Treatment for muscle weakness caused by alkaline phosphatase

Abstract

This disclosure features a method for treating or improving at least one symptom in a subject having or being prone to a muscle weakness disorder by administering a therapeutically effective dose of at least one recombinant polypeptide having alkaline phosphatase activity to the subject. The subject is characterized by lacking a loss-of-function mutation in the ALPL gene.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority and benefit of U.S. Provisional Patent Application No. 63 / 463,539, filed on 2 May 2023, which is incorporated herein by reference in its entirety.

[0002] (Sequence Listing) This application includes a sequence listing, which has been submitted electronically in XML format and is incorporated herein by reference in its entirety. A copy of said XML, created on 22 March 2024, is named 0467WO_SL.xml and is 31,813 bytes in size. [Background technology]

[0003] Hypophosphatasia (HPP)-like disorders are caused by unknown etiologies and result in a wide range of symptoms and severity, from rickets (osteomalacia) to near-complete absence of bone mineralization in the uterus. Most individuals exhibit skeletal changes, short stature, leg pain, gait disturbance, and premature tooth loss. Furthermore, these individuals present with muscle weakness and hypotonia, severely reducing their quality of life due to the physical impairments these types of disorders cause on a daily basis. [Overview of the project]

[0004] The characteristic feature of this method is that, in subjects with or at risk of developing muscle weakness, the administration of a therapeutically effective dose of at least one recombinant polypeptide having alkaline phosphatase activity to the subject is used to treat muscle weakness or reduce the risk of developing it, wherein the subject shows a decrease in tissue-nonspecific alkaline phosphatase (TNSALP) concentration and the subject does not have a loss-of-function mutation in the ALPL gene.

[0005] A decrease in TNSALP concentration may be caused by a decrease in ALPL transcription, a decrease in ALPL mRNA translation, an increase or decrease in TNSALP post-translational modification, or a decrease in TNSALP enzyme activity. These decreases can be measured in comparison to normal subjects.

[0006] The subjects may have mutations in the 3' untranslated region (UTR), 5'UTR, or intron region of the ALPL gene. The subjects may have mutations in one or more of the following genes: ATP1A3, ANKH, ENPP1, FGFR3, PHOSPHO1, PTH1R, PTH2R, SPP1, TNFRSF11A, TNFRSF11B, COL1A1, COL1A2, SOX9, PDXP, AOX1, PNPO, PDXK, ADCK3, MTRNR2, and S1PR1. The subjects may have the ATP1A3 variant c.357+1G>A. The subjects may have the ADCK3 variant m.1665G>A. The subjects may have the MTRNR2 variant m.1836A>G.

[0007] The muscles in question (e.g., leg muscles such as the soleus or extensor digitorum longus (EDL)) are not significantly different from normal muscles of subjects without muscle weakness disorders in at least one characteristic selected from the proportion of muscle fiber types and fiber contraction characteristics.

[0008] Muscle weakness disorders can be caused by decreased alkaline phosphatase activity. Subjects may have elevated serum pyrophosphate (PPi) concentrations, and / or muscle weakness disorders can be caused by elevated PPi concentrations. Elevated pyrophosphate (PPi) concentrations can exacerbate muscle weakness in subjects. Administration of recombinant polypeptides reduces PPi concentrations in subjects compared to untreated subjects (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%).

[0009] Recombinant polypeptides may be administered to subjects daily for at least one week, one month, three months, six months, or one year. Recombinant polypeptides may be administered subcutaneously, intravenously, intramuscularly, sublingually, intrathecally, or intradermally.

[0010] Recombinant polypeptides may comprise at least one of tissue-nonspecific alkaline phosphatases (TNALP), placental alkaline phosphatases (PALP), germ cell alkaline phosphatases (GCALP), intestinal alkaline phosphatases (IALP), and their biologically functional fragments, fusions, or chimeric constructs. Recombinant polypeptides may comprise at least one of soluble fragments of TNALP, PALP, GCALP, and IALP. Tissue-nonspecific alkaline phosphatases (TNALP) may contain amino acids 1-485 of Sequence ID No. 1.

[0011] Recombinant polypeptides can be fusion proteins. Recombinant polypeptides may contain immunoglobulin molecules, such as a fragment crystallizable (Fc) region. Fc may have the amino acid sequence of SEQ ID NO: 20. Recombinant polypeptides may contain negatively charged peptides. Negatively charged polypeptides may contain 1 to 50 negatively charged amino acids, such as aspartic acid or glutamic acid. Negatively charged peptides are D 10 , D 16 , E 10 , and E 16 It may include at least one of the following.

[0012] Recombinant polypeptides may include bone-targeting alkaline phosphatases having the following structure: Z-sALP-Y-Spacer-XW n -V In the formula, sALP is the extracellular domain of alkaline phosphatase. V is either absent or an amino acid sequence of at least one amino acid. X is either nonexistent or an amino acid sequence of at least one amino acid. Y is either absent or an amino acid sequence of at least one amino acid. Z is either non-existent or is an amino acid sequence of at least one amino acid, W n is polyaspartic acid or polyglutamic acid, and n = 10 - 16.

[0013] The spacer may include an Fc region (e.g., FC having the amino acid sequence of SEQ ID NO: 20). The recombinant polypeptide may have the structure of sALP-Fc-D 10 and may include.

[0014] The recombinant polypeptide may be administered at a dose of about 0.1 mg / kg / day to about 20 mg / kg / day (e.g., about 0.5 mg / kg / day to about 20 mg / kg / day, about 0.5 mg / kg / day to about 10 mg / kg / day, about 1 mg / kg / day to about 10 mg / kg / day, e.g., about 6 mg / kg / week), or an equivalent weekly dose. The recombinant polypeptide may be administered 6 times a week at a dose of about 1 mg / kg, 3 times a week at a dose of about 2 mg / kg, or 2 times a week at a dose of 3 mg / kg.

[0015] Prior to administration of the recombinant polypeptide, the subject may be characterized as having an average walking distance of about 350 meters or less in six minutes. Administration of the recombinant polypeptide may promote an increase in the average walking distance of at least 100 meters or more in six minutes by the subject. After administration of the recombinant polypeptide, the subject may exhibit an average walking distance of about 500 meters or more in six minutes. After administration of the recombinant polypeptide, the subject may show a decrease in dependence on assistive mobility devices (e.g., walkers, wheelchairs, braces, canes, and orthotic devices).

[0016] Prior to administration of the recombinant polypeptide, the subject may be characterized as having a plasma PPi concentration of about 4.5 μM or more. Administration of the recombinant polypeptide may promote a median decrease in the PPi concentration in the plasma sample from the subject of at least about 1 μM in the plasma sample. After administration of the recombinant polypeptide, the subject exhibits a plasma PPi concentration of about 2 μM to about 5 μM.

[0017] In some embodiments, i) The subjects are 0 to 14 days old and have a plasma ALP concentration of approximately 90 U / L or less before administration of recombinant polypeptide. ii) The subjects are 15 days old to less than 1 year old and have a plasma ALP concentration of approximately 134 U / L or less before administration of recombinant polypeptide. iii) The subjects are approximately 1 to under 10 years of age and have a plasma ALP concentration of approximately 156 U / L or less prior to administration of recombinant polypeptide. iv) The subjects are approximately 10 to 13 years old and have a plasma ALP concentration of approximately 141 U / L or less prior to administration of recombinant polypeptide. v) The subjects are female, approximately 13 to 15 years old, and have a plasma ALP concentration of approximately 62 U / L or less prior to administration of recombinant polypeptide. vi) The subjects are male, approximately 13 to 15 years of age, and have a plasma ALP concentration of approximately 127 U / L or less prior to administration of recombinant polypeptide. vii) The subjects are female, approximately 15 to 17 years old, and have a plasma ALP concentration of approximately 54 U / L or less prior to administration of recombinant polypeptide. viii) The subjects are male, approximately 15 to 17 years old, and have a plasma ALP concentration of approximately 89 U / L or less prior to administration of recombinant polypeptide. ix) The subjects are approximately 17 years of age or older and have a plasma ALP concentration of approximately 48 U / L or less prior to administration of recombinant polypeptide, or x) The subjects are approximately 17 years of age or older and have a plasma ALP concentration of approximately 59 U / L or less prior to administration of the recombinant polypeptide.

[0018] Administration of recombinant polypeptides may promote an increase in the median ALP concentration in plasma samples derived from the target organism, to at least approximately 100 U / L or higher.

[0019] In some embodiments, i) The subjects are 0 to 14 days old and have a plasma ALP concentration of approximately 273 U / L or higher after administration of recombinant polypeptide. ii) The subjects are 15 days old to less than 1 year old and are characterized by having a plasma ALP concentration of approximately 518 U / L or higher after administration of recombinant polypeptide. iii) The subjects are approximately 1 year old to less than 10 years old, and are characterized by having a plasma ALP concentration of approximately 369 U / L or higher after administration of recombinant polypeptide. iv) The subjects are approximately 10 to 13 years old and are characterized by having a plasma ALP concentration of approximately 460 U / L or higher after administration of recombinant polypeptide. v) The subjects are female, approximately 13 to 15 years old, and characterized by having a plasma ALP concentration of approximately 280 U / L or higher after administration of recombinant polypeptide. vi) The subjects are male, approximately 13 to 15 years old, and characterized by having a plasma ALP concentration of approximately 517 U / L or higher after administration of recombinant polypeptide. vii) The subjects are female, approximately 15 to 17 years old, and characterized by having a plasma ALP concentration of approximately 128 U / L or higher after administration of recombinant polypeptide. viii) The subjects are male, approximately 15 to 17 years old, and characterized by having a plasma ALP concentration of approximately 365 U / L or higher after administration of recombinant polypeptide. ix) The subjects are female, approximately 17 years of age or older, MTRNR2, and have a plasma ALP concentration of approximately 95 U / L or higher after administration of recombinant polypeptide, or x) The subjects are male, approximately 17 years of age or older, and have a plasma ALP concentration of approximately 164 U / L or higher after administration of recombinant polypeptide.

