mL). Mouse organs were then examined for microscopic lesions indicative of tumor formation. Organs were kept in a −80°C freezer until analysis.

We then monitored Akp2 ^−/− mice to determine whether TNALP delivery via intramuscular AAV8-TNALP-D10 injection could phenotypically correct the Akp2 −/− mice. For 2 months after AAV8-TNALP-D10 injection, plasma ALP activity in treated mice remained significantly higher than that in WT mice (14.8 ± 4.3 vs. 0.1 ± 0.004 U/mL at 2 months) (Fig. 1A). Furthermore, 5 of 7 treated mice survived for more than 18 months (Fig. 1B), demonstrating persistent expression of ALP activity (19.38 ± 5.02 U/mL) even at 18 months (Fig. 1A). ^{Akp2 −/−} mice survived healthily for 18 months, or nearly their entire survival, after a single intramuscular injection of just 2 μL of AAV8-TNALP-D10 administered during the neonatal period. The therapeutic effect of intramuscular injection was not inferior to that of systemic injection with respect to lifespan or physical phenotype.

Treatment failed in two mice: one died on day 25 and the second on day 393. Similarly, the efficacy of administering 3.0 × 10 ¹¹ vg/individual or 1.0 × 10 ¹¹ vg/individual AAV8-TNALP-D10 was tested to determine the lowest vector strength required for phenotypic correction in these mice. Akp2 ^−/− mice treated with 3.0 × 10 ¹¹ vg/individual also exhibited strong ALP activity (Fig. 1A) and survived for more than 12 months until sacrifice (n = 3/7 surviving) (Fig. 1B). In contrast, all but one mouse administered 1.0 × 10 ¹¹ vg/individual died within 3 weeks (n = 1/5 surviving). In the remaining mice, ALP activity reached 0.7 U/mL within 2 months, and the mice died shortly thereafter.

This prolongation of survival indicated that at vector doses of 3.0 × 10 ¹¹ vg/animal or higher, plasma ALP activity reached a plateau sufficient to sustain treated mice throughout their lifespan. No seizures were observed in treated Akp2 ^−/ − mice. No dose-dependence of plasma ALP activity was observed. Therefore, 3.0 × 10 ¹¹ vg/animal of AAV8-TNALP-D10 was determined to be safe and provide a sufficient effective vector titer for treating Akp2 ^−/− mice.

Example 3: Mature bone mineralization in Akp2 ^−/− mice treated with 1.0 × 10 ¹² vg/individual AAV8-TNALP-D10 at 18 months. Sustained ALP activity not only extended the lifespan of Akp2 ^−/− mice but also improved their maturation of bone mineralization. There was no significant difference in mean body weight between WT mice (n = 3) and 1.0 × 10 ¹² vg/individual AAV8-TNALP-D10-treated Akp2 ^−/− (n = 5) mice (Figures 2A and 2B).

Secondary ossification centers were detected by X-ray analysis. X-ray analysis was performed according to a previously described method. Matsumoto, T, et al. (2011) Hum Gene Ther 22: 1355-1364. Briefly, X-ray images of adult mice were acquired on μFX-1000 film (Fujifilm, Tokyo, Japan) at an energy level of 25 kV and an exposure time of 10 seconds. Secondary ossification centers were detected in all WT mice (n = 8/8) 10 days after birth. In contrast, the majority of untreated Akp2 ^−/− mice did not have secondary ossification centers at day 10 (n = 3/10) (Figure 2C).

After treatment, similar to WT mice, secondary ossification centers were found in 1.0 × 10 ¹² vg/individual AAV8-TNALP-D10-treated Akp2 ^−/− mice on day 10 (n = 9/10). Upon reaching adulthood on day 56, X-ray analysis of the knee joints showed mature bone in 1.0 × 10 ¹² vg/individual AAV8-TNALP-D10-treated Akp2 ^−/− mice, similar to WT mice (Fig. 2D).

