Methods and compositions for treating, ameliorating and / or preventing diffuse idiopathic hypertrophy (DISH)

Abstract

In one aspect, the present disclosure provides specific doses of ENPP1 agents for use in the treatment of diffuse idiopathic bone hypertrophy (DISH), ankylosing spondylitis, and / or spinal arthritis in vivo.

Description

[0001] CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 325,554, filed on March 30, 2022, the contents of which are hereby incorporated by reference in their entirety.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0004] This invention was made with government support under Grants DK121326-01, AR080416-01, and AG067347A1 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0005] In one aspect, the field of the invention relates to the treatment and / or amelioration of DISH, ankylosing spondylitis and / or spondyloarthritis problems by enzyme therapy.

[0006] Sequence Listing

[0007] The XML file named "047162-7377WO1(01806) Sequence Listing.xml" was created on September 29, 2022, has a size of 149KB, and is now incorporated herein by reference in its entirety. Technical Background

[0008] Ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENP1) is a type 2 transmembrane protein whose extracellular activity hydrolyzes extracellular nucleotides, such as the phosphodiester bond of adenosine triphosphate (ATP), to generate adenosine monophosphate (AMP) and inorganic pyrophosphate (PPi). Since PPi is the main physiological inhibitor of hydroxyapatite deposition, biallelic ENPP1 deficiency leads to ectopic mineralization in early life, resulting in high mortality in infancy due to arterial calcification and luminal narrowing (Ziegler et al., Generalized arterial calcification of infancy. In: Adam MP, Ardinger HH, Pagon RA, et al., (eds). Gene Reviews 2014). Surviving patients, as well as ENPP1-deficient individuals who do not show neonatal calcification, may develop fibroblast growth factor 23 (FGF23)-mediated hypophosphatemic rickets later in life (Ferreira et al., Prospective phenotyping of long-term survivors of generalized arterial calcification of infancy (GACI), Genet Med. 2020; 23: 396-407).

[0009] WO2014126965 relates to the treatment of pathological calcification and pathological ossification in diseases involving ENPP1 deficiency. Three proteins - ENPP1, tissue nonspecific alkaline phosphatase (TNAP) and ankylosing kinase or ankyrin (ANK) - play a role in regulating the extracellular balance of inorganic pyrophosphate (PPi) and phosphate (Pi). Inorganic pyrophosphate (PPi) is generated by cleavage of extracellular nucleotide triphosphates (NTPs) by ENPP1 or by transfer of PPi from the intracellular to the extracellular space by Ank. TNAP degrades PPi to generate Pi.

[0010] "ENPP1 deficiency" is characterized by reduced levels of ENPP1 enzyme activity in the serum and / or plasma of a subject. ENPP1 deficiency is a rare inherited disorder caused by an inactivating mutation in the ENPP1 gene encoding the ENPP1 enzyme. As described elsewhere herein, ENPP1 is an integral transmembrane protein whose extracellular domain has pyrophosphatase and phosphodiesterase activities. Thus, ENPP1 converts extracellular ATP into inorganic pyrophosphate (PPi) and AMP.

[0011] Calcification in biological systems is a complex process in which calcium salts are maintained in noncirculating matrices at concentrations higher than those in regional circulating body fluids or other flowing fluids. The main result of normal calcification is the accumulation of calcium and related inorganic salts in specialized intercellular matrices in crystalline patterns of similar arrangement and chemical composition, all of which may vary in different species. On the other hand, the net effect of pathological calcification is the accumulation of calcium and related inorganic salts with a greater diversity of chemical compositions or patterns than the normal range, not only in these specialized matrices but also in other intercellular, extracellular, and cellular materials, leading to several disease states.

[0012] Diffuse idiopathic skeletal hyperostosis (DISH) is a skeletal disorder characterized by unusual new bone formation (Resnick et al., Diffuse idiopathic skeletal hyperostosis (DISH): Forestier's disease with extraspinal manifestations, Radiology. 1975; 115: 513-524). New bone formation is most common where ligaments and tendons meet bones (entheseal area), but there is also widespread bone sclerosis and bone overgrowth (hyperostosis) (Pillai & Littlejohn, Metabolic Factors in Diffuse Idiopathic Skeletal Hyperostosis–A Review of Clinical Data. The Open Rheumatology Journal. 2014; 8: 116-128). The roles of ENPP1, TNAP, and ANK in relation to DISH are not clear. In addition, it is not known whether defects in one or more of these enzymes are part of the DISH phenotype. Summary of the invention

[0013] The present invention provides a method for treating, ameliorating and / or preventing diffuse idiopathic skeletal hyperostosis (DISH), ankylosing spondylitis and / or spondyloarthritis in a patient in need thereof. In certain embodiments, the method comprises administering to the patient a therapeutically effective amount of a compound of formula (I), or a salt or solvate thereof,

[0014] Protein-Z-domain-XY(I),

[0015] Wherein in (I):

[0016] The protein includes the catalytic domain of ENPP1;

[0017] The domain is absent or is at least one selected from the group consisting of human IgG Fc domain (Fc), human serum albumin (ALB) and fragments thereof;

[0018] X and Z are independently absent or are a polypeptide comprising 1-20 amino acids,

[0019] Y is absent or is a negatively charged bone targeting sequence,

[0020] Thereby treating, ameliorating and / or preventing DISH, ankylosing spondylitis and / or spondyloarthritis in the patient.

[0021] In certain embodiments, the compound lacks a negatively charged bone targeting sequence.

[0022] In certain embodiments, Y is absent.

[0023] In certain embodiments, the compound includes a negatively charged bone targeting sequence.

[0024] In certain embodiments, the patient has ENPP1 haploinsufficiency.

[0025] In certain embodiments, the patient does not have ENPP1 haploinsufficiency.

[0026] In certain embodiments, the patient is not ENPP1 -deficient.

[0027] In certain embodiments, the patient is ENPP1 deficient.

[0028] In certain embodiments, the patient is administered the compound by at least one route selected from the group consisting of oral, aerosol, inhalation, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ophthalmic, pulmonary, and topical.

[0029] In certain embodiments, the compound is administered to the patient intravenously or subcutaneously.

[0030] In certain embodiments, administration of a compound to a patient increases the patient's extracellular pyrophosphate concentration or prevents it from further decreasing.

[0031] In certain embodiments, administering a compound to a patient reduces or prevents further increase in one or more of Achilles tendon calcification, spinal calcification, hip joint calcification, and bilateral calcification in the patient.

[0032] In certain embodiments, the domain comprises albumin.

[0033] In certain embodiments, the domain comprises an IgG Fc domain.

[0034] In certain embodiments, the protein lacks the ENPP1 transmembrane domain.

[0035] In certain embodiments, the compound is administered to a patient as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

[0036] In certain embodiments, the patient is a mammal.

[0037] In certain embodiments, the mammal is a human.

[0038] In certain embodiments, the protein includes amino acid residues 99 (PSCAKE...) to 925 (...QED) of SEQ ID NO:1.

[0039] In certain embodiments, the protein includes amino acid residues 1 to 833 of SEQ ID NO:3.

[0040] In certain embodiments, the protein comprises the amino acid sequence depicted in SEQ ID NO:2.

[0041] In certain embodiments, the protein comprises the amino acid sequence depicted in SEQ ID NO:3 or 4 or 5.

[0042] In certain embodiments, the domain increases the circulating half-life of the compound relative to the circulating half-life of the compound lacking the domain.

[0043] In certain embodiments, the patient is further diagnosed with a disease or condition selected from the group consisting of early-onset osteoporosis, osteopenia, age-related osteopenia, OPLL, hereditary hypophosphatemic rickets, X-linked hypophosphatemia, autosomal recessive hypophosphatemic rickets type 2, autosomal dominant hypophosphatemic rickets, and hypophosphatemic rickets.

[0044] In certain embodiments, the patient has not been diagnosed with a disease or condition selected from the group consisting of early-onset osteoporosis, osteopenia, age-related osteopenia, OPLL, hereditary hypophosphatemic rickets, X-linked hypophosphatemia, autosomal recessive hypophosphatemic rickets type 2, autosomal dominant hypophosphatemic rickets, and hypophosphatemic rickets.

[0045] describe

[0046] In certain embodiments, the disease or disorder contemplated herein is diffuse idiopathic skeletal hyperostosis (DISH). In certain embodiments, the disease or disorder contemplated herein is ankylosing spondylitis. In certain embodiments, the disease or disorder contemplated herein is spondyloarthritis. In certain embodiments, the disease or disorder contemplated herein is diffuse idiopathic skeletal hyperostosis (DISH) and / or ankylosing spondylitis. In certain embodiments, the disease or disorder contemplated herein is DISH and / or spondyloarthritis. In certain embodiments, the disease or disorder contemplated herein is ankylosing spondylitis and / or spondyloarthritis. In certain embodiments, the disease or disorder contemplated herein is ankylosing spondylitis and / or spondyloarthritis.

[0047] The present invention provides a method for treating, ameliorating, preventing further development or progression of diffuse idiopathic skeletal hyperostosis (DISH), ankylosing spondylitis and / or spondyloarthritis in a patient in need thereof and / or preventing diffuse idiopathic skeletal hyperostosis (DISH), ankylosing spondylitis and / or spondyloarthritis in a patient in need thereof. The present invention further provides a method for treating and / or ameliorating DISH, ankylosing spondylitis and / or spondyloarthritis in a patient in need thereof.

[0048] In one aspect, the present disclosure relates to treating, ameliorating, preventing further development and / or progression of DISH, ankylosing spondylitis and / or spondyloarthritis, and / or preventing DISH, ankylosing spondylitis and / or spondyloarthritis by administering a therapeutically effective amount of an ENPP1 agent to a subject suffering from DISH, ankylosing spondylitis and / or spondyloarthritis.

[0049] In one aspect, the present disclosure relates to treating, ameliorating, preventing further development and / or progression of ENPP1 deficiency in a subject, and / or preventing ENPP1 deficiency in a subject by administering a therapeutically effective amount of an ENPP1 agent to the subject.

[0050] In one aspect, the disclosure relates to treating, ameliorating, preventing further development and / or progression of one or more symptoms of ENPP1 haploinsufficiency in a subject, and / or preventing one or more symptoms of ENPP1 haploinsufficiency in a subject by administering a therapeutically effective amount of an ENPP1 agent to the subject.

[0051] Treatment of DISH, ankylosing spondylitis and / or spondyloarthritis comprises administering a dose of about 0.1 mg / kg, about 0.2 mg / kg of the subject, about 0.3 mg / kg of the subject, about 0.4 mg / kg of the subject, about 0.5 mg / kg of the subject, about 0.6 mg / kg of the subject, about 0.7 mg / kg of the subject, about 0.8 mg / kg of the subject, about 0.9 mg / kg of the subject, about 1.0 mg / kg of the subject, about 1.1 mg / kg of the subject, about 1.2 mg / kg of the subject, about 1.3 mg / kg of the subject, about 1.6 mg / kg of the subject, about 1.7 mg / kg of the subject, about 1.8 mg / kg of the subject, about 1.9 mg / kg of the subject, about 2.0 mg / kg of the subject, about 2.1 mg / kg of the subject, about 2.2 mg / kg of the subject, about 2.3 mg / kg of the subject, about 2.4 mg / kg of the subject, about 2.5 mg / kg of the subject, about 2.6 mg / kg of the subject, about 2.7 mg / kg of the subject, about 2.8 mg / kg of the subject, about 2.9 mg / kg of the subject The invention relates to an agent of ENPP1 administered to a subject in an amount of about 1.2 mg per kg of the subject, about 1.3 mg per kg of the subject, about 1.4 mg per kg of the subject, about 1.5 mg per kg of the subject, about 1.6 mg per kg of the subject, about 1.7 mg per kg of the subject, about 1.8 mg per kg of the subject, about 1.9 mg per kg of the subject, or about 2.0 mg per kg of the subject, thereby treating, reducing and / or ameliorating one or more symptoms of DISH, ankylosing spondylitis and / or spondyloarthritis disease, wherein the subject does not have an ENPP1 deficiency.

[0052] In one aspect, the present disclosure relates to administering to a subject having DISH, ankylosing spondylitis and / or spondyloarthritis a dose of about 0.1 mg / kg, about 0.2 mg / kg of the subject, about 0.3 mg / kg of the subject, about 0.4 mg / kg of the subject, about 0.5 mg / kg of the subject, about 0.6 mg / kg of the subject, about 0.7 mg / kg of the subject, about 0.8 mg / kg of the subject, about 0.9 mg / kg of the subject, about 1.0 mg / kg of the subject, about 1.1 mg / kg of the subject. , about 1.2 mg / kg of the subject, about 1.3 mg / kg of the subject, about 1.4 mg / kg of the subject, about 1.5 mg / kg of the subject, about 1.6 mg / kg of the subject, about 1.7 mg / kg of the subject, about 1.8 mg / kg of the subject, about 1.9 mg / kg of the subject, or about 2.0 mg / kg of the subject of an ENPP1 agent, thereby treating, reducing and / or ameliorating one or more symptoms of DISH, ankylosing spondylitis and / or spondyloarthritis disease, wherein the subject has an ENPP1 deficiency.

[0053] In one aspect, the present disclosure relates to administering to a subject having DISH, ankylosing spondylitis and / or spondyloarthritis a dose of about 0.1 mg / kg, about 0.2 mg / kg of the subject, about 0.3 mg / kg of the subject, about 0.4 mg / kg of the subject, about 0.5 mg / kg of the subject, about 0.6 mg / kg of the subject, about 0.7 mg / kg of the subject, about 0.8 mg / kg of the subject, about 0.9 mg / kg of the subject, about 1.0 mg / kg of the subject, about 1.1 mg / kg of the subject, about 1.2 mg / kg of the subject, about 1.3 mg / kg of the subject, about 1.4 mg / kg of the subject, about 1.6 mg / kg of the subject, about 1.7 mg / kg of the subject, about 1.8 mg / kg of the subject, about 1.9 mg / kg of the subject, about 2.0 mg / kg of the subject, about 2.1 mg / kg of the subject, about 2.2 mg / kg of the subject, about 2.3 mg / kg of the subject, about 2.4 mg / kg of the subject, about 2.5 mg / kg of the subject, about 2.6 mg / kg of the subject, about 2.7 mg / kg of the subject, about 2.8 mg / kg of the subject, about 2.9 mg / kg of the subject About 1.2 mg / kg of the subject, about 1.3 mg / kg of the subject, about 1.4 mg / kg of the subject, about 1.5 mg / kg of the subject, about 1.6 mg / kg of the subject, about 1.7 mg / kg of the subject, about 1.8 mg / kg of the subject, about 1.9 mg / kg of the subject, or about 2.0 mg / kg of the subject of an ENPP1 agent to treat, reduce and / or ameliorate one or more symptoms of DISH, ankylosing spondylitis and / or spondyloarthritis disease, wherein the subject does not have ENPP1 haploinsufficiency.

[0054] In one aspect, the present disclosure relates to administering to a subject having DISH, ankylosing spondylitis and / or spondyloarthritis a dose of about 0.1 mg / kg, about 0.2 mg / kg of the subject, about 0.3 mg / kg of the subject, about 0.4 mg / kg of the subject, about 0.5 mg / kg of the subject, about 0.6 mg / kg of the subject, about 0.7 mg / kg of the subject, about 0.8 mg / kg of the subject, about 0.9 mg / kg of the subject, about 1.0 mg / kg of the subject, about 1.1 mg / kg of the subject, about 1.2 mg / kg of the subject, about 1.3 mg / kg of the subject, about 1.4 mg / kg of the subject, about 1.6 mg / kg of the subject, about 1.7 mg / kg of the subject, about 1.8 mg / kg of the subject, about 1.9 mg / kg of the subject, about 2.0 mg / kg of the subject, about 2.1 mg / kg of the subject, about 2.2 mg / kg of the subject, about 2.3 mg / kg of the subject, about 2.4 mg / kg of the subject, about 2.5 mg / kg of the subject, about 2.6 mg / kg of the subject, about 2.7 mg / kg of the subject, about 2.8 mg / kg of the subject, about 2.9 mg / kg of the subject About 1.2 mg / kg of the subject, about 1.3 mg / kg of the subject, about 1.4 mg / kg of the subject, about 1.5 mg / kg of the subject, about 1.6 mg / kg of the subject, about 1.7 mg / kg of the subject, about 1.8 mg / kg of the subject, about 1.9 mg / kg of the subject, or about 2.0 mg / kg of the subject of an ENPP1 agent to treat, reduce and / or ameliorate one or more symptoms of DISH, ankylosing spondylitis and / or spondyloarthritis disease, wherein the subject has ENPP1 haploinsufficiency.

[0055] In one aspect, the present disclosure relates to administering to a subject having DISH, ankylosing spondylitis and / or spondyloarthritis a dose of about 0.1 mg / kg, about 0.2 mg / kg of the subject, about 0.3 mg / kg of the subject, about 0.4 mg / kg of the subject, about 0.5 mg / kg of the subject, about 0.6 mg / kg of the subject, about 0.7 mg / kg of the subject, about 0.8 mg / kg of the subject, about 0.9 mg / kg of the subject, about 1.0 mg / kg of the subject, about 1.2 mg / kg of the subject, about 1.3 mg / kg of the subject, about 1.4 mg / kg of the subject, about 1.6 mg / kg of the subject, about 1.7 mg / kg of the subject, about 1.8 mg / kg of the subject, about 1.9 mg / kg of the subject, about 2.0 mg / kg of the subject, about 2.1 mg / kg of the subject, about 2.2 mg / kg of the subject, about 2.3 mg / kg of the subject, about 2.4 mg / kg of the subject, about 2.5 mg / kg of the subject, about 2.6 mg / kg of the subject, about 2.7 mg / kg of the subject, about 2.8 mg / kg of the subject, about 2.9 mg / kg of the subject, about 3. About 1.1 mg per kg of subject, about 1.2 mg per kg of subject, about 1.3 mg per kg of subject, about 1.4 mg per kg of subject, about 1.5 mg per kg of subject, about 1.6 mg per kg of subject, about 1.7 mg per kg of subject, about 1.8 mg per kg of subject, about 1.9 mg per kg of subject, or about 2.0 mg per kg of subject of ENPP1 agent to restore physiological levels of ENPP1 protein and / or activity in the subject's plasma and / or tissues. As used herein, physiological levels of ENPP1 protein and / or activity in plasma and / or tissues refers to an amount or concentration of ENPP1 agent sufficient to achieve and maintain physiological levels of PPi in human serum. In certain embodiments, the ENPP1 agent is ENPP1 and / or an ENPP1 construct comprising ENPP1 activity.

[0056] In one aspect, the present disclosure relates to administering to a subject having ENPP1 haploinsufficiency a dose of about 0.1 mg / kg, about 0.2 mg / kg of the subject, about 0.3 mg / kg of the subject, about 0.4 mg / kg of the subject, about 0.5 mg / kg of the subject, about 0.6 mg / kg of the subject, about 0.7 mg / kg of the subject, about 0.8 mg / kg of the subject, about 0.9 mg / kg of the subject, about 1.0 mg / kg of the subject, about 2.0 mg / kg of the subject, about 3.0 mg / kg of the subject, about 4.0 mg / kg of the subject, about 5.0 mg / kg of the subject, about 6.0 mg / kg of the subject, about 7.0 mg / kg of the subject, about 8.0 mg / kg of the subject, about 9.0 mg / kg of the subject, about 10.0 mg / kg of the subject, about 11.0 mg / kg of the subject, about 12.0 mg / kg of the subject, about 13.0 mg / kg of the subject, about 14.0 mg / kg of the subject, about 15.0 mg / kg of the subject, about 16.0 mg / kg of the subject, about 17.0 mg / kg of the subject, about 18.0 mg / kg of the subject, about 19.0 mg / kg of the subject, about 20.0 mg / kg of the subject, about 21.0 mg / kg of the subject, about 22.0 mg / kg of the subject, about 24.0 mg / kg of the subject In some embodiments, the ENPP1 agent is about 1.1 mg per kg of the subject, about 1.2 mg per kg of the subject, about 1.3 mg per kg of the subject, about 1.4 mg per kg of the subject, about 1.5 mg per kg of the subject, about 1.6 mg per kg of the subject, about 1.7 mg per kg of the subject, about 1.8 mg per kg of the subject, about 1.9 mg per kg of the subject, or about 2.0 mg per kg of the subject to restore physiological levels of ENPP1 protein and / or activity in the subject's plasma and / or tissues. As used herein, physiological levels of ENPP1 protein and / or activity in plasma and / or tissues refers to an amount or concentration of ENPP1 agent sufficient to achieve and maintain physiological levels of PPi in human serum. In certain embodiments, the ENPP1 agent is ENPP1 and / or an ENPP1 construct comprising ENPP1 activity.