[0020] Prior to administration of recombinant polypeptide, subjects may be characterized by having an average Blueinx-Oseletzky Scale of Motor Skills, Second Edition (BOT-2) intensity score of approximately 10 or less. Prior to administration of recombinant polypeptide, subjects may be characterized by having an average BOT-2 running speed and agility score of approximately 5 or less. Administration of recombinant polypeptide may result in subjects with an average BOT-2 intensity score of approximately 10 or higher. Administration of recombinant polypeptide may result in subjects with an average BOT-2 running speed and agility score of approximately 5 or higher.

[0021] Prior to administration of recombinant polypeptide, subjects may be characterized by having a mean Childhood Health Assessment Questionnaire (CHAQ) score of approximately 0.8 or higher. Administration of recombinant polypeptide may result in subjects with a mean CHAQ score of approximately 0.5 or lower.

[0022] Prior to administration of recombinant polypeptide, subjects may be characterized by having an average Pediatric Outcome Data Acquisition Inventory (PODCI) score of approximately 40 or less. Administration of recombinant polypeptide may result in subjects with an average PODCI score of approximately 40 or higher.

[0023] Prior to administration of recombinant polypeptide, subjects may be characterized by having an average muscle strength grade of less than approximately 5. Administration of recombinant polypeptide may result in an average increase of approximately 1 or more in muscle strength grade in the subjects.

[0024] Prior to administration of recombinant polypeptide, subjects may be characterized by having an average handheld dynamometer (HHD) value of less than approximately 80% of their predicted HHD value. Administration of recombinant polypeptide may result in an average HHD value of 80% or more of the predicted HHD value in the subjects. HHD values ​​may represent the subject's grip strength, knee flexion, knee extension, hip flexion, hip extension, or hip abduction.

[0025] The subjects may have muscle weakness disorders selected from muscular dystrophy, myasthenia gravis, calcium pyrophosphate deposition disease (CPPD), amyotrophic lateral sclerosis (ALS), myositis, myotonic dystrophy, myosrigor, Guillain-Barré syndrome, Duchenne muscular dystrophy (DMD), and Lambert-Eaton myasthenic syndrome.

[0026] In some embodiments of any of the treatments and / or diagnostic methods described herein, subjects having familial hypophosphatemia (e.g., ADHR, autosomal recessive hypophosphatemic rickets, X-linked hypophosphatemic rickets, and XLH) or calcium pyrophosphate deposition disorder (CPPD) are explicitly excluded.

[0027] definition As used herein, “a” or “an” means “at least one” or “one or more” unless otherwise specified. Furthermore, the singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise.

[0028] As used herein, the term “about” means an amount that is ±10% of the listed value, preferably ±5% of the listed value, or more preferably ±2% of the listed value.

[0029] "Asfotase alpha" refers to a human TNALP (hTNALP) fusion protein containing two identical polypeptide chains of soluble glycoprotein, each polypeptide chain containing amino acid residues 1-726 of Sequence ID No. 1. The structure of each polypeptide chain contains the catalytic domain of hTNALP, the human immunoglobulin G1Fc domain, and the decaaspartate peptide used as the bone targeting domain (structure hTNALP-Fc-D 10The two polypeptide chains are covalently linked by two disulfide bonds. Asfotase alfa is approved in the United States, Europe, Japan, Canada, Israel, Australia, and South Korea under the trademark name STRENSIQ® (Alexion Pharmaceuticals, Inc., Boston, MA) for the treatment of subjects with HPP.

[0030] The terms “individual,” “subject,” and “patient” are used interchangeably and refer to any subject, in particular human, for whom diagnosis, treatment, or therapy is desired. As used herein, a subject “at risk” of disease, or a subject “susceptible” to disease, is a subject identified as having a risk of developing a disease, impairment, or condition associated with muscle weakness disorders.

[0031] As used herein, “mean” refers to a numerical value representing the mean or median of a dataset. The mean of a dataset is calculated by dividing the sum of the values ​​in the dataset by the number of values. The median of a dataset is calculated by determining the midpoint of an odd-numbered list of data, or by determining the average of the two midpoint data values ​​in an even-numbered list.

[0032] Where used in this disclosure for TNALP or other genes or proteins, the terms “wild-type” or “wild-type sequence” refer to the typical form of a gene or protein as naturally found in normal humans, non-human mammals, or other organisms. A wild-type sequence may refer to a standard, “normal” allele at a gene locus, or a standard, “normal” primary amino acid sequence of a polypeptide or protein (with optional standard, “normal” post-translational modifications to amino acid residues, and / or interchain bonds and / or interactions between amino acid residues), as opposed to a non-standard, “mutant” allele, or one produced by amino acid sequences / modifications / interactions. “Mutant” alleles can vary greatly and, if genetic shifts occur within a population, can even become wild-type. It is now understood that most, or all, gene loci (and for most polypeptide sequences, less frequently but still possible) exist in various allele forms with varying frequencies across the geographical range of a species, and that a uniform wild-type does not necessarily exist. However, generally speaking, the most dominant allele or amino acid sequence (i.e., the one that is most frequently observed in a normal individual human or other organism) is considered to be the wild type in this disclosure.

[0033] As used herein, the term “normal subject” means, unless otherwise specified, a subject, e.g., a human, who has no evidence of muscle weakness disorder and / or its symptoms or physiological consequences caused by or associated with abnormal alkaline phosphatase activity (e.g., which may result from a deficiency or absence of gene expression or protein levels, and / or loss of function of a gene or protein mutation). An example of a normal subject is a human who lacks muscle weakness or symptoms of muscle weakness and does not show mutations or modifications to the alkaline phosphatase gene or protein (e.g., TNSALP) that could result in HPP-related muscle weakness. A normal subject may also be a subject that does not show abnormal endogenous alkaline phosphatase activity (this can be tested, for example, by evaluating levels of PPi, PEA, and PLP compared to a healthy control).

[0034] As used herein, “increased” or “enhanced” concentration means that in a subject having or prone to a muscle weakness disorder as described herein, the concentration of a molecule (e.g., a substrate of TNSALP such as PPi) is higher than the concentration of the molecule in a normal subject, such as a subject without a muscle weakness disorder, or at a point in time when the subject does not have obvious symptoms of a muscle weakness disorder (e.g., after treatment). The “increase” in the concentration of the molecule may be determined in the cells, tissues, or organs of the subject, such as in the subject’s blood or serum.

[0035] As used herein, the term "Bayley Scales of Infant and Toddler Development, Third Edition," or "BSID-III," refers to a standardized set of measurements used to assess a subject's motor (fine and gross), language (receptive and expressive), and cognitive development. See Bayley, (2006). Bayley scales of infant and toddler development: administration manual, San Antonio, TX: Harcourt Assessment (which is hereby incorporated by reference in its entirety). The BSID-III measurements include a series of developmental performance tasks administered to the subject. The raw scores of successfully completed items are converted to scale scores. The scale scores are then used to determine the subject's performance compared to healthy age-adjusted subjects. The BSID-III may also include a social-emotional / adaptive behavior questionnaire completed by the parent / guardian to establish the range of the subject's adaptive behavior. For example, the measurements to determine a BSID-III score (e.g., BSID-III total motor function score) may include grasping, perceptual-motor integration, motor planning and speed, visual tracking, reaching, object grasping, object manipulation, functional hand skills, response to tactile information, limb and trunk movement, static positioning, dynamic movement, balance, and motor planning. These subject measurements are then converted to a BSID-III scale score in the range of 0-14 (e.g., BSID-III total motor function scale score), where scores of about 7-about 13 are considered the normal range for healthy subjects.

[0036] As used herein, the term "bone targeting moiety" means that the bone targeting moiety, alone, is about -6 M to about -15 M For example, -7 M, -8 M, -9 M, -10 M, -11 M, -12 M, -13 M, -14 M, or -15This refers to an amino acid sequence of 1 to 50 amino acid residues that has sufficient affinity to the bone matrix, such that M) has in vivo binding affinity to the bone matrix.

[0037] As used herein, the terms “Bruininks-Oseretsky Test of Motor Proficiency, Second Edition” or “BOT-2” refer to the second edition of the standardized test of overall and fine motor skills for subjects approximately 4 to 21 years of age. See Bruininks, RH (2005). Bruininks-Oseretsky Test of Motor Proficiency, (BOT-2). Minneapolis, MN: Pearson Assessment (the entirety of which is incorporated herein by reference). The BOT-2 is administered individually to assess overall and fine motor skills in a range of subjects. In particular, the BOT-2 may be used to assess physical impairment and mobility limitations in subjects with HPP. The BOT-2 provides a composite BOT-2 score in the following areas: strength, running speed and agility, fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance, and upper limb coordination. For example, the BOT-2 strength score can be determined by having the subject perform sit-ups, v-ups, standing long jumps, wall sits, and push-ups. Running speed and agility scores can be determined by having the subject step on a balance beam or perform shuttle runs, bipedal side hops, or single-leg side hops. The BOT-2 strength, running speed, and agility scores range from 0 to 25, where scores of approximately 10 to 20 are considered to represent healthy subjects.