Next, we analyzed the femoral structure of 18-month-old AAV8-TNALP-D10-treated Akp2 ^-/- mice (n = 4) at 1.0 x 10 ¹² vg/individual dose and control AAV8-GFP vector-treated WT mice (n = 3) using computed tomography (CT). CT was performed using a Latheta Laboratory Animal CT System (LCT-200; Hitachi Healthcare BU, Tokyo, Japan) as previously described (Okuda, T, et al. (2018) J Nippon Med Sch 85: 322-329). Bone morphometry was performed to determine bone mineral density (BMD) using Latheta software version 3.44 (Hitachi Healthcare BU). Quantitative assessment was performed using serial 48-μm slice images, and cortical BMD was assessed in 10 slices of the central portion of the femoral shaft.

Statistical analysis of the CT data revealed increased bone width and hyperplasia in the femurs of treated mice, with irregular epiphyseal cartilage structures. The diaphyseal volume was similar to that of WT femurs. At 18 months, the bone mineral density (BMD) in treated mice (673.7 ± 41.6 vs. 620.8 ± 52.9 mg/ ^cm3 : p = 0.26) was not significantly different from that of controls (Fig. 2E). Considering these results, although we observed accelerated bone width expansion similar to flaring in rickets, the trabecular bone quality in treated mice was similar to that in WT mice. Therefore, a single intramuscular AAV8-TNALP-D10 injection during the neonatal period appears safe and effective for the treatment of Akp2 ^-/- mice.

Example 4: ALP activity in bones of mice treated with 1.0 × 10 ¹² vg/individual AAV8-TNALP-D10. ALP activity was examined in the knee joints of untreated wild-type (WT), 1.0 × 10 ¹² vg/individual AAV8-TNALP-D10-treated Akp2 ^−/− mice, and 1.0 × 10 ¹¹ vg/individual AAV8-TNALP-D10-treated Akp2 ^−/− mice. Bones were directly stained without fixation or decalcification. Knee joint sections (10 μm thick) were cut using the Kawamoto film method. Matsumoto, T, et al. (2011) Hum Gene Ther 22: 1355-1364. ALP activity was assessed in the supernatant after biochemical tissue homogenization and histologically examined under a light microscope in tissues stained with Fast Blue (BX60; Olympus, Tokyo, Japan). Histological images of the knee joints are shown in Figure 3. Blue-stained areas were observed on the surface of the endosteal bone and in the resorption zone, and cartilage was mineralized in the resorption zone in WT mice and 1.0 × 10 ¹² vg/individual AAV8-TNALP-D10-treated Akp2 ^−/ mice.

Example 5: Vector distribution Although the vector was injected intramuscularly, vector volumes exceeding the muscle's capacity may leak into the blood and circulate throughout the body. The distribution of 1.0 x ¹⁰ vg/individual AAV-TNALP-D10 was analyzed in the liver, muscle, heart, and bone of treated mice.

The biodistribution of AAV vectors was determined in Akp2 ^−/− mice treated with AAV8-TNALP-D10. Genomic DNA was extracted from the heart, liver, bone, and muscle and then subjected to real-time PCR as previously described. Matsumoto, T, et al. (2011) Hum Gene Ther 22: 1355-1364. Quadriceps muscles from both legs were analyzed, and injected muscles were compared with untreated muscles on the contralateral side.

Using real-time PCR, AAV vector genomes were not detected in any organs other than the AAV-TNALP-D10-injected muscle. Because germline insertion of viral vectors in the testes and/or ovaries is a significant concern for clinical use, we analyzed the AAV vector genomes present in the testes and ovaries. AAV vector genomes were not detected in the testes or ovaries. Real-time PCR revealed the presence of AAV vectors only in the muscle at the vector injection site (data not shown). This suggests that 2 μL of vector injected into the quadriceps muscle of newborn mice was localized to the injection site. These results indicate that intramuscular injection was safer than intravenous injection because the vector remained in the muscle and was not distributed systemically.