[0057] In one aspect, the disclosure relates to a method of increasing circulating pyrophosphate (PPi) in a subject having DISH, ankylosing spondylitis, and / or spondyloarthritis, the method comprising administering to the subject a dose of about 0.1 mg / kg, about 0.2 mg / kg of the subject, about 0.3 mg / kg of the subject, about 0.4 mg / kg of the subject, about 0.5 mg / kg of the subject, about 0.6 mg / kg of the subject, about 0.7 mg / kg of the subject, about 0.8 mg / kg of the subject, about 0.9 mg / kg of the subject, about 10 mg / kg of the subject, about 20 mg / kg of the subject, about 30 mg / kg of the subject, about 40 mg / kg of the subject, about 50 mg / kg of the subject, about 60 mg / kg of the subject, about 70 mg / kg of the subject, about 80 mg / kg of the subject, about 90 mg / kg of the subject, about 100 mg / kg of the subject, about 150 mg / kg of the subject, about 10 ... The invention relates to an ENPP1 dose of about 0.9 mg per kg of the subject, about 1.0 mg per kg of the subject, about 1.1 mg per kg of the subject, about 1.2 mg per kg of the subject, about 1.3 mg per kg of the subject, about 1.4 mg per kg of the subject, about 1.5 mg per kg of the subject, about 1.6 mg per kg of the subject, about 1.7 mg per kg of the subject, about 1.8 mg per kg of the subject, about 1.9 mg per kg of the subject, or about 2.0 mg per kg of the subject, thereby increasing circulating PPi in the subject.

[0058] In one aspect, the disclosure relates to a method of increasing circulating pyrophosphate (PPi) in a subject having ENPP1 haploinsufficiency, the method comprising administering to the subject a dose of about 0.1 mg / kg, about 0.2 mg / kg of the subject, about 0.3 mg / kg of the subject, about 0.4 mg / kg of the subject, about 0.5 mg / kg of the subject, about 0.6 mg / kg of the subject, about 0.7 mg / kg of the subject, about 0.8 mg / kg of the subject, about 0.1 mg / kg of the subject, about 0.2 mg / kg of the subject, about 0.3 mg / kg of the subject, about 0.4 mg / kg of the subject, about 0.5 mg / kg of the subject, about 0.6 mg / kg of the subject, about 0.7 mg / kg of the subject, about 0.8 mg / kg of the subject, about 0. 9 mg per kg of subject, about 1.0 mg per kg of subject, about 1.1 mg per kg of subject, about 1.2 mg per kg of subject, about 1.3 mg per kg of subject, about 1.4 mg per kg of subject, about 1.5 mg per kg of subject, about 1.6 mg per kg of subject, about 1.7 mg per kg of subject, about 1.8 mg per kg of subject, about 1.9 mg per kg of subject, or about 2.0 mg per kg of subject of an ENPP1 dose, thereby increasing circulating PPi in the subject.

[0059] In one aspect, the disclosure relates to a method of ameliorating one or more symptoms of ENPP1 haploinsufficiency in a subject, the method comprising administering to the subject a dose of about 0.1 mg / kg, about 0.2 mg / kg of the subject, about 0.3 mg / kg of the subject, about 0.4 mg / kg of the subject, about 0.5 mg / kg of the subject, about 0.6 mg / kg of the subject, about 0.7 mg / kg of the subject, about 0.8 mg / kg of the subject, about 0.9 mg / kg of the subject, about 1.0 ... About 1.1 mg / kg of the subject, about 1.2 mg / kg of the subject, about 1.3 mg / kg of the subject, about 1.4 mg / kg of the subject, about 1.5 mg / kg of the subject, about 1.6 mg / kg of the subject, about 1.7 mg / kg of the subject, about 1.8 mg / kg of the subject, about 1.9 mg / kg of the subject, or about 2.0 mg / kg of the subject of an ENPP1 agent to improve one or more symptoms of ENPP1 deficiency or one or more symptoms of ENPP1 haploinsufficiency in the subject.

[0060] In one aspect, the present disclosure relates to preventing and / or reversing the progression of pathological calcification and / or ossification masses present in a subject with DISH, ankylosing spondylitis, and / or spondyloarthritis, or minimizing and / or reducing pathological calcification and / or ossification masses present in a subject with DISH, ankylosing spondylitis, and / or spondyloarthritis, the method comprising administering to the subject a dose of about 0.1 mg / kg, about 0.2 mg / kg of the subject, about 0.3 mg / kg of the subject, about 0.4 mg / kg of the subject, about 0.5 mg / kg of the subject, about 0.6 mg / kg of the subject, about 0.7 mg / kg of the subject, about 0.8 mg / kg of the subject, about 0.9 mg / kg of the subject, about 10 mg / kg of the subject, about 15 mg / kg of the subject, about 16 mg / kg of the subject, about 17 mg / kg of the subject, about 18 mg / kg of the subject, about 19 mg / kg of the subject, about 20 mg / kg of the subject, about 21 mg / kg of the subject, about 22 mg / kg of the subject, about 24 mg / kg of the subject, about 25 mg / kg of the subject, about 26 mg / kg of the subject, about 27 mg / kg of the subject, about 28 mg / kg of the subject, about 29 mg / kg of the subject, about 30 mg / kg of the subject, about 31 mg / kg of the subject, about 32 mg / kg of the subject, about 33 mg / kg of the subject, about 34 mg / kg of the subject, about 35 mg / kg of the subject, about 36 mg / kg of the subject, about 37 mg / kg of the subject, about 38 mg / kg of the subject, about 39 mg / kg of the subject, about About 0.9 mg / kg of the subject, about 1.0 mg / kg of the subject, about 1.1 mg / kg of the subject, about 1.2 mg / kg of the subject, about 1.3 mg / kg of the subject, about 1.4 mg / kg of the subject, about 1.5 mg / kg of the subject, about 1.6 mg / kg of the subject, about 1.7 mg / kg of the subject, about 1.8 mg / kg of the subject, about 1.9 mg / kg of the subject, or about 2.0 mg / kg of the subject of the ENPP1 agent to prevent and / or reverse the progression of pathological calcification and / or ossification of bone mass in the subject, or minimize and / or reduce pathological calcification and / or ossification of bone mass in the subject.

[0061] In some embodiments of any of the methods described above, the method comprises administering to the patient a therapeutically effective amount of an ENPP1 agent.

[0062] In some embodiments, the ENPP1 agent is a compound of formula (I), or a salt or solvate thereof.

[0063] W-protein-Z-domain-XY

[0064] (I),

[0065] Wherein in (I):

[0066] W is absent or includes a signal sequence that allows export of the compound into the extracellular space;

[0067] The protein includes the catalytic domain of ENPP1;

[0068] The domain is absent or is at least one selected from the group consisting of human IgG Fc domain (Fc), human serum albumin (ALB), and biologically active fragments thereof.

[0069] X and Z are independently absent or are a polypeptide comprising 1-20 amino acids; and

[0070] Y is absent or is a "bone targeting" sequence group

[0071] wherein m is independently an integer from 1 to 15, and

[0072] wherein n is independently an integer from 1 to 10.

[0073] In some embodiments, W is absent. In some embodiments, W comprises a signal sequence that allows export of the compound into the extracellular space.

[0074] In some embodiments, Y is a "bone targeting" sequence group selected from the following: m (SEQ ID NO:11), (DSS) n (SEQ ID NO:12), (ESS) n (SEQ ID NO: 13), (RQQ) n (SEQ ID NO: 14), (KR) n (SEQ ID NO: 15), R m E m (SEQ ID NO: 117). BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1A-1B Spine CT and bone scintigraphy images of Patient 1 are shown. Figure 1A Indicates the presence of multiple compression fractures in the spine as detected by spine CT, shown as white arrows. Figure 1B A bone scintigraphy image is shown, revealing multiple accumulations in the ribs.

[0076] Figure 1C-1H Radiographs of the hip, knee, and Achilles tendon of Patient 1 are shown. Figure 1C : Right hip joint, Figure 1D : Left hip joint, Figure 1E : Right knee joint, Figure 1F : Left knee joint, Figure 1G : Right Achilles tendon, Figure 1H : Left Achilles tendon. No evidence of heterotopic ossification.

[0077] Figures 2A-2C The pedigree of the family with ENPP1 mutation is shown, corresponding to patients 1, 2, and 3. The arrow indicates the proband.

[0078] Figures 3A-3G Patient 2's spine CT, and radiographs of the hip, knee, and Achilles tendon are shown. Figure 3A : CT of the spine shows ossification of the paraspinal ligaments (white arrows) and multiple compression fractures (white arrows). Figure 3B-3C Corresponding to the right hip joint ( Figure 3B ) and left hip joint ( Figure 3C ). The images showed no signs of heterotopic ossification. Figure 3D-3E Corresponding to the right knee joint ( Figure 3D ) and left knee joint ( Figure 3E ). The image showed no signs of heterotopic ossification. Figure 3F and Figure 3G Corresponding to the right Achilles tendon ( Figure 3F ) and left Achilles tendon ( Figure 3G ). Evidence of enthesopathy was noted in the right Achilles tendon (shown as white arrows).

[0079] Figures 4A-4I Patient 3's spine CT, and radiographs of the hip, knee, and Achilles tendon are shown. Figures 4A-4C Corresponding to the spine CT, which shows the cervical spine ( Figure 4A ),thoracic( Figure 4B ) and lumbar spine ( Figure 4C ) with multiple paraspinal ossifications (white arrows). Figure 4D-4E The right hip joint detected in the X-ray is shown ( Figure 4D ) and left hip joint ( Figure 4E ) with prominent ossification around the anterior cruciate ligament (white arrow). Figure 4F-4G The right knee joint is shown ( Figure 4F ) and left knee joint ( Figure 4G ) showed no evidence of heterotopic ossification. Figures 4H-4I Shown in the right Achilles tendon ( Figure 4H ) and left Achilles tendon ( Fig. 4I ) in the X-ray image of the posterior iliac spine (white arrow).

[0080] Figure 5A-5G CT of the spine and radiographs of the hip, knee, and Achilles tendon of Patient 1's son are shown. Figure 5A ) Spine CT, ( Figure 5B )Right hip joint, ( Figure 5C )Left hip joint, ( Figure 5D )Right knee joint, ( Figure 5E )Left knee joint, ( Fig. 5F )Right Achilles tendon, ( Figure 5G ) Left Achilles tendon. No evidence of heterotopic ossification.

[0081] Figures 6A-6G Shown is a CT scan of the spine and radiographs of the hip, knee, and Achilles tendon of one of Patient 3's sons. Fig. 6A ) Spine CT, ( Figure 6B )Right hip joint, ( Figure 6C )Left hip joint, ( Fig.6D )Right knee joint, ( Fig. 6E )Left knee joint, ( Fig. 6F )Right Achilles tendon, ( Figure 6G ) Left Achilles tendon. There is mild enthespathy in the left Achilles tendon (white arrow).

[0082] Figures 7A-7G CT scan of the spine and X-rays of the hip, knee, and Achilles tendon of another son of Case 3. Fig. 7A ) Spine CT, ( Figure 7B )Right hip joint, ( Figure 7C )Left hip joint, ( Fig.7D )Right knee joint, ( Fig. 7E )Left knee joint, ( Figure 7F )Right Achilles tendon, ( Figure 7G ) Left Achilles tendon. The left Achilles tendon has mild enthesopathy (white arrow).

[0083] Figure 8 Sanger sequencing of complementary DNA confirmed compound heterozygosity for ENPP1 variants in patient 3. While clonotype 1 contained the ENPP1 variant c.536A>G, ​​another ENPP1 variant c.1352A>G was located in clonotype 2, indicating compound heterozygosity.

[0084] Fig. 9 A non-limiting schematic diagram of the ENPP1 structure and the location of the present variants are shown. ENPP1 consists of a cytoplasmic domain (CD), a transmembrane domain (TM), and an extracellular domain consisting of two somatomedin domains (SMB1 and SMB2), a catalytic domain, and a nuclease-like domain. N179S and Y451C are located in the SMB2 and catalytic domains, respectively.

[0085] Fig.10 Comparison of the enzyme activities of WT, N179S and Y451C is shown. When compared to WT ENPP1, the N179S and Y451C variants showed a 55% and 70% decrease in the enzymatic reaction rate, respectively. The bars represent median and quartile values. ****: p<0.0001.

[0086] Fig.11 The complete, unprocessed amino acid sequence of the wild-type ENPP1 precursor protein (SEQ ID NO: 1) is shown. The cytoplasmic and transmembrane regions are underlined. Potential N-glycosylation sites are in bold. PSCAKE (residues 99-104; boxed) is the start of the soluble ENPP1 protein portion, which includes SMB1 (residues 104-144) and SMB2 (residues 145-189).

[0087] Fig.12 Shown are the results of weekly subcutaneous administration of vehicle or ENPP1 to WT and Enpp1 cells. asj Plasma PPi responses in mice ( Fig.12 ). *P<0.05, **P<0.01; (analysis of variance, Kruskal-Wallis test).

[0088] Fig.13 Shown are 17-week-old WT and 17-week-old Enpp1 mice dosed weekly with the indicated subcutaneous doses of vehicle or ENPP1 constructs #1118 and #2000. asj Paraspinal osteophytes and ankylosis responses in male mice. Construct #1118 was dosed weekly starting at week 3, while construct #2000 was dosed weekly starting at week 5. Micro-CT images showed preferential attenuation of paraspinal osteophytes and ankylosis in construct #2000 and less attenuation of paraspinal osteophytes and ankylosis in Enpp1 given at 1 mg / Kg weekly. asj This was evident in both male and female mice.

[0089] Fig.14 Shown are 17-week-old WT and 17-week-old Enpp1 mice dosed weekly with the indicated subcutaneous doses of vehicle or ENPP1 constructs #1118 and #2000. asjParaspinal osteophytes and ankylosis responses in female mice. Construct #1118 was dosed weekly starting at week 3, while construct #2000 was dosed weekly starting at week 5. Micro-CT images showed preferential attenuation of paraspinal osteophytes and ankylosis in construct #2000 and less attenuation of paraspinal osteophytes and ankylosis in Enpp1 given at 1 mg / Kg weekly. asj This was evident in both male and female mice.

[0090] Figures 15A-15B Shown are 17-week-old WT and 17-week-old Enpp1 mice dosed weekly with the indicated subcutaneous doses of vehicle or ENPP1 constructs #1118 and #2000. asj Auditory brainstem responses in mice. Construct #1118 was dosed once a week starting from week 3, while construct 2000 was dosed once a week starting from week 5. Stimulus frequency measurements showed that ENPP1 construct #1118 at a dose of 2 mg / Kg per week, as well as ENPP1 construct #2000 at doses of 0.5 and 1 mg / Kg per week, could prevent hearing loss in the low frequency (8kHz) range. It was also noted that the hearing of ENPP1 deficient animals was improved in the high frequency range (32kHz), preferably in ENPP1 construct #2000 dosed at 1 mg / Kg per week.

[0091] Fig.16 Shown are WT and 17-week-old ENPP1 cells treated weekly with indicated subcutaneous doses of vehicle or ENPP1 constructs #1118 and #2000. asj Complete FGF23 levels in mice. Construct #1118 was administered once a week starting from week 3, while construct #2000 was administered once a week starting from week 5. The data show that when administered at 1 mg / Kg and 4 mg / Kg per week, ENPP1 construct #2000 preferably inhibits complete FGF23. Statistical significance was assessed by ANOVA Kruskal-Wallis test, followed by Dunn's post hoc analysis to assess differences from WT levels (one-way ANOVA). Statistical significance is represented by p value, with the following annotations: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0092] definition

[0093] The terms used in this specification generally have their ordinary meaning in the art in the context of the present disclosure and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to practitioners describing the compositions and methods of the present disclosure and how to make and use them. Any scope of use or meaning of a term will be apparent from the specific context in which the term is used.

[0094] As used herein, the term "DISH" refers to diffuse idiopathic skeletal hyperostosis, also known as Forestier disease, a skeletal disorder characterized by unusual new bone formation (Resnick et al., Diffuse idiopathic skeletal hyperostosis (DISH). Forestier's disease with extraspinal manifestations, Radiology. 1975; 115: 513-524). DISH causes calcification (hardening) of ligaments and tendons in the body and / or new bone growth (ossified masses) in areas around the spine and bones. These calcified areas may also form bone spurs (abnormal new bone growth) in some instances, which can cause pain, stiffness, and decreased mobility. New bone formation is most common where ligaments and tendons attach to bones (tendon terminal areas), but the relationship between calcification of tendon terminals and ossified masses around the spine is unclear. There is also widespread bone sclerosis and bone overgrowth (osteophytes) (Pillai & Littlejohn, Metabolic Factors in Diffuse Idiopathic Skeletal Hyperostosis-A Review of Clinical Data, The Open Rheumatology Journal. 2014; 8: 116-128). Another aspect of the disease is the formation of large, mobile osteophytes due to abnormal bone growth. These ossifications are more common in the axial skeleton, with the thoracic region being the main location. In addition, the peripheral tendon ends, such as the peripatellar ligament, Achilles tendon insertion, plantar fascia, shoulder, olecranon, and metacarpophalangeal joints may also have calcification. In some instances, calcification is seen in the hip, knee, ankle, foot, shoulder, hand, and rib (Helfgott, Diffuse idiopathic skeletal hyperostosis (DISH). Up To Date. June 7, 2017). Common features and / or symptoms of DISH include focal and diffuse calcification and ossification of the anterior longitudinal ligament, calcification of the paraspinal connective tissue and annulus fibrosus, degeneration of the peripheral annulus fibrosus fibers, anterolateral extension of fibrous tissue, hypervascularity, chronic inflammatory cell infiltration, periosteal new bone formation on the anterior surface of vertebral bodies, ossification of the metacarpophalangeal joints, pain in the thoracic, lumbar and / or cervical regions, radiculopathy, polyarticular pain, monoarticular synovitis and / or dysphagia.

[0095] DISH is characterized by the accumulation of calcium salts (calcification) and abnormal new bone growth (ossification) in tendons and ligaments, but the cause is unknown (Mader et al., Diffuse idiopathic skeletal hyperostosis: clinical features and pathogenic mechanisms, Nat Rev Rheumatol. December 2013; 9(12): 741-50 & Nascimento et al., Diffuse idiopathic skeletal hyperostosis: A review, Surgical Neurology International. 2014; 5(Suppl 3): S122-S125). Common risk factors for developing DISH include, but are not limited to, large waist circumference, BMI / obesity, hyperinsulinemia, diabetes, hyperuricemia, dyslipidemia, hypertension, coronary artery disease, and gout. DISH can be asymptomatic, and in these cases, the diagnosis is usually made based on radiographic images.

[0096] DISH is a progressive musculoskeletal disease associated with aging. In two large Midwestern hospital populations, the prevalence of DISH was 25% and 15% in men and women over 50 years of age, respectively. In addition, up to 6.3% in the Japanese population and 25% of patients with degenerative cervical spondylosis in North America develop progressive calcification in the posterior longitudinal ligament of the spine in a condition known as OPLL. Progressive calcium deposition in the enthesinus leads to spinal stenosis and spinal cord compression caused by a growing paraspinal mass. The resulting myelopathy is often severely painful and debilitating. Treatment usually consists of conservative chronic pain management with NSAIDs, as there are no effective therapies to prevent the progressive ossification that leads to the symptomatic process, and little is known about the factors that lead to the initiation and promotion of ectopic mineralization. Surgical decompression is used to provide short-term relief in acute cases, but enthesinus progression in OPLL occurs more rapidly after surgery (laminoplasty) compared with conservatively managed patients (70% vs. 24%, respectively), and therefore, surgical intervention is discouraged except in cases with severe symptoms.

[0097] As used herein, a subject with DISH refers to a subject diagnosed with DISH. The diagnosis of DISH is based on radiological and / or clinical findings and is defined by Resnick and Niwayana. Radiography of the thoracic and lumbar spine is the single most useful imaging modality for diagnosing DISH. Computed tomography (CT) scans can be used to assess complications such as fractures, or symptoms caused by pressure on the trachea, esophagus, and veins. This can distinguish this entity from ankylosing spondylitis or OPLL (Artner et al., Diffuse idiopathic skeletal hyperostosis: current aspects of diagnostics and therapy, 2012, Orthopade. 2012 Nov; 41(11): 916-22; Olivieri et al., Diffuse idiopathic skeletal hyperostosis may give the typical postural abnormalities of advanced Ankylosing Spondylitis Rheumatology 2007 Nov 1; 46(11): 1709-11). The presence of flowing calcification and ossification predominantly along the anterolateral aspect (anterior longitudinal ligament) of at least 4 adjacent vertebrae (spanning 3 disc spaces) with preserved disc height indicates DISH. The spinal and extraspinal features visible on radiographs and CT images of DISH patients (Radswiki & Baba, Diffuse idiopathic skeletalhyperostosis. Reference article, Radiopaedia.org) are as follows.

[0098] Non-restrictive spinal features:

[0099] -Flowing ossification: florid-like flowing ossification along the anterior or right anterolateral aspect of at least four adjacent vertebrae

[0100] -Disc space is usually well preserved

[0101] - Ankylosis is more common in the thoracic spine than in the cervical or lumbar spine

[0102] - Often incomplete

[0103] - There may be alternating areas of prominent disc material in the fluid ossification

[0104] - No sacroiliitis or ankylosis of the sacroiliac joint, although anterior and posterior sacroiliac bridges and tendon end bridges may be present10.

[0105] Non-restrictive extraspinal features:

[0106] - Enthesopathy of the iliac crest, ischial tuberosity, and greater trochanter

[0107] - Osteoarthritis of the accessory skeleton (olecranon, calcaneus, patellar ligament) is often present.

[0108] - "Whisker-like" marginal spurs (enthesophytes)

[0109] Non-limiting clinical features:

[0110] -pain

[0111] - Reduced range of motion

[0112] - Some patients are at increased risk of spinal fractures

[0113] In some instances, DISH is symptomatic, with the primary clinical features including one or more of pain, stiffness and decreased mobility (range of motion), dysphagia (caused by osteophyte compression), esophageal obstruction, hoarseness, cervical spondylosis, atlantoaxial subluxation, spinal stenosis, ossification of the posterior longitudinal ligament, spinal cord injury, dyspnea, foreign body sensation, neurologic manifestations due to spinal cord compression, hypercholesterolemia (leading to cardiovascular complications), and / or peripheral joint disease.