[0038] As used herein, the terms “Child Health Assessment Questionnaire” or “CHAQ” refer to a questionnaire used to assess the health status (e.g., ability to perform activities of daily living (ADL) and pain incidence) of subjects aged 1 to 19 years, including subjects with HPP-like disorders. For a description of the CHAQ index, see Bruce & Fries (J. Rheumatol. 30(1):167-178, 2003) (which is incorporated herein by reference in its entirety). For children aged 8 years and older, the CHAQ may be administered through interviews or self-report. The CHAQ includes eight subscales: grooming, getting up, eating, walking, hygiene, reaching, grasping, and activity. The score ranges from 0 to 3 within each category, with a score of 0 indicating no problem; a score of 1 indicating some difficulty; a score of 2 indicating significant difficulty; and a score of 3 indicating the subject is unable to perform the activity. The CHAQ index may also be used to determine the presence and severity of pain.

[0039] The term "extracellular domain" refers to the extracellular portion of a natural protein, such as alkaline phosphatase. In particular, the extracellular domain lacks a signal peptide.

[0040] "Fc" refers to the fragment crystallizable region of an immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4, containing the CH2 and CH3 domains of the immunoglobulin heavy chain. Fc may also include any portion of the hinge region linking the Fab region and the Fc region. Fc may be derived from any mammal, including humans, and may be post-translationally modified (e.g., by glycosylation). In a non-limiting example, Fc may be the fragment crystallizable region of human IgG-1 having the amino acid sequence of SEQ ID NO: 20.

[0041] "Fragment" preferably means a portion of a polypeptide or nucleic acid molecule that contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the total length of the reference nucleic acid molecule or polypeptide. The fragments may contain, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 400, 500, 600, 700, or more amino acid residues, up to the full length of the polypeptide. Exemplary soluble alkaline phosphatase (sALP) fragments have amino acid residues 18-498, 18-499, 18-500, 18-501, 18-502, 18-503, 18-504, 18-505, 18-506, 18-507, 18-508, 18-509, 18-510, 18-511, or 18-512 of ALP (e.g., SEQ ID NOs: 2-6), and may include additional C-terminal and / or N-terminal portions.

[0042] As used interchangeably herein, the terms “handheld dynamometer” and “HHD” refer to methods for measuring grip strength and muscle strength in subjects, particularly subjects with or prone to muscle weakness disorders. Dynamometers may be used to assess grip strength, knee flexion, knee extension, hip flexion, hip extension, and hip abduction in subjects (e.g., subjects with or prone to muscle weakness disorders). For example, knee flexion and extension, as well as hip flexion, extension, and abduction in subjects with or prone to muscle weakness disorders, may be measured using, for example, a MICROFET2® dynamometer, while the subject's grip strength may be measured using, for example, a Jamar Grip dynamometer. Specifically, the administrator holds the dynamometer in a stationary position, and the subject applies maximum force to the dynamometer. Peak force data is collected in pounds and then converted to Newtons (N). The torque value is then calculated using the limb length in N meters. Next, the torque value can be compared to, for example, the value of a normal subject of approximately the same age, same sex, and / or same height, and expressed as a percentage value to generate the subject's HHD value.

[0043] As used herein, the terms “hypophosphatasia” or “HPP” refer to a rare hereditary skeletal disorder caused by one or more loss-of-function mutations in the ALPL (alkaline phosphatase, liver / bone / kidney) gene, which encodes tissue-nonspecific alkaline phosphatase (TNALP). HPP may be further characterized as infantile HPP, childhood HPP, perinatal HPP (e.g., benign perinatal HPP or severe perinatal HPP), or dentitionally localized HPP.

[0044] As used herein, the terms “hypophosphatasia-like disorder” or “HPP-like disorder” refer to a disorder that presents with symptoms similar to HPP but is not caused by a loss-of-function mutation in the ALPL gene.

[0045] "Untreated patient" or "untreated subject" means a patient or subject with a muscle weakness disorder as described herein who has never been treated with alkaline phosphatase or with a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alpha).

[0046] As used herein, “pain” means physical distress or discomfort caused by a muscle weakness disorder as described herein, such as muscle pain. For example, symptoms of pain may include, for example, pain, tightness, or stiffness. The severity of pain may vary among subjects (e.g., chronic pain or acute pain). In particular, chronic pain refers to pain that lasts longer than 3 to 6 months, or pain that persists beyond the expected period of recovery. In contrast, acute pain typically refers to pain that lasts less than 3 to 6 months. As described herein, therapeutic compositions (e.g., sALPs such as asfotase alfa) may be administered to subjects suffering from pain (e.g., muscle pain) in an amount sufficient to alleviate, or at least partially alleviate, the symptoms of pain (e.g., discomfort, pain, tightness, or stiffness) and its complications (e.g., fatigue, insomnia, weakened immune system, depression, anxiety, stress, irritability, or physical impairment).

[0047] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to any chain of two or more natural or non-natural amino acid residues, whether post-translational modifications (e.g., glycosylation or phosphorylation), constituting all or part of a naturally occurring or non-natural polypeptide or peptide, as described herein.

[0048] "Pharmacologically acceptable carrier" or "pharmaceutically acceptable excipient" each means at least one carrier or excipient that is physiologically acceptable to a subject (e.g., a human) and that maintains the therapeutic properties of the molecule administered with it. One exemplary pharmaceutically acceptable carrier substance is physiological saline. For example, pharmaceutically acceptable carriers may include sodium chloride (e.g., 150 mM sodium chloride) and sodium phosphate (e.g., 25 mM sodium phosphate). Other physiologically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (Remington: The Science and Practice of Pharmacy, 22nd Ed., Allen, Ed. 2012).

[0049] "Pharmaceutical composition" means a composition containing a polypeptide or nucleic acid molecule as described herein, formulated with at least one pharmaceutically acceptable excipient, diluent, or carrier. Pharmaceutical compositions are manufactured or marketed with the approval of a government regulatory body as part of a therapeutic regimen for the treatment or prevention of a disease or event in a subject. Pharmaceutical compositions may be formulated, for example, for subcutaneous administration, intravenous administration (e.g., as a sterile solution in a solvent system suitable for intravenous use and free from particulate embolisms), oral administration (e.g., tablets, capsules, caplets, gel caps, or syrups), or for any other formulation as described herein in unit dosage forms. In one embodiment, a pharmaceutical composition of the present disclosure is administered subcutaneously or formulated for subcutaneous administration.

[0050] As used herein, the term “disability” refers to a physiological condition, such as osteoporosis and muscle weakness, as described herein, that may limit or eliminate a subject’s ability to walk, functional endurance, and activities of daily living (ADL). In particular, disability may limit or eliminate a subject’s ability to perform ADL, which are routine activities that healthy subjects perform on a daily basis without assistance, such as functional mobility or movement (e.g., walking), bathing and showering, grooming, self-feeding, and personal hygiene and grooming. As described herein, therapeutic compositions (e.g., compositions containing sALP, such as asfotase alfa) may be administered to a subject to reduce the severity and / or frequency of a disability associated with muscle weakness.

[0051] As used herein, the terms “Pediatric Outcome Data Acquisition Device” or “PODCI” refer to a questionnaire used to assess the overall health, pain incidence, and ability to perform activities of daily living (ADL) in subjects under 19 years of age, particularly in subjects with chronic health conditions such as HPP-like disease. For a description of PODCI, see Plint et al. (J. Pediatr. Orthop. 23(6):788-790, 2003), which is incorporated herein by reference in its entirety. The questionnaire may be completed by the subject or by a parent / guardian of the subject who is aware of the subject's condition. The eight scales generated from PODCI include: 1) Upper limb and physical function scales to measure difficulties encountered in performing daily personal care and student activities; 2) Mobility and basic mobility scales to measure difficulties experienced in performing routine movements and motor activities in daily activities; 3) Sport / physical function scales to measure difficulties or limitations encountered when participating in more vigorous activities or sports; 4) Pain / comfort scales to measure the level of pain experienced in the past week; 5) Treatment expectancy scales to measure long-term expectations of treatment; 6) Well-being scales to measure overall satisfaction with one's sense of similarity to friends and others of one's age, and one's appearance; 7) Symptom satisfaction scales to measure acceptance of the subject to current limitations if this is a lifelong condition; and 8) Overall function scales, a general combined scale calculated from the first four scales above. Standardized scores are generated from a set of questions in PODCI and converted to a scale of 0 to 100, where 0 represents significant impairment and 100 represents less impairment.

[0052] As used herein, the terms “Peabody Developmental Motor Scale, 2nd Edition” or “PDMS-2” refer to an early childhood motor development program that provides an assessment of overall and fine motor skills in subjects (e.g., infants and children) from birth through childhood. For a description of the PDMS-2 scale, see van Hartingsveldt et al. (Occup.Ther.Int.12(1):1-13, 2005), which is incorporated herein by reference in its entirety. The PDMS-2 consists of six subtests that measure interrelated motor abilities in early development. The six subtests include: 1) a motor subtest that measures the ability of subjects to move from one place to another (measurements include crawling, walking, running, hopping, and jumping forward); 2) a reflex subtest that measures the ability of subjects to react automatically to environmental events; and 3) a static subtest that measures the ability of subjects to maintain control of the body within the center of gravity and maintain balance. 4) Object Manipulation Subtest: Measures the subject's ability to manipulate objects, such as catching, throwing, and kicking a ball. 5) Grasping Subtest: Measures the subject's ability to use their hands, such as the ability to hold an object with one hand and movements involving controlled use of both hands' fingers. 6) Vision-Motor Integration Subtest: Measures the subject's ability to use their visual perception skills to perform complex eye-hand coordination tasks, such as picking up and grasping an object, building with blocks, and copying a design. PDMS-2 scores from each subtest are converted to PDMS-2 scores, such as the PDMS-2 Motor Standard Score, ranging from 0 to 13, with a healthy range of approximately 7 to 13.