The dose-dependence of the response to systemic treatment of Akp2 ^−/− mice has been previously described ( Matsumoto, T, et al. (2011) Hum Gene Ther 22: 1355-1364 ; Yadav, MC, et al. (2011) Bone 49: 250-256 ). However, as shown herein, distribution of intramuscularly injected AAV8-TNALP-D10 vectors was limited to the injected muscle; i.e., there was no significant leakage of AAV8-TNALP-D10 from the muscle. Thus, although the CAG promoter is not tissue-specific, physical targeting to muscle was successfully achieved by injecting a relatively small volume of AAV8-TNALP-D10. These results indicate that a relatively small volume (e.g., approximately 100 μL) can be used with modified vectors containing non-tissue-specific promoters, allowing for higher expression.

Example 6: Ectopic calcification on the limbs of C57BL/6 mice due to extremely high plasma ALP activity. Constitutively high plasma ALP activity is not a physiological state. Therefore, to evaluate the safety and adverse effects of constitutively high plasma ALP activity, a large amount of AAV8-TNALP-D10 was injected into 6-week-old wild-type C57BL/6 mice.

To analyze the potential effects of excessively elevated plasma TNALP, 6-week-old male C57BL/6 mice were injected with a high dose of AAV8-TNALP-D10 (3.0 × 10 ¹² , 5.5 × 10 ¹² , 3.0 × 10 ¹³ , or 5.5 × 10 ¹³ vg/individual in 50 μL of PBS) into the right quadriceps using a 29-gauge insulin syringe. The mice were then monitored for the appearance of ectopic calcification in their bodies and analyzed for plasma ALP activity, plasma transaminases, and renal function.

Mice (n = 3) injected with 5.5 × 10 ¹³ vg/animal grew to approximately 4 months of age and ultimately died by approximately 6 months of age with high plasma ALP activity (8347.3 ± 5738.4 U/mL) (Fig. 4A). All three mice receiving this high vector dose developed stones. One mouse had stones in each volar pad of its forepaws 2 months after injection and a small stone in the pad of its left hind paw by 6 months. Another mouse had two stones in each volar pad by 2 months, while the third mouse had a small stone in the pad of its left forepaw by 3 months. When mice (n = 3) were injected with a lower dose of vector (2.8 × 10 ¹³ vg/animal), the ALP activity level was approximately one order of magnitude lower (373.0 ± 215.3 U/mL) than that of the higher dose. In these mice, ectopic calcifications began to appear approximately 4 months after injection. The stones were solid and radiopaque (FIGS. 4B and 4C).

Infrared spectroscopy analysis of the stones by SRL (Tokyo, Japan) showed that they were composed primarily of calcium phosphate (55%) and calcium carbonate (45%). X-ray analysis revealed no vascular calcification. Alizarin red staining showed no ectopic calcification in the liver, heart, muscle, kidney, or blood vessels of these treated mice. AAV8-TNALP-D10-injected Akp2 ^-/- mice did not induce ectopic or vascular calcification, even at the highest dose (1.0 × 10 ¹² vg/animal) (data not shown). C57BL/6 mice injected with 2.8 × 10 ¹² and 1.1 × 10 ¹³ vg/animal did not develop stones on their limbs or any other body surface throughout the experimental period.

Preliminary experiments showed that extremely high ALP activity (10,000 U/mL) caused ectopic calcification regardless of the route of administration or the age of the mice at the time of administration (data not shown). Mice with the highest ALP activity developed calcification in the tips of their noses, but X-ray examination showed no evidence of ectopic calcification elsewhere, such as in the kidneys or blood vessels.