[0114] The "enthesis" is the site where a tendon or ligament attaches to bone, and its structure is divided into four regions: a dense fibrous connective tissue zone, filled by fibroblast-type cells (tenocytes) and composed of type I and type III collagen and decorin; unmineralized fibrocartilage, filled by fibrochondrocytes and composed of type I and type II collagen and aggrecan; mineralized fibrocartilage, filled by hypertrophic chondrocytes and composed of type II and type X collagen and aggrecan; and bone, filled by osteoblasts, osteocytes, and osteoclasts and composed of type I collagen (Calejo et al., Enthesis tissue engineering: biologicalrequirements meet at the interface, Tissue Eng Part B Rev. 2019; 25(4): 330-356). Therefore, the tendon end represents a musculoskeletal structure that can achieve a smooth transition between two distinct tissues, tendons or ligaments (compliant soft tissue) and bones (rigid hard tissue) (Calejo et al., Enthesis tissue engineering: biological requirements meet at the interface, Tissue Eng Part BRev. 2019; 25 (4): 330-356). The abrupt transition at this interface can lead to stress concentration between regions, increasing the risk of failure; in contrast, the gradual transition of composition and structure above the tendon end reduces stress concentration. (Genin et al., Functional grading of mineral and collagen in the attachment of tendon to bone, Biophys J. 2009; 97 (4): 976-985). From the tendon to the bone, it can be seen that the collagen fiber arrangement gradually decreases and the mineral content increases, which produces a gradient of tissue stiffness (Genin et al., Functional grading of mineral and collagen in the attachment of tendon to bone, Biophys J. 2009; 97 (4): 976-985).Reduced mineralization of the tendon attachment results in decreased strength of the structure (Deymier et al., Micro-mechanical properties of the tendon-to-bone attachment, Acta Biomater. 2017; 1(56): 25-35), and animal models of mineralized fibrocartilage expansion also show decreased strength of the tendon attachment (Marinovich et al., The role of bone sialoprotein in the tendon-bone insertion, Matrix Biol. 2016; 52–54: 325-338). Therefore, mineralization must be properly regulated to achieve optimal mechanical properties of the tendon attachment.

[0115] As used herein, "ankylosing spondylitis" refers to a type of arthritis characterized by long-term inflammation of the joints of the spine, usually where the spine joins the pelvis. Affected areas may include other joints, such as the shoulders or hips, and eye and intestinal problems as well as back pain may occur. Joint mobility in the affected area generally worsens over time.

[0116] Although the cause of ankylosing spondylitis is unknown, it is thought to involve a combination of genetic and environmental factors. Many affected people have a specific human leukocyte antigen, called the HLA-B27 antigen. The underlying mechanism is thought to be autoimmune or autoinflammatory. Diagnosis is usually based on symptoms, with support from medical imaging and blood tests. Ankylosing spondylitis is a seronegative spondyloarthropathy, meaning that tests show no presence of rheumatoid factor (RF) antibodies. There is no known cure for ankylosing spondylitis. Treatment may include medication, exercise, physical therapy, and, in rare cases, surgery. Medications used include NSAIDs, steroids, DMARDs (e.g., sulfasalazine), and biologics (e.g., TNF inhibitors). Approximately 0.1% and 0.8% of all humans are affected, with onset usually occurring in young adults. Men and women are equally affected; however, women are more likely than men to present with inflammation rather than fusion.

[0117] As used herein, "spondyloarthritis" or "SpA" is characterized by inflammation of the axial skeleton (sacroiliitis, spondylitis), peripheral joints, and tendinous ends. Extraskeletal manifestations can occur, such as anterior uveitis, psoriasis, and inflammatory bowel disease. HLA-B27 is the main genetic risk factor. The incidence of the entire SpA group is 0.1% to 1.9% worldwide, with differences between different countries and ethnic groups. Nonsteroidal anti-inflammatory drugs (commonly referred to as NSAIDs) provide symptom relief for most patients by reducing pain and swelling. Other drugs known as biologics, including anti-TNF drugs (TNF blockers) and anti-IL-17 drugs (IL-17 blockers), are effective for patients who do not respond well enough to NSAIDs.

[0118] "ENPP1 deficiency" is characterized by reduced levels of ENPP1 enzyme activity in the serum and / or plasma of a subject. ENPP1 deficiency is a rare inherited disorder caused by an inactivating mutation in the ENPP1 gene encoding the ENPP1 enzyme. ENPP1 is an integral transmembrane protein whose extracellular domain has pyrophosphatase and phosphodiesterase activities. ENPP1 converts extracellular ATP into inorganic pyrophosphate (PPi) and AMP.

[0119] "Enzymatically active" with respect to an ENPP1 polypeptide, or as used herein, "enzymatically active" with respect to an ENPP1 polypeptide is defined as having activity that hydrolyzes ATP to AMP and PPi and / or hydrolyzes AP3A to ATP. ENPP1 readily hydrolyzes ATP to AMP and PPi. The steady-state Michaelis-Menten enzyme constant for ENPP1 is determined using ATP as a substrate. ENPP1 cleavage of ATP can be demonstrated by HPLC analysis of the enzymatic reaction, and the identity of the substrate and reaction products can be confirmed by using ATP, AMP, and ADP standards. In the presence of ENPP1, the ATP substrate degrades over time, with accumulation of the enzymatic product AMP. Using varying concentrations of the ATP substrate, the initial rate velocity of ENPP1 in the presence of ATP is derived, and the data is fit to a curve to derive the enzyme rate constant. At physiological pH, the kinetic rate constant for NPP1 is K m = 2 μM and k cat =3.4±0.4s -1 .

[0120] As used herein, the term "plasma pyrophosphate (PPi) level" refers to the amount of pyrophosphate present in the plasma of an animal. In certain embodiments, the animal includes rats, mice, cats, dogs, humans, cattle and horses. Due to the release from platelets, it is necessary to measure PPi in plasma rather than serum. There are several methods for measuring PPi, one of which is an enzymatic test using uridine-diphosphoglucose (UDPG) pyrophosphorylase (Lust & Seegmiller, 1976, Clin. Chim. Acta 66: 241-249; Cheung & Suhadolnik, 1977, Anal. Biochem. 83: 61-63), with modifications.

[0121] Typically, the plasma PPi level of healthy human subjects ranges from about 1 μM to 3 μM, and in some cases between 1-2 μM. The normal level of ENPP1 in plasma refers to the amount of ENPP1 protein required to maintain normal levels of plasma pyrophosphate (PPi) in healthy subjects. The normal level of PPi in healthy people corresponds to 2-3 μM. Subjects with ENPP1 deficiency exhibit low PPi levels ranging from at least 10% lower than normal levels, at least 20% lower than normal levels, at least 30% lower than normal levels, at least 40% lower than normal levels, at least 50% lower than normal levels, at least 60% lower than normal levels, at least 70% lower than normal levels, at least 80% lower than normal levels, and combinations thereof. In patients with GACI, PPi levels were found to be below 1 μM, and in some cases below detectable levels. In patients with PXE, PPi levels are below 0.5 μM (Arterioscler Thromb Vasc Biol. 2014 Sep; 34(9): 1985-9; Braddock et al., Nat Commun. 2015; 6: 10006).

[0122] As used herein, the term "pathological calcification" refers to the abnormal deposition of calcium salts in the soft tissues, secretory and excretory channels of the body, causing them to harden. There are two types: malnutrition calcification that occurs in dying and dead tissues, and metastatic calcification, which is characterized by elevated extracellular calcium levels (hypercalcemia) that exceed the homeostatic capacity of cells and tissues. Calcification can involve cells as well as extracellular matrix components, such as collagen in the basement membrane and elastic fibers in the arterial wall. Examples of some tissues that are prone to calcification include: gastric mucosa - the inner epithelial layer of the stomach, kidneys and lungs, cornea, body arteries and pulmonary veins.

[0123] The term "pathological ossification" as used herein refers to a pathological condition in which bone is produced in tissues other than the bone system, or in connective tissues that do not normally exhibit osteogenic properties. Ossification is divided into three types according to the nature of the tissue or organ affected: endochondral ossification is ossification that occurs in cartilage and replaces cartilage; intramembranous ossification is ossification that occurs in connective tissue and replaces bone in connective tissue; metaplastic ossification is the development of bony material in normal soft tissue structures; also called heterotrophic ossification.

[0124] "Deficiency" of ENPP1 refers to a condition in which ENPP1 in a subject's plasma is less than or equal to 5%-10% of normal levels. Normal levels of ENPP1 in healthy human subjects are approximately between 10 and 30 ng / mL (Am J Pathol. 2001 Feb; 158(2):543-554).

[0125] "Ectopic calcification" refers to a condition characterized by the pathological deposition of calcium salts in tissues or bone growth in soft tissues.

[0126] “Ectopic calcification of soft tissue” refers to inappropriate biomineralization, typically composed of calcium phosphate, hydroxyapatite, calcium oxalate, and octacalcium phosphate, that occurs in soft tissue resulting in soft tissue sclerosis and loss. “Arterial calcification” refers to ectopic calcification that occurs in arteries and heart valves resulting in arteriosclerosis and / or narrowing. Arterial calcification is associated with an increased burden of atherosclerotic plaque and risk of myocardial infarction, increased ischemic episodes of peripheral vascular disease, and an increased risk of dissection following angioplasty.

[0127] "Venous calcification" refers to ectopic calcification that occurs in the veins, which reduces the elasticity of the veins and restricts blood flow, which can then lead to increased blood pressure and coronary artery defects.

[0128] "Vascular calcification" refers to the pathological deposition of minerals in the vasculature. It has many forms, including intimal calcification and medial calcification, but can also be found in heart valves. Vascular calcification is associated with atherosclerosis, diabetes, certain genetic conditions, and kidney disease (particularly CKD). Patients with vascular calcification are at higher risk for adverse cardiovascular events. Vascular calcification affects a wide variety of patients. Idiopathic infantile arterial calcification is a rare form of vascular calcification in which the arteries of newborns are calcified.

[0129] "Brain calcification" (BC) refers to a nonspecific neuropathology in which deposition of calcium and other minerals occurs in the blood vessel walls and tissue parenchyma, leading to neuronal death and gliosis. Brain calcification is often associated with a variety of chronic and acute brain disorders, including Down syndrome, Lewy body disease, Alzheimer's disease, Parkinson's disease, vascular dementia, brain tumors and / or a variety of endocrine disorders.

[0130] Cardiac calcification refers to the accumulation of calcium deposits (and possibly other minerals) in cardiac tissues, such as the aorta and coronary arteries.

[0131] As used herein, "mineral bone disorder (MBD)" refers to a disorder characterized by abnormal hormone levels leading to an imbalance in calcium and phosphorus levels in a person's blood. Mineral and bone disorders commonly occur in people with CKD and affect the majority of people with kidney failure who are receiving dialysis.

[0132] As used herein, the term "early-onset osteoporosis" refers to the beginning stages of osteoporosis, which are usually characterized by back pain, stooped posture, and / or slow bone loss. Common causes include a low calcium diet, smoking, and age-related hormonal changes.

[0133] "Osteopenia" is a skeletal disorder characterized by a decrease in bone density, which leads to weakened bones and an increased risk of fractures. Osteomalacia is a skeletal disorder characterized by a decrease in the mineralization of newly formed bones. Osteomalacia is caused by severe vitamin D deficiency (which can be nutritional or caused by a genetic syndrome) and conditions that lead to very low blood phosphate levels. Both osteomalacia and osteopenia increase the risk of broken bones. Symptoms of osteomalacia include bone pain and muscle weakness, bone tenderness, difficulty walking, and muscle cramps.

[0134] As used herein, "age-related osteopenia" refers to a condition in which bone mineral density is lower than normal. Generally, patients with osteopenia have a bone mineral density T score between -1.0 and -2.5. If osteopenia is not treated, it can progress to osteoporosis, in which bones become very brittle and easily fractured.

[0135] As used herein, "ossification of the posterior longitudinal ligament (OPLL)" refers to a bone hypertrophy (excessive bone growth) condition that causes ectopic calcification of the posterior longitudinal ligament. The posterior longitudinal ligament connects and stabilizes the bones of the spine. The thickened or calcified ligament may compress the spinal cord, producing myelopathy. Symptoms of myelopathy include difficulty walking and difficulty with bowel and bladder control. OPLL may also lead to radiculopathy, or compression of nerve roots. Symptoms of cervical radiculopathy include pain, tingling, or numbness in the neck, shoulders, arms, or hands. OPLL is different from DISH because with OPL, ossification occurs only in the posterior longitudinal ligament, unlike DISH where ossification also occurs in the thoracic area and anterior longitudinal ligament.

[0136] Clinical signs and symptoms caused by OPLL can be categorized as: (1) myelopathy, or spinal cord injury, with motor and sensory deficits in the upper and lower extremities, spasticity, and bladder dysfunction; (2) cervical radiculopathy, with pain and sensory deficits in the upper extremities; and (3) axial discomfort, with pain and stiffness around the neck. The most common early symptoms of OPLL include dysesthesia and tingling in the hands, as well as clumsiness. As neurological deficits progress, lower extremity symptoms, such as gait disturbances, may develop. OPLL is detected on lateral plain radiographs, and magnetic resonance imaging (MRI) and computed tomography (CT) have definitively demonstrated the diagnosis and morphological details of OPLL in the neck.

[0137] OPLL is common in Americans with cervical spondylosis and is even more widespread in Asian populations. Myelopathy and decreased mobility progressively worsen with age, and there are currently no effective measures to prevent the progression of paraspinal ossification that leads to myelopathy and ossification. Current treatments focus on relieving symptoms, and while surgery may help in the short term, this approach is often complicated by the rapid progression of enthesopathy and recurrence of symptoms.

[0138] Patients with rapidly progressive OPLL show elevations in circulating FGF23 (Kawaguchi, Y., et al., Serum biomarkers in patients with ossification of the posterior longitudinal ligament (OPLL): Inflammation in OPLL. PLoS One, 2017. 12(5): p. e0174881; Kawaguchi, Y., et al., Increase of the Serum FGF-23 in Ossification of the Posterior Longitudinal Ligament. Global Spine J, 2019. 9(5): p. 492-498), a core finding in the rare disorders X-linked hypophosphatemia (XLH) and autosomal recessive hypophosphatemic rickets (ARHR), both of which also exhibit enthesopathy similar to DISH and OPLL. These findings suggest that FGF23 (or resulting hypophosphatemia) plays a pathogenic role in the pathogenesis of enthesopathy; however, the mechanisms by which elevated FGF23 and / or decreased phosphate might induce enthesopathy or spondylosis are unclear.

[0139] As used herein, "hereditary hypophosphatemic rickets" refers to a disorder (hypophosphatemia) associated with low phosphate levels in the blood. Phosphate is a mineral that is essential for the normal formation of bones and teeth. Most commonly, it is caused by mutations in the PHEX gene. Other genes that may cause this condition include CLCN5, DMP1, ENPP1, FGF23 and SLC34A3 genes. Other signs and symptoms of hereditary hypophosphatemic rickets may include premature fusion of the skull bones (craniosynostosis) and tooth deformities. This disorder may also lead to abnormal bone growth at the attachment of ligaments and tendons to joints (entheses). In adults, hypophosphatemia is characterized by a softening of the bones called osteomalacia. Another rare type of disorder is called hereditary hypophosphatemic rickets with hypercalciuria (HHRH), in which, in addition to hypophosphatemia, this condition is also characterized by the excretion of high levels of calcium in the urine (hypercalciuria).

[0140] As used herein, "X-linked hypophosphatemia (XLH)", the term X-linked hypophosphatemia (XLH), also known as X-linked dominant hypophosphatemic rickets, or X-linked vitamin D-resistant rickets, is an X-linked dominant form of rickets (or osteomalacia) that is different from most cases of rickets in that vitamin D supplementation does not cure it. It can cause skeletal deformities, including short stature and genu varum (bowed legs). It is associated with mutations in the PHEX gene sequence (Xp.22) and subsequent inactivation of the PHEX protein.

[0141] As used herein, "autosomal recessive hypophosphatemic rickets type 2 (ARHR2)" refers to an inherited renal phosphate wasting disorder characterized by hypophosphatemia, rickets and / or osteomalacia and slow growth. Autosomal recessive hypophosphatemic rickets type 2 (ARHR2) is caused by a homozygous loss-of-function mutation in the ENPP1 gene.

[0142] As used herein, "autosomal dominant hypophosphatemic rickets (ADHR)" refers to a rare genetic disease in which excessive loss of phosphate in the urine leads to poor bone formation (rickets), bone pain and dental abscesses. ADHR is caused by mutations in fibroblast growth factor 23 (FGF23). ADHR is characterized by impaired bone mineralization, rickets and / or osteomalacia, suppressed levels of calcitriol (1,25-dihydroxyvitamin D3), renal phosphate depletion and low serum phosphate. Mutations in FGF23 make the protein more stable and non-cleaved by proteases, thereby enhancing the biological activity of FGF23. The enhanced activity of FGF23 mutants reduces the expression of sodium-phosphate co-transporters NPT2a and NPT2c on the apical surface of proximal tubular cells, leading to renal phosphate depletion.

[0143] "Hypophosphatemic rickets" (formerly known as vitamin D-resistant rickets) is a disorder in which bones become painfully soft and easily bend due to low phosphate levels in the blood. Symptoms may include bowing of the legs and other skeletal deformities; bone pain; joint pain; poor bone growth; and short stature. In some affected infants, the spaces between the skull bones close prematurely, resulting in craniosynostosis. Most patients show abnormalities in calcium and phosphate metabolism, abnormal tooth enamel, delayed tooth eruption, and a long, narrow head (dolichocephaly).

[0144] As used herein, the term "ENPP1 haploinsufficiency" refers to a genetic disorder in which one copy of the ENPP1 gene is inactivated or deleted, and the remaining functional copy of the ENPP1 gene is insufficient to produce the required gene product to maintain normal function. Since ENPP1 haploinsufficiency can, but does not necessarily have to, manifest in the form of low PPi levels and pathological calcification. Diseases like DISH or early-onset osteoporosis can be associated with, and / or caused by, ENPP1 haploinsufficiency in certain embodiments.

[0145] For most genes, a single copy is sufficient to support normal growth and development in diploid organisms, but a small subset of genes, termed haploinsufficient (HI) genes, exhibit extreme sensitivity to reductions in gene dosage. Given the relatively high frequency of gene-inactivating mutations during an organism's life cycle and the variability in gene expression between cells, haploinsufficiency represents a significant barrier to organismal fitness. Haploinsufficiency in genetics describes a model of dominant gene action in diploid organisms in which the heterozygous combination of a single copy of the wild-type allele at a locus with a variant allele is insufficient to produce a wild-type phenotype. Haploinsufficiency can result from de novo or inherited loss-of-function mutations in the variant allele, rendering it to produce little or no gene product.

[0146] For example, it has been found that the N179S mutation of ENPP1 results in a loss of function of the ENPP1 protein. The presence of a single copy of a mutant ENPP1 gene with the N179S mutation results in decreased PPi production. The present invention discloses that the N179S mutation is found in certain patients with DISH or early-onset osteoporosis and can be used as a genetic marker for the presence or future development and / or progression of DISH or osteoporosis in certain embodiments.

[0147] Likewise, it has been found that the Y451C mutation of ENPP1 results in a loss of function of the ENPP1 protein. The presence of a single copy of a mutant ENPP1 gene with the Y451C mutation results in decreased PPi production. The present invention discloses that the Y451C mutation is found in certain patients with DISH or early-onset osteoporosis and can be used as a genetic marker for the presence or future development and / or progression of DISH or osteoporosis in certain embodiments.

[0148] As used herein, "pretreatment" refers to treatment prior to initiating the treatment methods described herein.

[0149] As used herein, the term "subject" refers to an individual, such as a mammal, such as a human, a non-human primate (such as chimpanzees and other apes and monkeys), a farm animal (such as birds, fish, cattle, sheep, pigs, goats, and horses), a domestic mammal (such as dogs and cats), or a laboratory animal (such as a rodent, such as mice, rats, and guinea pigs). The term includes subjects of any age or sex. In another embodiment, the subject is a mammal, preferably a human.

[0150] A disease or disorder is "relieved" if there is a decrease in the severity of the symptoms of the disease or disorder, the frequency with which the patient experiences such symptoms, or both.

[0151] As used herein, the terms "alteration", "defect", "variation" or "mutation" refer to a mutation in a gene in a cell that affects the function, activity, expression (transcription or translation) or configuration of the polypeptide encoded thereby, including missense and nonsense mutations, insertions, deletions, frameshifts and premature terminations.

[0152] "Disease" is a health condition in an animal in which the animal is unable to maintain homeostasis and in which the animal's health will continue to deteriorate if the disease is not ameliorated.

[0153] A "disorder" in an animal is a health state in which the animal is able to maintain homeostasis, but in which the animal's health state is unfavorable compared to a state in the absence of the disorder. If untreated, the disorder will not necessarily result in a further decline in the animal's health state.

[0154] As used herein, the term "ENPP" or "NPP" refers to ectonucleotide pyrophosphatase / phosphodiesterase.