[0053] The terms "sALP," "soluble alkaline phosphatase," and "extracellular domain of alkaline phosphatase" are used interchangeably and refer to soluble non-membrane-bound alkaline phosphatases, or their domains, biologically active fragments, or biologically active variants. sALP includes, for example, alkaline phosphatases lacking a C-terminal glycolipid anchor (GPI signal sequence, e.g., a polypeptide containing or consisting of amino acid residues 18-502 of human TNALP (SEQ ID NO: 2, 3, 4, 5, or 6)). In particular, TNALP may include polypeptides containing or consisting of amino acid residues 1-485 of SEQ ID NO: 1, such as asfotase alpha, or polypeptide variants having at least 95% sequence identity to amino acid residues 1-485 of SEQ ID NO: 1 (SEQ ID NO: 21 has 95.8% sequence identity to SEQ ID NO: 1, and SEQ ID NO: 22 has 96.08% sequence identity to SEQ ID NO: 1). sALP further includes, for example, mammalian orthologues of human TNALP, such as rhesus monkey TNALP (SEQ ID NO: 7), rat TNALP (SEQ ID NO: 8), canine TNALP (SEQ ID NO: 9), porcine TNALP (SEQ ID NO: 10), mouse TNALP (SEQ ID NO: 11), bovine TNALP (SEQ ID NOs: 12-14), or feline TNALP (SEQ ID NO: 15). sALP also includes soluble, non-membrane-bound forms of human PALP (e.g., a polypeptide containing or comprising amino acid residues 18-502 of SEQ ID NO: 16 or 17), GCALP (e.g., a polypeptide containing or comprising amino acid residues 18-502 of SEQ ID NO: 18), and IALP (e.g., a polypeptide containing or comprising amino acid residues 18-502 of SEQ ID NO: 19), as well as further variants and analogs thereof that retain alkaline phosphatase activity, such as the ability to hydrolyze PPi. sALP, in particular, lacks an N-terminal signal peptide (e.g., amino acids 1-17 of SEQ ID NOs. 2-6, 8, 11-13, or 15, or amino acids 1-25 of SEQ ID NO. 7).

[0054] "sALP polypeptide" means a polypeptide having the structure A-sALP-B, where sALP is as defined herein, and each of A and B is an amino acid sequence of either no amino acids or at least one amino acid. An exemplary sALP polypeptide has an amino acid sequence containing or consisting of amino acids 1-485 of SEQ ID NO: 1. Other exemplary sALP polypeptides include any sALP fusion polypeptide described herein (e.g., the sALP fusion polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha).

[0055] A "signal peptide" refers to a short peptide (5–30 amino acids long) at the N-terminus of a polypeptide that directs the polypeptide towards a secretory pathway (e.g., the extracellular space). Signal peptides are typically cleaved during polypeptide secretion. Signal sequences can direct polypeptides towards intracellular compartments or organelles, such as the Golgi apparatus. Signal sequences can be identified by homology or biological activity to peptides with known functions that target polypeptides to specific regions of the cell. Those skilled in the art can identify signal peptides by using readily available software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, or PILEUP / PRETTYBOX programs). Signal peptides may be substantially identical to, for example, amino acid residues 1–17 of SEQ ID NOs. 2–6, or amino acid residues 1–25 of SEQ ID NOs. 7.

[0056] As used herein, when a polypeptide or nucleic acid sequence is referred to as having "at least X% sequence identity" with respect to a reference sequence (where "X" is a real number), it means that at least X percent of amino acid residues or nucleotides in the polypeptide or nucleic acid are identical to those of the reference sequence when the sequences are optimally aligned. Optimal alignment of sequences can be determined by various methods within the skill of those skilled in the art, for example, by the Smith-Waterman alignment algorithm (Smith et al., J.Mol.Biol.147:195-7, 1981) and BLAST (Basic Local Alignment Search Tool; Altschul et al., J.Mol.Biol.215:403-10, 1990). These and other alignment algorithms are accessible using publicly available computer software such as "Best Fit" (Smith and Waterman, Advances in Applied Mathematics, 482-489, 1981) (incorporated in GeneMatcher Plus (Schwarz and Dayhoff, Atlas of Protein Sequence and Structure, Dayhoff, MO, Ed pp 353-358, 1979)), BLAST, BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, Megalign (DNASTAR), or other software / hardware for alignment. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve optimal alignment across the entire length of the sequences being compared.

[0057] "Therapeutic dose" means an amount of polypeptide or nucleic acid molecule described herein that is sufficient to substantially improve, treat, prevent, delay, suppress, or cessate at least one symptom of muscle weakness. The therapeutic dose of the compositions described herein may depend on the severity of the disorder being treated, as well as the subject's condition, weight, and overall health, and can be determined by a person skilled in the art, taking such factors into consideration. The therapeutic dose of the compositions described herein may be administered to a subject in a single dose or in multiple doses administered over a period of time.

[0058] "To treat," "to treat," or "treatment" means, for example, the medical management of a subject with the intention of curing, improving, stabilizing, reducing the likelihood of, or preventing a muscle weakness disorder (e.g., in a subject with hypotonia) by administering a pharmaceutical composition (e.g., sALP such as asfotase alfa), and / or the management of a subject (e.g., in a subject with hypotonia) that exhibits or may exhibit a muscle weakness disorder.

[0059] This term includes active treatment, i.e., treatment specifically directed towards or related to the improvement of a disease, condition, disorder, or event, and causal treatment, i.e., treatment directed towards the elimination of the cause of the related disease, condition, disorder, or event. Furthermore, this term includes palliative treatment, i.e., treatment designed not to cure the disease, condition, disorder, or event, but to alleviate or improve at least one symptom; symptomatic treatment, i.e., treatment directed towards the systemic symptoms of the related disease, condition, disorder, or event; preventive treatment, i.e., treatment aimed at minimizing, partially or completely inhibiting, the onset of the related disease, condition, disorder, or event in a subject who is not yet diseased but is susceptible to or otherwise at risk of developing a particular disease, condition, disorder, or event; and supportive treatment, i.e., treatment used to complement another specific treatment aimed at improving the related disease, condition, disorder, or event.

[0060] As used herein, “walking ability” refers to the ability of an individual to lift and lower each leg in sequence (for example, an individual with a muscle weakness disorder as described herein). Walking ability may be assessed by tests, in particular by the six-minute walk test (6MWT). For guidelines on the six-minute walk test, see the statement by the American Thoracic Society (American Journal of Respiratory and Critical Care Medicine, 166(1):111-7, 2002) (which is incorporated herein by reference in its entirety). [Brief explanation of the drawing]

[0061] [Figure 1] This graph shows the RNA sequencing results for the ATP1A3 transcript. The x-axis represents the position within the transcript, and the y-axis represents the number of reads. The results for patient A, her mother, father, and three siblings are shown. [Figure 2A] This graph shows the ALPL transcriptions of patient A (the proband), his mother, father, and three siblings. Transcription is measured by total transcripts per million (TPM). [Figure 2B] This is a Western blot graph showing TNAP translation for patient A (proband), mother, father, and three siblings. Vinculin is used as the standard. [Figure 3] This shows the Seahorse mitochondrial respiratory stress test on extensor digitorum longus (EDL) muscle fibers of age- and sex-matched HPP and WT mice. [Figure 4] This indicates the reserve respiratory volume of HPP mouse muscle after vehicle or in vivo treatment as described in Sequence ID No. 21. [Figure 5] This shows the branched-chain amino acid (BCAA) pathway in adenosine triphosphate (ATP) production via the electron transport chain (ETC) and oxidative phosphorylation (Ox-Phos). [Figure 6]The following shows the levels of BCAAs (valine, leucine, and isoleucine) in the tibialis muscle of tissue-nonspecific alkaline phosphatase (TNSALP) knockout mice (Akp2- / -(KO) mice) treated with SEQ ID NO: 1 for 35 days followed by a vehicle for 12 days (discontinuation of SEQ ID NO: 1), or treated with SEQ ID NO: 1 continuously for 47 days, similar to those of wild-type untreated mice. [Figure 7] The following shows the levels of BCAAs (valine, leucine, and isoleucine) in the tibialis muscle of tissue-nonspecific alkaline phosphatase (TNSALP) knockout mice (Akp2- / -(KO) mice) treated with SEQ ID NO: 1 for 35 days followed by a vehicle for 12 days (discontinuation of SEQ ID NO: 1), or treated with SEQ ID NO: 1 continuously for 47 days, similar to those of wild-type untreated mice. [Figure 8] The following shows the levels of BCAAs (valine, leucine, and isoleucine) in the tibialis muscle of tissue-nonspecific alkaline phosphatase (TNSALP) knockout mice (Akp2- / -(KO) mice) treated with SEQ ID NO: 1 for 35 days followed by a vehicle for 12 days (discontinuation of SEQ ID NO: 1), or treated with SEQ ID NO: 1 continuously for 47 days, similar to those of wild-type untreated mice. [Modes for carrying out the invention]

[0062] Muscle weakness is a prominent symptom of several diseases and disorders. One such disease is hypophosphatasia (HPP), a congenital defect caused by a loss-of-function mutation in the ALPL gene encoding tissue-nonspecific alkaline phosphatase (TNSALP). Asfotase alfa (STRENSIQ®, Alexion Pharmaceuticals, Inc.), a TNSALP fusion protein, is the first and only treatment available for subjects diagnosed with HPP. Surprisingly, the inventors have discovered that subjects who do not have a loss-of-function mutation in the ALPL gene but exhibit symptoms similar to HPP, including muscle weakness (e.g., hypotonia), also respond to asfotase alfa treatment. Therefore, the present invention features a method for treating muscle weakness in subjects without a loss-of-function mutation in ALPL, characterized by, for example, a decrease in the concentration of TNSALP in their serum, by administering a recombinant polypeptide having alkaline phosphatase activity (e.g., asfotase alfa).

