Methods and compositions for treating, ameliorating, and / or preventing mineralization disorders

Abstract

Bone-targeting ENPP1 constructs fused to a human IgG Fc domain, or human serum albumin protein, and a bone-targeting sequence are disclosed. Methods relating to hearing loss comprising administering the constructs are further disclosed.

Description

[0001] Attorney Docket No. 047162-7564W01(02906)

[0002] TITLE

[0003] Methods and Compositions for Treating, Ameliorating, and / or Preventing Mineralization Disorders

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims priority under 35 U. S. C. § 119(e) to U. S. Provisional Patent Application No. 63 / 788,948 filed April 15, 2025, which is incorporated herein by reference in its entirety.

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0007] This invention was made with government support under AR080416-01 awarded by National Institutes of Health. The government has certain rights in the invention.

[0008] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0009] The XML file named “047162-7564WO1 created on April 14, 2026, comprising 153,061 bytes, is incorporated herein by reference in its entirety.

[0010] BACKGROUND

[0011] Ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) is a type-2 transmembrane protein whose extracellular activity hydrolyzes phosphodi ester bonds of extracellular nucleotides, such as adenosine triphosphate (ATP), to generate adenosine monophosphate (AMP) and inorganic pyrophosphate (PPi). Because PPi is the main physiologic inhibitor of hydroxyapatite deposition, biallelic ENPP1 deficiency leads to ectopic mineralization in early life, with high mortality in infancy resulting from arterial calcification and luminal narrowing. Patients who survive, as well as ENPP1 -deficient individuals who do not exhibit neonatal calcifications, can develop fibroblast growth factor 23 (FGF23)-mediated hypophosphatemic rickets in later life.

[0012] PCT Application Publication No. WO2014126965 relates to treatment of pathological calcification and pathological ossification in diseases involving ENPP1 deficiency. Three proteins — ENPP1, tissue-nonspecific alkaline phosphatase (TNAP), and progressive ankylosis protein or ankyrin (ANK) — play a role in regulating the extracellular balance of inorganic pyrophosphate (PPi) and phosphate (Pi). Inorganic pyrophosphate (PPi) is generated by the cleavage of extracellular nucleotide triphosphates (NTPs) by ENPP1 or the - 1 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0013] transfer of PPi from the intracellular to extracellular space by Ank. TNAP degrades PPi to generate Pi.

[0014] ■‘ENPP1 Deficiency” is characterized by a reduced level of ENPP1 enzymatic activity in serum and / or plasma of a subject. ENPP1 Deficiency is a rare genetic disorder caused by inactivating mutations in the ENPP1 gene that encodes the ENPP1 enzy me. As described elsewhere herein, ENPP1 is an integral transmembrane protein whose extracellular domains carry pyrophosphatase and phosphodiesterase activities. As such, ENPP1 converts extracellular ATP to inorganic pyrophosphate (PPi) and AMP.

[0015] Calcification in biological systems is a complex process by which calcium salts are maintained at higher concentrations in noncirculating matrices than in regional circulating humoral or other mobile fluids. The principal result of normal calcification is the accumulation of calcium and associated inorganic salts in crystalline patterns of similar arrangement and chemical composition in specialized intercellular matrices, all of which might vary among species. On the other hand, the net effect of pathological calcification is the accumulation of calcium and associated inorganic salts with a greater-than-normal range in chemical composition or diversity of pattern, not only in these specialized matrices, but also in other intercellular, extracellular, and cellular materials thereby leading to several disease states.

[0016] BRIEF DESCRIPTION

[0017] The disclosure provides a compound comprising formula (I):

[0018] W-PROTEIN-Z-DOMAIN-X-Y (I), wherein in (I):

[0019] W is absent or comprises a signal sequence which allows for export of the compound into extracellular space;

[0020] PROTEIN comprises the catalytic region of ENPP1;

[0021] DOMAIN is absent or at least one selected from the group consisting of a human IgG Fc domain (Fc), human serum albumin protein (ALB), and a biologically active fragment thereof;

[0022] X and Z are independently absent or a polypeptide comprising 1-20 amino acids; and

[0023] Y is a “bone targeting” sequence group.

[0024] The disclosure further provides a method of restoring, improving, and / or preventing further loss of hearing in a subject. In certain embodiments, the method comprises

[0025] - 2 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0026] administering to the subject a therapeutically effective amount of any compound contemplated in the disclosure.

[0027] BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, non-limiting illustrative embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0029] FIGs. 1 A-1D: Study design and definition of the region of interest for analyzing the auditory ossicles (malleus). (FIG. 1 A) Schematic representation of the enzyme replacement therapy (ERT) regimen. (FIG. IB) Representative pCT scan of the ossicles within the temporal bone. (FIG. 1C) Three-dimensional and (FIG. ID) cross-section view of the malleus showing the defined region of interest (malleal processus brevis).

[0030] FIGs. 2A-2F: Blood plasma measurements in Enppl^''^ mice with different ERT formulations compared to untreated Enpplasj'asJmice and WT mice. (FIG. 2A) Quantification of alkaline phosphatase (ALP), (FIG. 2B) phosphorus, (FIG. 2C) calcium, (FIG. 2D) PPi, (FIG. 2E) FGF23 and (FIG. 2F) PTH. Statistical analysis was conducted with ANOVA with significance reported by Sidak’s multiple comparisons tests comparing WT controls with untreated Enpplasj / asjmice and WT or untreated Enpplasj / asjmice with all treatment groups. *p < 0.05, **p < 0.01, ***p < 0.001. WT: w=24, Enpplasj,as}+ veh: n=17, Enpplasj,asj+ 1 mg / kg 1118: 17=14, Enpplasj,asj+ 2 mg / kg 1118: w=12, Enpplasi'asj+ 4 mg / kg 1118: w=13, Enpplas} / as}+ 0.5 mg / kg 2000: w=16, Enpplas} asj+ 1 mg / kg 2000: n= 8.

[0031] FIGs. 3A-3C: Functional hearing tests (ABR thresholds) in Enpplasj / asjmice with different ERT formulations compared to untreated Enpplas] as]and WT mice. ABR measurements of 17-week old wild-type, untreated and treated Enpplast / as}mice at (FIG. 3 A) 8, (FIG. 3B) 16, and (FIG. 3C) 32 kHz. Statistical analysis was conducted with ANOVA with significance reported by Sidak’s multiple comparisons tests comparing WT controls with untreated Enpplasj / as]mice and WT or untreated Enpplusj / as]mice with all treatment groups. *p < 0.05, **p < 0.01, ***p < 0.001. WT: w=5, Enpplas' / as]+ veh: w=10, Enpplas} / asi+ 1 mg / kg 1118: n=l, Enpplas} / as]+ 2 mg / kg 1118: n=1, Enpplas] / as]+ 4 mg / kg 1118: n=8, Enpplasj'asj+ 0.5 mg / kg 2000: =8, Enpplasj / asj+ 1 mg / kg 2000: n=l.

[0032] FIGs. 4A-4E: Mineralization properties in the different groups. (FIG. 4A) Representative quantitative backscattered electron images of the malleus from 17-week old - 3 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0033] wild-type, untreated and treated Enpplas] / as]mice displaying the mineralization levels in pseudocolors (high and low mineralization are represented by bright and dark pixels, respectively). Scale bars: 100 pm. Quantitative analysis of (FIG. 4B) mean mineralization (CaMean), (FIG. 4C) peak mineralization properties (CaPeak) and (FIG. 4D) mineralization heterogeneity (CaWidth) of the malleus. Statistical analysis in FIGs. 4B-4D was conducted with ANOVA with significance reported by Sidak’s multiple comparisons tests comparing WT controls with untreated Enp ■ mice and WT or untreated Enpplasj as]mice with all treatment groups. WT: 77=12, Enpplas>asi+ veh: n=5, Enpplasi / / asJ + 1 mg / kg 1118: n=5, Enpplasj / asj+ 2 mg / kg 1118: n=5, Enpplasj / as}+ 4 mg / kg 1118: n=5, Enpplas] / as]+ 0.5 mg / kg 2000: 77=13, Enpplasi / asj+ 1 mg / kg 2000: 77=13. FIG. 4E: Comparison of CaMean between the malleus and femur of WT and Enppl -deficient mice (ANOVA (Sidak). WT: malleus, 77=12, femur, «=7, Enpplas] asjmalleus, n=5, femur, n=7). *p < 0.05, **p < 0.01, ***p < 0.001.

[0034] FIGs. 5A-5I: Osteocyte lacunar phenotype in Enpplasi / as}mice with different ERT formulations compared to untreated Enpplasj / aSj' and WT mice. (FIG. 5 A) Representative quantitative backscattered electron images of the malleus from 17-week old wild-type, untreated and treated Enpplas^asjmice osteocyte lacunar morphology. Quantification of (FIG.

[0035] 5B) osteocyte lacunar size, (FIG. 5C) density of osteocyte lacunae as well as (FIG. 5D) density and (FIG. 5E) proportion of mineralized lacunae. Statistical analysis in FIGs. 5B-5E was conducted with ANOVA with significance reported by Sidak’s multiple comparisons tests comparing WT controls with untreated Enpplasj / asjmice and WT or untreated Enpplasj / asjmice with all treatment groups. WT: 77=12, Enpplas},asj+ veh: 77=5, Enpplas' / as}+ 1 mg / kg 1118: 77=5, Enpplasj / asj+ 2 mg / kg 1118: 77=5, Enpplas} / as]+ 4 mg / kg 1118: 7 =5, Enpplasj / asj+ 0.5 mg / kg 2000: 77=13, Enpplas} as]+ 1 mg / kg 2000: 77=13. FIG. 4E: Comparison of CaMean between the malleus and femur of WT and Enppl -deficient mice (ANOVA (Sidak), WT: malleus, 77=12, femur, n=7. Enpp1asj / asj-. malleus, n=5, femur, n=7). *p < 0.05, **p < 0.01, ***p < 0.001. (FIG. 5F) High-magnification qBEI images of mineralized lacunae in the malleus and (FIG. 5G) quantification of mean lacunar mineralization (CaMean, wt%) of Enppl -deficient untreated and soluble ERT mice (upper panel). Enpplas] as}+ veh: n=5, EnpplUSJ'“SJ+ Img / kgl 118: n=5, Enpplasj,asj+ 2mg / kgl 118: n=5, Enpp1asj / asj+ 4mg / kg 1118: n=4. Pooled BMD distribution of osteocyte lacunae of all analyzed groups indicates a low' and inhomogeneous mineralization pattern of mineralized osteocyte lacunae compared to the bone matrix (lower panel). (FIG. 5H) Representative layered EDX image of a mineralized osteocyte lacuna (encircled by a dotted line) and adjacent bone in a malleus of an Enppl -deficient mouse treated with 1 mg / kg ERT1118. The pseudo-colored image represents the - 4 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0036] weight percentage distribution of calcium (Ca), phosphate (P), oxide (O), carbon (C), magnesia (Mg), and sodium (Na) and indicates a similar proportion in the matrix and lacuna. (FIG. 51) Quantification of lacunar mineral composition (Ca / P ratio) by EDX in the malleus of Enppl -deficient untreated and soluble ERT mice (upper panel) and pooled comparison of all lacunae with the surrounding bone matrix. Veh, vehicle (untreated). Enpp lasj / asj+ veh: n=5. Enpplasj / asj+ 1 mg / kg 1118: n=5. Enpp1asj / asj+ 2 mg / kg 1118: n=5, Enpplasi'asj+ 4 mg / kg 1118: n=4. *p < 0.05, **p < 0.01, ***p < 0.001.

[0037] DESCRIPTION

[0038] Hearing loss is common in conditions caused by ENPP1 deficiency, such as generalized arterial calcification of infancy and autosomal recessive hypophosphatemic rickets type 2. Mechanistically, it is hypothesized that poor mineralization of the auditory ossicles leads to impaired sound transmission in the middle ear (i.e. conductive hearing loss). Unfortunately, therapeutic options remain underexplored. For example, oral phosphate supplementation has been investigated but did not provide sufficient improvements regarding the skeletal abnormalities of the patients and was further associated with nephrocalcinosis.

[0039] Hearing loss is defined by an average pure-tone threshold detection exceeding 20 dB, affecting approximately 466 million people worldwide. According to the value of pure tone thresholds, it can be classified as mild (20-35 dB), moderate (35-50 dB). moderately severe (50-65 dB), severe (65-80 dB), profound (80-95 dB), and total (>95 dB) hearing loss. As used herein, ‘'neurological hearing loss” refers to hearing loss caused by damage to the inner ear or the auditory nerve pathway. As used herein, “conductive hearing loss” occurs when sound waves are blocked from reaching the inner ear, typically due to problems in the outer or middle ear.

[0040] The present study investigated whether enzyme replacement therapy (ERT) improves hearing loss in an Enppl deficient mouse model and whether this is associated with corrected bone properties in the ossicles. For this purpose, Enpplasj as}mice were subjected to either a soluble or bone-targeted ERT of ENPPl-Fc. Hearing function and bone properties of the malleus, the first bone of the ossicular chain, were evaluated compared to untreated and wild-type mice. The hearing deficits observed in Enpp las-l / asJ mice were partially or fully corrected by both soluble and bone-targeted ERT, as evidenced by restoration of auditory brainstem response thresholds, with dose dependence and generally stronger effect of bone-targeted ERT. This was associated not only with corrected blood plasma markers of mineral metabolism, but also improved matrix mineralization and restored osteocyte properties in the - 5 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0041] malleus. The present study further evidenced mineralized osteocyte lacunae in mice for the first time, fully corrected by the bone-targeted, but not soluble, ERT. In contrast to the human phenomenon of osteocyte lacunar mineralization (micropetrosis), the intra-lacunar mineral was overall less mineralized than the adjacent bone. In conclusion, the present results point toward the potential benefit of correcting ossicular mineralization to prevent hearing loss in ENPP1 deficiency.

[0042] As described elsewhere herein, the present study aimed to investigate newly developed ERT formulations to ameliorate functional hearing impairments and to correct both structural and cellular alterations of the auditory ossicles in the Enpplas, / as]mouse model. Specifically, the present study examined the effects of a soluble (ERT 1118) and bone-targeted (ERT 2000) form of ENPP1, aiming to decipher how these treatments differentially influence hearing capacity and middle ear bone matrix properties.

[0043] In certain embodiments, the present invention allows for restoring osteocyte activity in the malleus. In certain embodiments, the present invention allows for restoring and / or improving mineralization of the auditor)’ ossicles. In certain embodiments, the present invention allows for restoring and / or improving sound transmission in the middle ear (i.e. conductive hearing loss). In certain embodiments, the present invention allows for restoring, improving, and / or preventing further loss of hearing in a subject. In certain embodiments, the subject has ENPP1 haploinsufficiency. In certain embodiments, the subject does not have ENPP1 haploinsufficiency. In certain embodiments, the subject is not ENPP1 deficient. In certain embodiments, the subject is ENPP1 deficient. In certain embodiments, the hearing loss is conductive in nature. In certain embodiments, the hearing loss is neurological in nature. In certain embodiments, the hearing loss is conductive and / or neurological in nature.

[0044] Treatment of the diseases and / or disorders contemplated herein comprises administering an ENPP1 agent at a dose of about 0.1 mg per kilogram, about 0.2 mg per kilogram of the subject, about 0.3 mg per kilogram of the subject, about 0.4 mg per kilogram of the subject, about 0.5 mg per kilogram of the subject, about 0.6 mg per kilogram of the subject, about 0.7 mg per kilogram of the subject, about 0.8 mg per kilogram of the subject, about 0.9 mg per kilogram of the subject, about 1.0 mg per kilogram of the subject, about 1.1 mg per kilogram of the subject, about 1.2 mg per kilogram of the subject, about 1.3 mg per kilogram of the subject, about 1.4 mg per kilogram of the subject, about 1.5 mg per kilogram of the subject, about 1.6 mg per kilogram of the subject, about 1.7 mg per kilogram of the subject, about 1.8 mg per kilogram of the subject, about 1.9 mg per kilogram of the subject, or about 2.0 mg per kilogram of the subject, thereby treating, reducing, and / or ameliorating - 6 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0045] one or more symptoms of the diseases and / or disorders contemplated herein, wherein the subject does not have ENPP1 Deficiency.

[0046] In one aspect, the disclosure relates to administering an ENPP1 agent to a subject having a disease and / or disorder contemplated herein at a dose of about 0.1 mg per kilogram, about 0.2 mg per kilogram of the subject, about 0.3 mg per kilogram of the subject, about 0.4 mg per kilogram of the subject, about 0.5 mg per kilogram of the subject, about 0.6 mg per kilogram of the subject, about 0.7 mg per kilogram of the subject, about 0.8 mg per kilogram of the subject, about 0.9 mg per kilogram of the subject, about 1.0 mg per kilogram of the subject, about 1.1 mg per kilogram of the subject, about 1.2 mg per kilogram of the subject, about 1.3 mg per kilogram of the subject, about 1.4 mg per kilogram of the subject, about 1.5 mg per kilogram of the subject, about 1.6 mg per kilogram of the subject, about 1.7 mg per kilogram of the subject, about 1.8 mg per kilogram of the subject, about 1.9 mg per kilogram of the subject, or about 2.0 mg per kilogram of the subject, thereby treating, reducing, and / or ameliorating one or more symptoms of a disease and / or disorder contemplated herein, wherein the subject has ENPP1 Deficiency.

[0047] In one aspect, the disclosure relates to administering to a subject having a disease and / or disorder contemplated herein an ENPP1 agent at a dose of about 0.1 mg per kilogram, about 0.2 mg per kilogram of the subject, about 0.3 mg per kilogram of the subject, about 0.4 mg per kilogram of the subject, about 0.5 mg per kilogram of the subject, about 0.6 mg per kilogram of the subject, about 0.7 mg per kilogram of the subject, about 0.8 mg per kilogram of the subject, about 0.9 mg per kilogram of the subject, about 1.0 mg per kilogram of the subject, about 1.1 mg per kilogram of the subject, about 1.2 mg per kilogram of the subject, about 1.3 mg per kilogram of the subject, about 1.4 mg per kilogram of the subject, about 1.5 mg per kilogram of the subject, about 1.6 mg per kilogram of the subject, about 1.7 mg per kilogram of the subject, about 1.8 mg per kilogram of the subject, about 1.9 mg per kilogram of the subject, or about 2.0 mg per kilogram of the subject, thereby treating, reducing, and / or ameliorating one or more symptoms of a disease and / or disorder contemplated herein, wherein the subject does not have ENPP1 haploinsufficiency.

[0048] In one aspect, the disclosure relates to administering to a subject having a disease and / or disorder contemplated herein an ENPP1 agent at a dose of about 0.1 mg per kilogram, about 0.2 mg per kilogram of the subject, about 0.3 mg per kilogram of the subject, about 0.4 mg per kilogram of the subject, about 0.5 mg per kilogram of the subject, about 0.6 mg per kilogram of the subject, about 0.7 mg per kilogram of the subject, about 0.8 mg per kilogram of the subject, about 0.9 mg per kilogram of the subject, about 1.0 mg per kilogram of the - 7 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0049] subject, about 1.1 mg per kilogram of the subject, about 1.2 mg per kilogram of the subject, about 1.3 mg per kilogram of the subject, about 1.4 mg per kilogram of the subject, about 1.5 mg per kilogram of the subject, about 1.6 mg per kilogram of the subject, about 1.7 mg per kilogram of the subject, about 1.8 mg per kilogram of the subject, about 1.9 mg per kilogram of the subject, or about 2.0 mg per kilogram of the subject, thereby treating, reducing, and / or ameliorating one or more symptoms of a disease and / or disorder contemplated herein, wherein the subject has ENPP1 haploinsufficiency.

[0050] In one aspect, the disclosure relates to administering to a subject having a disease and / or disorder contemplated herein an ENPP1 agent at a dose of about 0.1 mg per kilogram, about 0.2 mg per kilogram of the subject, about 0.3 mg per kilogram of the subject, about 0.4 mg per kilogram of the subject, about 0.5 mg per kilogram of the subject, about 0.6 mg per kilogram of the subject, about 0.7 mg per kilogram of the subject, about 0.8 mg per kilogram of the subject, about 0.9 mg per kilogram of the subject, about 1.0 mg per kilogram of the subject, about 1.1 mg per kilogram of the subject, about 1.2 mg per kilogram of the subject, about 1.3 mg per kilogram of the subject, about 1.4 mg per kilogram of the subject, about 1.5 mg per kilogram of the subject, about 1.6 mg per kilogram of the subject, about 1.7 mg per kilogram of the subject, about 1.8 mg per kilogram of the subject, about 1.9 mg per kilogram of the subject, or about 2.0 mg per kilogram of the subject, in order to restore a physiological level of ENPP1 protein and / or activity in the plasma and / or tissues of the subject. A physiological level of ENPP1 protein and / or activity in the plasma and / or tissues, as used herein, is an amount or concentration of ENPP1 agent sufficient to achieve and maintain a physiological level of PPi in human serum. In certain embodiments, the ENPP1 agent is ENPP1 and / or an ENPP1 construct comprising ENPP1 activity.

[0051] In once aspect, the disclosure relates to a method for increasing circulating pyrophosphate (PPi) in a subject with the diseases and / or disorders contemplated herein, the method comprising administering to the subject an ENPP1 agent at a dose of about 0.1 mg per kilogram, about 0.2 mg per kilogram of the subject, about 0.3 mg per kilogram of the subject, about 0.4 mg per kilogram of the subject, about 0.5 mg per kilogram of the subject, about 0.6 mg per kilogram of the subject, about 0.7 mg per kilogram of the subject, about 0.8 mg per kilogram of the subject, about 0.9 mg per kilogram of the subject, about 1.0 mg per kilogram of the subject, about 1.1 mg per kilogram of the subject, about 1.2 mg per kilogram of the subject, about 1.3 mg per kilogram of the subject, about 1.4 mg per kilogram of the subject, about 1.5 mg per kilogram of the subject, about 1.6 mg per kilogram of the subject, about 1.7 mg per kilogram of the subject, about 1.8 mg per kilogram of the subject, about 1.9

[0052] - 8 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0053] mg per kilogram of the subject, or about 2.0 mg per kilogram of the subject, to thereby increase circulating PPi in the subject.