[0155] As used herein, the term "ENPP1 protein" or "ENPP1 polypeptide" refers to an ectonucleotide pyrophosphatase / phosphodiesterase-1 protein encoded by the ENPP1 gene. The encoded protein is a type II transmembrane glycoprotein and cleaves a variety of substrates, including phosphodiester bonds of nucleotides and nucleotide sugars and pyrophosphate bonds of nucleotides and nucleotide sugars. The ENPP1 protein has a transmembrane domain and a soluble extracellular domain. The extracellular domain is further subdivided into a somatomedin B domain, a catalytic domain (residues 186 to 586 of SEQ ID NO: 1) and a nuclease domain (residues 524 to 885 of SEQ ID NO: 1). The sequence and structure of wild-type ENPP1 are described in detail in PCT Application Publication No. WO 2014 / 126965 by Braddock et al., which is incorporated herein by reference in its entirety.

[0156] Mammalian ENPP1 polypeptides, mutants, or mutant fragments thereof have been previously disclosed in the following International PCT Application Publication Nos.: WO / 2014 / 126965 to Braddock et al., WO / 2016 / 187408 to Braddock et al., WO / 2017 / 087936 to Braddock et al., and WO2018 / 027024 to Braddock et al., all of which are incorporated herein by reference in their entirety.

[0157] As used herein, the term "ENPP1 precursor protein" refers to ENPP1 having its signal peptide sequence at the N-terminus of ENPP1. After proteolysis, the signal sequence (in certain non-limiting embodiments, represented by W in the compound of formula (I)) is cleaved from ENPP1 to provide ENPP1 protein. Signal peptide sequences useful in the present invention include, but are not limited to, albumin signal sequence, azurocidin signal sequence, ENPP1 signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence and / or ENPP5 signal peptide sequence.

[0158] As used herein, the term "ENPP1-Fc construct" refers to ENPP1 recombinantly fused and / or chemically conjugated (including covalent and non-covalent conjugation) to the FcR binding domain of an IgG molecule (preferably, human IgG). In certain embodiments, the C-terminus of ENPP1 is fused or conjugated to the N-terminus of the FcR binding domain.

[0159] As used herein, the term "Fc" refers to a human IgG (immunoglobulin) Fc domain. IgG subtypes such as IgG1, IgG2, IgG3, and IgG4 are considered for use as the Fc domain.

[0160] As used herein, "Fc region or Fc polypeptide" is a part of an IgG molecule that is associated with a crystallizable fragment obtained by papain digestion of an IgG molecule. The Fc region comprises the C-terminal half of the two heavy chains of the IgG molecule connected by a disulfide bond. It has no antigen binding activity, but comprises a carbohydrate moiety and binding sites for complement and Fc receptors (including FcRn receptors). The Fc fragment comprises the entire second constant domain CH2 (residues 231-340 of human IgG1 according to the Kabat numbering system) and the third constant domain CH3 (residues 341-447). The term "IgG hinge-Fc region" or "hinge-Fc fragment" refers to the region of the IgG molecule consisting of the Fc region (residues 231-447) and the hinge region (residues 216-230) extending from the N-terminus of the Fc region. The term "constant domain" refers to a part of an immunoglobulin molecule that has a more conservative amino acid sequence relative to another part of the immunoglobulin, i.e., the variable domain containing the antigen binding site. The constant domains comprise the CH1, CH2 and CH3 domains of the heavy chain and the CHL domain of the light chain.

[0161] As used herein, the term "fragment" when applied to nucleic acids refers to a subsequence of a larger nucleic acid. A "fragment" of a nucleic acid can be at least about 15, 50-100, 100-500, 500-1000, 1000-1500 nucleotides, 1500-2500 or 2500 nucleotides (and any integer value therebetween). As used herein, the term "fragment" when applied to a protein or peptide refers to a subsequence of a larger protein or peptide, and the length can be at least about 20, 50, 100, 200, 300 or 400 amino acids (and any integer value therebetween).

[0162] "Isolated" means changed or removed from its natural state. For example, a nucleic acid or polypeptide naturally present in a living animal is not "isolated", but the same nucleic acid or polypeptide partially or completely separated from the coexisting materials of its natural state is "isolated". An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-natural environment, such as a host cell.

[0163] As used herein, the term "patient," "individual," or "subject" refers to a human.

[0164] As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound useful in the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient. There are a variety of techniques for administering compounds in the art, including but not limited to subcutaneous, intravenous, oral, aerosol, inhalation, rectal, vaginal, transdermal, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ophthalmic, pulmonary, and topical administration.

[0165] As used herein, the term "pharmaceutically acceptable" refers to a relatively nontoxic material, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compound, i.e., the material can be administered to an individual without causing adverse biological effects or interacting in a deleterious manner with any components contained in the composition; for example, phosphate buffered saline (PBS)

[0166] As used herein, the term "plasma pyrophosphate (PPi) level" refers to the amount of pyrophosphate present in the plasma of an animal. In certain embodiments, the animals include rats, mice, cats, dogs, humans, cows, and horses. Due to release from platelets, it is necessary to measure PPi in plasma rather than serum. There are a variety of ways to measure PPi, one of which is an enzymatic assay using uridine diphosphoglucose (UDPG) pyrophosphorylase (Lust and Seegmiller, 1976, Clin. Chim. Acta 66: 241-249; Cheung and Suhadolnik, 1977, Anal. Biochem 83: 61-63), with modifications. Normal PPi levels in healthy subjects typically range from about 1 μM to about 3 μM, and in some cases between 1-2 μM. In some embodiments, a "low" level of PPi refers to a condition in which a subject's plasma pyrophosphate (PPi) is less than or equal to 2%-5% of normal levels (Arthritis and Rheumatism, Vol. 22, No. 8 (August 1979)).

[0167] Subjects with ENPP1 expression defects tend to show lower PPi levels, ranging from at least 10% lower than normal, at least 20% lower than normal, at least 30% lower than normal, at least 40% lower than normal, at least 50% lower than normal, at least 60% lower than normal, at least 70% lower than normal, at least 80% lower than normal, and combinations thereof. In patients with GACI, PPi levels were found to be below 1 μM, in some cases below detectable levels. In patients with PXE, PPi levels were below 0.5 μM (Arterioscler Thromb Vasc Biol. 2014 Sep; 34 (9): 1985-9; Braddock et al., Nat Commun. 2015; 6: 10006).

[0168] As used herein, the term "polypeptide" refers to a polymer composed of amino acid residues linked by peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof.

[0169] As used herein, the term "PPi" refers to pyrophosphate.

[0170] As used herein, the term "prevent" or "preventing" means that if a disorder or disease does not occur, then the disorder or disease does not develop, or if a disorder or disease has already developed, then there is no further development of the disorder or disease. Also contemplated is the ability of a person to prevent some or all of the symptoms associated with a disorder or disease.

[0171] As used herein, "sample" or "biological sample" refers to biological material isolated from a subject. A biological sample may include any biological material suitable for detecting mRNA, polypeptides, or other markers of a physiological or pathological process in a subject, and may include fluid, tissue, cells, and / or non-cellular material obtained from an individual.

[0172] As used herein, "substantially purified" means substantially free of other components. For example, a substantially purified polypeptide is one that has been separated from other components with which it is normally associated in its naturally occurring state. Non-limiting embodiments include 95% purity, 99% purity, 99.5% purity, 99.9% purity, and 100% purity.

[0173] As used herein, the term "treatment" or "treatment" is defined as the application or administration of a therapeutic agent, i.e., a compound useful in the present invention (alone or in combination with another agent), to a patient, or the application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnostic or ex vivo applications), who has a disease or disorder and / or symptoms of a disease or disorder, with the intent to treat, cure, alleviate, mitigate, alter, remedy, modify, ameliorate, affect and / or prevent and / or minimize the progression of a disease or disorder and / or symptoms of a disease or disorder. Such treatments can be specifically tailored or modified based on knowledge gained from the field of pharmacogenomics.

[0174] As used herein, the terms "prevent," "preventative," and "preventing" refer to inhibiting the onset of a disease in a subject or reducing its occurrence. Prevention can be complete (such as the complete absence of pathological cells in a subject) or partial. Prevention also refers to a reduction in susceptibility to a clinical condition. In certain embodiments, "preventing" includes preventing the onset of a disease or disorder.

[0175] As used herein, the term "wild type" refers to a gene or gene product isolated from a naturally occurring source. The wild type gene is the most common in the human population and is therefore arbitrarily designed to be the "normal" or "wild type" form of the human ENPP1 gene. In contrast, the term "functionally equivalent" refers to an ENNP1 gene or gene product that exhibits an altered sequence and / or functional property (i.e., altered properties) compared to the wild type gene or gene product. Naturally occurring mutants can be isolated; they are identified by the fact that they have altered properties (including altered nucleic acid sequences) compared to the wild type gene or gene product.

[0176] As used herein, the term "functionally equivalent variant" relates to a polypeptide that is substantially homologous to the sequence of ENPP1 (defined above) and retains the enzymatic and biological activity of ENPP1. Methods for determining whether a variant retains the biological activity of native ENPP1 are widely known to the skilled person and include any experimental methods used in the experimental part of this application. In particular, functionally equivalent variants of ENPP1 delivered by viral vectors are encompassed by the present invention.

[0177] Functionally equivalent variants of ENPP1 are polypeptides that are substantially homologous to native ENPP1. "Substantially homologous" relates to protein sequences when the degree of identity of the protein sequence to the above ENPP1 sequence is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, respectively.

[0178] The degree of identity between two polypeptides is determined using computer algorithms and methods widely known to those skilled in the art. The identity between two amino acid sequences is preferably determined using the BLASTP algorithm (BLAST Manual, Altschul et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), but other similar algorithms may also be used. BLAST and BLAST 2.0 are used to determine the sequence identity percentage using the parameters described herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

[0179] "Functionally equivalent variants" of ENPP1 can be obtained by replacing nucleotides within the polynucleotide, which take into account the codon preference in the host cell used to produce ENPP1. Such "codon optimization" can be determined via a computer algorithm that incorporates a codon frequency table, such as the "Humanhigh.cod" of codon preferences provided by the University of Wisconsin Package Version 9.0, Genetics Computer Group, Madison, Wis.

[0180] As used herein, "about" when referring to a measurable value, such as an amount, a time period, etc., is meant to include variations of ±10% or 5%, in certain embodiments ±1-5%, in certain embodiments ±5%, in certain embodiments ±4%, in certain embodiments ±4%, in certain embodiments ±2%, and in certain embodiments ±1% from the specified value (2 mg / kg or 0.6 mg / kg or 1.8 mg / kg), as such variations are appropriate for performing the disclosed methods.

[0181] The disclosure provides examples of representative protein sequences. The protein sequences can be converted to nucleic acid sequences by reverse transcription and codon optimization. There are several tools that can perform this conversion, such as Expasy (https: / / www.expasy.org / ) and bioinformatics servers (www dot bioinformatics dotorg).

[0182] Range: Throughout this disclosure, aspects according to the present invention may be presented in a range format. It should be understood that the description in a range format is only for convenience and brevity, and should not be interpreted as an inflexible limitation on the scope according to the present invention. Therefore, the description of a range should be considered to have explicitly disclosed all possible subranges within the range as well as single values. For example, a range description such as from 1 to 6 should be considered to have explicitly disclosed subranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0183] Preferred methods and materials are described herein, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the disclosed methods and compositions. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. DETAILED DESCRIPTION

[0184] 1. ENPP1 dose

[0185] An ENPP1 agent is an ENPP1 polypeptide. The ENPP1 polypeptides disclosed herein include, but are not limited to, naturally occurring polypeptides of the ENPP1 family and any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain biological activity (such as, but not limited to, the catalytic activity of ENPP1).

[0186] The ENPP1 agent can be expressed in the form of the formula shown below.

[0187] W-protein-Z-domain-XY

[0188] (I)

[0189] Wherein in (I):

[0190] W is absent or includes a signal sequence that allows export of the compound into the extracellular space;

[0191] The protein includes the catalytic domain of ENPP1;

[0192] The domain is absent or is at least one selected from the group consisting of human IgG Fc domain (Fc), human serum albumin (ALB), and biologically active fragments thereof;

[0193] X and Z are independently absent or are a polypeptide comprising 1-20 amino acids; and

[0194] Y is absent or is a sequence selected from the following "bone targeting" sequences: D m (SEQ ID NO:11), (DSS)n (SEQ ID NO: 12), (ESS) n (SEQ ID NO: 13), (RQQ) n (SEQ ID NO: 14), (KR) n (SEQ ID NO: 15), R m E m (SEQ ID NO: 117),

[0195] wherein m is independently an integer from 1 to 15, and

[0196] wherein n is independently an integer from 1 to 10.

[0197] In some embodiments, W is absent. In some embodiments, W comprises a signal sequence that allows export of the compound to the extracellular space.

[0198] In some embodiments, the ENPP1 agent comprises a bone targeting domain. In some embodiments, the bone targeting domain is a negatively charged amino acid sequence. In some embodiments, the negatively charged bone targeting domain is polyaspartic acid.

[0199] The term "ENPP1" or "ENPP1 polypeptide" refers to an ectonucleotide pyrophosphatase / phosphodiesterase 1 protein (NPP1 / ENPP1 / PC-1) and ENPP1-related proteins from any species. ENPP1 proteins include type II transmembrane glycoproteins that form homodimers. Each monomer of ENPP1 protein includes a short intracellular N-terminal domain, a transmembrane domain, and a large extracellular region comprising several domains that participate in targeting the plasma membrane. The large extracellular region includes SMB1 and SMB2 domains, which are reported to participate in ENPP1 dimerization (Gijsbers et al., Biochem. J. 371; 2003: 321-330). Specifically, the SMB domain contains eight cysteine ​​residues, each arranged into four disulfide bonds, and has been shown to mediate homodimerization of ENPP1 by covalent cysteine ​​intermolecular and intramolecular bonds. Protein cleavage of a variety of substrates, including phosphodiester bonds of nucleotides and nucleotide sugars and pyrophosphate bonds of nucleotides and nucleotide sugars. The function of the ENPP1 protein is to hydrolyze nucleotide 5' triphosphatases to the corresponding monophosphates and also hydrolyze diadenosine polyphosphates. The ENPP1 protein plays a role in purinergic signaling, which is involved in the regulation of cardiovascular, neurological, immune, musculoskeletal, hormonal and hematological functions. Fig.11 An exemplary amino acid sequence (SEQ ID NO: 1) of a human ENPP1 precursor protein (NCBI accession number NP_006199) is shown. The human ENPP1 precursor protein includes an endogenous ENPP1 signal peptide sequence at the N-terminus of ENPP1. The numbering of the amino acids of all ENPP1-related polypeptides described herein is based on the numbering of the human ENPP1 precursor protein sequence provided in Figure 2, unless otherwise specifically specified. In certain embodiments, the ENPP1 precursor protein further includes an endogenous or heterologous signal peptide sequence. During protein hydrolysis, the signal peptide sequence is cleaved from the ENPP1 precursor protein to provide a mature ENPP1 protein. See, for example, Jansen et al., J Cell Sci. 2005; 118 (Pt 14): 3081-9. Exemplary signal peptide sequences that can be used with the polypeptides disclosed herein include, but are not limited to, an ENPP1 signal peptide sequence, an ENPP2 signal peptide sequence, an ENPP7 signal peptide sequence, and / or an ENPP5 signal peptide sequence. A non-limiting processed (mature) extracellular ENPP1 polypeptide sequence is shown in SEQ ID NO:2.

[0200] It is generally known in the art that ENPP1 is very conserved in vertebrates, and most of the sequences of the extracellular domain are basically conserved. Figure 7A-7BA multiple sequence alignment of the extracellular structure of human ENPP1 compared to a variety of ENPP1 orthologs is depicted. ENPP1 binding to a variety of nucleotide triphosphates (such as ATP, UTP, GTP, TTP and CTP), pNP-TMP, 3',5'-cAMP and 2'-3'-cGAMP is also highly conserved (e.g., see Kato et al., Proc Natl Acad Sci USA. 2012; 109(42):16876-81 and Mackenzie et al., Bone. 2012; 51(5):961-8). Therefore, from these alignments, key amino acid positions in the extracellular domain that are important for normal ENPP1 activity can be predicted, as can amino acid positions that may tolerate substitution without significantly changing normal ENPP1 activity. Therefore, a human ENPP1 polypeptide having enzymatic activity useful according to the presently disclosed compositions may include one or more amino acids at corresponding positions in another vertebrate ENPP1 sequence, or may include residues similar to those in human or other vertebrate sequences. Substitution of one or more amino acids at corresponding positions may include conservative changes or substitutions that are unlikely to change the shape of the polypeptide chain or alter normal ENPP1 activity. Examples of conservative changes or substitutions include replacing one hydrophobic residue such as isoleucine, valine, leucine or methionine with another, or replacing one polar residue with another, such as replacing lysine with arginine, replacing aspartic acid with glutamic acid, or replacing asparagine with glutamine. For example, ENPP1 polypeptides include polypeptides derived from the sequence of any known ENPP1 polypeptide, whose sequence is at least about 80% identical to the sequence of the ENPP1 polypeptide, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identical.

[0201] 2. Enzyme activity of ENPP1

[0202] ENPP1 proteins have been characterized in the art in terms of structure and biological properties. In certain embodiments, the soluble ENPP1 proteins disclosed herein include pyrophosphatase and / or phosphodiesterase activity. For example, in some embodiments, the ENPP1 protein binds nucleotide triphosphates (such as ATP, UTP, GTP, TTP and CTP), pNP-TMP, 3', 5'-cAMP and 2'-3'-cGAMP; and converts nucleotide triphosphates into inorganic pyrophosphates [see, e.g., Kato et al., Proc Natl Acad Sci USA. 2012; 109(42): 16876-81; Li et al., Nat Chem Biol. 2014; 10(12): 1043-8; Jansen et al., Structure. 2012; 20(11): 1948-59; and Onyedibe et al., Molecules. 2019; 24(22)].

[0203] "Enzymatically active" or "biologically active" ENPP1 polypeptides exhibit pyrophosphatase and / or phosphodiesterase activity (e.g., capable of binding ATP and / or hydrolyzing it to AMP and PPi, and / or binding AP3a and / or hydrolyzing it to ATP). For example, the pyrophosphatase / phosphodiesterase domain of the ENPP1 protein hydrolyzes extracellular nucleotide triphosphates to produce inorganic pyrophosphate (PPi) and is generally soluble. This activity can be measured using the pNP-TMP assay described previously (Saunders et al., 2008, Mol. Cancer Ther. 7(10):3352-62; Albright et al., 2015, Nat Comm. 6:10006). In certain embodiments, the k of a soluble ENPP1 polypeptide for the substrate ATP is cat Value greater than or equal to about 3.4 (± 0.4) s -1 Enzymes -1 , where k cat is determined by measuring the rate at which the polypeptide hydrolyzes ATP. In certain embodiments, the K of a soluble ENPP1 polypeptide for the substrate ATP is M value is less than or equal to about 2 μM, where K M It is determined by measuring the rate at which the polypeptide hydrolyzes ATP. In addition to the teachings herein, these references also provide sufficient guidance on how to generate soluble ENPP1 proteins that retain one or more biological activities (such as converting nucleotides to inorganic pyrophosphate).

[0204] 3. Soluble ENPP1

[0205] In one embodiment, the present disclosure relates to an ENPP1 agent, such as, but not limited to, an ENPP1 polypeptide. As described herein, the term soluble ENPP1 polypeptide includes any naturally occurring extracellular domain of an ENPP1 protein and any variant thereof (including mutants, fragments, and peptide mimetic forms) that retains biological activity (e.g., enzymatic activity). Examples of soluble ENPP1 polypeptides include, for example, an ENPP1 extracellular domain (SEQ ID NO: 2). In certain embodiments, the soluble ENPP1 polypeptide further includes a signal sequence in addition to the extracellular domain of the ENPP1 polypeptide. Exemplary signal sequences include the native signal sequence of the ENPP1 polypeptide, or a signal sequence from another protein, such as a hENPP7 signal sequence. Examples of variant soluble ENPP1 polypeptides are provided in International Patent Application Publication Nos. WO 2012 / 125182, WO 2014 / 126965, WO 2016 / 187408, WO 2018 / 027024, WO2020206302, and WO 2020 / 047520, all of which are incorporated herein by reference in their entireties.

[0206] 4. ENPP1 fusion protein

[0207] In some embodiments, the ENPP1 polypeptide is a fusion protein comprising an ENPP1 polypeptide domain and one or more heterologous protein portions (i.e., polypeptide domains that are heterologous to ENPP1). If the amino acid sequence is not found exclusively in the form of ENPP1 represented by SEQ ID NO: 1, it is understood to be heterologous to ENPP1. In some embodiments, the heterologous protein portion comprises an Fc domain of an immunoglobulin. In some embodiments, the Fc domain of an immunoglobulin is an Fc domain of an IgG1 immunoglobulin. In certain embodiments, the soluble ENPP1 polypeptide is fused at the C-terminus to the Fc domain of human immunoglobulin 1 (IgG1), human immunoglobulin 2 (IgG2), human immunoglobulin 3 (IgG3), and / or human immunoglobulin 4 (IgG4). In other embodiments, the soluble ENPP1 polypeptide is fused at the N-terminus to the Fc domain of human immunoglobulin 1 (IgG1), human immunoglobulin 2 (IgG2), human immunoglobulin 3 (IgG3), and / or human immunoglobulin 4 (IgG4). In some embodiments, the presence of the Fc domain improves half-life, solubility, reduces immunogenicity, and increases the activity of soluble ENPP1 polypeptides. In certain embodiments, portions of native human IgG proteins (IgG1, IgG2, IgG3, and IgG4) can be used for the Fc portion (e.g., ENPP1-Fc). For example, the fusion protein provided by the present disclosure includes ENPP1 fused to a polypeptide comprising an immunoglobulin constant domain, such as a CH1, CH2, or CH3 domain derived from human IgG1, IgG2, IgG3, and / or IgG4. The Fc fragment may include regions of native IgG, such as the hinge region (residues 216-230 of human IgG1 according to the Rabat numbering system), the entire second constant domain CH2 (residues 231-340), and the third constant domain CH3 (residues 341-447). As used herein, the term "ENPP1-Fc construct" refers to a soluble form of ENPP1 (e.g., an extracellular domain of an ENPP1 polypeptide) that is recombinantly fused and / or chemically conjugated (including covalent and non-covalent conjugations) to the FcR binding domain of an IgG molecule (preferably human IgG). In certain embodiments, the C-terminus of ENPP1 is fused or conjugated to the N-terminus of the FcR binding domain.