[0063] Subjects that can be treated using the compositions and methods described herein This method is characterized by a treatment for muscle weakness in subjects with reduced TNSALP levels by administering a polypeptide possessing alkaline phosphatase activity. The subjects are those who do not have loss-of-function mutations in ALPL but may have mutations in the non-coding region of the ALPL gene. The reduction in TNSALP levels is characterized by decreased ALPL transcription (e.g., a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), decreased ALPL mRNA translation (e.g., a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), and increased post-translational modifications of TNSALP (e.g., a decrease of 10%, 20%, 30%, 40%, 5%). It may be caused by one or more factors, such as an increase of 0%, 60%, 70%, 80%, 90%, or 100% (e.g., a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), and / or a decrease in TNSALP enzyme activity (e.g., a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). Decreased ALPL transcription, decreased ALPL mRNA translation, increased or decreased TNSALP post-translational modification, and / or decreased TNSALP enzyme activity may be measured against the mean, normalized values, baseline, pre-disease control values, or healthy subjects.

[0064] Loss-of-function mutations in ALPL are publicly known in the art and are disclosed, for example, in PCT Publication WO2011134084, which is incorporated herein by reference in its entirety. Accordingly, this invention features a method for treating subjects with muscle weakness who do not have one of the mutations described herein or otherwise known. Non-loss-of-function mutations, such as mutations in the regulatory region, 3' or 5' UTR, enhancer, promoter, or intron region of ALPL, may contribute to a decrease in TNSALP levels. Alternatively, or furthermore, subjects may have mutations (e.g., single nucleotide polymorphisms (SNPs)) somewhere in the genome, for example, in the regulatory region, 3' or 5' UTR, enhancer, promoter, or intron region of non-ALPL genes.

[0065] The subject may have one or more structural variants in its genome that result in reduced TNSALP activity, leading to muscle weakness. Examples of structural variants include copy number variants, insertions, duplications, inversions, translocations, and break ends. The subject may have mutations or structural variants in the coding region(s) of proteins, enzymes, or regulatory RNAs (e.g., non-coding RNAs) other than TNSALP. For example, other proteins or RNAs that directly or indirectly regulate the function, activity, and / or expression of TNSALP, or that interact with the TNSALP or ALPL genes, may have mutations or structural variants that confer downstream effects that reduce TNSALP levels. For example, the subject may have mutations or structural variants in one or more of the following genes: ATP1A3, ANKH, ENPP1, FGFR3, PHOSPHO1, PTH1R, PTH2R, SPP1, TNFRSF11A, TNFRSF11B, COL1A1, COL1A2, SOX9, PDXP, AOX1, PNPO, PDXK, ADCK3, MTRNR2, or S1PR1. Mutations or structural variants may be located in the coding region, 5'UTR, 3'UTR, or intron sequences. The subject may have the ATP1A3 variant c.357+1G>A. The subject may have the ADCK3 variant m.1665G>A. The subject may have the MTRNR2 variant m.1836A>G.

[0066] The ATP1A3 gene encodes the alpha subunit of the Na+ / K+ ATPase pump, which uses energy from ATP to transport ions into and out of cells, maintaining the electrochemical gradient. It also plays a role in neuronal function, muscle contraction, and neurotransmitter reuptake in the central nervous system. All reported adverse ATP1A3 mutations exhibit a dominant inheritance pattern with variability in penetrance. Furthermore, adverse mutations are associated with a rapidly developing dystonia-parkinsonian syndrome phenotype characterized by motor delay and ataxia, alternating hemiplegia in children, and cognitive problems. The phenotype may vary in children and may include hypotonia, ataxia, dysarthria, and slurred speech. Subjects with mutations in ATP1A3 may exhibit decreased ALP levels or related symptoms, as described herein. Subjects with mutations in ATP1A3 and muscle weakness may benefit from treatment with polypeptides having alkaline phosphatase activity, according to the methods described herein.

[0067] The ADCK3 gene encodes a mitochondrial protein that functions in the electron transport membrane protein complex in the respiratory chain. Mutations in this gene impair coenzyme Q10 production. PLP is also a necessary cofactor for the enzyme in the Q10 biosynthesis pathway. Mutations in the ADCK3 gene are associated with autosomal recessive cerebellar ataxia. Subjects with ADCK3 mutations may exhibit decreased ALP levels or related symptoms, as described herein. Subjects with ATP1A3 mutations and muscle weakness may benefit from treatment with a polypeptide having alkaline phosphatase activity, according to the methods described herein.

[0068] Subjects treated according to the methods described herein may be characterized by having one or more of the following: elevated PPi concentration, decreased alkaline phosphatase concentration and / or activity, e.g., mean BOT-2 strength score less than 10, e.g., mean BOT-2 running speed and agility scores less than 5, e.g., mean CHAQ index score greater than about 0.8, or e.g., mean PODCI score less than about 40, e.g., mean 6MWT less than about 80% of the predicted 6MWT value (e.g., the predicted 6MWT value is the 6MWT value of an age-matched and / or sex-matched normal subject), e.g., muscle strength grade less than 5, and / or mean HHD value less than about 50% of the predicted HHD value (e.g., mean HHD muscle or grip strength value) (e.g., the predicted HHD value is the HHD value of an age-matched and / or sex-matched normal subject). In particular, subjects may be subjects that have muscle weakness or have been identified as being prone to muscle weakness.

[0069] Subjects may be identified as requiring treatment. For example, subjects identified as having or being prone to muscle weakness disorders (e.g., disorders not caused by loss-of-function mutations in ALPL, but characterized by, for example, decreased ALP levels or activity, or increased ALP substrate levels). Such subjects may be treated by administration of recombinant polypeptides with alkaline phosphatase activity (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or polypeptide variants having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa). The subjects may be characterized by having elevated PPi concentration, decreased ALP concentration, for example, an average BOT-2 strength score of less than 10, for example, an average BOT-2 running speed and agility score of less than 5, for example, an average CHAQ index score greater than approximately 0.8, for example, an average PODCI score of less than approximately 40, for example, an average 6MWT of less than approximately 80% of the predicted 6MWT value, for example, a muscle strength grade of less than 5, and / or for example, an average HHD value (e.g., average HHD muscle or grip strength value) of less than approximately 80% of the predicted HHD value. For example, elevated PPi concentrations of approximately 5.71 μM or higher in samples (e.g., plasma samples) from infants or children (e.g., subjects under approximately 12 years of age), elevated PPi concentrations of approximately 4.78 μM or higher in samples (e.g., plasma samples) from adolescents (e.g., subjects between approximately 13 and 18 years of age), and elevated PPi concentrations of approximately 5.82 μM or higher in samples (e.g., plasma samples) from adults (e.g., subjects over approximately 18 years of age) can be used to identify subjects requiring treatment.In particular, a decrease in ALP concentration in samples from subjects (e.g., plasma samples) can be used to identify subjects requiring treatment, for example, by using U / L values ​​such as: ≤90 U / L for subjects 0-14 days old, ≤134 U / L for subjects 15 days old to under 1 year old, ≤156 U / L for subjects 1 year to under 10 years old, ≤141 U / L for subjects 10 years to under 13 years old, ≤62 U / L for female subjects 13 years to under 15 years old, ≤127 U / L for male subjects 13 years to under 15 years old, ≤54 U / L for female subjects 15 years to under 17 years old, ≤89 U / L for male subjects 15 years to under 17 years old, ≤48 U / L for female subjects 17 years and older, and ≤59 U / L for male subjects 17 years and older.

[0070] Subjects with or prone to muscle weakness disorders (e.g., subjects without loss-of-function mutations in ALPL) may be identified by elevated PPi concentration, decreased alkaline phosphatase concentration, and / or decreased grip strength or muscle strength (e.g., assessed using BOT-2, 6MWT, CHAQ, PODCI, muscle strength grade, and / or HHD). Subjects may have, for example, decreased ALPL transcription, decreased ALPL mRNA translation, increased or decreased TNSALP post-translational modification, and / or decreased TNSALP enzyme activity compared to the mean, normalized value, baseline, pre-disease control value, or healthy subjects.

[0071] The invention also features a method for identifying subjects who have or are prone to muscle weakness disorders, wherein the subjects have elevated PPi concentration, decreased ALP concentration, and / or decreased grip strength or muscle strength; and a method for subsequently treating, improving, or reducing the risk thereof of at least one symptom of a muscle weakness disorder in the subject by, for example, administering ALP (e.g., asfotase alfa).