[0054] In some embodiments of any of the aforesaid methods, the method comprises administering to the patient a therapeutically effective amount of an ENPP1 agent.

[0055] In some embodiments, the ENPP1 agent is a compound of formula (I), or a salt or solvate thereof:

[0056] W-PROTEIN-Z-DOMAIN-X-Y

[0057] (I),

[0058] wherein in (I):

[0059] W is absent or comprises a signal sequence which allows for export of the compound into extracellular space;

[0060] PROTEIN comprises the catalytic region of ENPP 1;

[0061] DOMAIN is absent or at least one selected from the group consisting of a human IgG Fc domain (Fc), human serum albumin protein (ALB), and a biologically active fragment thereof;

[0062] X and Z are independently absent or a polypeptide comprising 1-20 amino acids; and Y is absent or a “bone targeting” sequence group.

[0063] In some embodiments, W is absent. In some embodiments, W comprises a signal sequence which allows for export of the compound into extracellular space.

[0064] In some embodiments. Y is a “bone targeting” sequence group selected from the group consisting of: Dm(SEQ ID NO: 1 1), (DSS)n(SEQ ID NO: 12), (ESS)n(SEQ ID NO: 13), (RQQ)n (SEQ ID NO: 14), (KR)„ (SEQ ID NO: 15), Rm(SEQ ID NO: 16), DSSSEEKFLRRIGRFG (SEQ ID NO: 17), EEEEEEEPRGDT (SEQ ID NO: 18), APWHLSSQYSRT (SEQ ID NO: 19), STLPIPHEFSRE (SEQ ID NO: 20), VTKHLNQISQSY (SEQ ID NO: 116), and Em(SEQ ID NO: 117),

[0065] wherein m is independently an integer ranging from 1 to 15, and wherein n is independently an integer ranging from 1 to 10.

[0066] DETAILED DESCRIPTION

[0067] Definitions

[0068] As used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which - 9 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0069] this disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in animal pharmacology, pharmaceutical science, separation science, and organic chemistry are those well-known and commonly employed in the art. It should be understood that the order of steps or order for performing certain actions is immaterial, so long as the present teachings remain operable. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

[0070] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components.

[0071] In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0072] In this document, the terms ““a.” "an." or '“the” are used to include one or more than one unless the context clearly dictates otherwise. The term ‘“or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B.”

[0073] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in certain embodiments ±5%, in certain embodiments ±1%, in certain embodiments ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0074] The following notation conventions are applied to the present disclosure for the sake of clarity. In any case, any teaching herein that does not follow this convention is still part of the present disclosure, and can be fully understood in view of the context in which the teaching is disclosed. Protein symbols are disclosed in non-italicized capital letters. As nonlimiting examples, ‘ENPP1’ refer to the protein. In certain embodiments, if the protein is a human protein, an “h‘ is used before the protein symbol. In other embodiments, if the protein is a mouse protein, an ‘m’ is used before the symbol. Hence, human ENPP1 is referred to as - 10 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0075] ‘hENPPT, and mouse ENPP1 is referred to as ‘mENPPl'. Eluman gene symbols are disclosed in italicized capital letters. As a non-limiting example, the human gene corresponding to the protein hENPPl is ENPP1. Mouse gene symbols are disclosed with the first letter in upper case and the remaining letters in lower case; further, the mouse gene symbol is italicized. As a non-limiting example, the mouse gene that makes the protein mEnppl is Enppl. Notations about gene mutations are shown as uppercase text.

[0076] The contents of PCT Application Publications No. W02020047520 and W02020206302 are herein incorporated in their entireties by reference.

[0077] “ENPP1 Deficiency’' is characterized by a reduced level of ENPP1 enzymatic activity in serum and / or plasma of a subject. ENPP1 Deficiency is a rare, genetic disorder caused by inactivating mutations in the ENPP1 gene that encodes the ENPP1 enzyme. ENPP1 is an integral transmembrane protein whose extracellular domains carry pyrophosphatase and phosphodiesterase activities. ENPP1 converts extracellular ATP to inorganic pyrophosphate (PPi) and AMP.

[0078] “Enzy matically active” with respect to an ENPP1 polypeptide, or, as used herein, “enzymatic activity” with respect to an ENPP1 polypeptide, is defined as possessing ATP hydrolytic activity into AMP and PPi and / or AP3A hydrolysis to ATP. ENPP1 readily hydrolyze ATP into AMP and PPi. The steady-state Michaelis-Menten enzymatic constants of ENPP1 are determined using ATP as a substrate. ENPP1 can be demonstrated to cleave ATP by HPLC analysis of the enzymatic reaction, and the identity of the substrates and products of the reaction are confirmed by using ATP, AMP, and ADP standards. The ATP substrate degrades over time in the presence of ENPP1, with the accumulation of the enzymatic product AMP. Using varying concentrations of ATP substrate, the initial rate velocities for ENPP1 are derived in the presence of ATP, and the data is fit to a curve to derive the enzymatic rate constants. At physiologic pH, the kinetic rate constants of ENPP1 are Km= 2 pM and kcat=3.4 ± 0.4 s’1.

[0079] As used herein the term “plasma pyrophosphate (PPi) levels” refers to the amount of pyrophosphate present in plasma of animals. In certain embodiments, animals include rat, mouse, cat, dog, human, cow and horse. It is necessary to measure PPi in the plasma rather than serum because of release from platelets. There are several ways to measure PPi, one of which is by enzymatic assay using uridine-diphosphoglucose (UDPG) pyrophosphorylase (Lust & Seegmiller, 1976, Clin. Chim. Acta 66:241-249; Cheung & Suhadolnik, 1977, Anal. Biochem. 83:61-63) with modifications.

[0080] Typically, plasma PPi levels in healthy human subjects range from about 1 pM to - 11 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0081] about 3 pM, in some cases between 1-2 pM. A normal level of ENPP1 in plasma refers to the amount of ENPP1 protein required to maintain a normal level of plasma pyrophosphate (PPi) in a healthy subject. A normal level of PPi in healthy humans corresponds to 2-3 pM.

[0082] Subjects who have a deficiency of ENPP1 exhibit low PPi levels which range from at least 10% below normal levels, at least 20% below normal levels, at least 30% below normal levels, at least 40% below normal levels, at least 50% below normal levels, at least 60% below normal levels, at least 70% below normal levels, at least 80% below normal levels and combinations thereof. In patients afflicted with GACI, the PPi levels are found to be less than 1 pM and in some cases are below a detectable level. In patients afflicted with PXE, the PPi levels are below 0.5 pM (Arterioscler Thromb Vase Biol. 2014 Sep;34(9): 1985-9; Braddock et al.. Nat Commun. 2015; 6: 10006).

[0083] A “deficiency” of ENPP1 refers to a condition in which the subject has less than or equal to 5%- 10% of normal levels of ENPP1 in blood plasma. Normal levels of ENPP1 in healthy human subjects is approximately between 10 to 30 ng / ml (Am J Pathol. 2001 Feb; 158(2): 543-554).

[0084] “Mineral bone disorders (MBD)” as used herein refers to a disorder characterized by abnormal hormone levels cause calcium and phosphorus levels in a person’s blood to be out of balance. Mineral and bone disorder commonly occurs in people with CKD and affects most people with kidney failure receiving dialysis.

[0085] As used herein, the term “ENPP1 haploinsufficiency” refers to a genetic condition wherein one copy of ENPP1 gene is inactivated or deleted and the remaining functional copy of the ENPP1 gene is not adequate to produce the needed gene product to preserve normal function. As a result of ENPP1 haploinsufficiency can, but does not necessarily have to, manifest in the form of low PPi levels and pathological calcifications.

[0086] For most genes, a single copy is enough to support normal grow th and development of diploid organisms, but a small subset of genes known as haploinsufficient (HI) genes exhibit extreme sensitivity to decreased gene dosage. Given the relatively high frequency of gene-inactivating mutations over the lifespan of an organism, and cell-to-cell variability in gene expression, haploinsufficiency represents a significant barrier to organismal fitness. Haploinsufficiency in genetics describes a model of dominant gene action in diploid organisms, in which a single copy of the wild-ty pe allele at a locus in heterozygous combination with a variant allele is insufficient to produce the wild-ty pe phenotype.

[0087] Haploinsufficiency may arise from a de novo or inherited loss-of-function mutation in the variant allele, such that it produces little or no gene product.

[0088] - 12 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0089] For example, N179S mutation in ENPP1 has been found to result in the loss of function of ENPP1 protein. The presence of a single copy of the mutated ENPP1 gene having N179S mutation results in decreased PPi production.

[0090] Likewise, Y451C mutation in ENPP1 has been found to result in the loss of function of ENPP1 protein. The presence of a single copy of the mutated ENPP1 gene having Y451C mutation results in decreased PPi production.

[0091] ”Pre-treatment“. as used herein, means treatment prior to commencement of a treatment method described herein.

[0092] The term “subject”, as used herein, refers to an individual, such as a mammal, such as a human, a non-human primate (e.g. chimpanzees and other apes and monkey species), a farm animal (e.g. birds, fish, cattle, sheep, pigs, goats, and horses), a domestic mammal (e.g. dogs and cats), or a laboratory animal (e.g. rodents, such as mice, rats and guinea pigs). The term includes a subject of any age or sex. In another embodiment the subject is a mammal, preferably a human.

[0093] A disease or disorder is “alleviated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.

[0094] 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 conformation of the polypeptide it encodes, including missense and nonsense mutations, insertions, deletions, frameshifts and premature terminations.

[0095] As used herein, the terms “conservative variation” or “conservative substitution” as used herein refers to the replacement of an amino acid residue by another, biologically similar residue. Conservative variations or substitutions are not likely to change the shape of the peptide chain. Examples of conservative variations, or substitutions, include the replacement of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.

[0096] As used herein, a “construct” of the disclosure refers to a fusion polypeptide comprising an ENPP1 polypeptide, or a fragment or site directed mutant thereof.

[0097] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.

[0098] A “disorder” in an animal is a state of health in which the animal is able to maintain - 13 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0099] homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0100] As used herein, the terms “effective amount,” “pharmaceutically effective amount” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0101] As used herein, the term “ENPP” or “NPP” refers to ectonucleotide pyrophosphatase / phosphodiesterase.

[0102] As used herein, the term “ENPP1 protein” or “ENPP1 polypeptide” refers to 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. ENPP1 protein has a transmembrane domain and soluble extracellular domain. The extracellular domain is further subdivided into somatomedin B domain, catalytic domain (residues 186 to 586 of SEQ ID NO: 1) and the nuclease domain (residues 524 to 885 of SEQ ID NO: 1). The sequence and structure of wild-type ENPP1 is described in detail in PCT Application Publication No. WO 2014 / 126965 to Braddock et al., which is incorporated herein in its entirety by reference.

[0103] Mammal ENPP1 polypeptides, mutants, or mutant fragments thereof, have been previously disclosed in International PCT Application Publications No. WO / 2014 / 126965 to Braddock et al., WO / 2016 / 187408 to Braddock et al., WO / 2017 / 087936 to Braddock et al., and W02018 / 027024 to Braddock et al., all of which are incorporated by reference in their entireties herein.

[0104] As used herein, the term “ENPP1 precursor protein” refers to ENPP1 with its signal peptide sequence at the ENPP1 N-terminus. Upon proteolysis, the signal sequence (in certain non-limiting embodiments, denoted by W in the compound of formula (I)) is cleaved from ENPP1 to provide the ENPP1 protein. Signal peptide sequences useful within the invention include, but are not limited to, albumin signal sequence, azuroci din signal sequence, ENPP1 signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence, and / or ENPP5 signal peptide sequence.

[0105] As used herein, the term “ENPPl-Fc construct” refers to ENPP1 recombinantly fused - 14 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0106] and / or chemically conjugated (including both covalent and non-covalent conjugations) to an FcR binding domain of an IgG molecule (preferably, a human IgG). In certain embodiments, the C-terminus of ENPP1 is fused or conjugated to the N-terminus of the FcR binding domain.

[0107] As used herein, the term “Fc” refers to a human IgG (immunoglobulin) Fc domain. Subtypes of IgG such as IgG1. IgG2, IgG3, and IgG4 are contemplated for use as Fc domains.

[0108] As used herein, the “Fc region or Fc polypeptide” is the portion of an IgG molecule that correlates to 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 an IgG molecule that are linked by disulfide bonds. It has no antigen binding activity but contains the carbohydrate moiety and the binding sites for complement and Fc receptors, including the FcRn receptor. The Fc fragment contains the entire second constant domain CH2 (residues 231-340 of human IgGl, 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 a region of an IgG molecule consisting of the Fc region (residues 231 -447) and a hinge region (residues 216-230) extending from the N-terminus of the Fc region. The term “constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain contains the CHI. CH2 and CH3 domains of the heavy chain and the CHL domain of the light chain.

[0109] As used herein, the term “fragment,” as applied to a nucleic acid, 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 in between). As used herein, the term “fragment,” as applied to a protein or peptide, refers to a subsequence of a larger protein or peptide, and can be at least about 20, 50, 100, 200, 300 or 400 amino acids in length (and any integer value in between).

[0110] The term “functional equivalent” or “functional derivative” denotes, in the context of a functional derivative of an amino acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of sequences of ENPPl-Fc constructs shown herein. A functional derivative or equivalent may be a natural derivative or is prepared synthetically. The functionally-equivalent polypeptides of the disclosure can also be polypeptides identified using one or more techniques of structural and or sequence alignment known in art.

[0111] - 15 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0112] Exemplary functional derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved. The substituting amino acid desirably has chemico-physical properties which are similar to that of the substituted amino acid. Desirable similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophilicity, and the like. Typically, greater than 30% identity between two polypeptides is considered to be an indication of functional equivalence. Preferably, functionally equivalent polypeptides of the disclosure have a degree of sequence identity with the ENPPl-Fc constructs of greater than 80%. More preferred polypeptides have degrees of identity of greater than 85%, 90%, 95%, 98% or 99%, respectively. Method for determining whether a functional equivalent or functional derivative has the same or similar or higher biological activity than the ENPPl-Fc construct can be determined by using the Enzymology assays involving ATP cleavage described in WO2016 / 187408.

[0113] “Instructional material,” as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression that can be used to communicate the usefulness of the nucleic acid, peptide, and / or compound of the disclosure in the kit for identifying or alleviating or treating the various diseases or disorders recited herein.

[0114] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a 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 can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0115] An “isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, i.e., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids that have been substantially purified from other components which naturally accompany the nucleic acid, i. e.. RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokary ote or eukary ote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

[0116] - 16 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0117] The term “operably linked'’ refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0118] As used herein, the term “patient,” “individual” or “subject” refers to a human. As used herein, the term “pharmaceutical composition” or “composition” refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient. Multiple techniques of administering a compound exist in the art including, but not limited to, subcutaneous, intravenous, oral, aerosol, inhalational, rectal, vaginal, transdermal. intranasal, buccal, sublingual, parenteral, intrathecal, intragastrical. ophthalmic, pulmonary, and topical administration.

[0119] As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained; for example, phosphate-buffered saline (PBS).

[0120] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the patient such that it may perform its intended function. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the disclosure, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful - 17 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0121] within the disclosure, and are physiologically acceptable to the patient. The “pharmaceutically acceptable carrier’ may further include a pharmaceutically acceptable salt of the compound useful within the disclosure. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the disclosure are known in the art and described, for example in Remington’s Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0122] As used herein, the language “pharmaceutically acceptable salt” refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, hydrates, and clathrates thereof.

[0123] As used herein the term “plasma pyrophosphate (PPi) levels” refers to the amount of pyrophosphate present in plasma of animals. In certain embodiments, animals include rat, mouse, cat, dog, human, cow and horse. It is necessary to measure PPi in plasma rather than serum because of release from platelets. There are several w ays to measure PPi, one of which is by enzymatic assay using uridine-diphosphoglucose (UDPG) pyrophosphorylase (Lust & Seegmiller, 1976, Clin. Chim. Acta 66:241-249; Cheung & Suhadolnik, 1977, Anal.

[0124] Biochem. 83:61-63) with modifications. Typically, normal PPi levels in healthy subjects range from about 1 pM to about 3 pM, in some cases between 1-2 pM. In some cases, a “low” level of PPi refers to a condition in which the subject has less than or equal to 2%-5% of normal levels of plasma pyrophosphate (PPi) (Arthritis and Rheumatism, Vol. 22. No. 8 (August 1979)).

[0125] Subjects who have defective ENPP1 expression tend to exhibit low PPi levels which range from at least 10% below normal levels, at least 20% below normal levels, at least 30% below normal levels, at least 40% below normal levels, at least 50% below normal levels, at least 60% below normal levels, at least 70% below normal levels, at least 80% below normal levels and combinations thereof. In patients afflicted with GACI, the PPi levels are found to be less than 1 pM and in some cases are below a detectable level. In patients afflicted with PXE, the PPi levels are below 0.5 pM (Arterioscler Thromb Vase Biol. 2014 Sep;34(9): 1985-9; Braddock et al., Nat Commun. 2015; 6: 10006).

[0126] As used herein, the term “polypeptide” refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds.

[0127] As used herein, the term “PPi” refers to pyrophosphate.

[0128] As used herein, the term “prevent” or “prevention” means no disorder or disease - 18 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0129] development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease.

[0130] The term “recombinant polypeptide” as used herein is defined as a polypeptide produced by using recombinant DNA methods.

[0131] The term “recombinant DNA” as used herein is defined as DNA produced by joining pieces of DNA from different sources.

[0132] “Sample” or “biological sample” as used herein means a biological material isolated from a subject. The biological sample may contain any biological material suitable for detecting a mRNA, polypeptide or other marker of a physiologic or pathologic process in a subject, and may comprise fluid, tissue, cellular and / or non-cellular material obtained from the individual.

[0133] As used herein, the term “signal peptide” refers to a sequence of amino acid residues (ranging in length from, for example, 10-30 residues) bound at the amino terminus of a nascent protein of interest during protein translation. The signal peptide is recognized by the signal recognition particle (SRP) and cleaved by the signal peptidase following transport at the endoplasmic reticulum. (Lodish, et al., 2000, Molecular Cell Biology, 4thedition).

[0134] As used herein, “substantially purified” refers to being essentially free of other components. For example, a substantially purified polypeptide is a polypeptide 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.

[0135] “Variant” as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide may differ in amino acid sequence by one or more substitutions, additions, or deletions in any combination. A variant of a nucleic acid or peptide may be a naturally occurring such as an allelic variant, or may be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or - 19 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0136] by direct synthesis.

[0137] As used herein, the term “treatment” or “treating” is defined as the application or administration of a therapeutic agent, i.e., a compound useful within the invention (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a disease or disorder and / or a symptom of a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, affect, and / or prevent and / or minimize progression of the disease or disorder and / or the symptoms of the disease or disorder. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.

[0138] The terms “prevent,” “preventing,” and “prevention”, as used herein, refer to inhibiting the inception or decreasing the occurrence of a disease in a subject. Prevention may be complete (e.g. the total absence of pathological cells in a subject) or partial. Prevention also refers to a reduced susceptibility' to a clinical condition. In certain embodiments, “preventing” comprises preventing onset of a disease or disorder.

[0139] As used herein, the term "wild-type” refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-ty pe” form of the human ENPP1 genes. In contrast, the term “functionally equivalent” refers to a ENPP1 gene or gene product that displays modifications in sequence and / or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. Naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics (including altered nucleic acid sequences) when compared to the wild-type gene or gene product.

[0140] The term “functional equivalent variant”, as used herein, relates to a polypeptide substantially homologous to the sequences of ENPP1 (defined above) and that preserves the enzymatic and biological activities of ENPP1. Methods for determining whether a variant preserves the biological activity of the native ENPP1 are widely known to the skilled person and include any of the assays used in the experimental part of the application. Particularly, functionally equivalent variants of ENPP1 delivered by viral vectors is encompassed by the present invention.

[0141] The functionally equivalent variants of ENPP1 are polypeptides substantially homologous to the native ENPP1. The expression “substantially homologous” relates to a protein sequence when the protein sequence has a degree of identity with respect to the ENPP1 sequences described above of at least 80%, at least 85%, at least 90%, at least 91%, at - 20 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0142] 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.

[0143] The degree of identity between two polypeptides is determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences is preferably determined by 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)), though other similar algorithms can also be used. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0144] “Functionally equivalent variants” of ENPP1 may be obtained by replacing nucleotides within the polynucleotide accounting for codon preference in the host cell that is to be used to produce the ENPP1 respectively. Such “codon optimization” can be determined via computer algorithms which incorporate codon frequency tables such as “Human high, cod” for codon preference as provided by the University of Wisconsin Package Version 9.0, Genetics Computer Group, Madison, Wis.

[0145] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass 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 ±3%, in certain embodiments ±2%, and in certain embodiments ±1 % from the specified value (0.2 mg / kg or 0.6 mg / kg or 1.8 mg / kg), as such variations are appropriate to perform the disclosed methods.

[0146] The disclosure provides a representative example of protein sequences. The protein sequences described can be converted into nucleic acid sequences by performing revere translation and codon optimization. There are several tools available in art such as Expasy (www dot expasy dot org) and bioinformatics servers (www dot bioinformatics dot org)that enable such conversions

[0147] Ranges: throughout this disclosure, various aspects according to the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope according to the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to - 21 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0148] 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2. 2.7, 3, 4. 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0149] Preferred methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the presently disclosed methods and compositions. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0150] 1. ENPP1 Agent

[0151] An ENPPl agent is an ENPPl polypeptide. ENPP1 polypeptides disclosed herein include, but are not limited to, naturally occurring polypeptides of the ENPP 1 family as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a biological activity, such as but not limited to ENPPl’s catalytic activity.