[0208] An example of an amino acid sequence that can be used for the Fc portion of human IgG1 (G1 Fc) is SEQ ID NO: 6 (Table 1). In part, the present disclosure provides a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence that is 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 6.

[0209] In some embodiments, the heterologous protein portion includes one or more domains selected from the group consisting of polyhistidine, FLAG tag, Glu-Glu, glutathione S-transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin. The fusion domain can be selected to confer the desired properties. For example, some fusion domains are particularly useful for separating fusion proteins by affinity chromatography. For the purpose of affinity purification, a relevant affinity chromatography matrix is ​​used, such as a glutathione, amylase, nickel, or cobalt conjugated resin. Many such matrices are provided in "kit" form, such as the Pharmacia GST purification system and QIAexpress TM System (Qiagen), available (HIS 6 ) fusion partner. As another example, the fusion domain can be selected to facilitate the detection of the ENPP1 polypeptide. Examples of such detection domains include a variety of fluorescent proteins (such as GFP) and "epitope tags," which are generally short peptide sequences for which specific antibodies are available. Well-known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus hemagglutinin (HA), and c-myc tags. In some embodiments, the fusion domain has a protease cleavage site, such as factor Xa or thrombin, which allows the associated protease to partially digest the fusion protein, thereby releasing the recombinant protein therefrom. The released protein can then be separated from the fusion domain by subsequent chromatographic separation.

[0210] 5. Connector

[0211] In some embodiments, the ENPP1 fusion protein further comprises a linker (Z) positioned between the ENPP1 polypeptide domain and one or more heterologous protein portions (e.g., an Fc immunoglobulin domain). In certain embodiments, the soluble ENPP1 polypeptide is fused directly or indirectly to the Fc domain. In some embodiments, the soluble ENPP1 fusion protein comprises a linker between the Fc domain and the ENPP1 polypeptide. In some embodiments, the linker can be an amino acid spacer comprising 1-200 amino acids. Suitable peptide spacers are known in the art, for example, including peptide linkers containing flexible amino acid residues such as glycine, alanine, and serine.

[0212] In some embodiments, the linker comprises a polyglycine linker or a Gly-Ser linker. In some embodiments, the spacer can comprise a motif, such as a plurality or repeating motif: GA (SEQ ID NO: 21), GS (SEQ ID NO: 22), GG (SEQ ID NO: 23), GGA (SEQ ID NO: 24), GGS (SEQ ID NO: 25), GGG (SEQ ID NO: 26), GGGA (SEQ ID NO: 27), GGGS (SEQ ID NO: 28), GGGG (SEQ ID NO: 29), GGGGA (SEQ ID NO: 30), GGGGS (SEQ ID NO: 31), GGGGG (SEQ ID NO: 32), GGAG (SEQ ID NO: 33), GGSG (SEQ ID NO: 34), AGGG (SEQ ID NO: 35), SGGGG (SEQ ID NO: 36), or SGGG (SEQ ID NO: 37). In some embodiments, the spacer can contain 2 to 12 amino acids, including a motif of GA or GS, such as GA, GS, GAGA (SEQ ID NO:38), GSGS (SEQ ID NO:39), GAGAGA (SEQ ID NO:40), GSGSGS (SEQ ID NO:41), GAGAGAGA (SEQ ID NO:42), GSGSGSGS (SEQ ID NO:43), GAGAGAGAGA (SEQ ID NO:44), GSGSGSGSGS (SEQ ID NO:45), GAGAGAGAGAGA (SEQ ID NO:46), and GSGSGSGSGSGS (SEQ ID NO:47). In some embodiments, the spacer can comprise 3 to 12 amino acids, including a motif of GGA or GGS, such as GGA, GGS, GGAGGA (SEQ ID NO:48), GGSGGS (SEQ ID NO:49), GGAGGAGGA (SEQ ID NO:50), GGSGGSGGS (SEQ ID NO:51), GGAGGAGGAGGA (SEQ ID NO:52), and GGSGGSGGSGGS (SEQ ID NO:53).In some other embodiments, the spacer can include 4 to 12 amino acids, including the motifs of GGAG (SEQ ID NO: 54), GGSG (SEQ ID NO: 55), such as GGAG (SEQ ID NO: 56), GGSG (SEQ ID NO: 57), GGAGGGAG (SEQ ID NO: 58), GGSGGGSG (SEQ ID NO: 59), GGAGGGAGGGAG (SEQ ID NO: 60), and GGSGGGSGGGSG (SEQ ID NO: 61). In some embodiments, the spacer can include the motifs of GGGGA (SEQ ID NO: 62) or GGGGS (SEQ ID NO: 63), such as GGGGAGGGGAGGGGA (SEQ ID NO: 64), and GGGGSGGGGSGGGGS (SEQ ID NO: 65). In some embodiments of the invention, the amino acid spacer between the heterologous protein portion (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin binding peptide, a fibronectin domain, or human serum albumin) and the soluble ENPP1 polypeptide can be GGG, GGGA (SEQ ID NO: 27), GGGG (SEQ ID NO: 29), GGGAG (SEQ ID NO: 66), GGGAGG (SEQ ID NO: 67), or GGGAGGG (SEQ ID NO: 68).

[0213] In some embodiments, the spacer can also include amino acids other than glycine, alanine, and serine, for example, LIN (SEQ ID NO:69), TGGGG (SEQ ID NO:70), AAAL (SEQ ID NO:71), AAAK (SEQ ID NO:72), AAAR (SEQ ID NO:73), EGKSSGSGSESKST (SEQ ID NO:74), GSAGSAAGSGEF (SEQ ID NO:75), AEAAAKEAAAKA (SEQ ID NO:76), KESGSVSSEQLAQFRSLD (SEQ ID NO:77), GENLYFQSGG (SEQ ID NO:78), SACYCELS (SEQ ID NO:79), RSIAT (SEQ ID NO:80), RPACKIPNDLKQKVMNH (SEQ ID NO:81), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO:82), AAANSSIDLISVPVDSR (SEQ ID NO:83), NO:83), GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO:84), NSS (SEQ ID NO:87), ESS (SEQ ID NO:88), RQQ (SEQ ID NO:89), KR (SEQ ID NO:90), (R) m ; m=0-15 (SEQ ID NO:91), DSSSEEKFLRRIGRFG (SEQ ID NO:92), EEEEEEEPRGDT (SEQ ID NO:93), APWHLSSQYSRT (SEQ ID NO:94), STLPIPHEFSRE (SEQ ID NO:95), VTKHLNQISQSY (SEQ ID NO:96), (E) m ; m=1-15 (SEQ ID NO:97), RSGSGGS (SEQ ID NO:98), (D) m; m=1-15 (SEQ ID NO:99), LVIMSLGLGLGLGLRK (SEQ ID NO:100), VIMSLGLGLGLGLRK (SEQ ID NO:101), IMSLGLGLGLGLRK (SEQ ID NO:102), MSLGLGLGLGLRK (SEQ ID NO:103), SLGLGLGLGLRK (SEQ ID NO:104), LGLGLGLRK (SEQ ID NO:105), GLGLGLRK (SEQ ID NO:106), LGLGLGLRK (SEQ ID NO:107), GLGLGLRK (SEQ ID NO:108), LGLGLRK (SEQ ID NO:109), GLGLRK (SEQ ID NO:110), LGLRK (SEQ ID NO:111), GLRK (SEQ ID NO:112), LRK (SEQ ID NO:113), RK (SEQ ID NO:114) or (K) m ; m=1-15 (SEQ ID NO: 115). In some embodiments, the spacer can comprise a motif of EAAAK (SEQ ID NO: 85), such as multiple or repeated motifs. In some embodiments, the spacer can comprise a motif of a praline-rich sequence, such as multiple or repeated motifs, such as (XP)n, where X can be any amino acid (e.g., A, K, or E), n is 1-5, and PAPAP (SEQ ID NO: 86).

[0214] The length of the peptide spacer and the amino acids used can be adjusted depending on the two proteins involved and the degree of flexibility required in the final protein fusion polypeptide. The length of the spacer can be adjusted to ensure proper protein folding and avoid aggregate formation.

[0215] In some embodiments, the different elements of the fusion protein (such as an immunoglobulin Fc fusion protein) can be arranged in any manner consistent with the desired function. For example, the soluble ENPP1 polypeptide domain can be placed at the C-terminus of the heterologous protein portion, or alternatively, the heterologous protein portion can be placed at the C-terminus of the soluble ENPP1 polypeptide domain. The soluble ENPP1 polypeptide domain and the heterologous protein portion can be directly or indirectly connected in the fusion protein, and additional domains or amino acid sequences can be included at the C-terminus or N-terminus of any domain or between domains. Preferred fusion proteins include the amino acid sequence listed in any one of SEQ ID NOs: 3-5.

[0216] In some embodiments, the soluble ENPP1 polypeptides of the present disclosure contain one or more heterologous moieties. Optionally, the soluble ENPP1 polypeptides include one or more heterologous moieties selected from the group consisting of glycosylated amino acids, PEGylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids conjugated to a lipid moiety, and amino acids conjugated to an organic derivatizing agent. In some embodiments, the soluble ENPP1 polypeptides disclosed herein are further modified. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Thus, the soluble ENPP1 polypeptides may contain non-amino acid elements, such as polyethylene glycol, lipids, polysaccharides or monosaccharides, and phosphates. The effects of such non-amino acid elements on the function of the soluble ENPP1 polypeptides may be tested as described herein for other soluble ENPP1 polypeptides. When the polypeptides of the present disclosure are produced in cells by cleaving the nascent form of the polypeptide, post-translational processing may also be important for the correct folding and / or function of the protein. Different cells (such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) have specific cellular machinery and characteristic mechanisms for this post-translational activity, and different cells can be selected to ensure the correct modification and processing of soluble ENPP1 polypeptides.

[0217] As used herein, the "identity" percentage between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence after the sequences are aligned and a gap is introduced (if necessary) to achieve the maximum sequence identity percentage. The comparison for the purpose of determining the amino acid sequence identity percentage can be achieved in a variety of ways by those skilled in the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW or CLUSTAL OMEGA software. In some embodiments, CLUSTAL OMEGA software is used for comparison. Those skilled in the art can determine the appropriate parameters for the comparison sequence, including any algorithm required for achieving the maximum comparison on the full length of the sequence being compared.

[0218] 6. Determine Solubility

[0219] In some embodiments, the activity of soluble ENPP1 polypeptides can also be tested in cell-based or in vivo assays. For example, the effect of soluble ENPP1 polypeptides on the production of inorganic pyrophosphate (PPi) can be measured. Specifically, the pyrophosphatase / phosphodiesterase domain of ENPP1 protein hydrolyzes extracellular nucleotide triphosphates to produce inorganic pyrophosphate (PPi), and is generally soluble. This activity can be measured using pNP-TMP assays and ATP hydrolysis assays based on HPLC, as previously described (Saunders et al., Mol. Cancer Ther. 7 (10): 3352-62; Albright et al., 2015, Nat Comm. 6: 10006). The effect of soluble ENPP1 polypeptides on gene expression involved in ENPP1-related diseases such as ARHR2 (e.g., transcription of fibroblast growth factor 23 in osteoblasts and osteoclasts) can be assessed. This can be performed in the presence of one or more nucleotide triphosphates or other ENPP1 substrates as needed, and cells can be transfected to produce soluble ENPP1 polypeptides. Likewise, soluble ENPP1 polypeptides can be administered to mice or other animals, and the effects on ENPP1-related diseases can be assessed using art-recognized methods.

[0220] In some embodiments, the ENPP1 polypeptide used according to the methods described herein is an isolated polypeptide. As used herein, an isolated protein or polypeptide refers to a protein or polypeptide that has been separated from a component of its natural environment. In some embodiments, the polypeptide of the present invention is purified to a purity greater than 95%, 96%, 97%, 98% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed phase HPLC) analysis. Methods for assessing purity are well known in the art [see, e.g., Flatman et al., 2007, J. Chromatogr. B 848:79-87]. In some embodiments, the soluble ENPP1 polypeptide used according to the methods described herein is a recombinant polypeptide.

[0221] 7. ENPP1 production

[0222] The ENPP1 polypeptides of the present disclosure can be produced by a variety of techniques known in the art. For example, the polypeptides of the present disclosure can be synthesized using standard protein chemistry techniques, such as those described in Bodansky, Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant GA (ed.), Synthetic Peptides: A User's Guide, WH Freeman and Company, New York (1992). In addition, automatic polypeptide synthesizers are commercially available (e.g., Advanced ChemTech Model 396; Milligen / Biosearch 9600). Alternatively, the polypeptides of the present disclosure, including fragments or variants thereof, can be recombinantly produced using a variety of expression systems [e.g., Escherichia coli, Chinese hamster ovary (CHO) cells, COS cells, baculovirus, Pichia pastoris], which are well known in the art. The protein can be produced in adherent cells or suspension cells. In some embodiments, the fusion protein is expressed in CHO cells. In order to establish a stable cell line, the nucleic acid sequence encoding the ENPP1 construct is cloned into an appropriate vector for large-scale protein production. In certain embodiments, modified or unmodified polypeptides of the present disclosure can be produced by digesting the recombinantly produced full-length ENPP1 polypeptide using, for example, a protease, such as trypsin, thermolysin, chymotrypsin, pepsin, or paired alkaline amino acid converting enzyme (PACE). Computer analysis (using commercially available software, such as MacVector, Omega, PCGene, Molecular Simulation, Inc.) can be used to identify protease hydrolysis cleavage sites. Alternatively, such polypeptides can be produced from recombinantly produced full-length ENPP1 polypeptides using chemical cleavage methods (e.g., cyanogen bromide, hydroxylamine, etc.).

[0223] 8. Expression system

[0224] Many expression systems are known and can be used to produce ENPP1 fusion proteins, including bacteria (e.g., E. coli and Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae, Kluyveromyces lactis, and Pichia pastoris), filamentous fungi (e.g., Aspergillus), plant cells, animal cells, and insect cells. The desired protein can be produced in a conventional manner, for example, from a coding sequence inserted into the host chromosome or on an episomal plasmid.

[0225] Yeast can be transformed with the coding sequence of the desired protein by any common means (e.g., electroporation). Methods for transforming yeast by electroporation are disclosed in Becker and Guarente, 1990, Methods Enzymol. 194: 182. Successfully transformed cells, i.e., cells containing a DNA construct of the present disclosure, can be identified by well-known techniques. For example, cells produced by the introduction of an expression construct can be grown to produce ENPP1 polypeptides. Cells can be harvested and lysed, and the DNA content of the cells can be examined using methods such as Southern, 1975, J. Mol. Biol. 98: 503 and / or Berent et al., 1985, Biotech 3: 208, to detect the presence of DNA. Alternatively, antibodies can be used to detect the presence of protein in the supernatant.

[0226] Useful yeast plasmid vectors include pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are yeast integrating plasmids (Yip) and incorporate yeast selective markers I-11S3, TRP1, LEU2 and IJRA3. Plasmids pRS413-416 are yeast centromere plasmids (YCp).

[0227] A variety of methods have been developed to operably link DNA to vectors through complementary cohesive ends. For example, complementary homopolymer tracts can be added to DNA segments for insertion into vector DNA. The vector and DNA segments are then linked by hydrogen bonds between the complementary homopolymer tails to form recombinant DNA molecules.

[0228] Synthetic linkers containing one or more restriction sites provide an alternative method for ligating DNA segments to vectors. DNA segments generated by endonuclease restriction digestion are treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, which are enzymes that remove protruding 3'-single-stranded ends with their 3'-5'-exonuclease activity and fill in recessed 3'-ends using their polymerization activity.

[0229] The combination of these activities therefore generates a flat-ended DNA segment. Then, in the presence of an enzyme (e.g., bacteriophage T4 DNA ligase) that can catalyze the connection of the flat-ended DNA molecule, the flat-ended segment is incubated with a large amount of molar excess of the linker molecule. As a result, the product of the reaction is a DNA segment with a polymerized linker sequence at its end. These DNA segments can be cleaved with appropriate restriction endonucleases and connected to an expression vector that has been cleaved by an enzyme that produces a terminal compatible with the DNA segment end.

[0230] Then, clones of single stably transfected cells are established and clones with high expression of the desired ENPP1 fusion protein are screened. Single cell clones for screening ENPP1 protein expression can be completed in a high-throughput manner in 96-well plates using the synthase substrate pNP-TMP (Albright et al., 2015, Nat. Commun. 6: 10006) as described above. After screening and identifying high-expressing clones, protein production can be completed in shake flasks or bioreactors, as described in Albright et al., 2015, Nat. Commun. 6: 10006.

[0231] 9. ENPP1 Purification

[0232] Purification of ENPP1 can be accomplished using a combination of standard purification techniques known in the art. After purification, ENPP1-Fc can be dialyzed into a column supplemented with Zn 2+ and Mg 2+ The PBS (PBSplus) was concentrated to between 5 and 7 mg / ml and frozen in aliquots of 200-500 pl at -80° C. Just before use, the aliquots were thawed and the specific activity of the solution was adjusted to 31.25 au / ml (or about 0.7 mg / ml, depending on the formulation) by dilution in PBSplus.

[0233] 10. Route and frequency of administration

[0234] The polypeptide may be administered to the subject acutely or chronically. In certain embodiments, a second dose of a soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein is administered to the subject after an appropriate time interval of about two days, four days, one week, or one month, or even less frequently, such as once every few months or even once a year or less. The frequency of dosage will be apparent to the skilled person and will depend on any number of factors, such as, but not limited to, the type and severity of the disease being treated, and the type and age of the patient.

[0235] The amount or frequency of the dose can be selected to maintain the steady-state level of plasma PPi at a constant or steady-state level, and / or to provide a sustained level of plasma PPi that is close to normal PPi levels (2-3 μM) or above normal PPi levels (30-50% higher than normal PPi levels), and does not return to the lower PPi level that the subject had before administration of the first dose construct disclosed herein.

[0236] Alternatively, the ENPP1 dose may be administered at appropriate time intervals, such as every 2 days, or every 4 days, weekly or monthly, to achieve a constant level of ENPP1 enzyme activity.

[0237] Optionally, an ENPP1 agent according to the present disclosure is administered at appropriate intervals of every 2 days, or every 4 days, or weekly or monthly by monitoring the subject for one or more symptoms of the disease or disorder.

[0238] Without wishing to be bound by theory, it is believed that maintaining steady-state concentrations of plasma PPi at normal levels reduces and / or prevents the progression of pathological calcification in a subject.

[0239] In certain embodiments, the polypeptide is administered to the subject topically, regionally, parenterally or systemically. In some embodiments, the polypeptide is administered subcutaneously.

[0240] As used herein, "parenteral administration" of a formulation includes any route of administration characterized by administration of the ENPP1 agent with physical disruption of a subject's tissue and through the disruption in the tissue. Thus, parenteral administration includes, but is not limited to, administration of the ENNP1 agent by injection of the composition, application of the composition through a surgical incision, application of the composition through a non-surgical wound that penetrates the tissue, and the like. Specifically, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrastemal injection, and renal dialysis infusion techniques.

[0241] The administration regimen may affect what constitutes an effective amount. For example, several divided doses and staggered doses may be administered over a given period of time (daily) or sequentially, or the dose may be a continuous infusion, or may be a bolus injection. In addition, the choice of the dose of the ENPP1 agent may be dictated by the urgency of the therapeutic or preventive situation.

[0242] Administration of the compositions of the present disclosure (e.g., soluble ENPP1 polypeptides and fusion proteins thereof) to a patient, such as a mammal (i.e., a human) can be performed using known procedures at dosages and for periods of time effective to treat the patient's disease or disorder. The effective amount of the dosage of the ENPP1 agent required to achieve a therapeutic effect may vary depending on factors such as the activity of the specific compound employed; the time of administration; the excretion rate of the compound; the duration of treatment; other drugs, compounds, or materials used in combination with the compound; the disease or disorder state, age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical field. The dosage regimen can be adjusted to provide the optimal therapeutic response. The selected dose is determined based on the biological activity of the therapeutic compound, which in turn depends on the half-life and the area under the plasma time curve of the therapeutic compound.

[0243] 11. Preventive administration

[0244] From the disclosure herein, one skilled in the art will appreciate that preventing a disease or disorder in a subject includes administering an ENPP1 polypeptide to the subject as a preventative measure for the disease or disorder.