[0072] Subjects may be identified regardless of whether they have previously been diagnosed with hypophosphatasia (HPP), calcium pyrophosphate deposition disorder (CPPD), or familial hypophosphatemia (e.g., autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets, X-linked hypophosphatemic rickets, X-linked hypophosphatemia (XLH), etc.). Subjects are identified, for example, based on elevated PPi concentrations. Causes of elevated PPi concentrations include, for example, deficiencies in signaling molecules that regulate PPi production, degradation, or stability, or mutations in genes encoding such signaling molecules. For example, deficiencies or mutations in signaling molecules may result in PPi overexpression or decreased degradation or hydrolysis of PPi. Deficiencies in signaling molecules also include deficiencies in cofactors or other molecules that promote the function of the signaling molecules. For example, in CPPD, a deficiency of Mg, which acts as a cofactor for various phosphatases, results in elevated PPi levels.

[0073] Targeted muscle weakness diseases Muscle weakness, myopathy, and myasthenia gravis, which can be treated by the methods described herein, include any disease or disorder that causes, results from, or is associated with, at least one symptom of muscle weakness. In this disclosure, the terms “muscle weakness,” “myopathy,” and “myasthenia gravis,” or other similar expressions, refer to conditions associated with impaired muscle function, such as a lack of muscle strength or deficiency, compared to another subject lacking the condition, or to the same subject at a time prior to having the condition. Muscle weakness can be classified into true muscle weakness or conditions having perceived muscle weakness. True muscle weakness may include conditions in which the force exerted by the muscles is less than expected. For example, true muscle weakness includes various skeletal muscle diseases, including muscular dystrophy and inflammatory muscle diseases. Other examples include neuromuscular junction disorders, such as myasthenia gravis. Muscle weakness can also be caused by low levels of potassium and other electrolytes in muscle cells, in which the force exerted by the muscles is less than expected. Perceived muscle weakness (or non-neuromuscular weakness) is a condition in which an object feels more effort is required to exert a certain amount of force than normal (i.e., compared to another object lacking the condition, or the same object at a time before the condition was acquired), but the actual muscle strength is normal, such as chronic fatigue syndrome.

[0074] In some conditions, such as myasthenia gravis, muscle strength is normal at rest, but a true decrease occurs after the muscles are subjected to exercise. This also applies to some cases of chronic fatigue syndrome, where objective post-exercise weakness with delayed recovery time is measured and is a feature of some published definitions. These diseases, or disorders, are also examples of “muscle weakness disorders” within the scope of this disclosure.

[0075] Muscle weakness can also be classified as either "proximal" or "distal" based on the location of the muscles it affects. Proximal weakness affects the muscles closest to the body's midline, while distal weakness affects the muscles further lateral to the limbs. Proximal weakness can be seen in Cushing's syndrome and hyperthyroidism.

[0076] Other categories of muscle weakness do exist. For example, neuromuscular fatigue can be classified as either "central" or "peripheral" depending on its cause. Central muscle fatigue manifests as a general feeling of energy deficiency, while peripheral muscle fatigue manifests as localized, muscle-specific inability to function.

[0077] The severity of muscle weakness can be classified into different "grades" based on the following exemplary criteria: Grade 0: No contraction or muscle movement. Grade 1: There are signs of contraction, but there is no movement in the joint. Grade 2: Joint movement free from gravity. Grade 3: Moves against gravity, but does not move against added resistance. Grade 4: Moves against external resistance with less force than usual. Grade 5: Normal strength. Subjects classified as having muscle weakness within any of the grades 0 to 4 may be treated according to the methods disclosed herein.

[0078] Hypophosphatasia (HPP) and muscle weakness Hypophosphatasia (HPP) is a rare hereditary metabolic disorder resulting from loss-of-function mutations (or multiple mutations) in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. The inventors identified a subset of subjects diagnosed with HPP but who, upon sequencing analysis, did not exhibit loss-of-function mutations in ALPL, the gene encoding TNSALP. Therefore, several other mechanisms contribute to the development of HPP-like symptoms. The biochemical feature in these subjects is subnormal serum ALP activity (hypophosphatasia), which results in elevated blood and / or urine levels of three phosphorus compound substrates: inorganic pyrophosphate (PPi), phosphoethanolamine (PEA), and pyridoxal 5'-phosphate (PLP). TNSALP deficiency can lead to a range of complications, including early tooth loss, rickets, stunted growth, muscle weakness, impaired physical function, and pain. Asfotase alpha treatment dramatically improves bone mineralization in subjects with HPP, but it was unclear whether it was effective in treating muscle weakness. As described herein, the inventors have discovered that asfotase alpha has a therapeutic effect on the muscles of subjects (e.g., subjects with muscle weakness disorders, such as muscle weakness in subjects with HPP-like disease) even in subjects without loss-of-function mutations in ALPL.

[0079] Calcium pyrophosphate deposition disorder (CPPD or CPDD) and muscle weakness Calcium pyrophosphate deposition disease (CPPD or CPDD), or calcium pyrophosphate dihydrate crystal deposition disease, is a metabolic joint disease caused by the deposition of calcium pyrophosphate dihydrate crystals in and around the joints, particularly in the articular cartilage and fibrocartilage. These diseases can be present in individuals who do not have loss-of-function mutations in ALPL and who show reduced levels of TNSALP. CPPD is often asymptomatic, with only radiographic changes (i.e., chondrocalcinosis) being observed, but a variety of clinical symptoms can occur, including acute (pseudogout) and chronic arthritis. The crystal deposits can cause inflammation in the joint and destroy the articular cartilage. This disease can take several different arthritis-related forms: osteoarthritis, rheumatoid arthritis (RA)-like inflammatory arthritis, or an acute painful inflammatory condition called pseudogout. The name pseudogout comes from the fact that it resembles another acute painful condition called gout. The main difference is the type of crystal involved in the inflammation and damage. The knees, wrists, and hips are most frequently affected, but almost all joints can be affected by CPPD. This condition is the most common cause of secondary metabolic osteoarthritis. Individuals with CPPD may experience significant pathological conditions due to the pain of acute pseudogout attacks or the symptoms of chronic arthritis. Treatment of symptomatic CPPD is important to prevent further end-organ damage, but it cannot reverse the joint disease.

[0080] The precise mechanism of CPPD development remains unclear. Due to aging, genetic factors, or both, subjects experience increased adenosine triphosphate degradation and increased inorganic pyrophosphate concentrations in the joints. Changes in the cartilage matrix may play a significant role in promoting the deposition of calcium pyrophosphate dihydrate crystals. Overactivity of triphosphate-degrading enzymes, such as nucleoside triphosphate pyrophosphohydrolase, is observed in the cartilage of subjects with CPPD. Therefore, inorganic pyrophosphate can bind with calcium, leading to deposition in cartilage and synovial membrane. Hyalin cartilage is most commonly affected, but fibrocartilage, such as the meniscus cartilage of the knee, can also be involved (Pritzker et al., 1988 J Rheumatol. 15(5):828-835).

[0081] Other diseases and muscle weakness Similar to HPP and CPPD (or CPDD), other diseases or disorders may include at least one symptom of muscle weakness. Among these, certain muscle weakness disorders have a characteristic elevation of inorganic pyrophosphate (PPi) levels. These muscle weakness disorders with elevated PPi levels are also targets of treatment with asfotase alfa in this disclosure.

[0082] For example, subjects diagnosed with familial hypophosphatemia (e.g., autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets, X-linked hypophosphatemic rickets, and X-linked hypophosphatemia (XLH)) typically exhibit a muscle weakness phenotype. Hypophosphatemia, or hypophosphatemic rickets, is a form of rickets characterized by low serum phosphate levels and resistance to treatment with ultraviolet light or vitamin D supplementation. X-linked hypophosphatemia (XLH) is a dominant disorder and accounts for over 80% of all familial hypophosphatemia cases. XLH is considered a systemic disorder due to mutations in the phosphate regulatory gene (PHEX) homologous to endopeptidase on the X chromosome. Subjects with XLH show normal or low serum concentrations of 1,25-dihydroxyvitamin D3, suggesting insufficient formation of this vitamin D metabolite. The remaining 20% ​​of those with familial hypophosphatemia have autosomal dominant hypophosphatemic rickets from gain-of-function autosomal recessive hypophosphatemic rickets, and hereditary hypophosphatemic rickets with hypercalciuria.

[0083] Other diseases characterized by frailty that can be treated according to the methods described herein include, for example, myasthenia gravis, amyotrophic lateral sclerosis (ALS), myositis, myotonic dystrophy, muscle rigidity, Guillain-Barré syndrome, Duchenne muscular dystrophy (DMD), and Lambert-Eaton myasthenic syndrome.

[0084] Treatment method This specification provides methods for treating or improving at least one symptom in subjects having or being prone to muscle weakness disorders (e.g., children, adolescents, or adults, e.g., subjects without loss-of-function mutations in ALPL and with reduced levels of TNSALP). Such treatment may include administering alkaline phosphatase or a polypeptide having alkaline phosphatase activity to reduce elevated PPi concentrations in such subjects. For example, soluble alkaline phosphatases (sALP such as TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha) may be administered to children, adolescents, or adults.