[0152] The ENPP1 agent can be represented by the formula shown below:

[0153] W-PROTEIN-Z-DOMAIN-X-Y

[0154] (I),

[0155] wherein in (I):

[0156] W is absent or comprises a signal sequence which allows for export of the compound into extracellular space;

[0157] PROTEIN comprises the catalytic region of ENPP 1;

[0158] DOMAIN is absent or at least one selected from the group consisting of a human IgG Fc domain (Fc), human serum albumin protein (ALB), and a biologically active fragment thereof;

[0159] X and Z are independently absent or a polypeptide comprising 1-20 amino acids; and Y is absent or a sequence selected from the ‘'bone targeting” sequence group consisting of: Dm(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), Rm(SEQ ID NO: 16), DSSSEEKFLRRIGRFG (SEQ ID NO: 17), EEEEEEEPRGDT (SEQ ID NO: 18), APWHLSSQYSRT (SEQ ID NO: 19), STLPIPHEFSRE (SEQ ID NO: 20), VTKHLNQISQSY (SEQ ID NO: 116), and Em(SEQ ID NO: 117),

[0160] wherein m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, and

[0161] wherein n is independently 1, 2, 3, 4. 5, 6, 7. 8, 9, or 10.

[0162] In some embodiments, W is absent. In some embodiments, W comprises a signal - 22 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0163] sequence which allows for export of the compound into extracellular space.

[0164] In some embodiments, the ENPP1 agent comprises a bone targeting domain. In some embodiments, the bone targeting domain is negatively charged sequence of amino acids. In some embodiments, the negatively charged bone targeting domain is polyaspartic acid.

[0165] In some embodiments, Y is Di. In some embodiments, Y is D2. In some embodiments, Y is D3. In some embodiments, Y is D4. In some embodiments, Y is D8. In some embodiments, Y is De. In some embodiments, Y is D7. In some embodiments, Y is D8. In some embodiments, Y is Ds>. In some embodiments, Y is Dio. In some embodiments, Y is Du. In some embodiments, Y is D12. In some embodiments, Y is Dis. In some embodiments, Y is Du. In some embodiments, Y is D15.

[0166] In some embodiments, Y is (DSS)i. In some embodiments, Y is (DSS)2. In some embodiments, Y is (DSS)s. In some embodiments, Y is (DSS)4. In some embodiments, Y is (DSS)s. In some embodiments, Y is (DSS)e. In some embodiments, Y is (DSS)?. In some embodiments, Y is (DSS)s. In some embodiments, Y is (DSS)s>. In some embodiments, Y is (DSS)io.

[0167] In some embodiments, Y is (ESS)i. In some embodiments, Y is (ESS)2. In some embodiments, Y is (ESS)s. In some embodiments, Y is (ESS)4. In some embodiments, Y is (ESS)5. In some embodiments, Y is (ESS)e. In some embodiments, Y is (ESS)?. In some embodiments, Y is (ESS)s. In some embodiments, Y is (ESS)9. In some embodiments, Y is (ESS)io.

[0168] In some embodiments, Y is (RQQ)i. In some embodiments, Y is (RQQ)2. In some embodiments, Y is (RQQ)3. In some embodiments, Y is (RQQ)4. In some embodiments, Y is (RQQ)s. In some embodiments, Y is (RQQ)e. In some embodiments, Y is (RQQ)?. In some embodiments, Y is (RQQ)s. In some embodiments, Y is (RQQ)9. In some embodiments, Y is (RQQ)io.

[0169] In some embodiments, Y is (KR)i. In some embodiments, Y is (KR)2. In some embodiments, Y is (KR)s. In some embodiments, Y is (KR)4. In some embodiments, Y is (KR)5. In some embodiments, Y is (KR)g. In some embodiments, Y is (KR)7. In some embodiments, Y is (KR)s. In some embodiments, Y is (KR)9. In some embodiments, Y is (KR)io.

[0170] In some embodiments, Y is Ri. In some embodiments, Y is R2. In some embodiments, Y is R3. In some embodiments, Y is R4. In some embodiments, Y is R5. In some embodiments, Y is Re. In some embodiments. Y is R7. In some embodiments, Y is Rs. In some embodiments, Y is R9. In some embodiments, Y is Rio. In some embodiments, Y is R11.

[0171] - 23 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0172] In some embodiments, Y is R12. In some embodiments, Y is R13. In some embodiments, Y is Ri4. In some embodiments, Y is R15.

[0173] In some embodiments, Y is Ei. In some embodiments, Y is E2. In some embodiments, Y is E3. In some embodiments, Y is E4. In some embodiments, Y is E5. In some embodiments, Y is Ee. In some embodiments, Y is E7. In some embodiments, Y is Es. In some embodiments, Y is E$>. In some embodiments, Y is E10. In some embodiments, Y is E11. In some embodiments, Y is E12. In some embodiments, Y is E13. In some embodiments, Y is E14. In some embodiments, Y is E15.

[0174] In some embodiments, Y is DSSSEEKFLRRIGRFG.

[0175] In some embodiments, Y is EEEEEEEPRGDT.

[0176] In some embodiments, Y is APWHLSSQYSRT.

[0177] In some embodiments, Y is STLPIPHEFSRE.

[0178] In some embodiments, Y is VTKHLNQISQSY.

[0179] In certain embodiments, the ENPP1 agent comprises the amino acid sequence of SEQ ID NO: 119.

[0180] In certain embodiments, the ENPP1 agent comprises the amino acid sequence of SEQ ID NO: 119, wherein the signal sequence (amino acid residues 1-22) is missing.

[0181] In certain embodiments, the ENPP1 agent comprises the amino acid residues 23-1086 of the sequence of SEQ ID NO: 119.

[0182] In certain embodiments, the ENPP1 agent comprises the amino acid residues 23-1078 of the sequence of SEQ ID NO: 119.

[0183] In certain embodiments, the ENPP1 agent comprises the amino acid residues 23-1078 of the sequence of SEQ ID NO: 119, further comprising a bone targeting domain.

[0184] In certain embodiments, the bone targeting domain is fused through a chemical bond to the amino acid residues 23-1078 of the sequence of SEQ ID NO: 119.

[0185] In certain embodiments, the bone targeting domain is fused through any linker contemplated herein to the amino acid residues 23-1078 of the sequence of SEQ ID NO: 119.

[0186] In certain embodiments, the ENPP1 agent comprises the amino acid residues 23-1078 of the sequence of SEQ ID NO: 119, further comprising a bone targeting domain fused to the N-terminus of the amino acid residues 23-1078 of the sequence of SEQ ID NO: 119.

[0187] In certain embodiments, the ENPP1 agent comprises the amino acid residues 23-1078 of the sequence of SEQ ID NO: 119, further comprising a bone targeting domain fused to the C-terminus of the amino acid residues 23-1078 of the sequence of SEQ ID NO: 119.

[0188] In certain embodiments, the ENPP1 agent can be an ENPPl-Fc polypeptide. The - 24 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0189] disclosure contemplates that the polypeptide of the disclosure can have one or more of the mutations described herein.

[0190] In another aspect, the disclosure provides an ENPP1 mutant polypeptide comprising at least one amino acid substitution at position 256 corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the amino acid substitution is the substitution of isoleucine (I) for threonine (T) at position 256 relative to SEQ ID NO: 120. In certain embodiments, the amino acid substitution is the substitution of isoleucine (I) for serine (S) at position 256 relative to SEQ ID NO: 120.

[0191] In certain embodiments, the ENPP1 mutant polypeptide comprises the catalytic domain of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide comprises the endonuclease domain of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide lacks the nuclease domain of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide lacks the transmembrane domain of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide lacks the intracellular domain of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide lacks both the intracellular domain and the transmembrane domains of ENPP1. In certain embodiments, the ENPP1 mutant polypeptide lacks a signal sequence. In certain embodiments, the ENPP1 mutant polypeptide comprises an amino acid sequence that is at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to amino acids 23-849 of SEQ ID NO: 120.

[0192] In another aspect, the disclosure provides an ENPP1 mutant polypeptide comprising an amino acid sequence that is at least about 90% (e.g., at least about 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to amino acids 23-849 of SEQ ID NO: 120, wherein the mutant polypeptide comprises an amino acid substitution at position 256 corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the amino acid substitution is I256T. In certain embodiments, the amino acid substitution is I256S.

[0193] In yet another aspect, the disclosure provides an ENPP1 mutant polypeptide comprising amino acids 23-849 of SEQ ID NO: 120 in which no more than ten (10) (e.g., no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than one) amino acid substitution(s) relative to amino acids 23-849 of SEQ ID NO: 120 are present. In certain embodiments, the ENPP1 mutant polypeptide comprises an amino acid substitution at position 256 relative to SEQ ID NO: 120. In certain embodiments, the amino acid substitution is I256T. In certain embodiments, the amino acid substitution is I256S.

[0194] In certain embodiments, the ENPP1 polypeptide comprises at least one mutation in - 25 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0195] the signal sequence region.

[0196] In certain embodiments, the polypeptide comprises mutation I256T corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0197] In certain embodiments, the mutation is selected from the group consisting of C25N, K27T, and V29N corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is C25N corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is K27T corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is V29N corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of C25N / K27T and V29N corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0198] In certain embodiments, the ENPP1 polypeptide comprises at least one mutation in the catalytic region. In certain embodiments, the mutation is selected from the group consisting of I256T, K369N. and 137 IT corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is I256Y corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is K369N corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is 137 IT corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of I256T and K369N / I371T corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0199] In certain embodiments, the ENPP1 polypeptide comprises at least one mutation in the endonuclease domain. In certain embodiments, the mutation is selected from the group consisting of P534N, V536T, R545T, P554L, E592N, R741D, and S766N corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is P534N corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is V536T corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is R545T corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is P554L corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is E592N corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is R741D corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the - 26 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0200] mutation is S766N corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of P534N / V536T, P554L / R545T, E592N, E592N / R741D, and S766N corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0201] In certain embodiments, the ENPP1 polypeptide comprises at least one mutation in the linker region. In certain embodiments, the mutation is selected from the group consisting of E864N and L866T corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the ENPP1 polypeptide comprises at least the mutation E864N / L866T corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is E864N corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is L866T corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0202] In certain embodiments, the polypeptide comprises an ENPP1 polypeptide and an FcRn binding domain, wherein the FcRn binding domain comprises at least one mutation. In certain embodiments, the mutation is selected from the group consisting of M883Y, S885N, S885T, T887E, H1064K, and N1065F corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is M883Y corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is S885N corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is S885T corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is T887E corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is H1064K corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the mutation is N1065F corresponding to the respectively numbered residues in SEQ ID NO: 120. In certain embodiments, the FcRn binding domain comprises at least one mutation selected from the group consisting of S885N, M883Y, M883Y / S885T / T887E, and H1064K / N1065F corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0203] In certain embodiments, the ENPP1 polypeptide comprises at least one mutation selected from the group consisting of C25N, K27T, V29N, C25N / K27T, I256T, K369N, I371T, K369N / I371T, P534N, V536T, R545T, P554L, E592N, R741D, S766N, P534N / V536T, P554L / R545T, E592N / R741D, E864N, L866T, E864N / L866T, M883Y, S885N, S885T, T887E, H1064K, N1065F, M883Y / S885T / T887E, H1064K / N1065F corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0204] - 27 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0205] In certain embodiments, the polypeptide comprises at least one mutation selected from the group consisting of S885N, S766N, M883Y / S885T / T887E, E864N / L866T, P534N / V536T / H1064K / N1065F, P554L / R545T, S766N / H1064K / N1065F, E592N / H1064K / N1065F, and P534N / V536T / M883Y / S885T / T887E corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0206] In certain embodiments, the polypeptide comprises an ENPP1 polypeptide and an FcRn binding domain, the polypeptide comprising mutations M883Y, S885T, and T887E corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0207] In certain embodiments, the polypeptide comprises an ENPP 1 polypeptide and an FcRn binding domain, the polypeptide comprising mutations P534N, V536T, M883Y, S885T. and T887E corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0208] In certain embodiments, the polypeptide comprises an ENPP1 polypeptide and an FcRn binding domain, the polypeptide comprising mutations E592N, H1064K, and N1065F corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0209] In certain embodiments, the polypeptide comprises an ENPP1 mutant polypeptide, wherein the mutant polypeptide comprises an ENPP1 mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0210] In certain embodiments, the ENPP1 mutant polypeptide comprises at least one mutation selected from the group consisting of S766N, P534N / V 536T, P554L / R545T. and E592N corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0211] In certain embodiments, the polypeptide further comprises an FcRn binding domain of an IgG.

[0212] In certain embodiments, the polypeptide comprises mutations selected from the group consisting of: S885N, S766N, M883Y / S885T / T887E, P534N / V536T / H1064K / N1065F, P554L / R545T, S766N / H1064K / N1065F, E592N / H1064K / N1065F, and P534N / V536T / M883Y / S885T / T887E corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0213] In certain embodiments, the polypeptide comprises an S885N mutation in the FcRn binding domain corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0214] In certain embodiments, the polypeptide comprises an S766N mutation in the ENPP1 mutant polypeptide corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0215] In certain embodiments, the polypeptide comprises mutations M883Y, S885T, and T887E in the FcRn binding domain corresponding to the respectively numbered residues in - 28 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0216] SEQ ID NO: 120.

[0217] In certain embodiments, the polypeptide comprises mutations P534N and V536T in the ENPP1 mutant polypeptide and mutations H1064K and N1065F in the FcRn binding domain corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0218] In certain embodiments, the polypeptide comprises mutations P554L and R545T in the ENPP1 mutant polypeptide corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0219] In certain embodiments, the polypeptide comprises mutation S766N in the ENPP1 mutant polypeptide and mutations H1064K and N1065F in the FcRn binding domain corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0220] In certain embodiments, the polypeptide comprises mutation E592N in the ENPP1 mutant polypeptide and mutations H1064K and N1065F in the FcRn binding domain corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0221] In certain embodiments, the polypeptide comprises mutations P534N and V536T in the ENPP1 mutant polypeptide and mutations M883Y, S885T, and T887E in the FcRn binding domain corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0222] In certain embodiments, the polypeptide comprises mutation I256T corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0223] In certain embodiments, the polypeptide comprises mutations I256T, M883Y, S885T, and T887E corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0224] In certain embodiments, the polypeptide comprises mutations V29N, I256T, P534N, V536T, M883Y, S885T, and T887E corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0225] In another aspect, the disclosure features an ENPP1 mutant polypeptide comprising an amino acid sequence that is at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to amino acids 23-849 of SEQ ID NO: 120, wherein the mutant polypeptide comprises mutation I256T corresponding to the respectively numbered residues in SEQ ID NO: 120, and further comprises a mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0226] In certain embodiments, any of the mutant polypeptides described herein comprises at least one amino acid substitution selected from the group consisting of S766N, P534N / V536T, P554L / R545T, and E592N corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0227] - 29 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0228] In certain embodiments, any of the mutant polypeptides described herein comprises the amino acid substitution V29N.

[0229] Also featured are ENPP1 mutant polypeptide fusions comprising any of the ENPP1 mutant polypeptides described herein and a heterologous protein, such as an FcRn binding domain. In certain embodiments, the heterologous protein is carboxy-terminal to the ENPP1 mutant polypeptide portion of the fusion. In certain embodiments, the heterologous protein is amino-terminal to the ENPP1 mutant polypeptide portion of the fusion.

[0230] In certain embodiments of any of the fusions described herein, the FcRn binding domain is an albumin polypeptide. In certain embodiments, the FcRn binding domain is a Fc portion of an immunoglobulin molecule, such as an IgGl immunoglobulin molecule.

[0231] In certain embodiments of any of the fusions described herein, the FcRn binding domain comprises one more amino acid substitutions relative to a wild type FcRn binding domain. In certain embodiments the FcRn binding domain is the Fc portion of a human IgGl molecule and comprises the following amino acid substitutions: M883Y, S885T, and T887E (the MST / YTE substitutions), each relative to SEQ ID NO: 120.

[0232] In certain embodiments, a fusion described herein comprises one or more of the following substitutions: S885N, S766N, M883Y / S885T / T887E, P534N / V536T / H1064K / N1065F, P554L / R545T, S766N / H1064K / N1065F, E592N / H1064K / N1065F, or P534N / V536T / M883Y / S885T / T887E, each corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0233] Also featured herein are conjugates of any one of the ENPP1 mutant polypeptides or ENPP1 mutant polypeptide fusions described herein and a heterologous moiety, such as but not limited to a small molecule. In certain embodiments, the heterologous moiety increases or further increases the pharmacokinetic and / or bioavailability of the mutant polypeptides in a mammal. In certain embodiments, the heterologous moiety is an oligomer of ethylene glycol and / or propylene glycol, such as but not limited to polyethylene glycol (PEG) and / or polypropylene glycol (PPG).

[0234] In certain embodiments, any of the ENPP1 mutant polypeptide fusions or conjugates described herein comprises the S885N mutation corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0235] In certain embodiments, any of the ENPP1 mutant polypeptides, fusions, or conjugates described herein comprises the S766N mutation corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0236] In certain embodiments, any of the ENPP1 mutant polypeptide fusions or conjugates - 30 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0237] described herein comprises mutations M883Y, S885T, and T887E corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0238] In certain embodiments, any of the ENPP1 mutant polypeptides, fusions, or conjugates described herein comprises mutations P534N, V536T, H1064K, and N1065F corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0239] In certain embodiments, any of the ENPP1 mutant polypeptides, fusions, or conjugates described herein comprises mutations P554L and R545T corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0240] In certain embodiments, any of the ENPP1 mutant polypeptides, fusions, or conjugates described herein comprises mutation S766N, H1064K, and N1065F corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0241] In certain embodiments, any of the ENPP1 mutant polypeptides, fusions, or conjugates described herein comprises mutation E592N, H1064K, and N1065F corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0242] In certain embodiments, any of the ENPP1 mutant polypeptides, fusions, or conjugates described herein comprises mutations P534N, V536T, M883Y, S885T, and T887E corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0243] In another aspect, the disclosure provides an ENPP1 mutant polypeptide fusion comprising an ENPP1 mutant polypeptide fused to a Fc region of an immunoglobulin, wherein the ENPP1 mutant polypeptide comprises a substitution at position 256 relative to SEQ ID NO: 120.

[0244] In certain embodiments of any of the fusions described herein, the Fc region comprises at least one mutation selected from the group consisting of M883Y, S885N, S885T. T887E, H1064K, and N1065F corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0245] In certain embodiments of any of the fusions described herein, the Fc region comprises at least one mutation selected from the group consisting of S885N, M883Y, M883Y / S885T / T887E, and H1064K / N1065F corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0246] In certain embodiments, the ENPP1 mutant polypeptide further comprises at least one mutation selected from the group consisting of C25N, K27T, and V29N corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0247] In certain embodiments, an ENPP1 mutant polypeptide, or a fusion described herein comprises at least one mutation selected from the group consisting of C25N / K27T and V29N - 31 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0248] corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0249] In certain embodiments, an ENPP1 mutant polypeptide described herein further comprises at least one mutation selected from the group consisting of K369N and I371T corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0250] In certain embodiments, an ENPP1 mutant polypeptide, or a fusion comprising such a mutant polypeptide, described herein comprises the mutation K369N / I371T corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0251] In certain embodiments, an ENPP1 mutant polypeptide, or fusion comprising such a mutant polypeptide, described herein further comprises at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, E592N, R741D, and S766N corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0252] In certain embodiments, an ENPP1 mutant polypeptide, or a fusion comprising such a mutant polypeptide, described herein comprises at least one mutation selected from the group consisting of P534N / V536T, P554L / R545T, E592N, E592N / R741D, and S766N corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0253] In certain embodiments, any of the ENPP1 mutant polypeptides or fusions described herein further comprises at least one mutation selected from the group consisting of E864N and L866T corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0254] In certain embodiments, any of the ENPP1 mutant polypeptides or fusions described herein comprises at least the mutation E864N / L866T corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0255] In certain embodiments, any of the ENPP1 mutant polypeptides or fusions described herein comprise at least one mutation selected from the group consisting of C25N, K27T, V29N, C25N / K27T, K369N, I371T, K369N / I371T, P534N, V536T, R545T, P554L, E592N, R741D, S766N, P534N / V536T, P554L / R545T, E592N / R741D, E864N, L866T, E864N / L866T, M883Y, S885N, S885T, T887E, H1064K, N1065F, M883Y / S885T / T887E, H1064K / N1065F corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0256] In certain embodiments, any of the fusions described herein comprise an Fc region of an IgG. such as IgG1.

[0257] In certain embodiments, any of the ENPP1 mutant polypeptides described herein, or the fusion proteins comprising such ENPP1 mutant polypeptides, comprise at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, S766N, and E592N corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0258] In certain embodiments, any of the ENPP1 mutant polypeptides described herein, or - 32 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0259] the fusion proteins comprising such ENPP1 mutant polypeptides, comprise at least one mutation selected from the group consisting of S766N, P534N / V536T, P554L / R545T, and E592N corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0260] In certain embodiments, any of the ENPP1 mutant polypeptides described herein, or the fusion proteins comprising such ENPP1 mutant polypeptides, comprise at least one mutation selected from the group consisting of S885N, S766N, M883Y / S885T / T887E, E864N / L866T, P534N / V536T / H1064K / N1065F, P554L / R545T, S766N / H1064K / N1065F, E592N / H1064K / N1065F, and P534N / V536T / M883Y / S885T / T887E corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0261] In certain embodiments, any of the fusions described herein comprise mutations I256T, M883Y, S885T, and T887E corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0262] In certain embodiments, any of the fusions described herein comprise an ENPP1 polypeptide and a Fc region of an immunoglobulin, the polypeptide fusion comprising mutations I256T, P534N, V536T, M883Y, S885T, and T887E corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0263] In certain embodiments, any of the fusions described herein comprise the ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and a Fc region of an immunoglobulin, the polypeptide fusion comprising mutations I256T, E592N, H1064K, and N1065F corresponding to the respectively numbered residues in SEQ ID NO: 120.