[0245] The relative amounts of active ingredients (e.g., soluble ENPP1 polypeptides and fusion proteins thereof), pharmaceutically acceptable carriers, and any additional ingredients in the formulations disclosed herein will vary, depending on the identity, size, and condition of the subject being treated, and further on the route by which the composition is administered. For example, the composition may include between about 0.1% and about 100% (w / w) of the active ingredient.

[0246] 12. ENPP1 polypeptide sequence

[0247] Table 1: Sequences

[0248]

[0249]

[0250]

[0251] For SEQ ID NOs: 118 and 119:

[0252] X = output sequence

[0253] Y = bone targeting tag or stop codon (ie no amino acids, as marked by *)

[0254] Non-limiting examples of bone targeting sequences include, but are not limited to, DDDDDDDD and / or EEEEEEEE.

[0255] Non-bold, non-underlined font = linker region, which can be of various lengths, etc. The amino acid composition of the linker can be variable. In certain non-limiting embodiments, the linker region can include residues such as histidine to prevent non-specific protease cleavage (i.e. HS or HG instead of RS or RG).

[0256] Bold font = human IgG1 (optimized in BL-1118 with M883Y S885T T887E mutations highlighted in bold italics)

[0257] Underlined font = Extracellular domain of human ENPP1 (optimized in BL-1118, with the substitution of I256T highlighted in bold underline)

[0258] SEQ ID NO:7 (mouse ENPP1 - NCBI accession number NP_001295256.1)

[0259]

[0260]

[0261] SEQ ID NO:8 (Bovine ENPP1 - NCBI Accession No. NP_001193141.1)

[0262]

[0263] SEQ ID NO:9 (rabbit ENPP1 - NCBI accession number NP_001162404.1)

[0264]

[0265] SEQ ID NO: 10 (Baboon ENPP1 - NCBI Accession No. NP_001076211.2)

[0266]

[0267] SEQ ID NO: 118 (Construct 1118 - soluble ENPP1-Fc)

[0268]

[0269] SEQ ID NO: 119 (Construct 2000 - bone-targeted ENPP1-Fc)

[0270]

[0271] Experimental Examples

[0272] The present disclosure is further described in detail by reference to the following experimental examples. These examples are provided for illustration only and are not meant to be limiting unless otherwise specified. Therefore, the present disclosure should never be construed as being limited to the following examples, but should be construed as including any and all variations that become apparent due to the teachings provided herein.

[0273] Without further description, it is believed that one of ordinary skill in the art can utilize the foregoing description and the following illustrative examples to make and utilize the compounds of the present disclosure and to practice the claimed methods. Therefore, the following working examples specifically point out the preferred embodiments of the present disclosure and should not be construed as limiting the remainder of the present disclosure in any way.

[0274] The materials and methods used in the following examples are now described.

[0275] Human subjects

[0276] Patients' data, including laboratory data, radiographs, computed tomography (CT), and bone mineral density (BMD), were collected retrospectively, and skeletal and biochemical data of patients with ENPP1 mutations were reported.

[0277] Mutation analysis

[0278] Mutational analysis of genes associated with hereditary hypophosphatemic rickets, osteogenesis imperfecta (OI), and osteoporosis-pseudoglioma syndrome (OPPG) (Table 2) was performed by next-generation sequencing using the MiSeqSequencing System at Kazusa DNA Research Institute as previously reported (Fujiki et al., 2018). Direct sequencing of the detected ENPP1 variants was performed in family members of the proband as previously reported (Fujiki et al., 2018) or by using the primers listed in Table 2. The pathogenicity of the detected variants was assessed by the in silico tools PolyPhen-2, SIFT, and Mutation Taster (Adzhubei et al., 2010; Kumar et al., 2009; Schwarz et al., 2010).

[0279] Table 2.

[0280] Genes tested in this case series (A) and primers targeting sequences for ENPP1 gene variants (B).

[0281]

[0282] OI, osteogenesis imperfecta; OPPG, osteoporosis-pseudoglioma syndrome

[0283] Cloning of complement DNA

[0284] In the case of patient 3, total RNA from peripheral blood mononuclear cells was used to synthesize ENPP1 cDNA using reverse transcription and polymerase chain reaction (RT-PCR). ENPP1 cDNA and pT7blue T-vector (Merck, Darmstadt, Germany) were ligated before transformation into Escherichia coli. The amplified vector was purified using the QIAprep Spin Miniprep Kit (Qiagen, Redwood City, CA, USA) and sequenced in both alleles.

[0285] Measurement of intact FGF23

[0286] Intact FGF23 was measured by Determinar CL FGF23 (CL) according to the manufacturer's protocol (Minaris Medical, Tokyo, Japan). CL is a sandwich chemiluminescent enzyme immunoassay (CLEIA) using an anti-human FGF23 mouse monoclonal antibody. Using this method, the reference range for FGF23 is 16.1-49.3 pg / ml, with a cutoff value of 30 pg / ml for FGF23-related hypophosphatemia (Ito et al., 2021; Kato et al., 2021).

[0287] Measurement of plasma PPi

[0288] Plasma was collected from participants to measure plasma pyrophosphate (PPi) concentration. After plasma separation, samples were filtered through a 30 kDa membrane (PALL, Port Washington, NY, USA) via centrifugation to remove platelets and frozen at -80 ° C within 1 hour after blood collection for single use. Plasma [PPi] was measured using ATP sulfurylase, as previously described with slight modifications (Jansen et al., 2013; Jansen et al., 2014). The luminescent signal was read at room temperature by an EnSpire multimode plate reader (PerkinElmer, Waltham, MA, USA). The final plasma [PPi] was normalized by subtracting [PPi] from the background of the appropriate control. The reference range of [PPi] for healthy children and adolescents using the ATP sulfurylase method has recently been reported to be 2,360 to 4,440 (Bernhard et al., 2022). These values ​​are similar to the standard ranges for healthy adults previously reported (O'Neill et al., 2010).

[0289] Enzyme kinetics assay of ENPP1

[0290] Human mutations were engineered into the hENPP1-Fc construct using Quikchange II-XL site-directed mutagenesis (Agilent Technologies, Santa Clara, CA, USA). After sequence verification, the construct was transfected into CHO-K1 cells using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). After 48 hours of transfection, 10 μL of supernatant was mixed with 90 μL of assay buffer containing 250 mM Tris pH 8.0, 500 mM NaCl, 0.05% Triton X-100, and 1 mM thymidine 50-monophosphate p-nitrophenyl. The speed of p-nitrophenyl release from the chromogenic substrate was reported as a change in OD405 nM / minute, with at least 5 replicates per construct, and normalized relative to WT%.

[0291] Statistical analysis

[0292] PPi levels are expressed as mean ± standard deviation (SD). Comparison of enzyme kinetics assays of ENPP1 between WT and mutants was analyzed using analysis of variance. Significance was set at P < 0.05. Data analysis was performed using GraphPad Prism version 6.05 for Windows (GraphPad Software, San Diego, CA, USA).

[0293] Example 1: Identification of ENPP1 haploinsufficiency in patients

[0294] Patient 1 is a 47-year-old male with a history of upper and lower extremity fractures. He was a social drinker with a 20-pack-year smoking history. He had no history of ureteral calculi or malabsorption syndrome, no treatment history related to osteoporosis, and no family history of osteoporosis. At age 46, he developed back pain and was diagnosed with spinal compression fractures, and one month after the back pain, he also developed knee, wrist, and ankle pain. He was transferred to a local hospital, and dual-energy X-ray absorptiometry (DEXA) of the lumbar and proximal femur showed T-scores of -3.8 and -2.6, respectively. A CT scan of the spine showed compression fractures at the 7th and 11th thoracic vertebrae ( Figure 1A ).

[0295] Bone scintigraphy shows multiple uptakes in the ribs, indicating multiple fractures ( Figure 1B No evidence of heterotopic ossification was detected in the hip and knee joints, Achilles tendons, or paraspinal ligaments ( Figure 1C-1H). The patient was diagnosed with early-onset osteoporosis, and laboratory tests were performed to exclude secondary osteoporosis and osteomalacia. Although low-normal serum phosphorus was detected in one local hospital, and serum alkaline phosphatase and bone alkaline phosphatase were slightly elevated, no abnormalities in serum calcium, phosphorus, or FGF23 were detected in another hospital (Supplementary Table 1).

[0296] Other endocrine function tests, such as thyroid-stimulating hormone, free testosterone, and cortisol (measured in the morning), were within normal ranges (Supplementary Table 1). Therefore, he was referred to the University of Tokyo Hospitals for further evaluation, and blood chemistry was reassessed (Table 3). Laboratory data showed normal serum albumin level, normal corrected calcium (8.8 mg / dL), low-normal serum phosphorus (2.7 mg / dL, reference interval 2.7–4.6 mg / dL), normal FGF23 (28 pg / mL, reference interval 16.1–49.3 pg / mL), and low 25-hydroxyvitamin D (7.2 ng / mL).

[0297] A diagnosis of mild phenotype of OI or OPPG was considered, and mutation analysis of genes associated with OI or OPPG was performed (Table 2), but no pathogenic variants were identified. Based on the serum analytes measured at the initial hospital (Supplementary Table 1) and the presence of low-normal serum phosphorus at our hospital, a diagnosis of mild hypophosphatemic rickets was next considered, and mutation analysis of related genes was performed, revealing the presence of a missense variant in ENPP1 (c.536A>G, ​​p.Asn179Ser[N179S]) (Table 3). Direct sequencing of the detected ENPP1 variant was next performed in the patient's 18-year-old son, revealing the same heterozygous ENPP1 variant ( Figure 2A Although the son demonstrated low-normal BMD with low Z scores at the lumbar spine (-1.6) and proximal femur (-0.8), he had no history of spontaneous fractures or heterotopic ossification in the spine, hip, knee, or Achilles tendon ( Figure 5A-5G and Table 3).

[0298] Patient 2 was a 77-year-old female diagnosed with diffuse idiopathic skeletal hyperostosis (DISH) who was referred to the University of Tokyo hospital when she suffered compression fractures in the spine. During the evaluation, ossification of the paraspinal ligaments and multiple spinal compression fractures ( Figure 3A In addition, mild heterotopic ossification was detected in the Achilles tendon ( Figure 3F ), but the hip and knee joints are intact ( Figure 3B , 3C, 3D, and 3E). Ossifications detected on spinal CT were not evaluated histologically. The patient's biochemical profile showed low-normal serum phosphorus (3.1 mg / dL, reference interval 2.7-4.6 mg / dL) and high-normal FGF23 (43.3 pg / mL, reference interval 16.1-49.3 pg / mL). Due to the presence of heterotopic ossification in the paraspinal ligaments and Achilles tendon, the diagnosis of hereditary FGF23-related hypophosphatemia (specifically ARHR or XLH) was considered. Testing for genes associated with hereditary hypophosphatemic rickets (Table 2) revealed the presence of a heterozygous missense variant in ENPP1 (c.1352A>G, p.Tyr451Cys[Y451C]) (Table 3).

[0299] Patient 3 is a 54-year-old female who presented to a nearby hospital with complaints of hip, knee, and back pain. She subsequently developed ossification of the anterior / posterior longitudinal ligament (OALL / OPLL), was diagnosed with DISH, osteophytes around both hip joints, and enthesopathy in both Achilles tendons ( Figures 4A-4I ). Laboratory data showed low-normal serum phosphate (2.9 mg / dL) and high-normal serum FGF23 (38.4 pg / mL, reference range 16.1-49.3 pg / mL). The biochemical profile, combined with the findings of ectopic paraspinal ossification, led to the conclusion of FGF23-related hypophosphatemia, and genetic analysis (Table 2) revealed the same ENPP1 variants (c.536A>G and c.1352A>G) as in patients 1 and 2 (Table 3). Sanger sequencing was then performed on her sons (19 and 23 years of age), revealing that both had the heterozygous ENPP1 variant of c.1352A>G ( Figure 2C Notably, both young men presented with calcific enthesopathy of the Achilles tendinopathy without heterotopic ossification in the spine or hip ( Figures 6A-6G and 7A-7G).

[0300] Supplementary Table 1. Biochemical and hormonal data of case 1 before referral

[0301] RI, Adult Case 1 Serum calcium (mg / dL) 8.8-10.1 9.4 Serum phosphate (mg / dL) 2.7-4.6 2.8 <![CDATA[eGFR (mL / min / 1.73m 2 )]]> 25(OH)D (ng / mL) <4.0 <![CDATA[1,25(OH) 2 D(pg / mL)]]> 20-60 39.7 ALP(U / L) 38-113 152 BAP (μg / L) 3.7-20.9 25.6 TRACP-5b (mU / dL) 170-590 594 iPTH (pg / mL) 25.8-75.7 36 FGF23 (pg / mL) 16.1-49.3 28 TSH (μIU / mL) 0.38-4.31 2.46 Free testosterone (pg / mL) 4.7-21.6 5.6 Cortisol (μg / dL) 4.5-21.1 6.0

[0302] Abbreviations: RI, reference interval; eGFR, estimated glomerular filtration rate; 25(OH)D, 25-hydroxyvitamin D; 1,25(OH) 2 D, 1,25-dihydroxyvitamin D; ALP, alkaline phosphatase; BAP, bone alkaline phosphatase; TRACP-5b, tartrate-resistant acid phosphatase 5b; iPTH, intact parathyroid hormone; FGF23, fibroblast growth factor 23; TSH, thyroid-stimulating hormone.

[0303]

[0304]

[0305] Example 2: Characterization of the effects of ENPP1 haploinsufficiency in patients

[0306] Cloning of complementary DNA of ENPP1 in patient 3

[0307] The complementary DNA of ENPP1 was cloned and sequenced to assess whether the two ENPP1 variants were located in the same allele or in opposite alleles. Sequence data for each allele revealed that case 3 had compound heterozygosity for the ENPP1 variants ( Figure 8 ).

[0308] In silico prediction of pathogenicity of detected ENPP1 variants

[0309] The allele frequencies of N179S and Y451C detected in this case were reported to be 0.00010 and 0.00016, respectively, in the genome aggregation database (GnomAD). However, in the whole genome reference panel of 3552 general Japanese individuals constructed by the Tohoku Medical Megabank Organization (ToMMo), the allele frequencies of N179S and Y451C were reported to be 0.0071 and 0.0055, respectively (Table 4). Asn 179 is located in the somatomedin B domain 2 (SMB2), which is essential for protein dimerization and stability, while Tyr 451 is in the catalytic domain ( Fig. 9 ), Asn179 and Tyr 451 are highly conserved in all species. A combination of in silico tools described in the Methods marked the N179S and Y451C mutations as pathogenic (Table 4).

[0310] Table 4. Allele frequencies and in silico predictions of ENPP1 variants, c.536A>Gorc.1352A>G

[0311]

[0312] Measurement of plasma PPi

[0313] Plasma PPi was measured in three probands (cases 1-3) and their family members. Patients with heterozygous ENPP1 variants showed plasma PPi levels between 1,000 and 2,000 nM (Table 3), while plasma PPi in patients with biallelic ENPP1 variants showed a similar concentration of 1,866 nM.

[0314] Enzyme kinetics assay of ENPP1

[0315] To further evaluate the effect of the mutations on the catalytic activity of ENPP1, the enzymatic rates of all variants were evaluated together in an in vitro assay compared to the WT ENPP1 isoform. The N179S and Y451C variants reduced the catalytic rate of ENPP1 by 55% and 70%, respectively, compared to WT ENPP1. Fig.10 ). Therefore, according to the ACMG guidelines, the N179S and Y451C variants were classified as “likely pathogenic” (N179S: PM1+PP3+PS3, Y451C: PM1+PS3).

[0316] Selected results

[0317] The results showed clinical manifestations of two monoallelic ENPP1 deficiencies and one compound biallelic heterozygous ENPP1 deficiency in the primary subjects (patients 1-3), and three additional cases of ENPP1 haploinsufficiency in the children of these subjects.

[0318] Patient 1 presented with early-onset osteoporosis, whereas Patient 2 presented with a presumptive diagnosis of spondylosis and DISH and was additionally found to have osteoporosis (evidenced by compression fractures in the spine, Figure 1A-1B Patient 3, who had compound heterozygous ENPP1 mutations consisting of the single mutations present in patients 1 and 2, was homozygous for ENPP1 and showed prominent paraspinal ligament ossification, as well as calcifications around the hip joints and bilateral Achilles tendon calcification enthesopathy ( Figures 4A-4I ).

[0319] Furthermore, all children with the monoallelic ENPP1 variant inherited from the original patient manifested musculoskeletal disorders at surprisingly young ages, such as low bone mass (Z score of −1.6 at the lumbar spine) in the 18-year-old son of patient 1 and calcific enthesopathy of the Achilles tendon in the 19- and 23-year-old children of patient 3. The segregation of genotype and phenotype further supports the notion that ENPP1 deficiency plays a central role in the pathogenesis of the skeletal disorders described in this study.

[0320] Previously, an evaluation of families with homozygous GACI-inducing ENPP1 mutations reported that haploinsufficient ENPP1 carriers were asymptomatic but had serum biochemical parameters of hypocalcemia and hypophosphatemia, suggesting that ENPP1 may play a role in calcium and phosphate homeostasis (Kotwal et al., 2020). In contrast, Oheim et al. reported ENPP1 haploinsufficiency in adult males with early-onset osteoporosis, a phenotype also present in a family called ENPP1. asj / asjIn mice homozygous for ENPP1, ENPP1 is expressed in the bone marrow (Oheim et al., 2020), supporting the idea that ENPP1 regulates bone mass in mammals. Indeed, the skeletal phenotypes of patient 1 and her 18-year-old son support the association of ENPP1 haploinsufficiency with early-onset osteoporosis.

[0321] This study extends the association of ENPP1 haploinsufficiency to patients with DISH. DISH is a systemic disease characterized by ossification of ligaments and tendon ends, especially around the thoracic spine. DISH patients sometimes suffer from pain and reduced range of motion and are at increased risk of spinal fractures (Mader et al., 2013). Although the pathogenesis is unknown, DISH is associated with older age (over 50 years old), male sex, obesity, hypertension, and diabetes (Kuperus et al., 2020).

[0322] In the case of patient 2, her age was over 50 years, but no other risk factors for DISH were present; instead, the patient was found to be haploinsufficient for ENPP1. Given that clinical and preclinical studies in humans and mice have linked homozygous ENPP1 deficiency to spinal ligament ossification (Okawa et al., 1998; Nakamura et al., 1999; Saito et al., 2011; Hirao et al., 2016), impaired ENPP1 activity leading to decreased plasma PPi is a risk factor for the progressive paraspinal ossification and calcific enthesopathy present in this patient.

[0323] Both described ENPP1 variants (N179S and Y451C) are located in highly conserved sequences in a region of ENPP1 that is important for dimerization and stability (Asn 179) and catalytic activity (Tyr 451). Furthermore, N179S and Y451C were found to be deleterious by multiple in silico tools (Table 4), but have not been reported to be pathogenic to date.

[0324] Furthermore, N179S and Y451C reduced enzyme activity by 55% and 70%, respectively, compared to WT levels, which is similar to the residual enzyme activity present in other pathogenic variants of ENPP1 (Kotwal et al., 2020; Oheim et al., 2020; Rutsch et al., 2003; Stella et al., 2016; Thumbiger-Math et al., 2018). Taken together, these findings support that one or more ENPP1 variants, N179S and / or Y451C, are responsible for the skeletal phenotype and heterotopic ossification present in the three probands.

[0325] Although the mechanism by which ENPP1 regulates musculoskeletal mineralization is not fully understood, either through catalysis or catalysis-independent protein signaling, both patients described in Patients 1 and 2 exhibited low plasma [PPi] (1,646 nM and 1,748 nM, respectively, with a reference range of 2,360-4,440 nM), consistent with a role for the catalytic activity of ENPP1 in the observed abnormal phenotype. Both ENPP1 and ABCC6 deficiencies result in low plasma PPi levels (Lorenz-Depiereux et al., 2010; Levy-litan et al., 2010; Rutsch et al., 2001; Nitschke et al., 2012; Le Saux et al., 2000). Plasma PPi levels in patients with ENPP1 haploinsufficiency are intermediate between those without ENPP1 deficiency and those with homozygous ENPP1 deficiency (Kotwal et al., 2020; Oheim et al., 2020). PPi levels in our patients also fell within the range of 1,000 to 2,000 nM, but unexpectedly, patient 3 with compound heterozygous ENPP1 deficiency had PPi concentrations similar to those in haploinsufficient ENPP1 patients, suggesting the involvement of compensatory mechanisms (Kotwal et al., 2020).

[0326] Furthermore, secondary hyperparathyroidism was observed in patient 3, a finding also observed in other patients with homozygous ENPP1 deficiency (Kotwal et al., 2020, and Capelli et al., 2015) and in mouse models of ENPP1 deficiency. PTH is also elevated in other disorders induced by elevated FGF23, such as Hyp mice and humans with XLH and ARHR, which present with secondary hyperparathyroidism due to impaired vitamin D activation by the action of FGF23 (Carpenter JBMR, 2011). Similar mechanisms may explain the secondary hyperparathyroidism in case 3, who was noted to have vitamin D deficiency, as well as other cases of homozygous ENPP1 deficiency (Kotwal et al., 2020). Since the patient described in patient 3 did not develop osteomalacia, treatment with natural vitamin D rather than active vitamin D would be appropriate to improve and prevent the exacerbation of secondary hyperparathyroidism.