[0085] Subjects may be diagnosed with muscle weakness disorders (e.g., HPP or HPP-like disorders, CPPD, familial hypophosphatemia, etc.) prior to administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP such as TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa). Furthermore, subjects with or prone to muscle weakness disorders may be untreated subjects who have not been previously treated with sALP (TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa). Subjects may have muscle weakness disorders other than HPP, CPPD, or familial hypophosphatemia.

[0086] This method involves administering alkaline phosphatase, or a polypeptide having alkaline phosphatase activity (e.g., sALP such as TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa), to a subject with or prone to muscle weakness, in a single or multiple dose over a period of time. In particular, sALPs such as asfotase alfa may be administered to subjects who have been previously determined to have elevated inorganic pyrophosphate (PPi) concentrations or at least one predetermined biomarker / score of muscle weakness, e.g., an average BOT-2 strength score of less than 10, an average BOT-2 running speed and agility score of less than 5, an average CHAQ index score greater than about 0.8, and / or an average PODCI score of less than about 40, an average 6MWT of less than about 80% of the predicted 6MWT value, a muscle strength grade of less than 5, and / or an average HHD value of less than about 80% of the predicted HHD value (e.g., average HHD muscle, or grip strength value). For example, sALP may be administered to subjects previously determined to have a concentration of PPi in a sample (e.g., plasma sample) greater than approximately 5.71 μM for infants or children (e.g., subjects under approximately 12 years of age), a concentration of PPi in a sample (e.g., plasma sample) greater than approximately 4.78 μM for adolescents (e.g., subjects approximately 13 to approximately 18 years of age), or a concentration of PPi in a sample (e.g., plasma sample) greater than approximately 5.82 μM for adults (e.g., subjects over approximately 18 years of age). In other embodiments, the muscle weakness disorders described herein are caused by elevated concentrations of at least one alkaline phosphatase substrate (e.g., PPi, PLP, PEA, etc.).Alternatively, alkaline phosphatase, or a polypeptide having alkaline phosphatase activity (e.g., sALP such as TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa), may be administered to subjects with or prone to muscle weakness before determining muscle weakness scores (e.g., using BOT-2 strength score, BOT-2 running speed and agility scores, CHAQ index score, BSID-III scale score, PDMS-2 standard score, muscle strength score, 6MWT value, and / or HHD value). Treatment with ALP according to the methods described herein promotes, for example, increased ADL activity, reduced pain, and / or improved motor development.

[0087] Furthermore, the effectiveness of treatment with sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) can be evaluated using each of the described scores (e.g., BOT-2 strength score, BOT-2 running speed and agility score, CHAQ index score, BSID-III scale score, PDMS-2 standard score, 6MWT, 12-POMA-G, modified performance-oriented mobility assessment (mPOMA-G, e.g., Phillips et al. 2015 Bone Abstracts 4:P136), or HHD value) for subjects with or prone to the muscle weakness disorders described herein, either alone or in any combination. Herein, improvement in a particular test score demonstrates that sALP is effective in treating muscle weakness disorders.

[0088] For example, if administration of alkaline phosphatase or an alkaline phosphatase-containing polypeptide (e.g., sALP such as TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa) to a subject with or prone to muscle weakness, results in an average increase of approximately 10 or more in the BOT-2 intensity score, and the subject previously had an average BOT-2 intensity score of less than approximately 10, then treatment with alkaline phosphatase or an alkaline phosphatase-containing polypeptide is effective, for example, to treat physical impairment associated with muscle weakness. Alternatively, if administration of sALP does not result in an average increase of approximately 10 or more in the BOT-2 intensity score, the dose and / or frequency of administration of alkaline phosphatase or an alkaline phosphatase-containing polypeptide may be modified to determine an effective amount of alkaline phosphatase or an alkaline phosphatase-containing polypeptide for the subject. For example, the dose of sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) may be increased, for example, from about 3 mg / kg / week to about 6 mg / kg / week, or from about 6 mg / kg / week to about 9 mg / kg / week.

[0089] Furthermore, if the administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP such as TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa) to subjects with or prone to muscle weakness disorders results in one or more improvements in the subject's muscle strength grade classification (e.g., improvement from a previously lower muscle strength grade to a muscle strength grade of 1, 2, 3, 4, or 5), and if the subject previously had an average muscle strength grade of less than approximately 5, then treatment with alkaline phosphatase or a polypeptide with alkaline phosphatase activity is effective, for example, in treating physical disabilities associated with muscle weakness disorders. Alternatively, if sALP administration does not result in an improvement in one or more subject muscle strength grade classifications from a previously lower muscle strength grade, the dose and / or frequency of administration of alkaline phosphatase or an alkaline phosphatase-active polypeptide may be modified (e.g., increased) to determine an effective dose of alkaline phosphatase or an alkaline phosphatase-active polypeptide for the subject. For example, the dose of sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) may be increased, for example, from about 3 mg / kg / week to about 6 mg / kg / week, or from about 6 mg / kg / week to about 9 mg / kg / week.

[0090] Biomarkers / endpoints for the diagnosis and / or treatment of muscle weakness disorders Muscle weakness disorders (e.g., perinatal HPP, infant HPP, pediatric HPP, and HPP including endodontic hypophosphatasia, HPP-like diseases, CPPD, and familial hypophosphatemia, as described herein) can be treated with alkaline phosphatase or polypeptides having alkaline phosphatase activity (e.g., sALP such as TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1 or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alpha). The methods described herein are also useful for diagnosing subjects who have or are prone to muscle weakness disorders, for identifying subjects who have or are prone to muscle weakness disorders, or for testing the effectiveness of treatments for muscle weakness disorders. For example, if a subject is characterized by having certain characteristic biomarkers or metric scores, the subject may be diagnosed as having or being prone to muscle weakness disorders. The subject may be treated with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP such as TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha), and the efficacy or effect of the treatment may be analyzed using characteristic biomarkers or metric scores. Such biomarkers may include, for example, elevated inorganic pyrophosphate (PPi) concentration and / or decreased alkaline phosphatase (ALP) in the subject's serum, bone or muscle tissue, or urine. Useful exemplary metrics for determining the effectiveness of muscle weakness treatments in the methods described herein include: (1) the Blueinx-Oseletzky Motor Skills Scale, 2nd Edition (BOT-2) for motor skills; (2) the Child Health Assessment Questionnaire (CHAQ); (3) the Child Outcome Data Acquisition System (PODCI); (4) the Bailey Scale for Infant and Toddler Development, 3rd Edition (BSID-III); (5) the Peabody Developmental Motor Scale, 2nd Edition (PDMS-2); (6) the 6-Minute Walk Test (6MWT); (7) muscle strength grades; and (8) the Handheld Dynamometer (HHD), which are described in more detail below.

[0091] Plasma inorganic pyrophosphate (PPi) and alkaline phosphatase (ALP) concentrations Subjects with or prone to muscle weakness disorders may be identified for treatment with alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP such as TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with SEQ ID NO: 1, e.g., asfotase alpha) by determining the concentrations of inorganic pyrophosphate (PPi) and / or alkaline phosphatase (ALP) in samples such as plasma or urine samples from the subject. The concentrations of PPi and / or ALP in plasma or urine samples can be quantified using any method known to those skilled in the art (as described in Whyte et al., 1995 (J. Clin. Invest. 95(4):1440-1445) (the whole of which is incorporated herein by reference)). Methods for quantifying PPi concentration in plasma or urine samples are described in Cheung et al., 1977 (Anal. Biochem. 83:61-63), Cook et al., 1978 (Anal. Biochem. 91:557-565), and Johnson et al., 1968 (Anal. Biochem. 26:137-145), each of which is incorporated herein by reference in its entirety.

[0092] In particular, alkaline phosphatase, or polypeptides having alkaline phosphatase activity (e.g., sALP such as TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or polypeptide variants having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa), may be administered to subjects with or prone to muscle weakness (e.g., subjects without loss-of-function mutations in ALPL) who have been previously determined to have a maximum plasma PPi concentration of approximately 6 μM (e.g., plasma PPi concentrations in the range of approximately 4.5 μM, approximately 5 μM, or approximately 5.5 μM, or within the range of approximately 4.5 μM to approximately 6 μM). For example, alkaline phosphatase, or polypeptides having alkaline phosphatase activity, may be administered to infants or children (e.g., subjects under approximately 12 years of age) with a plasma PPi concentration of approximately 5.71 μM or higher, to adolescents (e.g., subjects between approximately 13 and 18 years of age) with a plasma PPi concentration of approximately 4.78 μM or higher, or to adults (e.g., subjects older than approximately 18 years of age) with a plasma PPi concentration of approximately 5.82 μM or higher. Furthermore, alkaline phosphatase, or polypeptides having alkaline phosphatase activity, may be administered to subjects (e.g., humans) who have or are prone to muscle weakness, and who have been previously determined to have plasma ALP concentrations of, for example, approximately 90 U / L or less for subjects 0 to 14 days old, approximately 134 U / L or less for subjects 15 days old to under 1 year old, approximately 156 U / L or less for subjects 1 to under 10 years old, approximately 141 U / L or less for subjects 10 to under 13 years old, approximately 62 U / L or less for female subjects 13 to under 15 years old, approximately 127 U / L or less for male subjects 13 to under 15 years old, approximately 54 U / L or less for female subjects 15 to under 17 years old, approximately 89 U / L or less for male subjects 15 to under 17 years old, approximately 48 U / L or less for female subjects 17 years and older, and approximately 59 U / L or less for male subjects 17 years and older.