Example 7: No Liver or Renal Dysfunction Detected in Treated Mice Biochemical data, liver and kidney function, and calcium levels were examined in mice with the highest plasma ALP activity. Plasma calcium metabolism in all treated mice was similar to that in WT mice (calcium: 9.5 ± 0.45 vs. 9.5 ± 0.63 mg/dL; P = 0.5). Liver and kidney function were within the normal range. Mice treated with 1.0 × 10 ¹² vg/individual AAV8-TNALP-D10 had plasma ALP activity (19.38 ± 5.02 U/mL) 397-fold higher than the endogenous level in WT mice, yet no obvious problems were observed in the AAV8-TNALP-D10-treated mice. Furthermore, no tumors were observed in any organs, and the organs appeared microscopically normal.

Example 8: Treatment of non-human primates with AAV-TNALP-D10 A study is conducted to determine the titer, volume, and injection multiplicity of AAV8-TNALP-D10 dose to achieve plasma ALP levels within the range of 10 U/mL in non-human primates. One-year-old cynomolgus monkeys weighing approximately 1 kg are injected with AAV8-TNALP-D10 or placebo. The AAV8-TNALP-D10 vector is formulated using phosphate-buffered saline. The placebo is phosphate-buffered saline (PBS) (137 mmol/L NaCl, _8.1 mmol/L _Na2HPO4 , 2.68 mmol/L KCl, 1.47 mmol/L _KH2PO4 , pH _7.4 ). The study design involves administering 100 μL of AAV-HPP as a single or multiple intramuscular injection into the right quadriceps (vastus lateralis) and/or right gastrocnemius. Vehicle (placebo) is administered into the same muscle group on the left side of the body with the same injection volume and number of sites. When multiple injections are administered, animals are lightly anesthetized. Before proceeding to the next animal, the dose group is monitored for tolerability, including liver enzymes and ALP activity, for 2 weeks; dose level is selected based on the tolerability of the dose in the animal.

During the study, Animal 1 is administered 100 μL of 1×10 ¹⁴ vg/mL AAV8-TNALP-D10 at a single intramuscular injection site for a total dose of 1×10 ¹³ vg. Animal 1 is monitored for tolerability for two weeks, and ALP activity is determined two weeks after administration. If Animal 1 cannot tolerate the 1×10 ¹³ vg dose, the total dose of AAV8-TNALP-D10 for Animal 2 is reduced and/or pretreated with corticosteroids. Animal 2 is monitored for tolerability for two weeks, and ALP activity is determined two weeks after administration.

If the ALP level in Animal 1 is >10 U/mL two weeks after administration, the total dose of AAV8-TNALP-D10 for Animal 2 will be reduced and ALP activity will be determined two weeks after administration.

If the ALP level in Animal 1 is 9 U/mL or less two weeks after administration, the total dose of AAV8-TNALP-D10 is increased for Animal 2. 100 μL of 1×10 ¹⁴ vg/mL AAV8-TNALP-D10 is injected into each of two intramuscular injection sites for a total dose of 2×10 ¹³ vg (total volume 200 μL). Animal 2 is monitored for tolerability for two weeks, and ALP activity is determined two weeks after administration.

If the ALP level in Animal 1 is 10 U/mL two weeks after administration, the total dose of AAV8-TNALP-D10 for Animal 2 is maintained. Animal 2 is administered 100 μL of 1×10 ¹⁴ vg/mL AAV8-TNALP-D10 at a single intramuscular injection site for a total dose of 1×10 ¹³ vg. Animal 2 is monitored for tolerability for two weeks, and ALP activity is determined two weeks after administration.

If ALP levels in Animal 2 two weeks after dosing are not within the target range, the total dose of AAV8-TNALP-D10 for Animal 3 will be increased or decreased as needed, and pretreatment with corticosteroids will be considered to mitigate any tolerability issues. Animal 3 will be monitored for tolerability for two weeks, and ALP activity will be determined two weeks after dosing.

If ALP levels in animals 1 and 2 are 10 U/mL two weeks after treatment, the total dose of AAV8-TNALP-D10 is maintained for animal 3, but the effect of injection multiplicity is examined. 100 μL of 2×10 ¹³ vg/mL AAV8-TNALP-D10 is injected into each of five intramuscular injection sites for a total dose of 1×10 ¹³ vg (total volume 500 μL). Animal 3 is monitored for tolerability for two weeks, and ALP activity is determined two weeks after treatment.