[0327] Although the patients in the study presented normal intact FGF23, the levels were interpreted as elevated in the context of their low normal serum phosphorus levels, raising the possibility of a genetically induced phosphate wasting disorder. In this regard, most of the patients described in the original report of ARHR2 had high-normal or slightly elevated intact FGF23 in the context of low serum phosphorus. In Levy-Litan's description of three rickets patients, two of the three had high-normal intact FGF23 (50 pg and 47 pg, normal reference range 10-50 pg), (Levy-Litan et al., 2010), and five of the seven ARHR2 patients reported by Lorenz-Depiereux had intact FGF23 at the upper end of the normal range or slightly elevated on repeated measurements. These ranges were also interpreted as high in the context of the low phosphate values ​​and the severe rickets phenotype presented by the patients.

[0328] Without wishing to be bound by any theory, the present study demonstrated that ENPP1 haploinsufficiency induced greater FGF23 elevations than homozygous ENPP1 deficiency, and the normal and high-normal levels of intact FGF23 in the context of normal or low-normal serum phosphorus (2.7-3.1 mg / dL, reference range 2.7-4.6 mg / dL) raised suspicion for genetically induced hypophosphatemia and initiated genetic testing. This study showed that increased circulating FGF23 and hypophosphatemia occur in patients with calcific enthesopathy with rare metabolic disorders such as ARHR, XLH, and hypophosphatemic tumor calcification (Okawa et al., 1998; Nakamura et al., 1999; Saito et al., 2011; Hirao et al., 2016; Maulding et al., 2021; Rutsch et al., 2001; Cheng et al., 2005; Albright et al., 2015), as well as in the general medical population of patients with rapidly progressive forms of OPL (Albright et al., 2015; Ferreira et al., 2021). In contrast, calcific enthesopathy is not associated with FGF23-independent forms of hypophosphatemic rickets such as SLC34A3 deficiency (Kotwal et al., 2020). Therefore, FGF23 appears to be closely related to the development of enthesopathy.

[0329] Previous transcriptome analysis in whole bone of ENPP1-deficient mice reported that inactivating mutations in Enpp1 increased Fgf23 transcription and decreased Wnt10b and Wnt16 transcription (Maulding et al., 2021), suggesting that ENPP1 deficiency induces abnormal bone mineralization in part through impaired Wnt signaling. Patient 1 did not exhibit overt hypophosphatemic rickets with elevated FGF23 levels, and the bone resorption marker tartrate-resistant acid phosphatase 5b (TRACP-5b) was within the normal range, indicating the development of osteoporosis without high bone turnover—a finding consistent with an anabolic defect induced by defective Wnt signaling. Thus, the human observations are consistent with the osteoporotic mechanism suggested by transcriptome analysis of mouse models of ENPP1 deficiency (Maulding et al., 2021).

[0330] Finally, it is important to note the differences in allele frequencies of the variants described in this study. The frequency of the allele (Y451C) detected in patient 2 was approximately 30-fold higher in Japanese (ToMMo: 0.0055) than in all other ethnic groups (GnomAD: 0.00016). Given the observation that monoallelic ENPP1 Y451C mutations are associated with OPL, this difference in variant frequency may explain the 10-40-fold higher incidence of OPLL in Japan (1.8-4.1%) compared to the United States (0.12%) and Germany (0.1%) (Stapleton et al., 2011).

[0331] Example 3: Production of ENPP1 fusion protein and treatment of DISH model mice

[0332] Generation of ENPP1

[0333] One example of an ENPP1 fusion protein is ENPP1-Fc. However, the illustration of ENPP1-Fc is also applicable to other ENNPP1 fusion proteins (such as ENPP1-albumin) as described herein.

[0334] ENPP1-Fc is a recombinant fusion protein containing the extracellular domain of human ENPP1 (soluble ENPP1) bound to the Fc fragment of IgG1 (rhENPP1-Fc). The recombinant extracellular domain of ENPP1-Fc contains its catalytic activity, identical to the native ENPP1 enzyme. ENPP1-Fc is a recombinant human protein produced in CHO cells via a fed-batch cell culture process that contains no animal-derived components. The molecular weight of the ENPP1-Fc dimer is approximately 290 kDa; ENPP1-Fc is highly glycosylated with a pI of approximately 6.0. Like endogenous ENPP1, the major substrate of ENPP1-Fc is ATP, which is cleaved into AMP and PPi.

[0335] In a specific embodiment, the soluble ENPP1 protein is fused to a human Fc domain with a linker via a linker including leucine, isoleucine and asparagine. Three ENPP1-Fc constructs are shown in Table 1 as SEQ ID NOs: 3, 4 and 5, as purified from CHO cells.

[0336] Purification of ENPP1-Fc can be achieved by a series of column chromatography steps, which include, for example, three or more of the following, in any order: protein A chromatography, Q Sepharose chromatography, phenyl Sepharose chromatography, size exclusion chromatography, and cation exchange chromatography. Purification can be accomplished by viral filtration and buffer exchange. After protein purification, the catalytic activity of the ENPP1-Fc protein can be assessed using pNP-TMP as a chromogenic substrate.

[0337] Treatment of DISH model mice with ENPP1 agents

[0338] The most established mouse model of DISH is the Enpp1-deficient mouse, termed ttw (for “tip toe walking mice”). ttw mice display severe myelopathy and extensive paraspinal ligament calcification and osteophyte formation. To assess the relationship between PPi and the severity of DISH, we performed a double-blind, ... asj The Achilles tendons of mice were examined while they were fed a normal diet.

[0339] Animals were dosed with vehicle (PBS) or human ENPP1-Fc that normalizes plasma [PPi] for more than one week after subcutaneous submilligram dosing. The administration regimen, radiographic analysis, and histological experiments of ENPP1-Fc are summarized in Ferreira et al., Musculoskeletal Comorbidities and Quality of Life in ENPP1-Deficient Adults and the Response of Enthesopathy to Enzyme Replacement Therapy in Murine Models. J Bone Miner Res, 2021). Optionally, the experiment can be repeated using an ENPP1 agent containing a bone targeting domain (such as, but not limited to, ENPP1-Fc) for better delivery and higher efficacy.

[0340] Between weeks 2 and 23, 0.3 mg / kg per week of Enpp1 asj Mice were administered ENPP1 agents (such as but not limited to ENPP1-Fc), and the effects of ENPP1 administration on theasj Plasma PPi in mice. Note that PPi was significantly increased relative to the WT pair, but not completely normalized (1358 vs 2235 nM, respectively, Table 6).

[0341] Table 6

[0342] 23-week-old Enpp1 WT and Enpp1 with and without drug administration asj / asj Mouse plasma analytes

[0343]

[0344] Enthesopathy in the Achilles tendon was analyzed histologically and red pixels were quantified in micrographs of Alizarin red-stained sections using custom MATLAB software. asj The Achilles tendons of mice are expected to show substantial calcification along the entire length of the tendon, and Enpp1 treated with ENPP1-Fc asj Tendon calcification in mice is expected to be inhibited.

[0345] Without being bound by theory, telogen activin may be dependent on plasma PPi and may be prevented by raising plasma PPi with enzyme-based biologics such as ENPP1 agents (eg, ENPP1-Fc). The results of the study suggest that complete suppression of telogen activin may be achieved following dose expansion.

[0346] Effects of PPi on DISH model mice

[0347] To evaluate the effects of plasma PPi on osteophyte formation, spinal fusion, and ossification in the DISH mouse model, Enpp1 was injected with vehicle or 1 mg / kg ENPP1-Fc between weeks 3 and 17. asj Mice were dosed and their spines were analyzed by micro-CT. Fig.12 : Mice dosed with ENPP1-Fc exhibited elevated plasma PPi levels (approximately 10 μM), which were several-fold higher than WT levels.

[0348] The presence of elevated plasma PPi and reduced paraspinal ossification in a DISH mouse model treated with an ENPP1 agent (eg, ENPP1-Fc) would suggest that such ENPP1 administration to human subjects with DISH would also reduce paraspinal osteophytes, ankylosing joints, and spinal fusions.

[0349] Example 4: Treatment regimen

[0350] The ENPP1 agent (e.g., but not limited to ENPP1, ENPP1-X, ENPP1-Fc and / or ENPP1-Fc-X, wherein X is a bone targeting tag as described elsewhere herein) is administered at one of the following selected doses: 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2 .0mg / kg, 2.1mg / kg, 2.2mg / kg, 2.3mg / kg, 2.4mg / kg, 2.5mg / kg, 2.6mg / kg, 2.7mg / kg, 2.8mg / kg, 2.9mg / kg, 3.0mg / kg, 3.1mg / kg, 3.2mg / kg, 3.3mg / k g, 3.4mg / kg, 3.5mg / kg, 3.6mg / kg, 3.7mg / kg, 3.8mg / kg, 3.9mg / kg, 4.0mg / kg, 4.1mg / kg, 4.2mg / kg, 4.3mg / kg, 4.4mg / kg, 4.5mg / kg, 4.6mg / kg, 4.7m g / kg, 4.8mg / kg, 4.9mg / kg, 5.0mg / kg, 5.1mg / kg, 5.2mg / kg, 5.3mg / kg, 5.4mg / kg, 5.5mg / kg, 5.6mg / kg, 5.7mg / kg, 5.8mg / kg, 5.9mg / kg, 6.0mg / kg, 6 .1mg / kg, 6.2mg / kg, 6.3mg / kg, 6.4mg / kg, 6.5mg / kg, 6.6mg / kg, 6.7mg / kg, 6.8mg / kg, 6.9mg / kg, 7.0mg / kg, 7.1mg / kg, 7.2mg / kg, 7.3mg / kg, 7.4mg / k g, 7.5mg / kg, 7.6mg / kg, 7.7mg / kg, 7.8mg / kg, 7.9mg / kg, 8.0mg / kg, 8.1mg / kg, 8.2mg / kg, 8.3mg / kg, 8.4mg / kg, 8.5mg / kg, 8.6mg / kg, 8.7mg / kg, 8.8m g / kg, 8.9 mg / kg, 9.0 mg / kg, 9.1 mg / kg, 9.2 mg / kg, 9.3 mg / kg, 9.4 mg / kg, 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, 10.0 mg / kg and / or fractions or multiples thereof.Administration is by subcutaneous injection (SC), at least once or twice every two months, at least once or twice a month, three times a month, at least once or twice a week.

[0351] The first dose of ENPP1 may be administered on day 1. On day 8 and thereafter, the subject is administered ENPP1 twice weekly at a dose of the selected dose of ENPP1 mg / kg. The dose may be administered at approximately the same time on each dosing day. The injection sites are alternated, with no injection site within 2 inches of any past injection site within the past 2 weeks.

[0352] A selected dose of ENPP1 agent is one of 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg or 1.8 mg / kg SC. Another selected dose of ENPP1 agent by SC is one of 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2.0 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5mg / kg, 2.6mg / kg, 2.7mg / kg, 2.8mg / kg, 2.9mg / kg, 3.0mg / kg, 3.1mg / kg, 3.2mg / kg, 3.3mg / kg, 3.4mg / kg, 3.5mg / kg, 3.6mg / kg, 3.7mg / k g, 3.8mg / kg, 3.9mg / kg, 4.0mg / kg, 4.1mg / kg, 4.2mg / kg, 4.3mg / kg, 4.4mg / kg, 4.5mg / kg, 4.6mg / kg, 4.7mg / kg, 4.8mg / kg, 4.9mg / kg, 5.0mg / kg, 5.1mg / kg, 5.2mg / kg, 5.3mg / kg, 5.4mg / kg, 5.5mg / kg, 5.6mg / kg, 5.7mg / kg, 5.8mg / kg, 5.9mg / kg, 6.0mg / kg, 6.1mg / kg, 6.2mg / kg, 6.3m g / kg, 6.4mg / kg, 6.5mg / kg, 6.6mg / kg, 6.7mg / kg, 6.8mg / kg, 6.9mg / kg, 7.0mg / kg, 7.1mg / kg, 7.2mg / kg, 7.3mg / kg, 7.4mg / kg, 7.5mg / kg, 7.6 .0 mg / kg, 7.7 mg / kg, 7.8 mg / kg, 7.9 mg / kg, 8.0 mg / kg, 8.1 mg / kg, 8.2 mg / kg, 8.3 mg / kg, 8.4 mg / kg, 8.5 mg / kg, 8.6 mg / kg, 8.7 mg / kg, 8.8 mg / kg, 8.9 mg / kg, 9.0 mg / kg, 9.1 mg / kg, 9.2 mg / kg, 9.3 mg / kg, 9.4 mg / kg, 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, 10.0 mg / kg and / or fractions or multiples thereof.The first dose of ENPP1 can be administered on day 1. After the first dose, the subject can be observed for 7 days to monitor safety and collect PK samples. On day 8 and thereafter, the subject receives the selected dose twice a week. Continue to administer ENPP1 at the selected dose as deemed appropriate by the medical professional.

[0353] A subject may receive 8 doses of ENPP1 over a 29-day period, for example, for doses of 0.2 mg / kg, 0.6 mg / kg, and 1.8 mg / kg, resulting in exposures of 1.6 mg, 4.8 mg, and 14.4 mg, respectively, every 29 days. Alternatively, a subject may receive more or less than 8 doses, as deemed appropriate by the medical professional.

[0354] Like the endogenous ENPP1 enzyme, ENPP1 agents cleave ATP to generate AMP and PPi, thereby increasing plasma PPi levels and converting to AMP, which is rapidly converted to adenosine by CD73. Replacing the endogenous human enzyme is intended to correct the inherent defect and allow for improved health outcomes and reduction of clinical complications associated with baseline ENPP1 patient, clinician and caregiver outcomes.

[0355] Example 5: Treatment of patients with ENPP1 haploinsufficiency

[0356] Patients identified as having ENPP1 haploinsufficiency were administered ENPP1 agents by subcutaneous injection on day 1 and twice weekly starting on day 8 using the selected doses as follows.

[0357] Table 7.

[0358]

[0359]

[0360] The ENPP1 agent is administered at a selected dose of one of 0.2 mg / kg, 0.6 mg / kg or 1.8 mg / kg SC, at least twice a week, for a period of time determined by a medical professional. The patient's response to enzyme replacement is monitored as needed, as determined by a medical professional, for example, by tracking the reduction of one or more symptoms of ENPP1 deficiency, and / or using the guidance provided herein.

[0361] Example 6: Treatment of Patients Diagnosed with DISH

[0362] DISH usually involves calcification of the tendons and ligaments around the spine. Once the tendons and ligaments become stiff, parts of these tissues turn to bone. This usually happens where the tissue joins the bone. As a result, bone spurs, which are outgrowths of bone that develop along the edges of bones, can occur. DISH usually affects the upper part of the back and neck, namely the thoracic and cervical spine. However, DISH can also affect the shoulders, elbows, hands, knees, hips, heels, and / or ankles.

[0363] In certain embodiments, a subject diagnosed with DISH is treated with an ENPP1 agent, the selected dose being one of 0.2 mg / kg, 0.6 mg / kg, or 1.8 mg / kg, the ENPP1 agent being administered SC, IV, and / or IP at least twice per week for a period of time determined by a medical professional.

[0364] In certain embodiments, a subject diagnosed with DISH is treated with an ENPP1 agent at a selected dose of 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2.0 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5mg / kg, 2.6mg / kg, 2.7mg / kg, 2.8mg / kg, 2.9mg / kg, 3.0mg / kg, 3.1mg / kg, 3.2mg / kg, 3.3mg / kg, 3.4mg / kg, 3.5mg / kg, 3.6mg / kg, 3.7mg / kg, 3.8mg / kg、3.9mg / kg、4.0mg / kg、4.1mg / kg、4.2mg / kg、4.3mg / kg、4.4mg / kg、4.5mg / kg、4.6mg / kg、4.7mg / kg、4.8mg / kg、4.9mg / kg、5.0mg / kg、5.1mg / kg、5 .2mg / kg, 5.3mg / kg, 5.4mg / kg, 5.5mg / kg, 5.6mg / kg, 5.7mg / kg, 5.8mg / kg, 5.9mg / kg, 6.0mg / kg, 6.1mg / kg, 6.2mg / kg, 6.3mg / kg, 6.4mg / kg, 6.5mg / kg, 6.6mg / kg, 6.7mg / kg, 6.8mg / kg, 6.9mg / kg, 7.0mg / kg, 7.1mg / kg, 7.2mg / kg, 7.3mg / kg, 7.4mg / kg, 7.5mg / kg, 7.6mg / kg, 7.7mg / kg, 7.8mg / kg, 7. 9mg / kg, 8.0mg / kg, 8.1mg / kg, 8.2mg / kg, 8.3mg / kg, 8.4mg / kg, 8.5mg / kg, 8.6mg / kg, 8.7mg / kg, 8.8mg / kg, 8.9mg / kg, 9.0mg / kg, 9.1mg / kg, 9.2mg / kg, 9.3mg / kg, 9.4mg / kg, 9.5mg / kg, 9.6mg / kg, 9.7mg / kg, 9.8mg / kg, 9.9mg / kg, 10.0mg / kg and / or one of its fractions or multiples, SC, IV and / or IP administration of ENPP1 doses at least twice a week for a period of time determined by a medical professional.

[0365] The response of the DISH patient to enzyme replacement is monitored as needed, as determined by a skilled physician, for example, by tracking the reduction of one or more symptoms of DISH, using the guidance provided herein.

[0366] Optionally, a subject suspected of being at risk for DISH is treated by administering a selected dose of an ENPP1 agent. In certain embodiments, common risk factors for developing DISH include, but are not limited to, large waist circumference, BMI / obesity, hyperinsulinemia, diabetes, hyperuricemia, dyslipidemia, hypertension, coronary artery disease, and / or gout. DISH can be asymptomatic, and in those cases, it is usually diagnosed based on radiological images. In certain embodiments, DISH is associated with older age (over 50 years old), male sex, obesity, hypertension, and / or diabetes.

[0367] Alternatively, subjects with radiographic evidence of DISH but lacking clinical manifestations of the disease can be treated with an ENPP1 agent to prevent or minimize an increase in spinal fractures, decreased mobility, myelopathy, and / or pain, which are all known phenotypes associated with DISH.

[0368] In the context of the entire specification, the following examples provide guidance for determining treatment regimens and therapeutic efficacy.

[0369] Example 7: Biomarkers associated with bone health

[0370] In addition to low plasma PPi, patients with ENPP1 deficiency are biochemically characterized by low serum phosphate, high urine phosphate, low renal TmP / GFR, normal calcium (Ca), low-normal urine Ca, normal 25-hydroxyvitamin D (25OH D), low-normal 1,25(OH)2D, high BAP, high intact FGF23, and normal and / or elevated PTH (IOF 2019).

[0371] Table 8 describes biomarkers that may be used in certain embodiments as additional determinants of bone health in treated patients.

[0372] Table 8. Clinical intermediates and biomarkers

[0373]

[0374] Example 8: Efficacy of treatment with ENPP1 agent

[0375] Treatment efficacy can be assessed by measuring plasma PPi as well as measuring other plasma analytes such as FGF23, Pi, FGF23, Pi, TmP / GFR, serum alkaline phosphatase (ALP), bone-specific ALP (BALP), carboxyl-terminal cross-linked telopeptide of type I collagen (CTx), and / or N-terminal propeptide of type 1 procollagen (P1NP). These analyte measurements can be used as PD markers associated with ENPP1 deficiency to determine the efficacy of ENPP1 agents. Changes in these analytes can be described as changes from baseline and in a time-dependent manner during treatment. Dose linearity of PK and PD parameters can be assessed.

[0376] Changes from baseline in plasma PPi levels, FGF23 levels, and urinary phosphate excretion per creatinine clearance were analyzed using paired differences t-tests.

[0377] Example 9: Measurement of drug concentration

[0378] In addition, blood samples can be obtained from patients to measure ENPP1 agent concentrations in plasma and for subsequent PK parameter determinations after the first dose (ie, single dose) and at / after multiple doses (ie, steady state).

[0379] Example 10: Immunogenicity (anti-drug antibodies)

[0380] If desired, immunogenicity to the ENPP1 agent can be measured using anti-drug antibodies (ADA). Immunogenicity testing can utilize a multi-tiered approach; if ADA is detected in the initial screening, confirmatory testing can be performed to determine specificity. Samples can also be used to evaluate and further establish assays for specificity confirmation (i.e., titer) and neutralizing antibodies.

[0381] Example 11: Pharmacokinetic, pharmacodynamic and exploratory biomarker analysis

[0382] Pharmacokinetic analysis can be performed on the PK population, and the PK parameters of the ENPP1 agent can be summarized by processing descriptive statistics. Dose linearity of PK and PD parameters can also be evaluated. PK / PD analysis, immunogenicity analysis; and exploratory biomarker analysis can be determined.

[0383] Example 12: Other Determinants of Efficacy

[0384] Although restoration of normal PPi levels is the primary indicator of efficacy of treatment with an ENPP1 agent, other physical measurements may be used to assist in determining efficacy of treatment if desired. These physical measurements may include one or more of the following.

[0385] 1. Radiography and Imaging

[0386] a. X-rays of the bones to determine severity. Standard x-rays may be obtained to detect rickets. For example, x-rays of the wrists and knees may be obtained.

[0387] b.DEXA scan. DEXA scans can be used to assess changes in bone density.