[0264] In certain embodiments, the ENPP1 mutant polypeptide fusions described herein comprise a linker amino acid sequence, for example, between the ENPP1 mutant polypeptide portion of the fusion and the heterologous protein moiety. In certain embodiments, the linker amino acid sequence comprises or consists of SEQ ID NO: 8. In certain embodiments, the linker amino acid sequence comprises or consists of SEQ ID NO: 9, wherein n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, or n=10.

[0265] In certain embodiments, the ENPP1 polypeptide lacks a nuclease domain. In other embodiments, the ENPP1 polypeptide is truncated to remove the nuclease domain. In yet other embodiments, the ENPP1 polypeptide is truncated to remove the nuclease domain from about residue 524 to about residue 885 relative to SEQ ID NO: 1, leaving only the catalytic domain from about residue 186 to about residue 586 relative to SEQ ID NO: 1, which serves to preserve the catalytic activity of the protein.

[0266] In certain embodiments, the ENPP1 polypeptide is modified with a segment of the extracellular region of ENPP1 containing a peptidase cleavage site after the signal peptide,

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[0268] and between the transmembrane and extracellular domain, as compared to SEQ ID NO: 1.

[0269] In certain embodiments, the ENPP1 polypeptide is modified with a segment of the extracellular region of ENPP1 containing a furin cleavage site between the transmembrane and extracellular domain, as compared to SEQ ID NO: 1. In other embodiments, the ENPP1 polypeptide is not modified with a segment of the extracellular region of ENPP1 containing a furin cleavage site between the transmembrane and extracellular domain, as compared to SEQ ID NO: 1.

[0270] In certain embodiments, the ENPP1 polypeptide is modified with a segment of the extracellular region of ENPP2 containing a signal peptidase cleavage site, as compared to SEQ ID NO: 1. In other embodiments, the ENPP1 polypeptide is not modified with a segment of the extracellular region of ENPP2 containing a signal peptidase cleavage site, as compared to SEQ ID NO: 1.

[0271] In yet another aspect, the disclosure provides an ENPP1 mutant polypeptide, a ENPP1 -containing polypeptide, or a fusion, which is expressed from a CHO cell line stably transfected with human ST6 beta-galactoside alpha-2.6-sialyltransferase (also known as ST6GAL1).

[0272] In yet another aspect, the disclosure provides an ENPP1 mutant polypeptide, a ENPP1 -containing polypeptide, or a fusion, which is grown in a cell culture supplemented with sialic acid and / or N-acetylmannosamine (also known as 1,3,4-O-Bu3ManNAc).

[0273] Also provided herein are pharmaceutical compositions comprising any one of the ENPP1 mutant polypeptides, the ENPP1 mutant polypeptide fusions, conjugates, or other polypeptides and proteins described herein and a pharmaceutically acceptable carrier.

[0274] In certain embodiments, the polypeptide is soluble. In other embodiments, the polypeptide is a recombinant polypeptide. In yet other embodiments, the polypeptide comprises an ENPP1 polypeptide that lacks the ENPP1 transmembrane domain. In yet other embodiments, the polypeptide comprises an ENPP1 polypeptide wherein the ENPP1 transmembrane domain has been removed (and / or truncated) and replaced with the transmembrane domain of another polypeptide, such as. by way of non-limiting example, ENPP2, ENPP5, or ENPP7.

[0275] In certain embodiments, the polypeptide comprises a signal peptide resulting in the secretion of a precursor of the ENPP1 polypeptide, which undergoes proteolytic processing to yield a polypeptide comprising the ENPP1 polypeptide. In other embodiments, the signal peptide is selected from the group consisting of signal peptides of ENPP2, ENPP5, and ENPP7. In yet other embodiments, the polypeptide comprises an ENPP1 polypeptide - 34 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0276] comprising transmembrane domains of ENPP1 and another polypeptide, such as, by way of non-limiting example, ENPP2. In yet other embodiments, the ENPP1 polypeptide comprises a cleavage product of a precursor ENPP1 polypeptide comprising an ENPP2 transmembrane domain. In yet other embodiments, the ENPP2 transmembrane domain comprises residues 12-30 of SEQ ID NO: 120, which corresponds to IISLFTFAVGVNICLGFTA.

[0277] In certain embodiments, the ENPP1 polypeptide is C-terminally fused to the Fc domain of human immunoglobulin 1 (IgGl), human immunoglobulin 2 (IgG2), human immunoglobulin 3 (IgG3), and / or human immunoglobulin 4 (IgG4). In other embodiments, the ENPP1 polypeptide is N-terminally fused to the Fc domain of human immunoglobulin 1 (IgGl), human immunoglobulin 2 (IgG2), human immunoglobulin 3 (IgG3), and / or human immunoglobulin 4 (IgG4). In yet other embodiments, the presence of IgFc domain improves half-life, solubility, reduces immunogenicity, and increases the activity of the ENPP1 polypeptide.

[0278] In certain embodiments, the ENPP1 polypeptide is C-terminally fused to human serum albumin. Human serum albumin may be conjugated to ENPP1 protein through a chemical linker, including but not limited to naturally occurring or engineered disulfide bonds, or by genetic fusion to ENPP1, or a fragment and / or variant thereof.

[0279] In certain embodiments, the polypeptide is further pegylated (fused with a poly(ethylene glycol) chain).

[0280] In certain embodiments, the polypeptide has a kcat value for the substrate ATP greater than or equal to about 3.4 (±0.4) s’1enzyme’1, wherein the kcat is determined by measuring the rate of hydrolysis of ATP for the polypeptide.

[0281] In certain embodiments, the polypeptide has a KM value for the substrate ATP less than or equal to about 2 pM. wherein the KM is determined by measuring the rate of hydrolysis of ATP for the polypeptide.

[0282] In certain embodiments, the polypeptide is formulated as a liquid formulation. In other embodiments, the disclosure provides a dry product form of a pharmaceutical composition comprising a therapeutic amount of a polypeptide of the disclosure, whereby the dry product is reconstitutable to a solution of the compound in liquid form.

[0283] The disclosure provides a kit comprising at least one polypeptide of the disclosure, or a salt or solvate thereof, and instructions for using the polypeptide within the methods of the disclosure.

[0284] In certain embodiments, the polypeptide lacks a negatively-charged bone-targeting sequence. In yet other embodiments, a polyaspartic acid domain (from about 2 to about 20 or - 35 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0285] more sequential aspartic acid residues) is a non-limiting example of a negatively -charged bone-targeting sequence. In other embodiments, the polypeptide has a negatively-charged bone-targeting sequence.

[0286] The terms " ENPP I " or “ENPP1 polypeptide” refers to ectonucleotide pyrophosphatase / phosphodiesterase 1 proteins (NPP1 / ENPP1 / PC-1) and ENPP 1 -related proteins, derived from any species. ENPP1 protein comprises a type II transmembrane glycoprotein that forms a homodimer. Each monomer of the ENPP1 protein comprises a short intracellular N-terminal domain involved in targeting to the plasma membrane, a transmembrane domain, and a large extracellular region comprising several domains. The large extracellular region comprises SMB1 and SMB2 domains, which have been reported to take part in ENPP1 dimerization (Gijsbers et al., Biochem. J. 371; 2003: 321-330).

[0287] Specifically, the SMB domains contain eight cysteine residues, each arranged in four disulfide bonds, and have been shown to mediate ENPP1 homodimerization through covalent cystine inter- and intramolecular bonds. The protein cleaves a variety of substrates, including phosphodi ester bonds of nucleotides and nucleotide sugars and pyrophosphate bonds of nucleotides and nucleotide sugars. ENPP1 protein functions to hydrolyze nucleoside 5’ triphosphatase to either corresponding monophosphates and also hydrolyzes diadenosine polyphosphates. ENPP1 proteins play a role in purinergic signaling which is involved in the regulation of cardiovascular, neurological, immunological, musculoskeletal, hormonal, and hematological functions. An exemplary amino acid sequence of the human ENPP1 precursor protein (NCBI accession NP_006199) is shown in SEQ ID NO: 1. The human ENPP 1 precursor protein includes an endogenous ENPP1 signal peptide sequence at the ENPP1 N-terminus. In certain embodiments, the ENPP1 precursor protein further comprises an endogenous or heterologous signal peptide sequence. Upon proteolysis, the signal peptide sequence is cleaved from the ENPP1 precursor protein to provide the mature ENPP1 protein. See, e.g., 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, ENPP1 signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence, and / or ENPP5 signal peptide sequence. A non-limiting processed (mature) extracellular ENPP1 polypeptide sequence is shown in SEQ ID NO: 2.

[0288] It is generally known in the art that ENPP1 is well-conserved among vertebrates, with large stretches of the extracellular domain substantially conserved. ENPP1 binding to various nucleotide triphosphates (e.g., ATP, UTP. GTP. TTP. and CTP), pNP-TMP, 3’,5'-cAMP, and 2’-3’-cGAMP is highly conserved (see, e.g., Kato et al., Proc Natl Acad Sci USA.

[0289] - 36 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0290] 2012;109(42): 16876-81 and Mackenzie et al. Bone. 2012;51(5):961-8). Accordingly, from these alignments, it is possible to predict key amino acid positions with the extracellular domain that are important for normal ENPP1 activities as well as to predict amino acid positions that are likely to be tolerant to substitution without significantly altering normal ENPP1 activities. Therefore, an enzymatically active, human ENPP1 polypeptide useful in accordance with the presently disclosed compositions may include one or more amino acids at corresponding positions from the sequence of another vertebrate ENPP1, or may include a residue that is similar to that in the human or other vertebrate sequences. Substitutions of one or more amino acids at corresponding positions may include conservative variations or substitutions that are not likely to change the shape of the polypeptide chain or alter normal ENPP1 activities. Examples of conservative variations, or substitutions, include the replacement of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. For example, ENPP1 polypeptides include polypeptides derived from the sequence of any known ENPP1 polypeptide having a sequence at least about 80% identical to the sequence of an ENPP1 polypeptide, and preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity.

[0291] It will be understood that an ENPP1 polypeptide according to the disclosure includes not only the native human proteins, but also any fragment, derivative, fusion, conjugate or mutant thereof having ATP hydrolytic activity of the native protein. As used herein in this disclosure, the phrase “an ENPP1 polypeptide, mutant, or mutant fragment thereof’ also includes any compound or polypeptide (such as, but not limited to, a fusion protein) comprising an ENPP1 polypeptide, mutant, or mutant fragment thereof. Fusion proteins according to the disclosure are considered biological equivalents of ENPP1, but are intended to provide longer half-life or greater potency due to increased in vivo biologic exposure, as judged by the “area under the curve” (AUC) or increased half-life in pharmacokinetic experiments.

[0292] 2. Enzymatic Activity of ENPP1

[0293] ENPP1 proteins have been characterized in the art in terms of structural and biological characteristics. In certain embodiments, soluble ENPP1 proteins disclosed herein comprise pyrophosphatase and / or phosphodiesterase activity. For instance, in some embodiments, the ENPP1 protein binds nucleotide triphosphates (e.g., ATP, UTP, GTP, TTP,

[0294] - 37 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0295] and CTP), pNP-TMP, 3’,5’-cAMP, and 2 -3’-cGAMP; and converts nucleotide triphosphates into inorganic pyrophosphate [see, e.g., Kato et al., Proc Natl Acad Sci USA.

[0296] 2012;109(42):16876-81; Li, et al., Nat Chem Biol. 2014;10(12):1043-8; Jansen et al..

[0297] Structure. 2012;20(11):1948-59; and Onyedibe et al., Molecules. 2019;24(22)].

[0298] “Enzymatically active” or “biologically active” ENPP1 polypeptides exhibit pyrophosphatase and / or phosphodiesterase activity (e.g., is capable of binding and / or hydrolyzing ATP into AMP and PPi and / or AP3a into ATP). For example, the pyrophosphatase / phosphodi esterase domain of an ENPP1 protein hydrolyzes extracellular nucleotide triphosphates to produce inorganic pyrophosphates (PPi) and is generally soluble. This activity can be measured using a pNP-TMP assay as previously described (Saunders et al., 2008. Mol. Cancer Ther. 7(10):3352-62; Albright et al., 2015. Nat Comm. 6: 10006). In certain embodiments, the soluble ENPP1 polypeptide has a kcat value for the substrate ATP greater than or equal to about 3.4 (±0.4) s-1enzyme-1, wherein the kcat is determined by measuring the rate of hydrolysis of ATP for the polypeptide. In certain embodiments, the soluble ENPP1 polypeptide has a KM value for the substrate ATP less than or equal to about 2 pM, wherein the K is determined by measuring the rate of hydrolysis of ATP for the polypeptide. In addition to the teachings herein, these references provide ample guidance for how to generate soluble ENPP1 proteins that retain one or more biological activities (e.g., conversion of nucleotides into inorganic pyrophosphate).

[0299] 3. Soluble ENPP1

[0300] In one embodiment, the disclosure relates to ENPP1 agents, such as but not limited to ENPP1 polypeptides. As described herein, the term soluble ENPP1 polypeptide includes any naturally occurring extracellular domain of an ENPP1 protein as well as any variants thereof (including mutants, fragments and peptidomimetic forms) that retain a biological activity (e.g., enzymatically active). Examples of soluble ENPP1 polypeptides include, for example, an ENPP1 extracellular domain (SEQ ID NO: 2). In certain embodiments, the soluble ENPP1 polypeptides further comprise a signal sequence in addition to the extracellular domain of an ENPP1 polypeptide. Exemplary signal sequences include the native signal sequence of an ENPP 1 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, WO 2020 / 206302 and WO 2020 / 047520. the contents of all of which are incorporated herein by reference in their entirety.

[0301] - 38 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0302] 4. ENPP1 Fusion Proteins

[0303] 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 heterologous to ENPP1). An amino acid sequence is understood to be heterologous to ENPP1 if it is not uniquely found in the form of ENPP1 represented by SEQ ID NO: 1. In some embodiments, the heterologous protein portion comprises an Fc domain of an immunoglobulin. In some embodiments, the Fc domain of the immunoglobulin is an Fc domain of an IgGl immunoglobulin. In certain embodiments, the soluble ENPP1 polypeptide is C-terminally fused to the Fc domain of human immunoglobulin 1 (IgGl), human immunoglobulin 2 (IgG2), human immunoglobulin 3 (IgG3), and / or human immunoglobulin 4 (IgG4). In other embodiments, the soluble ENPP1 polypeptide is N-terminally fused 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 an Fc domain improves half-life, solubility, reduces immunogenicity, and increases the activity of the soluble ENPP1 polypeptide. In certain embodiments, portions of the native human IgG proteins (IgGl, IgG2, IgG3, and IgG4), may be used for the Fc portion (e.g., ENPPl-Fc). For instance, the present disclosure provides fusion proteins comprising ENPP1 fused to a polypeptide comprising a constant domain of an immunoglobulin, such as a CH1, CH2, or CH3 domain derived from human IgGl, IgG2, IgG3, and / or IgG4. The Fc fragment may comprise regions of the native IgG such as the hinge region (residues 216- 230 of human IgGl, 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 “ENPPl-Fc construct” refers to a soluble form of ENPP1 (e.g.. the extracellular domain of an ENPP1 polypeptide) recombinantly fused and / or chemically conjugated (including both covalent and non-covalent conjugations) to an FcR binding domain of an IgG molecule (preferably, a human IgG). In certain embodiments, the C-terminus of ENPP1 is fused or conjugated to the N-terminus of the FcR binding domain.

[0304] An example of an amino acid sequence that may be used for the Fc portion of human IgGl (GIFc) is SEQ ID NO: 6 (Table 1). In part, the disclosure provides polypeptides comprising, consisting essential of, or consisting of amino acid sequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%, or 100% identity to SEQ ID NO: 6.

[0305] In some embodiments, the heterologous protein portion comprises one or more - 39 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0306] domains selected from the group consisting of polyhistidine, FLAG tag, Glu-Glu, glutathione S-transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy-chain constant region (Fc), maltose binding protein (MBP), or human serum albumin. A fusion domain may be selected so as to confer a desired property. For example, some fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography. For the purpose of affinity purification, relevant matrices for affinity chromatography, such as glutathione-, amylase-, and nickel- or cobalt- conjugated resins are used. Many of such matrices are available in “kit” form, such as the Pharmacia GST purification system and the QIAexpress™ system (Qiagen) useful with (HISe) fusion partners. As another example, a fusion domain may be selected so as to facilitate detection of the ENPP1 polypeptide. Examples of such detection domains include the various fluorescent proteins (e.g., GFP) as well as “epitope tags,” which are usually short peptide sequences for which a specific antibody is available. Well-known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus haemagglutinin (HA), and c-myc tags. In some cases, the fusion domains have a protease cleavage site, such as for Factor Xa or thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated proteins can then be isolated from the fusion domain by subsequent chromatographic separation.

[0307] 5. Linkers

[0308] In some embodiments, the ENPP1 fusion protein further comprises a linker (Z) positioned between the ENPP1 polypeptide domain and the one or more heterologous protein portions (e.g., an Fc immunoglobulin domain). In certain embodiments, the soluble ENPP1 polypeptide is directly or indirectly fused 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, a linker can be an amino acid spacer including 1-200 amino acids. Suitable peptide spacers are known in the art, and include, for example, peptide linkers containing flexible amino acid residues such as glycine, alanine, and serine.

[0309] In some embodiments, the linker comprises a polyglycine linker or a Gly-Ser linker. In some embodiments, a spacer can contain motifs, e.g, multiple or repeating motifs, of 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 - 40 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0310] (SEQ ID NO: 35), SGGGG (SEQ ID NO: 36), or SGGG (SEQ ID NO: 37). In some embodiments, a spacer can contain 2 to 12 amino acids including motifs of GA or GS, e.g., 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, a spacer can contain 3 to 12 amino acids including motifs of GGA or GGS, e g., 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 yet some embodiments, a spacer can contain 4 to 12 amino acids including motifs of GGAG (SEQ ID NO: 33), GGSG (SEQ ID NO: 34), e.g., GGAGGGAG (SEQ ID NO: 58), GGSGGGSG (SEQ ID NO: 59), GGAGGGAGGGAG (SEQ ID NO: 60), and GGSGGGSGGGSG (SEQ ID NO: 61). In some embodiments, a spacer can contain motifs of GGGGA (SEQ ID NO: 30) or GGGGS (SEQ ID NO: 31), e g., GGGGAGGGGAGGGGA (SEQ ID NO: 64) and GGGGSGGGGSGGGGS (SEQ ID NO: 65). In some embodiments of the invention, an amino acid spacer between a 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 a human serum albumin) and a soluble ENPP1 polypeptide may 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).

[0311] In some embodiments, a spacer can also contain amino acids other than glycine, alanine, and serine, e g., 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), 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), - 41 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0312] VTKHLNQISQSY (SEQ ID NO: 96), (E)m; m=l-15 (SEQ ID NO: 97), RSGSGGS (SEQ ID NO: 98), (D)m: m=l-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), LGLGLGLGLRK (SEQ ID NO: 105), GLGLGLGLRK (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=l-15 (SEQ ID NO: 115). In some embodiments, a spacer can contain motifs, e.g., multiple or repeating motifs, of EAAAK (SEQ ID NO: 85). In some embodiments, a spacer can contain motifs, e.g., multiple or repeating motifs, of praline-rich sequences such as (XP)n, in which X may be any amino acid (e.g., A, K, or E) and n is from 1-5, and PAPAP(SEQ ID NO: 86).

[0313] 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 desired in the final protein fusion polypeptide. The length of the spacer can be adjusted to ensure proper protein folding and avoid aggregate formation.

[0314] In some embodiments, different elements of the fusion proteins (e.g., immunoglobulin Fc fusion proteins) may be arranged in any manner that is consistent with desired functionality. For example, a soluble ENPP1 polypeptide domain may be placed C-terminal to a heterologous protein portion, or alternatively, a heterologous protein portion may be placed C-terminal to a soluble ENPP1 polypeptide domain. The soluble ENPP1 polypeptide domain and the heterologous protein portion may be directly or indirectly linked in a fusion protein, and additional domains or amino acid sequences may be included C- or N-terminal to either domain or between the domains. Preferred fusion proteins comprise the amino acid sequence set forth in any one of SEQ ID NOs: 3-5.

[0315] In some embodiments, soluble ENPP1 polypeptides of the present disclosure contain one or more heterologous moieties. Optionally, a soluble ENPP1 polypeptide includes one or more heterologous moieties selected from: a glycosylated amino acid, a PEGylated amino acid, a famesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, and an amino acid conjugated to an organic derivatizing agent. In some embodiments, a soluble ENPP1 polypeptide disclosed herein is further modified. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation. and acylation. As a result, the soluble ENPP1 polypeptide may contain non-amino acid elements, such as polyethylene - 42 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0316] glycols, lipids, polysaccharide or monosaccharide, and phosphates. Effects of such nonamino acid elements on the functionality of a soluble ENPP1 polypeptide may be tested as described herein for other soluble ENPP1 polypeptides. When a polypeptide of the disclosure is produced in cells by cleaving a nascent form of the polypeptide, post-translational processing may also be important for correct folding and / or function of the protein. Different cells (e.g., CHO, HeLa, MDCK, 293, WI38. NIH-3T3 or HEK293) have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the soluble ENPP1 polypeptides.