To achieve target ALP levels, administer AAV8-TNALP-D10 to Animal 4 at the appropriate concentration, volume, and number of injection sites based on the results observed in Animals 1-3.

Example 9: Sustained TNALP expression in rats intramuscularly injected with AAV8-TNALP-D10. TNSALP-D10 expression was analyzed in 5-6 week-old wild-type (wt) rats treated with three different doses of AAV8-TNALP-D10 ( ^1x10 , ^1x10 , and ^1x10 vg/animal, n=2 animals per dose cohort) administered by intramuscular injection into the quadriceps. Untreated wt rats served as controls (n=1).

A dose response in TNSALP-D10 activity was observed at week 1, with TNSALP-D10 levels of 192 U/mL, 841± ⁹⁴⁸ U/mL, 1373±1121 U/mL, and 3837±3318 after administration of no vector, 1.0×10 ¹¹ , 1.0×10 12 , or 1.0×10 ¹³ vg/animal, respectively (Figure 5 and Table 4). TNSALP-D10 activity peaked at week 1, and expression persisted through week 18 for the two high-dose groups. Serum chemistry analysis did not reveal any elevations in liver or muscle damage-related enzymes, and no other abnormalities were observed.

Similar to Alpl ^−/− mice treated with AAV8-TNALP-D10, TNSALP-D10 activity was measurable at the earliest time point (1 week after treatment) and persisted through week 18. TNSALP-D10 elevation was not associated with any elevation of liver enzymes.

Claims (13)

1. A pharmaceutical composition for intramuscular injection into a subject in need of treatment for hypophosphatasia (HPP) , comprising:
the pharmaceutical composition comprises a vector comprising the polynucleotide and a pharmaceutically acceptable carrier;
the polynucleotide comprises a promoter operably linked to a nucleic acid encoding a fusion protein comprising tissue non-specific alkaline phosphatase (TNALP) and a decaaspartic acid (D10) amino acid sequence, and comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 1;
The pharmaceutical composition . 1, wherein the nucleic acid encoding the fusion protein comprises the nucleic acid sequence of SEQ ID NO: 2.
The pharmaceutical composition described in 3. The pharmaceutical composition of claim 1, wherein the promoter comprises a CAG promoter. The pharmaceutical composition according to any one of claims 1 to 3, wherein the polynucleotide further comprises a CMV enhancer upstream of the promoter. The pharmaceutical composition of any one of claims 1 to 4, wherein the promoter comprises the nucleic acid sequence of SEQ ID NO: 3 or 21. The pharmaceutical composition according to any one of claims 1 to 5, wherein the vector is an adeno-associated virus (AAV) vector. The pharmaceutical composition of claim 6, wherein the AAV is of the AAV1 serotype, AAV2 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, or AAV10 serotype. 8. The pharmaceutical composition of any one of claims 1 to 4, or 6 or 7, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:1. 9. The pharmaceutical composition of claim 1, wherein the subject's plasma alkaline phosphatase (ALP) level is between 5 U/mL and 100 U/mL two weeks after administration. The pharmaceutical composition of any one of claims 1 to 9 , wherein the subject does not develop ectopic calcification or abnormal calcium metabolism up to 6 months after administration. 11. The pharmaceutical composition of any one of claims 1 to 10 , wherein the vector is not detectable in the liver, heart, bone, testes, ovaries, or any combination thereof of the subject up to 6 months after administration. 12. The pharmaceutical composition of any one of claims 1 to 11 , wherein administration of said pharmaceutical composition does not cause liver or kidney dysfunction in said subject up to six months after administration. 13. The pharmaceutical composition of any one of claims 1 to 12 , wherein administration of said pharmaceutical composition has no carcinogenic effect in said subject up to 6 months after administration.