[0388] c.Positron emission tomography.Computed tomography.Baseline Na 18 F-PET / HRpQCT (or HR-CT) may be a whole-body scan performed within 1 month of the first dose of ENPP1 to measure arterial and organ calcification and bone abnormalities at baseline and for future interventional assessments. 18 F-PET measures bone turnover and arterial microcalcification. High-resolution quantitative computed tomography (HRQCT) or HR-CT can determine bone microarchitecture in the non-dominant distal radius and tibia. Standard bone geometry parameters are calculated.

[0389] d. Doppler echocardiogram. A baseline echocardiogram can be obtained within 3 days before the first dose of ENPP1. Doppler ultrasound can be used to measure cardiac function [LVEF, blood flow], cardiac and valvular calcification, and arterial stiffness.

[0390] e. Optical coherence tomography. Optical coherence tomography can be used to visualize neointimal proliferation.

[0391] f. Peripheral arterial tonometry. Peripheral arterial tonometry (PAT) can be used to assess digital pulse wave amplitude (PWA), which corresponds to digital volume changes.

[0392] g. Renal Ultrasound. Renal ultrasound can be used, for example, within 1 week of starting ENPP1 to measure renal calcification.

[0393] h. Bone histomorphometry and bone biopsy. A bone biopsy may be performed as a baseline measurement. A 10-day tetracycline loading is preferred before a bone biopsy.

[0394] 2. Walking ability

[0395] The walking test can be used as a measure of submaximal exercise to measure functional capacity in ambulatory patients in combination with cardiopulmonary, neuromuscular and musculoskeletal function. The 6-minute walk test (6MWT) was originally developed by the American Thoracic Society (ATS2002) for adults and is now commonly used in adults and pediatric populations (Mylius et al., 2016), as well as children with neuromuscular diseases such as spinal muscular atrophy (Montes et al., 2018), Duchenne muscular dystrophy (McDonald et al., 2013) and infantile Pompe disease (van der Meijden et al., 2018). The 2-minute walk test (2MWT) is included in the NIH toolbox and is increasingly being used to measure the same properties.

[0396] 6MWT and 2MWT can be administered to patients at the discretion of a healthcare provider before and during treatment. If a subject cannot complete at least 2MWT at baseline, additional assessment during treatment can be determined by a healthcare provider. Resting heart rate is obtained before and after the test. The distance walked in the first 2 minutes of the 6MWT and the entire 6 minutes can be recorded. The distance walked in 2 minutes and 6 minutes can be compared with normal data matched for age and gender (percentage of predicted value).

[0397] 3. Muscle strength measurement

[0398] Strength may be assessed using dynamometers at the discretion of the healthcare provider before and / or during treatment. Handheld dynamometers are a direct measure of strength and are commonly used in children and adults. Muscle groups that may be assessed include: shoulder abduction, shoulder flexion, elbow flexion, elbow extension, hip abduction, hip flexion, hip extension, and knee extension. Each muscle group may be measured 2 times on each side.

[0399] a. Grip strength. Grip strength may be measured using a handgrip dynamometer at the discretion of the healthcare provider prior to and / or during treatment. The equipment and assessor instructions may be standardized across sites. Grip strength may be assessed bilaterally with one exercise and one maximal force measurement performed for each hand, and the results may be compared to age- and sex-matched normative data, if available.

[0400] b. Range of Motion. Range of motion can be assessed with a goniometer, an instrument that tests joint angles and measures the extent of movement at a joint. The fixed arm of the goniometer is aligned with designated bony landmarks on the fixed body segment, and the moving arm of the goniometer is aligned with designated bony landmarks on the limb that is moving. For each movement measured using an axis of motion and a bony landmark, a fulcrum of the goniometer is specified. Range of motion can assess one or more of the following: shoulder abduction, shoulder flexion, elbow flexion, elbow extension, hip abduction, hip flexion, hip extension, and knee extension.

[0401] 4. Hearing test

[0402] Moderate hearing loss is associated with ARHR2 (Brachet et al., 2014; Steichen-Gersdorf et al., 2015). Baseline hearing can be determined by one or more of the following: physical examination and otoscopy, immittance audiometry (often called tympanometry), pure tone audiometry (PTA) up to 8 kHz if possible (if PTA thresholds are >15 dB, the subject should also undergo bone conduction testing), high frequency audiometry (HFA) up to 16 kHz.

[0403] 5. Clinician Global Impression Scale

[0404] The Clinical Global Impression (CGI-S) scale was developed for use in clinical studies sponsored by the National Institute of Mental Health to provide a brief, independent assessment of the clinician's perception of the patient's overall functioning before and after initiation of a study drug (Guy 1976). The CGI provides an overall summary measure determined by the clinician that takes into account all available information, including knowledge of the patient's history, psychosocial environment, symptoms, behaviors, and the impact of symptoms on the patient's functional ability. The CGI-S may be administered before and / or during treatment, as determined by the healthcare provider, and provides an overall assessment of change using a seven-point scale ranging from -3 (severely worsened) to +3 (much improved).

[0405] 6. Gross Motor Function Classification System - Expanded and Revised

[0406] The Gross Motor Function Classification System-Expanded and Revised (GMFCS-E and R) can be administered before and / or during treatment, as determined by the healthcare provider. The GMFCS-E and R classify patient-initiated movements, focusing on movements that are rated 1 to 5 on a scale.

[0407] 7. Patient-Reported Outcomes Measurement Information System

[0408] The Patient-Reported Outcomes Measurement Information System (PROMIS) consists of a variety of questionnaires developed by the National Institutes of Health (NIH) to assess physical, mental, and social well-being from the patient's perspective (www.healthmeasures.net). These questionnaires have been used in clinical studies of people with chronic health conditions such as X-linked hypophosphatemia, arthritis, multiple sclerosis, and neurofibromatosis. Each questionnaire contains 8 to 10 items that are rated by the participant on a 5-point Likert scale ranging from 1 (never) to 5 (always). The scores for each questionnaire are summed, with higher scores indicating more of the domain being measured (e.g., more fatigue, more physical function). Raw scores are converted to T-scores based on a mean of 50 and a standard deviation of 10 to allow comparison of study samples with the general population. PROMIS scales may include pain interference (Short Form 8a), pain intensity (version 3a), physical function - upper extremity (customized short form), physical function - mobility (Short Form 13a FACIT fatigue), fatigue (Short Form), and cognitive impact (Short Form 8a) and may be administered before and / or during treatment at the discretion of the healthcare provider. These assessments may be completed by the subject without assistance.

[0409] 8. Caregiver Global Impression Scale

[0410] The Caregiver Global Impression of Status may be administered to the patient's caregiver prior to and / or during treatment at the discretion of the healthcare provider. The Caregiver Global Impression of Change provides an overall assessment of change using a seven-point scale ranging from -3 (severely worsened) to +3 (much improved).

[0411] 9. Western Ontario and McMaster Universities Osteoarthritis Index

[0412] WOMAC is the result of patient report, is used to assess the daily living activities, functional activities, gait, general health, pain and quality of life (www.sralab.org) of the patient with hip or knee pain. This assessment is made up of 24 items, takes about 12 minutes to apply. WOMAC can be applied before treatment and / or during treatment, and it is determined by the medical service provider. This assessment can be completed by the experimenter without help.

[0413] Example 13: Comparison of soluble (Construct 1118, SEQ ID NO: 118) and bone-targeted (Construct 2000, SEQ ID NO: 119) ENPP1-Fc on cervical spine and auditory phenotypes

[0414] Fig.13 Shown are 17-week-old WT and 17-week-old Enpp1 mice dosed weekly with the indicated subcutaneous doses of vehicle or ENPP1 constructs #1118 and #2000.asj Paraspinal osteophytes and ankylosis in male mice. Construct #1118 was administered weekly starting at week 3, while construct #2000 was administered weekly starting at week 5. Micro-CT images show that Enpp1 is preferentially expressed in the presence of construct #2000. asj Attenuation of paraspinal osteophytes and ankylosis in male mice.

[0415] Fig.14 Shown are 17-week-old WT and 17-week-old Enpp1 mice dosed weekly with the indicated subcutaneous doses of vehicle or ENPP1 constructs #1118 and #2000. asj Paraspinal osteophytes and ankylosis in female mice. Construct #1118 was administered weekly starting at week 3, while construct #2000 was administered weekly starting at week 5. Micro-CT images showed that Enpp1 was preferentially expressed in the presence of construct #2000. asj Attenuation of paraspinal osteophytes and ankylosis in female mice.

[0416] Figures 15A-15B Shown are 17-week-old WT and 17-week-old Enpp1 mice dosed weekly with the indicated subcutaneous doses of vehicle or ENPP1 constructs #1118 and #2000. asj Auditory brainstem responses in mice. Construct #1118 was dosed once a week starting from week 3, while construct 2000 was dosed once a week starting from week 5. Stimulus frequency measurements showed that ENPP1 construct #1118 at a dose of 2 mg / Kg per week, as well as ENPP1 construct #2000 at doses of 0.5 and 1 mg / Kg per week, could prevent hearing loss in the low frequency (8kHz) range. It was also noted that the hearing of ENPP1 deficient animals was improved in the high frequency range (32kHz), preferably in ENPP1 construct #2000 dosed at 1 mg / Kg per week.

[0417] Fig.16 Shown are WT and 17-week-old ENPP1 cells treated weekly with indicated subcutaneous doses of vehicle or ENPP1 constructs #1118 and #2000. asjComplete FGF23 levels in mice. Construct #1118 was administered once a week starting from week 3, while construct #2000 was administered once a week starting from week 5. The data show that when administered at 1 mg / Kg and 4 mg / Kg per week, ENPP1 construct #2000 preferably inhibits complete FGF23. Statistical significance was assessed by ANOVA Kruskal-Wallis test, followed by Dunn's post hoc analysis to assess differences from WT levels (one-way ANOVA). Statistical significance is represented by p value, with the following annotations: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

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[0472] Additional references for Examples 3-12

[0473] ATS. American Thoracic Society Statement: Guidelines for the six-minute walk test. Am J Respir, Crit Care Med. 2002 2002; 166(1): 111-7.

[0474] Brachet C, Mansbach AL, Clerckx A, Deltenre P, Heinrichs C. Hearing loss is part of the clinical picture of ENPP1 loss of function mutation. Horm Res Paediatr. 2014;81(1):63-6.

[0475] CTFG. Recommendations related to contraception and pregnancy testing in clinical trials. 2014[19 May 2020]; Available from:

[0476] http: / / www.hma.eu / fileadmin / dateien / Human_Medicines / 01-

[0477] About HMA / Working Groups / CTFG / 2014 09HMA CTFG Contraception.pdf.

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[0479] Guy W. The Clinical Global Impression Scale. In: Rush Jr AJ, First MB, Blacker D, editors. Handbook of Psychiatric Measures, 2008. Washington, DC: American Psychiatric Publishing, Inc; 1976. p.90-2.

[0480] IOF. Autosomal Recessive Hypophosphatemic Rickets Type 2 (ARHR2). 2019 [19 May 2020]; Available from: https: / / www.iofbonehealth.org / osteoporosis-musculoskeletal-disorders / skeletal-rare-disorders / autosomal-recessive-hypophosphatemi-0.

[0481] Mackenzie NC, Huesa C, Rutsch F, MacRae VE. New insights into NPP1 function: lessons from clinical and animal studies. Bone. 2012 Nov; 51(5): 961 - 8.

[0482] McDonald CM, Henricson EK, Abresch RT, Florence JM, Eagle M, Gappmaier E, et al. The 6 - minute walk test and other endpoints in Duchenne muscular dystrophy: longitudinal natural history observations over 48 weeks from a multicenter study. Muscle Nerve. 2013 Sep; 48(3): 343 - 56.

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[0484] Ambulatory function in spinal muscular atrophy: Age - related patterns of progression. PLoS One. 2018; 13(6): e0199657.

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[0490] Examples of implementation methods

[0491] The following exemplary embodiments are provided, and their numbering is not to be construed as designating a level of importance.

[0492] Embodiment 1. A method for treating, ameliorating, preventing further development and / or progression of diffuse idiopathic skeletal hyperostosis (DISH), ankylosing spondylitis and / or spondyloarthritis in a patient in need thereof, and / or preventing diffuse idiopathic skeletal hyperostosis (DISH), ankylosing spondylitis and / or spondyloarthritis in the patient, the method comprising administering to the patient a therapeutically effective amount of a compound of formula (I) or a salt or solvate thereof:

[0493] Protein-Z-Domain-XY

[0494] (I),

[0495] Wherein in (I):

[0496] The protein includes the catalytic domain of ENPP1;

[0497] The domain is absent or is at least one selected from the group consisting of human IgG Fc domain (Fc), human serum albumin (ALB) and fragments thereof;

[0498] X and Z are independently absent or are a polypeptide comprising 1-20 amino acids, and

[0499] Y is a negatively charged bone targeting sequence;

[0500] Thereby treating, ameliorating, preventing further development and / or progression of DISH, ankylosing spondylitis and / or spondyloarthritis in said patient and / or preventing DISH and / or spondyloarthritis in said patient.

[0501] Embodiment 2. The method according to embodiment 1, wherein the patient has ENPP1 haploinsufficiency.

[0502] Embodiment 3. The method of embodiment 1, wherein the patient does not have ENPP1 haploinsufficiency.

[0503] Embodiment 4. The method according to any one of embodiments 1-3, wherein the patient is not ENPP1 deficient.

[0504] Embodiment 5. The method according to any one of embodiments 1-3, wherein the patient is ENPP1 deficient.

[0505] Embodiment 6. The method of any one of embodiments 1-5, wherein the patient is administered the compound by at least one route selected from the group consisting of oral, aerosol, inhalation, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ophthalmic, pulmonary, and topical.

[0506] Embodiment 7. The method according to any one of embodiments 1-6, wherein the compound is administered to the patient by intravenous or subcutaneous injection.

[0507] Embodiment 8. The method of any one of embodiments 1-7, wherein administering the compound to the patient increases the patient's extracellular pyrophosphate concentration or prevents the patient's extracellular pyrophosphate concentration from further decreasing.

[0508] Embodiment 9. The method of any one of embodiments 1-8, wherein administering the compound to the patient reduces one or more of the patient's Achilles tendon calcification, spinal calcification, hip joint calcification, and bilateral calcification or prevents further increase in one or more of the patient's Achilles tendon calcification, spinal calcification, hip joint calcification, and bilateral calcification.

[0509] Embodiment 10. A method according to any one of embodiments 1-9, wherein the domain comprises albumin.

[0510] Embodiment 11. A method according to any one of embodiments 1-10, wherein the domain comprises an IgG Fc domain.

[0511] Embodiment 12. The method of any one of embodiments 1-11, wherein the protein lacks the ENPP1 transmembrane domain.

[0512] Embodiment 13. The method according to any one of embodiments 1-12, wherein the compound is administered to the patient as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

[0513] Embodiment 14. The method of any one of embodiments 1-13, wherein the patient is a mammal.

[0514] Embodiment 15. The method of embodiment 14, wherein the mammal is a human.

[0515] Embodiment 16. The method of any one of embodiments 1-15, wherein the protein comprises amino acid residues 99 (PSCAKE) to 925 (QED) of SEQ ID NO: 1.

[0516] Embodiment 17. The method of any one of embodiments 1-16, wherein the protein comprises amino acid residues 1 to 833 of SEQ ID NO:3.

[0517] Embodiment 18. A method according to any one of embodiments 1-16, wherein the protein comprises the amino acid sequence depicted in SEQ ID NO:2.

[0518] Embodiment 19. A method according to any one of embodiments 1-16, wherein the protein comprises the amino acid sequence depicted in SEQ ID NO: 3 or 4 or 5.

[0519] Embodiment 20. A method according to any one of embodiments 1-19, wherein the domain increases the circulating half-life of the compound relative to the circulating half-life of the compound lacking the domain.

[0520] Embodiment 21. A method according to any one of embodiments 1-20, wherein the patient is also diagnosed with a disease or condition selected from the group consisting of early-onset osteoporosis, osteopenia, age-related osteopenia, OPLL, hereditary hypophosphatemic rickets, X-linked hypophosphatemia, autosomal recessive hypophosphatemic rickets type 2, autosomal dominant hypophosphatemic rickets, and hypophosphatemic rickets.

[0521] Embodiment 22. A method according to any one of embodiments 1-20, wherein the patient has not been diagnosed with a disease or condition selected from the group consisting of early-onset osteoporosis, osteopenia, age-related osteopenia, OPLL, hereditary hypophosphatemic rickets, X-linked hypophosphatemia, autosomal recessive hypophosphatemic rickets type 2, autosomal dominant hypophosphatemic rickets, and hypophosphatemic rickets.

[0522] Embodiment 23. A method according to any one of embodiments 1-22, wherein the patient suffers from a disease or condition selected from the group consisting of early-onset osteoporosis, osteopenia, age-related osteopenia, OPLL, hereditary hypophosphatemic rickets, X-linked hypophosphatemia, autosomal recessive hypophosphatemic rickets type 2, autosomal dominant hypophosphatemic rickets, and hypophosphatemic rickets.

[0523] Embodiment 22. A method according to any one of embodiments 1-22, wherein the patient does not suffer from a disease or condition selected from the group consisting of early-onset osteoporosis, osteopenia, age-related osteopenia, OPLL, hereditary hypophosphatemic rickets, X-linked hypophosphatemia, autosomal recessive hypophosphatemic rickets type 2, autosomal dominant hypophosphatemic rickets, and hypophosphatemic rickets.

[0524] Incorporated by Reference

[0525] All publications and patents mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0526] Other Implementations

[0527] Although specific embodiments of the present subject matter have been discussed, the above description is illustrative rather than restrictive. Many variations will become apparent to those skilled in the art upon reading this specification and the claims that follow. The full scope of the invention should be determined by reference to the claims and their equivalents, as well as the specification and the full scope of these variations.

Claims

1. A method for treating, ameliorating, preventing the further development and / or progression of diffuse idiopathic skeletal hyperostosis (DISH) and / or spondyloarthritis in a patient in need thereof, and / or preventing diffuse idiopathic skeletal hyperostosis (DISH) and / or spondyloarthritis in the patient, the method comprising administering to the patient a therapeutically effective amount of a compound of formula (I) or a salt or solvate thereof: Protein-Z-Domain-XY (I), Wherein in (I): The protein includes the catalytic domain of ENPP1; The domain is absent or is at least one selected from the group consisting of human IgG Fc domain (Fc), human serum albumin (ALB) and fragments thereof; X and Z are independently absent or are a polypeptide comprising 1-20 amino acids, and Y is a negatively charged bone targeting sequence; Thereby treating, ameliorating, preventing further development and / or progression of DISH and / or spondyloarthritis in said patient and / or preventing DISH and / or spondyloarthritis in said patient.

2. The method of claim 1, wherein the patient has ENPP1 haploinsufficiency.

3. The method of claim 1, wherein the patient does not have ENPP1 haploinsufficiency.

4. The method of claim 1, wherein the patient is not ENPP1 deficient.

5. The method of claim 1, wherein the patient is ENPP1 deficient.

6. The method of claim 1, wherein the patient is administered the compound by at least one route selected from the group consisting of oral, aerosol, inhalation, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ophthalmic, pulmonary, and topical.

7. The method of claim 1, wherein the compound is administered to the patient by intravenous or subcutaneous injection.

8. The method of claim 1, wherein administering the compound to the patient increases the patient's extracellular pyrophosphate concentration or prevents the patient's extracellular pyrophosphate concentration from further decreasing.

9. The method of claim 1, wherein administering the compound to the patient reduces one or more of Achilles tendon calcification, spinal calcification, hip joint calcification, and bilateral calcification in the patient or prevents further increase in one or more of Achilles tendon calcification, spinal calcification, hip joint calcification, and bilateral calcification in the patient.

10. The method of claim 1, wherein the domain comprises albumin.

11. The method of claim 1, wherein the domain comprises an IgG Fc domain.

12. The method of claim 1, wherein the protein lacks the ENPP1 transmembrane domain.

13. The method of claim 1, wherein the compound is administered to the patient as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

14. The method of claim 1, wherein the patient is a mammal.

15. The method of claim 14, wherein the mammal is a human.

16. The method of any one of claims 1-15, wherein the protein comprises amino acid residues 99 (PSCAKE) to 925 (QED) of SEQ ID NO:

1.

17. The method of any one of claims 1-16, wherein the protein comprises amino acid residues 1 to 833 of SEQ ID NO:

3.

18. The method of any one of claims 1-16, wherein the protein comprises the amino acid sequence depicted in SEQ ID NO:

2.

19. The method of any one of claims 1-16, wherein the protein comprises the amino acid sequence depicted in SEQ ID NO: 3 or 4 or 5.

20. The method of any one of claims 1-19, wherein the domain increases the circulating half-life of the compound relative to the circulating half-life of the compound lacking the domain.

21. The method of claim 1, wherein the patient is further diagnosed with a disease or condition selected from the group consisting of early-onset osteoporosis, osteopenia, age-related osteopenia, OPLL, hereditary hypophosphatemic rickets, X-linked hypophosphatemia, autosomal recessive hypophosphatemic rickets type 2, autosomal dominant hypophosphatemic rickets, and hypophosphatemic rickets.

22. The method of claim 1, wherein the patient has not been diagnosed with a disease or condition selected from the group consisting of early-onset osteoporosis, osteopenia, age-related osteopenia, OPLL, hereditary hypophosphatemic rickets, X-linked hypophosphatemia, autosomal recessive hypophosphatemic rickets type 2, autosomal dominant hypophosphatemic rickets, and hypophosphatemic rickets.

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