[0317] As used herein, percent “identity ” 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 aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA software. In some embodiments, alignment is performed using the CLUSTAL OMEGA software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0318] 6. Determining Solubility

[0319] In some embodiments, the activity of soluble ENPP1 polypeptides may also be tested in a cell-based or in vivo assay. For example, the effect of a soluble ENPP1 polypeptide on the production of inorganic pyrophosphates (PPi) can be measured. Specifically, the pyrophosphatase / phosphodi esterase domain of an ENPP1 protein hydrolyzes extracellular nucleotide triphosphates to produce inorganic pyrophosphates (PPi) and is generally soluble. This activity can be measured using a pNP-TMP assay as well as an HPLC-based ATP hydrolysis assay, as previously described (Saunders et al., 2008, Mol. Cancer Ther.

[0320] 7(10):3352-62; Albright et al.. 2015. Nat Comm. 6:10006). The effect of soluble ENPP1 polypeptides on the expression of genes involved in ENPP1 associated diseases such as ARHR2 (e.g., transcription of fibroblast grow th factor 23 in osteoblasts and osteoclasts) can be assessed. This may as needed be performed in the presence of one or more nucleotide triphosphates or other ENPP1 substrates, and cells may be transfected so as to produce a soluble ENPP1 polypeptide. Likewise, a soluble ENPP1 polypeptide may be administered to - 43 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0321] a mouse or other animal and effects on ENPP1 associated diseases may be assessed using art-recognized methods.

[0322] In some embodiments, ENPP1 polypeptides to be used in accordance with the methods described herein are isolated polypeptides. As used herein, an isolated protein or polypeptide is one which has been separated from a component of its natural environment. In some embodiments, a polypeptide of the disclosure is purified to greater than 95%. 96%. 97%, 98%, or 99% purity as determined by, for example, electrophoretic (e.g, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) analyses. Methods for assessment of purity are well known in the art [see, e.g., Flatman et al., 2007, J. Chromatogr. B 848:79-87], In some embodiments, soluble ENPP1 polypeptides to be used in accordance with the methods described herein are recombinant polypeptides.

[0323] 7. ENPP1 Production

[0324] ENPP1 polypeptides of the disclosure can be produced by a variety of art-known techniques. For example, polypeptides of the 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 G. A. (ed.), Synthetic Peptides: A User's Guide, W. H. Freeman and Company, New York (1992). In addition, automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Model 396;

[0325] Milligen / Biosearch 9600). Alternatively, the polypeptides of the disclosure, including fragments or variants thereof, may be recombinantly produced using various expression systems [e.g., E. coli, Chinese Hamster Ovary (CHO) cells, COS cells, baculovirus, Yeast Pichia] as is well known in the art. The protein can be produced in either adherent or suspension cells. In some embodiments, the fusion protein is expressed in CHO cells. To establish stable cell lines the nucleic acid sequence encoding ENPP1 constructs are cloned into an appropriate vector for large scale protein production. In certain embodiments, the modified or unmodified polypeptides of the disclosure may be produced by digestion of recombinantly produced full-length ENPP1 polypeptides by using, for example, a protease, e.g., trypsin. thermolysin, chymotrypsin, pepsin, or paired basic amino acid converting enzyme (PACE). Computer analysis (using commercially available software, e.g., MacVector, Omega, PCGene, Molecular Simulation, Inc.) can be used to identify proteolytic cleavage sites. Alternatively, such polypeptides may be produced from recombinantly generated full-length ENPP1 polypeptides using chemical cleavage (e.g., cyanogen bromide,

[0326] - 44 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0327] hydroxylamine, and so forth).

[0328] ENPP1, or aENPPl polypeptide, is prepared as described in US 2015 / 0359858 Al, which is incorporated herein in its entirety by reference. ENPP1 is a transmembrane protein localized to the cell surface with distinct intramembrane domains. In order to express ENPP1 as a soluble extracellular protein, the transmembrane domain of ENPP1 may be swapped for the transmembrane domain of ENPP2, which results in the accumulation of soluble, recombinant ENPP1 in the extracellular fluid of the baculovirus cultures.

[0329] Signal sequences of any other known proteins may be used to target the extracellular domain of ENPP1 for secretion as well, such as but not limited to the signal sequence of the immunoglobulin kappa and lambda light chain proteins. Further, the disclosure should not be construed to be limited to the polypeptides described herein, but also includes polypeptides comprising any enzymatically active truncation of the ENPP1 extracellular domain.

[0330] ENPP1 is made soluble by omitting the transmembrane domain. Human ENPP1 (SEQ ID NO: 1) was modified to express a soluble, recombinant protein by replacing its transmembrane region (e.g., residues 77-98) with the corresponding subdomain of human ENPP2 (NCBI accession NP_00112433 5, e.g.. residues 12-30). The modified ENPP1 sequence was cloned into a modified pFastbac FIT vector possessing a TEV protease cleavage site followed by a C-terminus 9-F1IS tag, and cloned and expressed in insect cells, and both proteins were expressed in a baculovirus system as described previously (Albright, etal., 2012, Blood 120:4432-4440; Saunders, etal.. 2011, J. Biol. Chem. 18:994-1004; Saunders, etal., 2008, Mol. Cancer Ther. 7:3352-3362), resulting in the accumulation of soluble, recombinant protein in the extracellular fluid.

[0331] 8. Expression Systems

[0332] Many expression systems are known and can be used for the production of ENPP 1 fusion protein, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae, Kluyveronmyces lactis and Pichia pastoris), filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells. The desired protein can be produced in conventional ways, for example from a coding sequence inserted in the host chromosome or on a free plasmid.

[0333] The yeasts can be transformed with a coding sequence for the desired protein in any of the usual ways (e.g., electroporation). Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente. 1990, Methods Enzymol. 194: 182. Successfully transformed cells, i.e., cells that contain a DNA construct of the present - 45 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0334] disclosure, can be identified by well-known techniques. For example, cells resulting from the introduction of an expression construct can be grown to produce an ENPP1 polypeptide. Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method, such as that described by Southern, 1975, J. Mol. Biol, 98:503 and / or Berent et al., 1985, Biotech 3:208. Alternatively, the presence of the protein in the supernatant can be detected using antibodies.

[0335] Useful yeast plasmid vectors include pRS403 — 406 and pRS413 — 416 and are generally available front Stratagene Cloning Systems, La Jolla, CA, USA Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers I-11S3, TRP1, LEU2 and 1JRA3. Plasmids pRS413 — 416 are Yeast Centromere plasmids (YCps).

[0336] A variety of methods have been developed to operably link DNA to vectors via complementary' cohesive termini. For instance, complementary homopolymer tract can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

[0337] Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, which are enzymes that remove protruding, 3 ’-single-stranded termini with their 3’-5’ -exonucleolytic activities, and fill in recessed 3’-ends with their polymerizing activities.

[0338] The combination of these activities thus generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. As a result, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments can be cleaved with an appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.

[0339] Clones of single, stably transfected cells are then established and screened for high expressing clones of the desired ENPP1 fusion protein. Screening of the single cell clones for ENPP1 protein expression can be accomplished in a high-throughput manner in 96 well plates using the synthetic enzymatic substrate pNP-TMP as previously described (Albright et - 46 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0340] al., 2015, Nat. Commun. 6:10006). Upon identification of high expressing clones through screening, protein production can be accomplished in shaking flasks or bio-reactors are previously described in Albright et al., 2015, Nat. Commun. 6:10006.

[0341] Also provided herein are nucleic acids that encode any one of the ENPP1 mutant polypeptides, ENPP1 -containing polypeptides, or fusions described herein. The disclosure further provides vectors, such as expression vectors, that comprise such nucleic acids. Also provided are a cell, cells, or a plurality of cells (e.g., mammalian cells) that comprise any one of the nucleic acids, vectors, or expression vectors described herein. Also provided are methods for producing a protein (e.g., any one of the ENPP1 mutant polypeptides, ENPP1-containing polypeptides, or fusions described herein), the methods in certain embodiments comprising culturing the cell, cells, or plurality of cells under conditions suitable for expression of the protein by the cell or cells from the nucleic acid, vector, or expression vector. The methods can also include purifying the protein from the cell, cells, or plurality of cells, or from the media in which the cell, cells, or plurality of cells were cultured. In addition, the disclosure provides proteins purified by any such methods.

[0342] The disclosure further provides an autonomously replicating or an integrative mammalian cell vector comprising a recombinant nucleic acid encoding a polypeptide of the disclosure. In certain embodiments, the vector comprises a plasmid or a virus. In other embodiments, the vector comprises a mammalian cell expression vector. In yet other embodiments, the vector further comprises at least one nucleic acid sequence that directs and / or controls expression of the polypeptide. In yet other embodiments, the recombinant nucleic acid encodes a polypeptide comprising an ENPP1 polypeptide of the disclosure and to a signal peptide, wherein the polypeptide is proteolytically processed upon secretion from a cell to yield the ENPP1 polypeptide of the disclosure.

[0343] In yet another aspect, the disclosure provides an isolated host cell comprising a vector of the disclosure. In certain embodiments, the cell is a non-human cell. In other embodiments, the cell is mammalian. In yet other embodiments, the vector of the disclosure comprises a recombinant nucleic acid encoding a polypeptide comprising a ENPP1 polypeptide of the disclosure and a signal peptide. In yet other embodiments, the polypeptide is proteolytically processed upon secretion from a cell to yield the ENPP1 polypeptide of the disclosure.

[0344] 9. ENPP1 Purification

[0345] Purification of ENPP1 can be accomplished using a combination of standard purification techniques known in the art. Following purification, ENPPl-Fc can be dialyzed - 47 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0346] into PBS supplemented with Zn2+and Mg2+(PBSplus) concentrated to between 5 and 7 mg / ml, and frozen at -80 °C in aliquots of 200-500 pl. Aliquots can be thawed immediately prior to use and the specific activity of the solution can be adjusted to 31.25 au / ml (or about 0.7 mg / ml depending on the preparation) by dilution in PBSplus.

[0347] 10. Route and Frequency of Administration

[0348] The polypeptide may be administered acutely or chronically to the subject. In certain embodiments, a second dosage of a soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein is administered after a suitable time interval of about after two days, after four days, after a week, or after a month to the subject or even less frequently, such as once every several months or even once a year or less. The frequency of the dose is readily apparent to the skilled artisan and depends upon 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.

[0349] A dose amount or frequency may be selected so that the steady state level of plasma PPi is maintained at a constant or steady state level, and / or so as to achieve a continuous level of plasma PPi that is either close to the normal (2-3 pM) level or above (30-50% higher than) normal levels of PPi and does not return to the lower level of PPi that the subject had prior to the administration of first dosage of constructs disclosed herein.

[0350] Alternative, the ENPP1 agent may be administered at appropriate time intervals of either every 2 days, or every 4 days, every week or every month so as to achieve a constant level of enzymatic activity of ENPP1.

[0351] Alternatively, an ENPP1 agent according to the disclosure is administered at an appropriate time interval of every 2 days, or every 4 days, or every week or every7month by monitoring one or more symptoms of a subject’s disease or disorder.

[0352] Without wishing to be bound by theory, it is believed that maintaining a steady7state concentration of plasma PPi at normal levels reduces and / or prevents progression of pathological calcification of subjects.

[0353] In certain embodiments, the polypeptide is administered locally, regionally, parenterally or systemically to the subject. In some embodiments, the polypeptide is administered subcutaneously.

[0354] As used herein, “parenteral administration” of a formulation includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the ENPP1 agent through the breach in the tissue. Parenteral administration thus includes,

[0355] - 48 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0356] but is not limited to, administration of an ENPP1 agent by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrastemal injection, and kidney dialytic infusion techniques.

[0357] The regimen of administration may affect what constitutes an effective amount. For example, several divided dosages, as well as staggered dosages may be administered in a given time period (daily) or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, selection of a recited dose of an ENPP1 agent may be indicated by the exigencies of the therapeutic or prophylactic situation.

[0358] Administration of the compositions of the present disclosure (e.g, soluble ENPP1 polypeptides and fusion proteins thereof) to a patient, such as a mammal (z.e., a human), may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder in the patient. An effective amount of the recited dosages of an ENPP1 agent necessary’ to achieve a therapeutic effect may vary’ according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. A selected dosage 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 of the therapeutic compound curve.

[0359] 11. Gene therapy

[0360] The nucleic acids encoding the polypeptide(s) useful within the disclosure may’ be used in gene therapy protocols for the treatment of the diseases or disorders contemplated herein. The improved construct encoding the polypeptide(s) can be inserted into the appropriate gene therapy vector and administered to a patient to treat or prevent the diseases or disorder of interest.

[0361] Vectors, such as viral vectors, have been used in the prior art to introduce genes into a wide variety of different target cells. Typically the vectors are exposed to the target cells so that transformation can take place in a sufficient proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired polypeptide (e.g., a - 49 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0362] receptor). The transfected nucleic acid may be permanently incorporated into the genome of each of the targeted cells, providing long lasting effect, or alternatively the treatment may have to be repeated periodically. In certain embodiments, the (viral) vector transfects liver cells in vivo with genetic material encoding the polypeptide(s) of the disclosure.

[0363] A variety of vectors, both viral vectors and plasmid vectors are known in the art (see for example U. S. Patent No. 5.252,479 and WO 93 / 07282). In particular, a number of viruses have been used as gene transfer vectors, including papovaviruses, such as SV40, vaccinia virus, herpes viruses including HSV and EBV, and retroviruses. Many gene therapy protocols in the prior art have employed disabled murine retroviruses. Several recently issued patents are directed to methods and compositions for performing gene therapy (see for example U. S. Patent Nos. 6,168,916; 6,135,976; 5,965,541 and 6,129.705). Each of the foregoing patents is incorporated by reference in its entirety herein.

[0364] A A V-Mediaied Gene Therapy:

[0365] AAV, a parvovirus belonging to the genus Dependovirus, has several features that make it particularly well suited for gene therapy applications. For example. AAV can infect a wide range of host cells, including non-dividing cells. Furthermore. AAV can infect cells from a variety of species. Importantly, AAV has not been associated with any human or animal disease, and does not appear to alter the physiological properties of the host cell upon integration. Finally, AAV is stable at a wide range of physical and chemical conditions, which lends itself to production, storage, and transportation requirements.

[0366] The AAV genome, which is a linear, single-stranded DNA molecule containing approximately 4,700 nucleotides (the AAV-2 genome consists of 4,681 nucleotides, the AAV-4 genome 4,767), generally comprises an internal non-repeating segment flanked on each end by inverted terminal repeats (ITRs). The ITRs are approximately 145 nucleotides in length (AAV-1 has ITRs of 143 nucleotides) and have multiple functions, including serving as origins of replication, and as packaging signals for the viral genome.

[0367] The internal non-repeated portion of the genome includes tw o large open reading frames (ORFs), known as the AAV replication (rep) and capsid (cap) regions. These ORFs encode replication and capsid gene products, which allow for the replication, assembly, and packaging of a complete AAV virion. More specifically, a family of at least four viral proteins are expressed from the AAV rep region: Rep 78, Rep 68, Rep 52, and Rep 40, all of which are named for their apparent molecular weights. The AAV cap region encodes at least three proteins: VP1, VP2, and VP3.

[0368] AAV is a helper-dependent virus, that is, it requires co-infection with a helper virus - 50 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0369] (e.g., adenovirus, herpesvirus, or vaccinia virus) in order to form functionally complete AAV virions. In the absence of co-infection with a helper virus. AAV establishes a latent state in which the viral genome inserts into a host cell chromosome or exists in an episomal form, but infectious virions are not produced. Subsequent infection by a helper virus “rescues” the integrated genome, allowing it to be replicated and packaged into viral capsids, thereby reconstituting the infectious virion. While AAV can infect cells from different species, the helper virus must be of the same species as the host cell. Thus, for example, human AAV replicates in canine cells that have been co-infected with a canine adenovirus.

[0370] To produce infectious recombinant AAV (rAAV) containing a heterologous nucleic acid sequence, a suitable host cell line can be transfected with an AAV vector containing the heterologous nucleic acid sequence, but lacking the AAV helper function genes, rep and cap. The AAV -helper function genes can then be provided on a separate vector. Also, only the helper virus genes necessary for AAV production (i.e., the accessory function genes) can be provided on a vector, rather than providing a replication-competent helper virus (such as adenovirus, herpesvirus, or vaccinia).

[0371] Collectively, the AAV helper function genes (i.e., rep and cap) and accessory function genes can be provided on one or more vectors. Helper and accessory function gene products can then be expressed in the host cell where they will act in trans on rAAV vectors containing the heterologous nucleic acid sequence. The rAAV vector containing the heterologous nucleic acid sequence will then be replicated and packaged as though it were a wild-type (wt) AAV genome, forming a recombinant virion. When a patient s cells are infected with the resulting rAAV virions, the heterologous nucleic acid sequence enters and is expressed in the patient's cells. Because the patient’s cells lack the rep and cap genes, as well as the accessory function genes, the rAAV cannot further replicate and package their genomes. Moreover, without a source of rep and cap genes, wtAAV cannot be formed in the patient’s cells.

[0372] There are eleven known AAV serotypes. AAV-1 through AAV-11 (Mori, et al., 2004, Virology 330(2):375-83). AAV-2 is the most prevalent serotype in human populations; one study estimated that at least 80% of the general population has been infected with wt AAV-2 (Berns and Linden, 1995, Bioessays 17:237-245). AAV-3 and AAV-5 are also prevalent in human populations, with infection rates of up to 60% (Georg-Fries, et al., 1984, Virology 134:64-71). AAV-1 and AAV -4 are simian isolates, although both seroty pes can transduce human cells (Chiorini, et al., 1997, J Virol 71:6823-6833; Chou, et al., 2000, Mol Ther 2:619-623). Of the six known serotypes. AAV-2 is the best characterized. For instance, AAV-2 has been used in a broad array of in vivo transduction experiments, and has been - 51 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0373] shown to transduce many different tissue types including: mouse (U. S. Patent Nos.

[0374] 5,858,351; U. S. Patent No. 6,093,392), dog muscle; mouse liver (Couto, et al., 1999. Proc. Natl. Acad. Sci. USA 96:12725-12730; Couto, et al., 1997, J. Virol. 73:5438-5447; Nakai, et al., 1999, J. Virol. 73:5438-5447; and, Snyder, etal., 1997, Nat. Genet. 16:270-276); mouse heart (Su, et al., 2000, Proc. Natl. Acad. Sci. USA 97:13801-13806); rabbit lung (Flotte, et al., 1993, Proc. Natl. Acad. Sci. USA 90:10613-10617); and rodent photoreceptors (Flannery et al., 1997, Proc. Natl. Acad. Sci. USA 94:6916-6921).

[0375] The broad tissue tropism of AAV -2 may be exploited to deliver tissue-specific transgenes. For example, AAV-2 vectors have been used to deliver the following genes: the cystic fibrosis transmembrane conductance regulator gene to rabbit lungs (Flotte. et al., 1993, Proc. Natl. Acad. Sci. USA 90:10613-10617); Factor NIII gene (Burton, et al., 1999. Proc. Natl. Acad. Sci. USA 96:12725-12730) and Factor IX gene (Nakai, etal., 1999, J. Virol. 73:5438-5447; Snyder, et al., 1997, Nat. Genet. 16:270-276; U. S. Patent No. 6,093,392) to mouse liver, dog, and mouse muscle (U. S. Patent No. 6,093,392); erythropoietin gene to mouse muscle (U. S. Patent Nos. 5,858,351); vascular endothelial growth factor (VEGF) gene to mouse heart (Su, et al., 2000, Proc. Natl. Acad. Sci. USA 97:13801-13806); and aromatic 1 -amino acid decarboxylase gene to monkey neurons. Expression of certain rAAV-delivered transgenes has therapeutic effect in laboratory animals; for example, expression of Factor IX was reported to have restored phenotypic normalcy in dog models of hemophilia B (U. S. Patent No. 6.093,392). Moreover, expression of rAAV-delivered NEGF to mouse myocardium resulted in neovascular formation (Su, et al., 2000, Proc. Natl. Acad. Sci. USA 97:13801-13806), and expression of rAAV-delivered AADC to the brains of parkinsonian monkeys resulted in the restoration of dopaminergic function.

[0376] Delivery of a protein of interest to the cells of a mammal is accomplished by first generating an AAV vector comprising DNA encoding the protein of interest and then administering the vector to the mammal. Thus, the disclosure should be construed to include AAV vectors comprising DNA encoding the polypeptide(s) of interest. Once armed with the present disclosure, the generation of AAV vectors comprising DNA encoding this / these polypeptide(s)s will be apparent to the skilled artisan.

[0377] In certain embodiments, the rAAV vector of the disclosure comprises several essential DNA elements. In certain embodiments, these DNA elements include at least two copies of an AAV ITR sequence, a promoter / enhancer element, a transcription termination signal, any necessary 5’ or 3‘ untranslated regions which flank DNA encoding the protein of interest or a biologically active fragment thereof. The rAAV vector of the disclosure may also include a - 52 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0378] portion of an intron of the protein on interest. Also, optionally, the rAAV vector of the disclosure comprises DNA encoding a mutated polypeptide of interest.

[0379] In certain embodiments, the vector comprises a promoter / regulatory sequence that comprises a promiscuous promoter which is capable of driving expression of a heterologous gene to high levels in many different cell types. Such promoters include, but are not limited to the cytomegalovirus (CMV) immediate early promoter / enhancer sequences, the Rous sarcoma virus promoter / enhancer sequences and the like. In certain embodiments, the promoter / regulatory sequence in the rAAV vector of the disclosure is the CMV immediate early promoter / enhancer. However, the promoter sequence used to drive expression of the heterologous gene may also be an inducible promoter, for example, but not limited to, a steroid inducible promoter, or may be a tissue specific promoter, such as. but not limited to, the skeletal a-actin promoter which is muscle tissue specific and the muscle creatine kinase promoter / enhancer, and the like.

[0380] In certain embodiments, the rAAV vector of the disclosure comprises a transcription termination signal. While any transcription termination signal may be included in the vector of the disclosure, in certain embodiments, the transcription termination signal is the SV40 transcription termination signal.

[0381] In certain embodiments, the rAAV vector of the disclosure comprises isolated DNA encoding the polypeptide of interest, or a biologically active fragment of the polypeptide of interest. The disclosure should be construed to include any mammalian sequence of the polypeptide of interest, which is either known or unknown. Thus, the disclosure should be construed to include genes from mammals other than humans, which polypeptide functions in a substantially similar manner to the human polypeptide. Preferably, the nucleotide sequence comprising the gene encoding the polypeptide of interest is about 50% homologous, more preferably about 70% homologous, even more preferably about 80% homologous and most preferably about 90% homologous to the gene encoding the polypeptide of interest.

[0382] Further, the disclosure should be construed to include naturally occurring variants or recombinantly derived mutants of wild type protein sequences, which variants or mutants render the polypeptide encoded thereby either as therapeutically effective as full-length polypeptide, or even more therapeutically effective than full-length polypeptide in the gene therapy methods of the disclosure.

[0383] The disclosure should also be construed to include DNA encoding variants which retain the polypeptide’s biological activity. Such variants include proteins or polypeptides which have been or may be modified using recombinant DNA technology, such that the - 53 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0384] protein or polypeptide possesses additional properties which enhance its suitability for use in the methods described herein, for example, but not limited to. variants conferring enhanced stability on the protein in plasma and enhanced specific activity of the protein. Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. For example, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function.

[0385] The disclosure is not limited to the specific rAAV vector exemplified in the experimental examples; rather, the disclosure should be construed to include any suitable AAV vector, including, but not limited to, vectors based on AAV-1, AAV-3, AAV -4 and AAV-6, and the like.

[0386] Also included in the disclosure is a method of treating a mammal having a disease or disorder in an amount effective to provide a therapeutic effect. The method comprises administering to the mammal an rAAV vector encoding the polypeptide of interest.

[0387] Preferably, the mammal is a human.

[0388] Typically, the number of viral vector genomes / mammal which are administered in a single injection ranges from about 1×108to about 5×1016. Preferably, the number of viral vector genomes / mammal which are administered in a single injection is from about 1×1010to about 1×1015; more preferably, the number of viral vector genomes / mammal which are administered in a single injection is from about 5×1010to about 5×1015: and, most preferably, the number of viral vector genomes which are administered to the mammal in a single injection is from about 5×1011to about 5×1014.

[0389] When the method of the disclosure comprises multiple site simultaneous injections, or several multiple site injections comprising injections into different sites over a period of several hours (for example, from about less than one hour to about two or three hours) the total number of viral vector genomes administered may be identical, or a fraction thereof or a multiple thereof, to that recited in the single site injection method.

[0390] For administration of the rAAV vector of the disclosure in a single site injection, in certain embodiments a composition comprising the virus is injected directly into an organ of the subject (such as, but not limited to, the liver of the subject).

[0391] For administration to the mammal, the rAAV vector may be suspended in a pharmaceutically acceptable carrier, for example, HEPES buffered saline at a pH of about 7.8. Other useful pharmaceutically acceptable carriers include, but are not limited to. glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as - 54 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0392] phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (1991. Mack Publication Co., New Jersey).

[0393] The rAAV vector of the disclosure may also be provided in the form of a kit, the kit comprising, for example, a freeze-dried preparation of vector in a dried salts formulation, sterile water for suspension of the vector / salts composition and instructions for suspension of the vector and administration of the same to the mammal.

[0394] 12. Prophylactic Administration

[0395] Armed with the disclosure herein, one skilled in the art would thus appreciate that the prevention of a disease or disorder in a subject encompasses administering to a subject an ENPP1 polypeptide as a preventative measure against the disease or disorder.

[0396] The relative amounts of the active ingredient (e.g, soluble ENPP1 polypeptides and fusion proteins thereof), the pharmaceutically acceptable carrier, and any additional ingredients in a formulation disclosed herein will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between about 0.1% and about 100% (w / w) active ingredient.

[0397] 13. ENPP1 Polypeptide Sequences

[0398] Table 1: Sequences

[0399]

[0400]

[0401] - 55 - 576437472Attomey Docket No. 047162-7564W01(02906)

[0402]

[0403] - 56 - 576437472Attomey Docket No. 047162-7564W01(02906)

[0404]

[0405] - 57 - 576437472Attomey Docket No. 047162-7564W01(02906)

[0406]

[0407] For SEQ ID NOs: 118 and 119:

[0408] X= export sequences

[0409] Y= Bone targeting tag or a stop codon (i.e. no amino acid as marked by *) Non-limiting examples of bone targeting sequences include, but are not limited to, DDDDDDDD (SEQ ID NO: 125) and / or EEEEEEEE (SEQ ID NO: 126).

[0410] Non-bold, non-underlined font = linker region, which can be various lengths and so

[0411] - 58 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0412] forth. Amino acid composition of the linker can be variable. In certain non-limiting embodiments, the linker region can include residues such as histidine to discourage non-specific protease cleavage (i. e., HS or HG instead of RS or RG).

[0413] Bold font = Human IgGl (optimized in BL-1118 with the M883Y S885TT887E mutations highlighted in bold italics)

[0414] Underlined font = extracellular domain of human ENPP1 (optimized in BL-1118 with the 1256T substitution, highlighted in bold underlined)

[0415] SEQ ID NO: 7 (Mouse ENPP1- NCBI accession NP 001295256.1)

[0416] 1 MERDGDQAGH GPRHGSAGNG RELESPAAAS LLAPMDLGEE PLEKAERARP AKDPNTYKVL 61 SLVLSVCVLT TILGCTFGLK PSCAKEVKSC KGRCFERTFS NCRCDAACVS LGNCCLDFQE 121 TCVEPTHIWT CNKFRCGEKR LSRFVCSCAD DCKTHNDCCI NYSSVCQDKK SWVEETCESI 181 DTPECPAEFE SPPTLLFSLD GFRAEYLHTW GGLLPVISKL KNCGTYTKNM RPMYPTKTFP 241 NHYSIVTGLY PESHGIIDNK MYDPKMNASF SLKSKEKFNP LWYKGQPIWV TANHQEVKSG 301 TYFWPGSDVE IDGILPDIYK VYNGSVPFEE RILAVLEWLQ LPSHERPHFY TLYLEEPDSS 361 GHSHGPVSSE VIKALQKVDR LVGMLMDGLK DLGLDKCLNL ILISDHGMEQ GSCKKYVYLN 421 KYLGDVNNVK WYGPAARLR PTDVPETYYS FNYEALAKNL SCREPNQHFR PYLKPFLPKR 481 LHFAKSDRIE PLTFYLDPQW QLALNPSERK YCGSGFHGSD NLFSNMQALF IGYGPAFKHG 541 AEVDSFENIE VYNLMCDLLG LIPAPNNGSH GSLNHLLKKP IYNPSHPKEE GFLSQCPIKS 601 TSNDLGCTCD PWIVPIKDFE KQLNLTTEDV DDIYHMTVPY GRPRILLKQH RVCLLQQQQF 661 LTGYSLDLLM PLWASYTFLS NDQFSRDDFS NCLYQDLRIP LSPVHKCSYY KSNSKLSYGF 721 LTPPRLNRVS NHIYSEALLT SNIVPMYQSF QVIWHYLHDT LLQRYAHERN GINWSGPVF 781 DFDYDGRYDS LEILKQNSRV IRSQEILIPT HFFIVLTSCK QLSETPLECS ALESSAYILP 841 HRPDNIESCT HGKRESSWVE ELLTLHRARV TDVELITGLS FYQDRQESVS ELLRLKTHLP 901 IFSQED

[0417] SEQ ID NO: 8 (Cow ENPP1- NCBI accession NP 001193141.1)

[0418] 1 MERDSCAGGG SRGGEGGRGP REGLAGNGRD PGPGRAAEAS GEPQAAASLL APMDLGEEPL 61 ERAARARPAK DPNTYKVLSL VLSVCVLTTI LGCIFGLKPS CAKEIKSCKG RCFERTFGNC 121 RCDAACVDLG NCCLDYQETC IEPERIWTCT KFRCGEKRLS RSLCSCSDDC KDKGDCCINH 181 GSVCRGEKSW AEEECDSIDE PQCPAGFETP PTLLFSLDGF RAEYLHTWGG LLPVISKLKT 241 CGTYTKNMRP VYPTKTFPNH YSIVTGLYPE SHGIIDNNIY DPQMNANFAL KNKEKFNPEW 301 YKGEPIWLTA KYQGLKTGTF FWPGSDVKIN GIFPDIYKIY NVSVPFEERI LAILKWLQLP 361 KDERPHFYTL YLEEPDSSGH SYGPVSSEVI RALQRVDNMV GMLMDGLKEL NLHRCLNLIL 421 ISDHGMEQGS CKKYVYLNKY LGDTKDYKW YGPAARLRPS DVPDKYYSFD YEGIAKNLSC 481 QEPNQHFKPY LKHFLPKRLH FAKNDRIERL TFYLDPQWQL ALNPSERKYC GGGFHGSDNT 541 FLNMQALFIG YGPGFKHSTE VDSFENIEVY NLMCDLLNLT PAPNNGTHGS LNHLLSNPVY 601 TPKHPKEVRP LVQCPFTRAP RESLDCSCDP SILPIVDFQT QLNLTMAEEK TIKRGALPYG 661 RPRVLQNSTV CLLYQHQFVS GYSRDILMPL WTSYTIGRND SFSTEDFSNC LYQDLRIPLS - 59 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0419] 721 PVHKCSFYKN NAKLSYGLLS PPQLHKGSSQ VYSEALLTTN IVPMYQSFQV IWHYLHGTLL 781 QRYAEERNGL NWSGPVFDS DYDGRYDSLE TLKQNSKIIR NLEVLIPTHF FLVLTSCKNT 841 SQTPLQCENL DAMAFILPHK TDNSESCAHG KHESLWVEEL LKLHTARITD VEHITGLSFY 901 QERKEPISDI LKLKTHLPTF NQED

[0420] SEQ ID NO: 9 (Rabbit ENPP1-NCBI accession NP 001162404.1)

[0421] 1 MERDGCAGGG SRGGEGGRAP REGPAGNSRD PGRSHAAEAP GNPQAAASLL APMDVGEEPL 61 EKAARARTAK DPNTYKVLSL VLSVCVLTTI LGCIFGLKPS CAKEVKSCKG RCFERTFGNC 121 RCDAACVELG NCCLDYQETC IEPEHIWTCN KFRCGEKRLT RSLCACSDDC KDQGDCCINY 181 SSVCQGEKSW VEEPCESTNE PQCPAGFETP PTLLFSLDGF RAEYLHTWGG LLPVISKLKK 241 CGTYTKNMRP VYPTKTFPNH YSIVTGLYPE SHGIIDNKMY DPKMNASFSL KSKEKFNPEW 301 YKGEPIWTA KYQGLKSGTF FWPGSDVEIN GIFPDIYKMY NGSVPFEERI LAVLQWLQLP 361 KDERPHFYTL YLEEPDSSGH SYGPVSSEVI KALQRVDNMV GMLMDGLKEL NLHRCLNLIL 421 VSDHGMEQGS CKKYIYLNKY LGDVKNIKVI YGPAARLRPS DVPDKYYSFN YEGIARNLSC 481 REPNQHFKPY LKHFLPKRLH FAKSDRIEPL TFYLDPQWQL ALNPSERKYC GSGFHGSDNI 541 FSNMQALFVG YGPGFKHGIE VDTFENIEVY NLMCDLLNLT PAPNNGTHGS LNHLLKNPVY 601 TPKHPKEVHP LIQCPFTRNP RDNLGCSCNP SILPIEDFQT QFNLTVAEEK NIKHETLPYG 661 RPRVLQKKNT ICLLSQHQFM SGYSQDILMP LWTSYTVDRN DSFSTEDFSN CLYQDFRISL 721 SPVHKCSFYK NNTKVSYGFL SPPQLNKNSR GIYSEALLTT NIVPMYQSFQ VIWRYFHDTL 781 LRKYAEERNG VNWSGPVFD FDYDGRYDSL EILRQKRRVI RNQEILIPTH FFIVLTSCKD 841 ASQTPLHCEN LDTLAFILPH RTDNSESCLH GKHESSWVEE LLMLHRARIT DVEHITGLSF 901 YQQRKEPVSD ILKLKTHLPT FSQED

[0422] SEQ ID NO: 10 (Baboon ENPP1-NCBI accession NP 001076211.2)

[0423] 1 MERDGCAGGG SQGGGKGGRG PREGLAGNGR DPSHGQASEA PGDPQAAASL LAPMDLGEEP 61 LEKAAGARPA KDPNTYKVLS LVLSVCVLTT ILGCIFGLKP SCAKEVKSCK GRCFERTFGN 121 CRCDVACVDL GNCCLDYQET CIEPERIWTC NKFRCGEKRL SRSLCACSDD CKERGDCCIN 181 YSAVCQGEKS WVEETCENIN EPQCPEGFEM PPTLLFSLDG FRAEYLHTWG GLLPVISKLK 241 KCGTYAKNMR PVYPTKTFPN HYSIVTGLYP ESHGIIDNKM YDPKMNASFS LKSKEKFNPE 301 WYKGEPIWLT AKYQGLRSGT FFWPGSDVKI NGIFPDIYKI YNGSVPFEER ILAILKWLRL 361 PKDERPHFYT LYLEEPDSSG HSYGPVSSEV IKALQRVDNM VGMLMDGLKE LNLHQCLNLI 421 LISDHGMEQG SCKKYIYLNK YLGDTKNIKV IYGPAARLRP SDVPEKYYSF NYENIARNLS 481 CREPNQHFKP YLKHFLPKRL HFAKSDRIEP LTFYLDPQWQ LALSPSERKY CGSGFHGSDN 541 VFSNMQALFV GYGPGFQHGI EVDSFENIEV YNLMCDLLNL TPAPNNGTHG SLNHLLKNPI 601 YTPKHPKEVQ PSVQCPLAGS PRDSLGCSCN PSILPIVDFQ TQFNLTTAEE KNINRASLPY 661 GRPRLLQKKS SVCLLYQHQF VSGYSHDVLM PLWTSYTVNR NDSFSTEDFS NCLYQDLRIS 721 FSPIHNCSFY KNNAKLSYGF LSPPQLSKDS SQIYSEALLT SNIVPMYQSF QVIWRYFHDT 781 LLQRYAEERN SINWSGPVF DSDYDGRYDS SEALKRNRRV IRNQEILIPT HFFIVITSCK 841 NTSQTPLQCD NLDPLAFILP HRSDNSESCV HEKRESSWIE ELLMMHRARI MDVEHITGLS 901 FYQERKEPVS DILKLKTHLP TVSQED

[0424] - 60 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0425] SEQ ID NO: 118 (Construct 1118 - Soluble ENPPl-Fc) MRGPAVLLTVALATLLAPGAGAPS CAKEVKS CKGRCFERTFGNCRCDAACVELGNCCLDYQRTC1RPRH1WTCNK FR CGEKRL TRSL C CSDD CKDKGD CCINYSS VCQGEKS WVEEPCES INEPQ CP AGE RT P PTL L F S L DGFRAE YLH TWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNASFSLKSKEKFNPEWY KGEPTWVTAKYQGLKSGTFFWPGSDVEINGgFPDIYKMYNGSVPFEERILAVLQWLQLPKDERPHFYTLYLEEPD SSGHSYGPVSSEVIKALQRVDGMVGMLMDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIY GPAARLRPSDVPDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLALNPSE RKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGTEADTFENIEVYNLMCDLLNLTPAPNNGTHGSLNHLLKNPVYT PKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTOFNLTVAEEKTIKHETLPYGRPRVLQKENTTCLLSO HQFMSGYSQDILMPLWTS YTVDRNDSFSTEDFSNCLYQDFRIPLS PVHKCSFYKNNTKVS YGFLS PPQLNKNSSG IYSEALLTTNIVPMYOSFOVIWRYFHDTLLRKYAEERNGVNWSGPVFDFDYDGRCDSLENLROKRRVIRNQEIL IPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELLMLHRARITDVEHITGLSFY OOB. KRPVSDTLKLKTHLPTFSORDRS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHE DPE VKFNWYVDGVE VHNAKTKPREEQ YNS T YRVVS VL TVLHQDWLNGKE YKCKVSNKAL PAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW Q GNVFS CSVMHEALHNHYTQKSLSLS PGK*

[0426] Singly -underlined font: Signal sequence for export from human ENPP7 (Accession Q6UWV6)

[0427] Italics font: Somatomedin B domain 1

[0428] Singly -underlined italics font: Somatomedin B domain 2

[0429] Normal font: Catalytic domain

[0430] Doubly -underlined font: Endonuclease domain

[0431] Doubly -underlined italics font: human IgG Fc (amino acids 852-1078)

[0432] Known glycosylation sites in the catalytic domain are highlighted as singly -underlined bold font; Isoleucine 256 residue in human ENPP1 was mutated to a threonine (I256T; doubly- underlined bold font) to enhance bioavailability. Residues in the Fc IgG domain were additionally mutated to enhanced bioavailability (M883Y, S885T, T887E). * stop codon.

[0433] SEQ ID NO: 119 (Construct 2000 - Bone Targeted ENPPl-Fc) RGPA LLTVKLATLLAPGAGAPSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQRTCIRPREELWTCNK FR CGEKRL TRSL CA CSDD CKDKGD CCINYSS VCQGEKS WVEEPCES INEPQ CP AGE RY P PTL L F S L DGFRAE YLH TWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNASFSLKSKEKFNPEWY KGEPIWVTAKYQGLKSGTFFWPGSDVEINGgFPDIYKMYNGSVPFEERILAVLQWLQLPKDERPHFYTLYLEEPD SSGHSYGPVSSEVIKALQRVDGMVGMLMDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIY GPAARLRPSDVPDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLALNPSE RKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGTEADTFENTEVYNLMCDLLNLTPAPNNGTHGSLNHLLKNPVYT PKHPKEVHPLVQCPFTKNPRDNLGCSCNPSILPIEDFQTOFNLTVAEEKTIKHETLPYGRPRVLQKENTTCLLSO HQFMSGYSQDILMPLWTS YTVDRNDSFSTEDFSNCLYQDFRIPLS PVHKCSFYKNNTKVS YGFLS PPQLNKNSSG

[0434] - 61 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0435] IYSEALLTTNIVPMYOSFOVIWRYFHDTLLRKYAEERNGVNWSGPVFDFDYDGRCDSLENLROKRRVIRNQEIL IPTHFFIVLTSCKDTSOTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELLMLHRARITDVEHITGLSFY

[0436]

[0437] Singly-underlined font: Signal sequence for export from human ENPP7 (Accession Q6UWV6)

[0438] Italics font: Somatomedin B domain 1

[0439] Singly -underlined italics font: Somatomedin B domain 2

[0440] Normal font: Catalytic domain

[0441] Doubly -underlined font: Endonuclease domain

[0442] Doubly-underlined italics font: human IgG Fc (amino acids 852-1078)

[0443] Known glycosylation sites in the catalytic domain are highlighted as singly -underlined bold font; Isoleucine 256 residue in human ENPP1 was mutated to a threonine (I256T; doubly- underlined bold font) to enhance bioavailability. Residues in the Fc IgG domain were additionally mutated to enhanced bioavailability (M883Y, S885T, T887E). * stop codon. Bone targeting was achieved by adding 6 aspartic acid residues to the C-terminus of 1118 to form clone 2000.

[0444] SEQ ID NO: 120: ENPPl-Fc MRGPAVLLTVALATLLAPGAGAPSCAKEVKSCKGRCFERTFGNCRCDAAC’YELGNCCLDYQETCIEPEHIWTCNK FRCGEKRLTRSLCACSDDCKDKGDCCINYSSVCQGEKSWEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYLH TWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNASFSLKSKEKFNPEWY KGEPlWTJiKYQGLKSGTFFWPGSWEINGIFPDIYKMYNGSVPFEERILAVLQWLQLPKDERPHFYTLYLEEPD SSGHSYGPVSSEVIKALQRVDGMVGMLMDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIY GPAARLRPSDVPDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLALNPSE RKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEAOTFENIEVYNLMCDLLNLTPAPt'JNGTHGSLNHLLKNP VYT PKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQFNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQ HQFMSGYSQDILMPLWTSYTVDRNDSFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSG IYSEALLTTNIVPMYQSFQVIWRYFHDTLLRKYAEERNGVNWSGPVFDFDYDGRCDSLENLRQKRRVIRNQEIL IPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELLMLHRARITDVEHITGLSFY QQRKEPVSD1LKLKTHLPTESQEDRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMLSRTPEVTCVVVDVSHE DPE VKFNWYVDGVE VHNAKTKPREEQ YNS T YRVVS VL TVLHQDWLNGKE YKCKVSNKAL PAPLEKTLSKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDLAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK

[0445] Bold: signal sequence

[0446] Regular: ENPP1 extracellular domain

[0447] Underlined: linker sequence

[0448] - 62 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0449] Italics: Fc domain

[0450] Pharmaceutical Compositions and Formulations

[0451] The disclosure provides pharmaceutical compositions comprising a polypeptide of the disclosure within the methods described herein.

[0452] Such a pharmaceutical composition is in a form suitable for administration to a subject, or the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The various components of the pharmaceutical composition may be present in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0453] In an embodiment, the pharmaceutical compositions useful for practicing the method of the disclosure may be administered to deliver a dose of between 1 ng / kg / day and 100 mg / kg / day. In other embodiments, the pharmaceutical compositions useful for practicing the disclosure may be administered to deliver a dose of between 1 ng / kg / day and 500 mg / kg / day.

[0454] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the disclosure will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between about 0.1% and about 100% (w / w) active ingredient.

[0455] Pharmaceutical compositions that are useful in the methods of the disclosure may be suitably developed for inhalational, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, intravenous or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations. The route(s) of administration is readily apparent to the skilled artisan and depends upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.

[0456] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory7ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0457] As used herein, a '“unit dose’7is a discrete amount of the pharmaceutical composition - 63 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0458] comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g, about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0459] Administration / Dosing

[0460] The regimen of administration may affect what constitutes an effective amount. For example, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation. In certain embodiments, administration of the compound of the disclosure to a subject elevates the subject’s plasma PPi to a level that is close to normal, where a normal level of PPi in mammals is 1-3 pM. “Close to normal” refers to 0 to 1.2 LIM or 0-40% below or above normal, 30 nM to 0.9 pM or 1-30% below or above normal, 0 to 0.6 pM or 0-20% below or above normal, or 0 to 0.3 pM or 0-10% below or above normal.

[0461] Administration of the compositions of the present disclosure to a patient, such as a mammal, such as a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. Dosage 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 of the therapeutic compound curve. The polypeptide according to the disclosure is administered at an appropriate time interval of every 2 days, or every 4 days, or every week or every month so as to achieve a continuous level of plasma PPi that is either close to the normal (1-3 pM) level or above (30-50% higher than) normal levels of PPi. Therapeutic dosage of the polypeptides of the disclosure may also be determined based on half-life or the rate at which the therapeutic polypeptide is cleared out of the body. The polypeptide - 64 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0462] according to the disclosure is administered at appropriate time intervals of either every 2 days, or every 4 days, every week or every’ month so as to achieve a constant level of enzymatic activity of ENPP1.

[0463] For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A nonlimiting example of an effective dose range for a therapeutic compound of the disclosure is from about 0.01 and 50 mg / kg of body weight / per day. In certain embodiments, the effective dose range for a therapeutic compound of the disclosure is from about 50 ng to 500 ng / kg, preferably 100 ng to 300 ng / kg of body weight. One of ordinary' skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0464] The compound can be administered to a patient as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every' 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose is readily apparent to the skilled artisan and depends upon 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.

[0465] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0466] A medical doctor, e.g., physician, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0467] In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In other embodiments.

[0468] - 65 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0469] the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two. days, every’ three days to once a week, and once every two weeks. The frequency of administration of the various combination compositions of the disclosure varies from subject to subject depending on many’ factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient will be determined by the attending physical taking all other factors about the patient into account.

[0470] In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.

[0471] Routes of Administration

[0472] Routes of administration of any of the compositions of the disclosure include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g, sublingual, lingual, (trans)buccal, (trans (urethral, vaginal (e.g, trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrap ulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0473] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. The formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.

[0474] Parenteral Administration

[0475] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical - 66 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0476] composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrastemal injection, and kidney dialytic infusion techniques.

[0477] Additional Administration Forms

[0478] Additional dosage forms of this disclosure include dosage forms as described in U. S. Patents Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790.

[0479] Additional dosage forms of this disclosure also include dosage forms as described in U. S. Patent Applications Nos. 20030147952, 20030104062, 20030104053, 20030044466.

[0480] 20030039688, and 20020051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos. WO 03 / 35041, WO 03 / 35040, WO 03 / 35029, WO 03 / 35177, WO 03 / 35039, WO 02 / 96404, WO 02 / 32416, WO 01 / 97783, WO 01 / 56544, WO 01 / 32217, WO 98 / 55107, WO 98 / 11879, WO 97 / 47285, WO 93 / 18755, and WO 90 / 11757.

[0481] Controlled Release Formulations and Drug Delivery Systems

[0482] Controlled- or sustained-release formulations of a pharmaceutical composition of the disclosure may be made using conventional technology. In some cases, the dosage forms to be used can be provided as slow or controlled-release of one or more active ingredients therein using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions. Single unit dosage forms suitable for oral administration, such as tablets, capsules, gelcaps, and caplets, which are adapted for controlled-release are encompassed by the present disclosure.

[0483] In certain embodiments, the formulations of the present disclosure may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.

[0484] The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may. although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should - 67 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0485] be a release that is longer that the same amount of agent administered in bolus form. For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material that provides sustained release properties to the compounds. As such, the compounds for use the method of the disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation. In certain embodiments of the disclosure, the compounds of the disclosure are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

[0486] The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours. The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration. The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.

[0487] As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.

[0488] As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.

[0489] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this disclosure and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction and preparation conditions, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.

[0490] It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present disclosure. Moreover, all values that fall within these ranges, as well as - 68 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0491] the upper or lower limits of a range of values, are also contemplated by the present application.

[0492] The following examples further illustrate aspects of the present disclosure. However, they are in no way a limitation of the teachings or disclosure of the present disclosure as set forth herein.

[0493] EXPERIMENTAL EXAMPLES

[0494] The disclosure is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the disclosure should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0495] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present disclosure and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present disclosure, and are not to be construed as limiting in any way the remainder of the disclosure.

[0496] The materials and methods employed in the following Examples are now described.

[0497] Material & Methods

[0498] Animals

[0499] The present study used Enpplasj,as]mice generated as described previously (Oheim R, Zimmerman K, Maulding ND, Sturznickel J, von Kroge S, Kavanagh D, et al. Human Heterozygous ENPP1 Deficiency Is Associated With Early Onset Osteoporosis, a Phenotype Recapitulated in a Mouse Model of Enppl Deficiency. J Bone Miner Res. 2020;35(3):528-39) and wild-type (WT) littermates. Enpplasj / as}mice possess a missense mutation in Enppl (Val246Asp) (Li Q, Guo H, Chou DW, Berndt A, Sundberg JP, Uitto J. Mutant Enppl asj mice as a model for generalized arterial calcification of infancy. Dis Model Meeh.

[0500] 2013;6(5): 1227-35; Albright RA, Stabach P, Cao W, Kavanagh D, Mullen I, Braddock AA, et al. ENPPl-Fc prevents mortality and vascular calcifications in rodent model of generalized arterial calcification of infancy. Nat Commun. 2015;6: 10006). The mice had ad libitum access to food and water throughout the study. The colony was maintained under specific - 69 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0501] pathogen-free conditions to ensure a controlled environment.

[0502] Enzyme replacement therapy formulations

[0503] The design and characterization of Enppl-Fc clone 1118 has been previously described (Stabach PR, Zimmerman K, Adame A, Kavanagh D, Saeui CT, Agatemor C, et al. Improving the Pharmacodynamics and In Vivo Activity of ENPPl-Fc Through Protein and Glycosylation Engineering. Clin Transl Sci. 2021; 14( l):362-72). Soluble 1118 was targeted to the bone via a 6 aspartic-acid C-terminal tag (clone 2000) introduced via site-directed mutagenesis using the QuikChange XL Site-Directed Mutagenesis Kit (Agilent Technologies Catalog # 200516), following manufacturer's protocols.

[0504] The sense and anti-sense oligos were 5’- CTGTCTCCGGGTAAAGATGATGATGATGATGATGACGACTGAGTGCTAGCTGGC CAG-3’ (SEQ ID NO: 121) and 5'- GCCAGCTAGCACTCAGTCGTCATCATCATCATCATCATCTTTACCCGGAGACAGG GAG-3’ (SEQ ID NO: 122).

[0505] Plasmids were sequence- verified correctly through the Yale Keck DNA Sequencing core facility. Both proteins were purified as previously described (Albright RA, Stabach P, Cao W, Kavanagh D, Mullen I, Braddock AA, et al. ENPPl-Fc prevents mortality and vascular calcifications in rodent model of generalized arterial calcification of infancy. Nat Commun. 2015;6: 10006). Further, the ERT 1118 and ERT 2000 were formulated in a vehicle consisting of Bio-Rad Laboratories 10x TBS (stock number 1706435, Hercules, CA, USA) diluted to lx with endotoxin-free water and supplemented with 20 pM CaCh and 20 pM ZnCh.

[0506] Application of enzyme replacement therapy

[0507] After weaning, Enppla^ / asjmice were randomly assigned to one of six groups, receiving weekly subcutaneous injections of either vehicle, soluble ENPP1 (ERT 1118) at 1 mg / kg, 2 mg / kg, or 4 mg / kg body weight, or bone-targeted ENPP1 (ERT 2000) at 0.5 mg / kg or 1 mg / kg body weight (FIG. 1A). Unaffected wildtype (WT) siblings were used as controls and received weekly subcutaneous injections of the vehicle. All animals received weekly I. P. injections of anti-murine CD4 to suppress autoimmune rejection of the human biologies, dosed 24 hours prior to treatment with ENPP1 (75 pl prior to the first ENPP1 dose and 50 pl thereafter; GK 1.5. Life Technologies Corporation, Grand Island. NY, USA; 1 mg / ml soln.; cat #16-0041-95). At 17 weeks of age, blood samples w ere taken, the mice w ere tested for - 70 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0508] hearing function, sacrificed and prepared for further analysis.

[0509] Blood plasma measurements

[0510] Blood samples were separated by centrifuging at 1000g for 10 minutes at 4°C.

[0511] Alkaline phosphatase levels in the plasma were quantified using a colorimetric assay kit from Abeam Corporation (Cambridge, MA. USA; catalog number ab83369). ELISA kits for measuring mouse PTH1-84 and FGF-23 were sourced from Quidel Corporation (San Diego, CA, USA; catalog numbers 60-2305 and 60-6800) and used as per the manufacturer’s instructions. Plasma phosphate and calcium levels were assessed by the George M. O’Brien Kidney Center Animal Physiology and Phenotyping Core at Yale.

[0512] Hearing tests

[0513] Auditory brainstem response (ABR) testing and data collection was performed by TumerScientific LLC (Jacksonville, IL) using a trained, objective ABR reviewer who was blinded to the treatment conditions. The reviewer employed a compact auditor)’ electrodiagnostic system to conduct the testing. The acoustic stimulus presentation, ABR acquisition, equipment control, and data management w ere coordinated using Tucker Davis Technologies (TDT) Hardware consisting of an RV6 processing unit, Medusa4Z headstage preamplifier, and BioSigRZ and SiggenRZ Software. Calibration of the acoustic signals was conducted at the beginning of each day of testing with Turner Scientific’s Sensory Sentinel mobile environmental monitoring and acoustic measurement system (www dot tumerscientific dot com / products-1), consisting of a 1 / 4” microphone with a relatively flat response through the mouse hearing range, an acoustic calibrator, a Precision Data Acquisition Unit (192 kHz sampling rate, 24 bit, IEPE power), and custom Sensory Sentinel acoustics processing software. Acoustic calibration measurements were collected using an ear canal model such that the intensity was measured approximately 1 cm from the tip of the speaker output, using a semi-closed coupler to approximate the fit of the speaker output into the canal, to simulate the pressure present at the animal's tympanic membrane. Sound stimuli were calibrated in dB SPL (unweighted peak sound pressure level, re: 20 pPa).

[0514] Mice were anesthetized with ketamine / xylazine. ABRs were recorded unilaterally using three stainless steel needle electrodes placed sub-dermally over the vertex (noninverting electrode) and the ipsilateral (speaker side) mastoid. Ground electrodes w ere placed either at the opposite mastoid or on the back. Acoustic stimuli were generated by the TDT system and presented to one ear via a high-frequency transducer (TDT MFI, closed field) - 71 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0515] directed into the canal as close to the canal tissue as possible without sealing against tissue or blocking the canal. The sound stimuli consisted of tone bursts with rise-fall times of 0.1 ms and plateaus of 1.5 ms at frequencies of 8, 16, and 32 kHz. The biological signals were amplified, averaged, stored, and displayed on the computer screen. Sound intensities were presented at descending intensity steps from 80 to 5 dB in 5 dB steps. All electronic data were stored for off-line, blinded analysis.

[0516] Determination of ABR thresholds were conducted by an ABR reviewer who was blinded to the treatment conditions. A non-repeating, random number was assigned to each ABR wave file to be analyzed such that the reviewer could determine the threshold without knowing the subject's treatment condition. The blinded reviewer then determined the lowest intensity at which any discernible waveform could be identified. After all thresholds were determined, animal ID numbers were unmasked and threshold shifts (the change in hearing threshold from baseline to terminal ABR) at each test frequency were computed. The blinded ABR threshold determinations at Turner Scientific have previously been shown to be highly reliable, with duplicate thresholds correlating at r = +0.97 (Pearson’s r).

[0517] Sample preparation

[0518] The preparation and isolation of middle ears and auditory7ossicles were conducted using a stereomicroscope as reported previously (Delsmann MM, Peichl J, Yorgan TA, Beil FT. Amling M. Demay MB, et al. Prevention of Hypomineralization In Auditory Ossicles of Vitamin D Receptor (Vdr) Deficient Mice. Front Endocrinol (Lausanne). 2022;13:901265). To standardize the analyses, the malleus of the animals was extracted, fixed in 3.7% formaldehyde, subjected to a graded ethanol dehydration series, and embedded undecalcified in methyl methacrylate. Next, polishing of the embedded specimen was performed on an EXAKT 400 CS surface grinding machine (EXAKT, Norderstedt, Germany). The process began with 1200 grit silicon carbide wet sandpaper (Allied High Tech Products Inc., Rancho Dominguez, USA) adjusting the grinding time for each specimen to achieve the ideal crosssection. Upon achieving the optimal cross-section visually, further polishing was conducted for four minutes using 4000-grit silicon carbide wet sandpaper (Delsmann MM, Peichl J. Yorgan TA, Beil FT, Amling M, Demay MB, et al. Prevention of Hypomineralization In Auditory Ossicles of Vitamin D Receptor (Vdr) Deficient Mice. Front Endocrinol (Lausanne). 2022;13:901265).

[0519] Quantitative backscattered electron imaging

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[0521] To assess the mineralization of the embedded malleus its bone mineral density distribution (BMDD) was assessed using quantitative backscattered electron imaging (qBEI). The samples were polished, coated with carbon, and imaged using a scanning electron microscope (LEO 435 VP; LEO Electron Microscopy Ltd., Cambridge, England) attached with a backscattered electron detector (type 202; K. E. Developments Ltd., Cambridge, England; 20 kV, 680 pA, 20 mm working distance) as previously described (Alimy AR, Thiessen ML, Strahl A, Boese CK, von Kroge S, Beil FT. et al. Sex-Specific Association of Clinical Parameters and Components of Femoral Bone Quality in Patients Undergoing Total Hip Arthroplasty. Calcif Tissue Int. 2024; Danyukova T, Alimy AR, Velho RV, Yorgan TA, Di Lorenzo G, von Kroge S, et al. Mice heterozygous for an osteogenesis imperfecta-linked MBTPS2 variant display a compromised subchondral osteocyte lacunocanalicular network associated with abnormal articular cartilage. Bone. 2023; 177: 116927). Calibration of brightness and contrast in the qBEI images was performed using carbon (gray value: 5.0 ± 0.5) and aluminum standards (gray value: 224 ± 0.5). To standardize the assessment across specimens, all analyses were conducted on a standardized region of the malleus (malleal processus) (FIGs. 1B-1D). For the BMDD analysis, the magnification was 100x at a resolution of 1.1μm / pixel. The mean calcium content (CaMean, wt%), the peak calcium content (CaPeak, wt%), and the mineralization heterogeneity (CaWidth, wt%) were determined using a custom MATLAB-based script quantifying the acquired gray-value-based images (MathWorks, Inc., Natick. Massachusetts. USA). For the osteocyte analysis, independent sets of images were acquired with a magnification of 400x and a resolution of 0.096pm / pixel. The acquired images were further analyzed with respect to osteocyte lacunar parameters, i.e., the number of osteocyte lacunae per bone area (N. Ot. Lc / B. Ar, 1 / mm2) and the mean osteocyte lacunar area (Ot.Lc.Ar, μm2), and mineralized lacunae (N. Mn. Lc / B. Ar, 1 / mm2) were counted manually. For the analysis of intralacunar mineralization (CaMean), the images were manually processed to evaluate either the mineral of the corresponding mineralized lacunae or the surrounding bone using the MATLAB routine.

[0522] Energy dispersive X-ray absorptiometry

[0523] The composition of lacunar mineralization was further characterized by energy dispersive X-ray absorptiometry (EDX). Subsequent to qBEI measurements, samples of untreated (i.e., vehicle-treated) or ERT 1118-treated (1 mg / kg, 2 mg / kg, 4 mg / kg) Enpp1-deficient were analyzed using a Crossbeam 340 (Zeiss, Germany) system assembled with an EDX detector (X-Max 80, Oxford Instruments, UK). The system was operated at a voltage of - 73 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0524] 30 kV and an acceleration current of 400 pA. To assess the composition and the calcium-to-phosphate ratio of osteocyte lacunae and the surrounding matrix, point measurements were taken within mineralized lacunae (n=14) and the bone matrix (n=8) at a 5,000x magnification. Per measurements, one million counts were taken to get a representative amount of information. Data on lacunar mineralization in WT mice and Enppl -deficient mice treated with ERT2000 were not assessed due to the low number of mineralized lacunae in these mice.

[0525] Statistical analysis

[0526] Statistical analysis was performed using GraphPad Prism (version 10, GraphPad Software, La Jolla. USA). Data are shown as box plots with dots representing individual values and indicating the group size. To test for differences between groups, a one-way ANOVA was first performed, and in case of overall significant differences, then Sidak’s multiple comparisons tests between WT controls and untreated Enpp1asj / asjmice and between WT or untreated Enpplasj / asJ mice and all treatment groups were performed. Individual multiple comparisons tests among all different treatment groups were not performed for reasons of clarity. For the additional comparison of CaMean values, ANOVA with Sidak’s multiple comparisons tests was performed to test for differences between genoty pes (WT vs Enpp1asj / asj) and an atomic locations (malleus vs femur). All statistical tests were two-sided and a significance threshold of 0.05 was chosen.

[0527] Example 1: Most plasma biomarkers of mineral metabolism are corrected by bone-targeted ERT

[0528] Analyses of blood samples showed that the bone-targeted ERT successfully normalized alkaline phosphatase levels to physiological levels (FIG. 2A). However, while soluble ERT effectively normalized phosphate levels, bone-targeted ERT did not achieve this correction (FIG. 2B). Notably, calcium levels remained stable in Enpp1asj / asjmice, showing no significant changes with either form of ERT (FIG. 2C). Furthermore, neither bone-targeted nor soluble ERT corrected FGF23 levels, and while soluble ERT elevated the reduced PPi in Enpp1asj / asjmice to supraphysiologic levels, bone-targeted ERT at the higher dose normalized plasma PPi to WT levels (FIGs. 2D-2E). Interestingly, bone-targeted ERT resulted in suppressed PTH below WT levels compared to soluble ERT (FIG. 2F).

[0529] Example 2: Conductive hearing loss is prevented by bone-targeted ERT (2000)

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[0531] Based on the reported hearing loss in patients with ENPP1 variants, the auditory phenotype of Enpp1asj / asjmice and their WT littermates was assessed using ABR, which reflect the cumulative electrical activity of the auditory nerve and central auditory pathways. The present study found that untreated Enpp1-deficient mice exhibited an impaired hearing threshold at stimulus frequencies of 8 kHz, 16 kHz, and 32 kHz, indicating a general impairment across these frequencies (FIGs. 3A-3C). At 8 kHz, administration of the soluble form of the enzyme at a low dose (1 mg) did not restore hearing function. However, higher doses normalized hearing performance. Similarly, a dose of 0.5 mg or 1 mg of the bone-targeted form effectively normalized the hearing threshold (FIG. 3 A). At 16 kHz, the hearing threshold was normalized by administering the soluble form at higher doses (2 mg and 4 mg) and partly corrected the bone-targeted form at 1 mg (FIG. 3B). In the high-frequency hearing range (i.e., at 32 kHz), the soluble form did not consistently normalize hearing thresholds (FIG. 3C). However, the higher dose of the bone-targeted form (1 mg) was able to fully normalize auditory function, indicating that bone-targeted ERT restores hearing in the low and high frequency range.

[0532] Example 3: Bone-targeted ERT prevents mineralization defects

[0533] Mineralization of the auditory ossicles is a critical factor in conductive hearing performance (Delsmann MM, Seist R, Stiirznickel J, Schmidt FN, Mansour A, Kobelski MM, et al. Conductive Hearing Loss in the Hyp Mouse Model of X-Linked Hypophosphatemia Is Accompanied by Hypomineralization of the Auditory Ossicles. J Bone Miner Res.

[0534] 2021;36(12):2317-28). Therefore, the present study analyzed the mineralization characteristics of the malleus in WT, Enppl -deficient, and treated Enppl -deficient mice using qBEI (FIG. 4 A). Vehicle-treated Enppl -deficient mice showed a trend towards lower mean matrix mineralization (CaMean) and significantly lower peak matrix mineralization than WT controls (FIGs. 4B-4C). Unexpectedly, the administration of the soluble ERT (1118) partially rescued the mineralization defect, while the bone-targeted ERT resulted in full correction. The mineralization heterogeneity (CaWidth), which indicates skeletal remodeling activity, showed no differences between WT, / s' / ? / ? / ? / -deficient mice treated with vehicle or ERT 1118. Notably however, the bone-targeted ERT, administered at a dosage of 0.5 mg / kg body weight, was associated with an increased mineralization heterogeneity compared to the other groups including WT (FIG. 4D). Notably, while the matrix mineralization was generally lower in the femur of the same animals, it was not decreased in Enpp1asj / asjmice compared to WT (FIG. 4E).

[0535] - 75 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0536] Example 4: Osteocyte lacunar mineralization as a unique phenomenon in ossicles of Enpp1asj / asjmice

[0537] Osteocytes, the most abundant cell type in bone, play a crucial role in regulating bone remodeling. To further elucidate the effects of Enppl deficiency on the osteocytic lacunar system, this study analyzed the number, size, and morphology of osteocyte lacunae in the malleus of Enpp1asj / asjmice (FIG. 5A). Both the osteocyte lacunar area and the total number of osteocyte lacunae did not differ between WT, untreated Enpp1asj / asjmice, and treated Enpplas’ / as}mice (FIGs. 5B-5C). However, a significant reduction in the number of nonmineralized osteocyte lacunae in untreated Enpp1asj / asjmice compared to WT controls was found. Treatment with the soluble form of the enzyme did not normalize the number of nonmineralized osteocyte lacunae at any dosage tested (FIG. 5D). In contrast, the bone-targeted form successfully normalized the number of lacunae at both 0.5 mg and 1 mg / kg dosages. In untreated Enpp1asj / asjmice and in animals treated with the soluble ERT 1118, mineralized lacunae were apparent (FIGs. 5A, 5E). Lacunar mineralization was prevented only by bone-targeted ERT 2000 (FIG. 5E). Because the mineralization of lacunae has not been described previously in mice, the present study performed detailed analyses on the mineralization characteristics (FIG. 5F). Comparison of the mean calcium content (CaMean) of mineralized osteocyte lacunae between untreated Enpp1 -deficient mice and those treated with various doses of ERT1118 indicated no differences (FIG. 5G). Interestingly, however, the mineral within the osteocyte lacunae showed lower calcium content compared to the surrounding bone matrix, indicating a mechanism deviating from hypermineralization of human osteocyte lacunae (i.e., micropetrosis). Using EDX, the atomic composition of mineralized lacunae in these mice was assessed. Similar to the observation determined by qBEI measurements, a difference of the calcium-to-phosphate ratio between untreated Enpp1-deficient mice and those treated with different dosages of ERT1118 was not apparent (FIG. 5H). Although presenting with a lower calcium content, the calcium-to-phosphate ratio of the mineralized osteocyte lacunae was similar to that of the adjacent bone, indicating that the intralacunar mineral is composed mainly of hydroxyapatite.

[0538] Example 5:

[0539] Variants in the ENPP1 gene cause different conditions depending on the patients’ age, environmental factors, and mutation status, including GACI, ARHR2, EOOP and OPLL, which have been associated with progressive hearing loss. Although various hypotheses exist,

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[0541] the precise mechanisms underlying hearing impairment in these conditions are still not well understood.

[0542] This study investigated the auditory ossicles in Enpp1-deficient mice and found that mineralization defects and osteocyte dysfunction may contribute to the progressive hearing loss observed, as evidenced by ABR testing and in-depth histomorphometric analyses.

[0543] Interestingly, both bone-targeted and soluble ERT corrected hearing deficits in Enpp1asj / asjmice, while bone-targeted ERT also corrected bone abnormalities with a main effect on osteocyte properties. These findings provide a novel approach to treat hearing loss in patients with ENPP1 variants.

[0544] Notably, the administration of bone-targeted ERT was associated with normalized hearing thresholds across tested frequencies. More specifically, while bone-targeted ERT at the higher dose of 1 mg / kg resulted in a partial correction of hearing loss at 16 kHz, a complete correction was observed at 8 kHz and 32 kHz. Since this normalization was completely mirrored by trajectories of bone and osteocyte lacunar mineralization, the primary mechanism of hearing loss in Enppl deficiency may be related to dysfunction of the ossicular chain. While chronic inflammatory processes, such as chronic otitis media, have been associated with hearing loss both generally and specifically in the context of ENPP1 mutations, the present findings reinforce that ossicular chain dysfunction plays a critical role in the auditory deficits observed. The ABR measurement at 32 kHz provides the most sensitive measurement given that mice demonstrate peak auditory sensitivity at frequencies between 15-20 kHz (Zheng QY, Johnson KR, Erway LC. Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses. Hear Res. 1999;130(l-2):94-107). This frequency not only closely corresponds with the natural inclination of the mouse auditory system towards higher frequencies - distinct from human auditory capabilities - but is also critical for detecting subtle auditory changes not readily observable at lower frequencies. At 17 weeks, mice are fully mature yet typically do not exhibit signs of age-related hearing loss, preferentially affecting higher frequencies initially (Li HS, Borg E. Age-related loss of auditory sensitivity in two mouse genotypes. Acta Otolaryngol. 1991; 111 (5): 827-34; Zeng L, Zhou W, Bing D, Xie L, Wang X, Zhang G. Protocol for assessing auditory brainstem response in mice using a four-channel recording system. STAR Protoc.

[0545] 2022;3(2):101251). Consequently, the present setup allowed a precise assessment of hearing function before the onset of age-related decline.

[0546] Conductive hearing is accompanied by hypomineralization of the auditory ossicles. Of note, dietary vitamin D supplementation is associated with improved matrix

[0547] - 77 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0548] mineralization of auditory ossicles in Vdr knock out mice. However, further therapeutic aspects have remained unanswered in the art, in particular the question of whether an improvement in the mineralization of the auditory ossicles is accompanied by improved hearing function. The present study shows in the Enpp1asj / asjmodel that an improvement of hearing function by bone-targeted ERT was closely related to an improvement of matrix mineralization of the malleus measured by qBEI. In certain non-limiting embodiments, early treatment, as in this model after weaning, can be crucial, as the ossification and mineralization of ossicles is completed very early in both humans and mice.

[0549] The bone-targeted form of ERT also effectively normalized the number of nonmineralized osteocyte lacunae at both applied doses, demonstrating a strong potential to counter the cellular anomalies associated with Enppl deficiency. In particular, this form of ERT also significantly reduced the number of mineralized osteocyte lacunae, emphasizing its targeted action on key bone mineralization processes and further highlighting the therapeutic approach of targeting osteocytic pathways. In humans, the phenomenon of osteocyte lacunar mineralization (i.e., micropetrosis) has been associated with aging and immobilization and has been found to be indicative of osteocyte apoptosis, further highlighting the therapeutic potential of targeting these cellular processes. In human auditory ossicles, the near absence of bone resorption by osteoclasts has been associated with a nearly appearance of mineralized osteocyte lacunae. Importantly, the mineralization of the osteocyte lacunae in the Enppl -deficient mice studied here clearly differs from the phenomenon of human micropetrosis since the intralacunar mineral exhibited a lower calcium content than the surrounding bone matrix, while in human micropetrosis it tends to be more highly mineralized than the adjacent bone. Another difference is the absence of mineralized pearl (i.e., spherite) formation in murine Enppl deficiency compared to human micropetrosis. Of further interest is that osteocyte lacunar mineralization was observed exclusively in the ossicles but not in other bones, such as the femur. Since osteocytes represent terminally differentiated osteoblasts, another cause for the observed mineralized osteocyte lacunae could be the type II collagen-producing hypermineralising osteoblast subtypes (termed auditory osteoblasts) previously identified in murine ossicles (Kuroda Y, Kawaai K, Hatano N, Wu Y, Takano H, Momose A, et al. Hypermineralization of Hearing-Related Bones by a Specific Osteoblast Subtype. J Bone Miner Res. 2021;36(8):1535-47), are more susceptible to mineralization when differentiated into osteocytes under pathological conditions, such as in Enppl deficiency. Here, the formation and dissolution of mineralized osteocyte lacunae were characterized for the first time in mice. Interestingly, osteocyte lacunae in Enppl -deficient mice presented with - 78 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0550] smaller osteocyte lacunae in almost all analyzed weight-bearing bones. Although this was not associated with signs of osteocyte lacunar mineralization at other skeletal sites in most studies, one study indicated mineralization of osteocyte lacunae also in long bones of Enppl-deficient mice (Hajjawi MO, MacRae VE, Huesa C, et al. Mineralisation of collagen rich soft tissues and osteocyte lacunae in Enppl(- / -)mice. Bone, 2014;69:139-147).

[0551] The restoration of both structural, cellular, and functional parameters reflects the usefulness of bone-targeted ERT for clinical application. Patients with GACI often exhibit a range of hearing impairments, from mild to moderate conductive hearing loss, and less commonly, sensorineural hearing loss. The present findings indicate an increased risk for progressive hearing loss and thus underscore the need for ongoing audiologic and otolaryngologic evaluations in these patients. Early detection and intervention are critical, as they can significantly mitigate language and cognitive impairments in affected individuals. The promising results of bone-targeted ERT in reversing hearing loss and bone anomalies provide a solid foundation for integrating this therapy into treatment strategies for patients with ENPP1 -related disorders.

[0552] Taken together, the present results show an improvement in auditory function in Enppl -deficient mice by application of bone-targeted ERT, along the correction of an ossicular phenotype in the auditory ossicles in the form of a combination of low mineralization of the bone matrix and mineralized osteocyte lacunae. These results support the benefit of correcting bone mineralization to prevent hearing loss in ENPP1 deficiency.

[0553] ENUMERATED EMBODIMENTS

[0554] The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance:

[0555] Embodiment 1: A compound comprising formula (I):

[0556] W-PROTEIN-Z-DOMAIN-X-Y (I),

[0557] wherein in (I):

[0558] W is absent or comprises a signal sequence which allows for export of the compound into extracellular space;

[0559] PROTEIN comprises the catalytic region of ENPP 1;

[0560] DOMAIN is absent or at least one selected from the group consisting of a human IgG Fc domain (Fc), human serum albumin protein (ALB), and a biologically active fragment thereof;

[0561] X and Z are independently absent or a polypeptide comprising 1-20 amino acids; and - 79 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0562] Y is a “bone targeting’" sequence group.

[0563] Embodiment 2: The compound of Embodiment 1, wherein the DOMAIN comprises Albumin.

[0564] Embodiment 3: The compound of any one of Embodiments 1 -2, wherein the DOMAIN comprises an IgG Fc domain.

[0565] Embodiment 4: The compound of any one of Embodiments 1-3, wherein the PROTEIN lacks the ENPP1 transmembrane domain.

[0566] Embodiment 5: The compound of any one of Embodiments 1 -4, wherein the PROTEIN comprises amino acid residues 99 (PSCAKE) to 925 (QED) of SEQ ID NO: 1.

[0567] Embodiment 6: The compound of any one of Embodiments 1-4, wherein the PROTEIN comprises amino acid residues 1 to 833 of SEQ ID NO: 3.

[0568] Embodiment 7: The compound of any one of Embodiments 1 -4, wherein the PROTEIN comprises the amino acid sequence depicted in SEQ ID NO: 2.

[0569] Embodiment 8: The compound of any one of Embodiments 1-4, wherein the PROTEIN comprises the amino acid sequence depicted in SEQ ID NO: 3 or 4 or 5.

[0570] Embodiment 9: The compound of any one of Embodiments 1-8, wherein the DOMAIN increases the circulating half-life of the compound relative to the circulating halflife of the compound lacking the DOMAIN.

[0571] Embodiment 10: The compound of any one of Embodiments 1-9, wherein Y is D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, or D15.

[0572] Embodiment 11: The compound of any one of Embodiments 1-10, wherein Y is D8. Embodiment 12: The compound of any one of Embodiments 1-9, wherein Y is (DSS)1, (DSS)2, (DSS)3, (DSS)4, (DSS)5, (DSS)6, (DSS)7, (DSS)8, (DSS)9, or (DSS)10.

[0573] Embodiment 13: The compound of any one of Embodiments 1-9, wherein Y is (ESS)1, (ESS)2, (ESS)3, (ESS)4, (ESS)5, (ESS)6, (ESS)7, (ESS)8, (ESS)9, or (ESS)10.

[0574] Embodiment 14: The compound of any one of Embodiments 1-9, wherein Y is (RQQ)1, (RQQ)2, (RQQ)3, (RQQ)4, (RQQ)5, (RQQ)6, (RQQ)7, (RQQ)8, (RQQ)9, or (RQQ)10.

[0575] Embodiment 15: The compound of any one of Embodiments 1-9, wherein Y is (KR)1, (KR)2, (KR)3, (KR)4, (KR)5, (KR)6, (KR)7, (KR)8, (KR)9, or (KR)10.

[0576] Embodiment 16: The compound of any one of Embodiments 1-9, wherein Y is R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, or R15.

[0577] Embodiment 17: The compound of any one of Embodiments 1-9, wherein Y is E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11, E12, E13, E14, or E15.

[0578] Embodiment 18: The compound of any one of Embodiments 1-9, wherein Y is - 80 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0579] DSSSEEKFLRRIGRFG.

[0580] Embodiment 19: The compound of any one of Embodiments 1-9, wherein Y is EEEEEEEPRGDT.

[0581] Embodiment 20: The compound of any one of Embodiments 1-9, wherein Y is APWHLSSQYSRT.

[0582] Embodiment 21: The compound of any one of Embodiments 1-9, wherein Y is STLPIPHEFSRE.

[0583] Embodiment 22: The compound of any one of Embodiments 1-9, wherein Y is VTKHLNQISQSY.

[0584] Embodiment 23: The compound of any one of Embodiments 1 -22, comprising the amino acid sequence of SEQ ID NO: 119.

[0585] Embodiment 24: The compound of any one of Embodiments 1-23, comprising the amino acid residues 23-1086 of the sequence of SEQ ID NO: 119.

[0586] Embodiment 25: The compound of any one of Embodiments 1 -24, comprising the amino acid residues 23-1078 of the sequence of SEQ ID NO: 119.

[0587] Embodiment 26: The compound of any one of Embodiments 1-25, comprising the amino acid residues 23-1078 of the sequence of SEQ ID NO: 119, further comprising a bone targeting domain.

[0588] Embodiment 27: The compound of Embodiment 26, wherein the bone targeting domain is fused through a chemical bond or a linker to the amino acid residues 23-1078 of the sequence of SEQ ID NO: 119.

[0589] Embodiment 28: The compound of any one of Embodiments 26-27, wherein the bone targeting domain is fused to the N-terminus or the C-terminus of the amino acid residues 23-1078 of the sequence of SEQ ID NO: 119.

[0590] Embodiment 29: A method of restoring, improving, and / or preventing further loss of hearing in a subject, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of Embodiments 1-26.

[0591] Embodiment 30: The method of Embodiment 29, wherein the subject has ENPP1 haploinsufficiency.

[0592] Embodiment 31: The method of Embodiment 29, wherein the subj ect does not have ENPP1 haploinsufficiency.

[0593] Embodiment 32: The method of Embodiment 29, wherein the subject is not ENPP1 deficient.

[0594] Embodiment 33: The method of Embodiment 29, wherein the subject is ENPP1 - 81 - 576437472Attorney Docket No. 047162-7564W01(02906)

[0595] deficient.

[0596] Embodiment 34: The method of any one of Embodiments 29-33. wherein the administering restores osteocyte activity in the malleus of the subject.

[0597] Embodiment 35: The method of any one of Embodiments 29-34, wherein the administering restores and / or improves mineralization of the auditory ossicles of the subject.

[0598] Embodiment 36: The method of any one of Embodiments 29-35, wherein the administering restores and / or improves sound transmission in the middle ear.

[0599] Embodiment 37: The method of any one of Embodiments 29-36, wherein the subject is administered the compound by at least one route selected from the group consisting of oral, aerosol, inhalational, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastrical, ophthalmic, pulmonary, and topical.

[0600] Embodiment 38: The method of any one of Embodiments 29-37, wherein the compound is intravenously or subcutaneously administered to the subject.

[0601] Embodiment 39: The method of any one of Embodiments 29-38, wherein administering the compound to the subject increases, or prevents further decrease of. the patient’s extracellular pyrophosphate concentrations.

[0602] Embodiment 40: The method of any one of Embodiments 29-39, wherein the compound is administered to the subject as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

[0603] Embodiment 41: The method of any one of Embodiments 29-40. wherein the subject is a mammal.

[0604] Embodiment 42: The method of Embodiment 41, wherein the mammal is a human. Embodiment 43: The method of any one of Embodiments 29-42, wherein the hearing loss is conductive.

[0605] Embodiment 44: The method of any one of Embodiments 29-42, wherein the hearing loss is neurological.

[0606] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0607] While specific embodiments of the subject matter have been discussed, the above specification is illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

[0608] - 82 - 576437472

Claims

Attorney Docket No. 047162-7564W01(02906)CLAIMSWhat is claimed is:

1. A compound comprising formula (I):W-PROTEIN-Z-DOMAIN-X-Y(I),wherein in (I):W is absent or comprises a signal sequence which allows for export of the compound into extracellular space;PROTEIN comprises the catalytic region of ENPP 1;DOMAIN is absent or at least one selected from the group consisting of a human IgG Fc domain (Fc), human serum albumin protein (ALB), and a biologically active fragment thereof;X and Z are independently absent or a polypeptide comprising 1-20 amino acids; and Y is a ‘“bone targeting" sequence group.

2. The compound of claim 1, wherein the DOMAIN comprises Albumin.

3. The compound of any one of claims 1-2. wherein the DOMAIN comprises an IgG Fc domain.

4. The compound of any one of claims 1-3, wherein the PROTEIN lacks the ENPP1 transmembrane domain.

5. The compound of any one of claims 1-4, wherein the PROTEIN comprises amino acid residues 99 (PSC AKE ) to 925 ( QED) of SEQ ID NO: 1.

6. The compound of any one of claims 1-4. wherein the PROTEIN comprises amino acid residues 1 to 833 of SEQ ID NO: 3.

7. The compound of any one of claims 1-4, wherein the PROTEIN comprises the amino acid sequence depicted in SEQ ID NO: 2.

8. The compound of any one of claims 1-4, wherein the PROTEIN comprises the amino - 83 - 576437472Attomey Docket No. 047162-7564W01(02906)acid sequence depicted in SEQ ID NO: 3 or 4 or 5.

9. The compound of any one of claims 1-8, wherein the DOMAIN increases the circulating half-life of the compound relative to the circulating half-life of the compound lacking the DOMAIN.

10. The compound of any one of claims 1-9. wherein Y is Di. D2, D3, D4, Ds, De, D7, Ds, D9, Dio, Du, D12, D13, D14, or Dis.

11. The compound of any one of claims 1-10, wherein Y is D8.

12. The compound of any one of claims 1-9, wherein Y is (DSS)i, (DSS)2, (DSS)3, (DSS)4, (DSS)S, (DSS)6, (DSS)7, (DSS)S, (DSS)9, or (DSS)io.

13. The compound of any one of claims 1-9. wherein Y is (ESS)i, (ESS)2, (ESS)3, (ESS)4, (ESS)s, (ESS)e, (ESS)7, (ESS)S, (ESS)9, or (ESS)io.

14. The compound of any one of claims 1-9, wherein Y is (RQQ)i, (RQQ)2, (RQQ)s, (RQQ)4, (RQQ)S. (RQQ)e, (RQQ)7, (RQQ)s. (RQQ or (RQQ)io.

15. The compound of any one of claims I -9, wherein Y is (KR)i, (KR)2, (KR)3, (KR)4, (KR)s, (KR)6, (KR)7, (KR)S, (KR)9, or (KR)io.

16. The compound of any one of claims 1-9. wherein Y is Ri. R2, R3, R4, Rs, Re, R7, Rs, R9, Rio, R11, R12, R13, R14, or Ris.

17. The compound of any one of claims 1-9, wherein Y is Ei, E2, E3, E4, Es, Ee, E7, Es, E9, E10, E11, E12, E13, Ei4. or Eis.

18. The compound of any one of claims 1-9, wherein Y is DSSSEEKFLRRIGRFG.

19. The compound of any one of claims 1-9. wherein Y is EEEEEEEPRGDT.

20. The compound of any one of claims 1-9, wherein Y is APWHLSSQYSRT.- 84 - 576437472Attorney Docket No. 047162-7564W01(02906)21. The compound of any one of claims 1-9. wherein Y is STLPIPHEFSRE.

22. The compound of any one of claims 1-9, wherein Y is VTKHLNQISQSY.

23. The compound of any one of claims 1-22, comprising the amino acid sequence of SEQ ID NO: 119.

24. The compound of any one of claims 1-23, comprising the amino acid residues 23- 1086 of the sequence of SEQ ID NO: 119.

25. The compound of any one of claims 1-24, comprising the amino acid residues 23- 1078 of the sequence of SEQ ID NO: 119.

26. The compound of any one of claims 1-25, comprising the amino acid residues 23- 1078 of the sequence of SEQ ID NO: 119, further comprising a bone targeting domain.

27. The compound of claim 26, wherein the bone targeting domain is fused through a chemical bond or a linker to the amino acid residues 23-1078 of the sequence of SEQ ID NO: 119.

28. The compound of any one of claims 26-27, wherein the bone targeting domain is fused to the N-terminus or the C-terminus of the amino acid residues 23-1078 of the sequence of SEQ ID NO: 119.

29. A method of restoring, improving, and / or preventing further loss of hearing in a subject, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of claims 1-28.

30. The method of claim 29, wherein the subject has ENPP1 haploinsufficiency.

31. The method of claim 29, wherein the subj ect does not have ENPP 1 haploinsufficiency.- 85 - 576437472Attomey Docket No. 047162-7564W01(02906)32. The method of claim 29, wherein the subject is not ENPP1 deficient.

33. The method of claim 29, wherein the subject is ENPP1 deficient.

34. The method of any one of claims 29-33, wherein the administering restores osteocyte activity in the malleus of the subject.

35. The method of any one of claims 29-34, wherein the administering restores and / or improves mineralization of the auditory ossicles of the subject.

36. The method of any one of claims 29-35, wherein the administering restores and / or improves sound transmission in the middle ear.

37. The method of any one of claims 29-36, wherein the subject is administered the compound by at least one route selected from the group consisting of oral, aerosol, inhalational, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastrical, ophthalmic, pulmonary, and topical.

38. The method of any one of claims 29-37. wherein the compound is intravenously or subcutaneously administered to the subject.

39. The method of any one of claims 29-38, wherein administering the compound to the subject increases, or prevents further decrease of, the patient’s extracellular pyrophosphate concentrations.

40. The method of any one of claims 29-39, wherein the compound is administered to the subject as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

41. The method of any one of claims 29-40, wherein the subject is a mammal.

42. The method of claim 41, wherein the mammal is a human.- 86 - 576437472Attorney Docket No. 047162-7564W01(02906)43. The method of any one of claims 29-42, wherein the hearing loss is conductive.

44. The method of any one of claims 29-42, wherein the hearing loss is neurological.- 87 - 576437472

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