[0093] The plasma PPi concentration and / or plasma ALP concentration of subjects with or prone to muscle weakness disorders (e.g., humans) can be compared to the plasma PPi concentration and / or plasma ALP concentration of normal subjects to determine the treatment effect in subjects administered alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP such as TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa). In particular, alkaline phosphatase or a polypeptide with alkaline phosphatase activity may be administered over a treatment period of at least one year (e.g., at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, at least ten years, or longer than ten years, e.g., the lifetime of the subject). Alternatively, this method may include determining plasma PPi concentrations and / or plasma ALP concentrations before administering alkaline phosphatase or a polypeptide having alkaline phosphatase activity, and evaluating the effect of alkaline phosphatase or a polypeptide having alkaline phosphatase activity on the subject of treatment.

[0094] This method results in a decrease in PPi and / or an increase in ALP concentration in a sample (e.g., plasma sample) from a subject with or prone to muscle weakness, such as a human subject (e.g., a human subject without loss-of-function mutations in ALPL). For example, treatment with alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP such as TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa) results in a decrease in PPi concentration of approximately 1 μM, 1.5 μM, 2 μM, 2.5 μM, or 3 μM, or more than 25% (e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more than 60%) in a sample (e.g., plasma sample) from the subject. Therefore, after administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity, subjects exhibit plasma PPi concentrations of, for example, approximately 2 μM to 5 μM, approximately 3 μM to 5 μM, approximately 2 μM to 4 μM, or approximately 2 μM to 3 μM.

[0095] Similarly, treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity results in an increase of 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more than 60% in ALP concentrations in samples (e.g., plasma samples) from subjects (e.g., humans) with or prone to muscle weakness, compared to subjects before administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity. For example, administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity increases ALP concentrations in subject-derived samples (e.g., plasma samples) to, for example, approximately 273 U / L or more for subjects 0-14 days old, approximately 518 U / L or more for subjects 15 days old to under 1 year old, approximately 369 U / L or more for subjects approximately 1 year to under 10 years old, approximately 460 U / L or more for subjects approximately 10 years to under 13 years old, and approximately 1 The recommended daily intake levels will be increased to approximately 280 U / L or higher for females aged 3 to under 15 years, approximately 517 U / L or higher for males aged approximately 13 to under 15 years, approximately 128 U / L or higher for females aged approximately 15 to under 17 years, approximately 365 U / L or higher for males aged approximately 15 to under 17 years, approximately 95 U / L or higher for females aged approximately 17 years and older, and approximately 164 U / L or higher for males aged approximately 17 years and older.

[0096] In subjects with or prone to muscle weakness disorders (e.g., humans), a decrease in plasma PPi and / or an increase in ALP concentration may persist during administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP such as TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa). For example, during treatment with sALP, plasma PPi concentration decreases by approximately 25%, remaining within ±10% of the decreased plasma PPi concentration, and / or during treatment with alkaline phosphatase or a polypeptide with alkaline phosphatase activity, plasma ALP concentration increases by approximately 50%, remaining within ±10% of the increased plasma ALP concentration.

[0097] Alternatively, if administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP such as TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) does not result in an average decrease of approximately 25% or more in PPi concentration in plasma samples from subjects with or prone to muscle weakness (e.g., humans), the dose and / or frequency of sALP administration may be modified to determine an effective dose of sALP for the subject. Similarly, if administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity does not result in an average increase of approximately 50% or more in ALP concentration in plasma samples from the subject, the dose and / or frequency of administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity may be modified to determine an effective dose of alkaline phosphatase or a polypeptide having alkaline phosphatase activity for the subject. For example, the dose of alkaline phosphatase, or a polypeptide having alkaline phosphatase activity, may be increased from, for example, about 2.1 mg / kg / week or about 3.5 mg / kg / week to about 6 mg / kg / week or about 9 mg / kg / week.

[0098] Assay for identifying subjects with decreased alkaline phosphatase (ALP) levels Biochemical and diagnostic assays can be used to identify individuals with reduced ALP concentration, which may be caused by decreased ALPL transcription, decreased ALPL mRNA translation, increased or decreased post-translational modifications of TNSALP, and / or decreased TNSALP enzyme activity. These individuals may have mutations (e.g., single nucleotide polymorphisms (SNPs)) in regulatory regions of their genome, such as promoters, upstream open reading frames (uORFs), downstream ORFs, the 3' or 5' untranslated region of ALPL, or other genes that are related to (e.g., directly or indirectly interact with) or unrelated to ALPL or TNSALP. Individuals may also have mutations in the coding regions of proteins or enzymes other than TNSALP. For example, another protein that interacts with TNSALP or directly or indirectly regulates the function, activity, and / or expression of TNSALP may have mutations that have a downstream effect on TNSALP concentration. The genome of a subject with muscle weakness can be sequenced (for example, using whole-genome sequencing (WGS), intron sequencing, or exon sequencing) to identify the relevant mutation(s) outside the ALPL gene coding region that contribute to or cause the muscle weakness symptoms described herein.

[0099] Assays for measuring ALPL transcription, ALPL mRNA translation, TNSALP post-translational modification, and / or reduction in TNSALP enzyme activity are known to those skilled in the art. For example, assays for measuring transcription include quantitative PCR (qPCR), RT-PCR, RNA-Seq, and next-generation sequencing (NGS). These assays can generate information about transcriptional dysregulation of ALPL or other genes. Assays for measuring translation include, for example, Western blot analysis, ELISA, Bradford assay, and polysome profiling. These assays can be used to quantify protein levels. Assays for measuring TNSALP post-translational modification (e.g., phosphorylation or glycosylation, e.g., N-linked or O-linked) include, for example, Western blot analysis or ELISA assays using antibodies that bind to post-translational modified TNSALP. Assays for measuring TNSALP activity are well known in the art and, for example, are described in PCT Publication WO2005103263, which is incorporated herein by reference in its entirety. Assays for measuring TNSALP activity include fluorescence-based enzyme assays and hydroxyapatite binding assays. Furthermore, TNSALP localization assays can be used to determine whether TNSALP is present in the cell membrane. In addition, biochemical analyses measuring ALP serum substrate levels (e.g., PPi, PLP, and PEA) can be used as outputs for measuring TNSALP activity.

[0100] Blueinx-Oselecky Motor Skills Test, 2nd Edition (BOT-2) An exemplary Bruininks-Oseletzky Motor Skills Test, Second Edition (BOT-2) is described in Bruininks, RH (2005). The Bruininks-Oseletzky Motor Skills Test (BOT-2) is described in Minneapolis, MN: Pearson Assessment, which is incorporated herein by reference in its entirety. In particular, the BOT-2 may be used to assess physical impairment and motor limitations in subjects who have or are prone to muscle weakness disorders (e.g., HPP-like disorders) and to generate a BOT-2 score for the subject.

[0101] BOT-2 includes a series of tests to assess a subject's physical disability, which may be administered using a kit including the tests. BOT-2 provides a composite BOT-2 score in the following areas: strength, running speed and agility, fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance, and upper limb coordination. For example, a subject with or prone to muscle weakness may perform sit-ups, v-ups, standing long jumps, wall sits, and / or push-ups to determine their BOT-2 strength score. A subject with or prone to muscle weakness may perform balance beam jumps and / or shuttle runs, bipedal jumps, and / or unipedal jumps to determine their BOT-2 running speed and agility scores. A subject with or prone to muscle weakness may perform circle cutting and / or dotting to determine their BOT-2 fine motor precision score. Individuals with or prone to muscle weakness may have their BOT-2 fine motor integration score determined by copying stars and / or squares. Individuals with or prone to muscle weakness may have their manual dexterity score determined by moving pennies, sorting cards, and / or tying blocks together. Individuals with or prone to muscle weakness may have their BOT-2 bilateral coordination score determined by tapping their feet and fingers and / or performing jumping jacks. Individuals with or prone to muscle weakness may have their BOT-2 balance score determined by walking forward on a line and / or standing on one leg on a balance beam. Individuals with or prone to muscle weakness may have their BOT-2 upper limb coordination score determined by throwing a ball at a target and / or catching the thrown ball.

[0102] Subjects with or prone to muscle weakness disorders (e.g., HPP-like disorders) may perform tests in one or more of the listed domains (strength, running speed and agility, fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance, and upper limb coordination) to generate a BOT-2 score indicating the subject's physical impairment. Within each BOT-2 domain (strength, running speed and agility, fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance, and upper limb coordination), such subjects may perform one or more tests to determine their BOT-2 score. For example, a subject may perform one or more of the following to determine their BOT-2 strength score: sit-ups, v-ups, standing long jumps, wall sits, and push-ups. Therefore, subjects with or a tendency toward muscle weakness disorders (e.g., HPP-like disorders) can undergo only one test (e.g., one test selected from groups of sit-ups, v-ups, standing long jumps, wall sits, and push-ups) to determine their BOT-2 score (e.g., BOT-2 strength score).

[0103] For subjects with or a tendency toward muscle weakness disorders (e.g., HPP-like disorders), each of the BOT-2 scores (strength, running speed and agility, fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance, and upper limb coordination) can be compared to the BOT-2 scores of subjects without muscle weakness disorders (e.g., HPP-like disorders) to determine, for example, a baseline comparison of BOT-2 scores. For subjects with or a tendency toward muscle weakness disorders (e.g., HPP-like disorders), each of the BOT-2 scores (e.g., strength, running speed and agility, fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance, and upper limb coordination) can be compared to the BOT-2 scores of other subjects with or a tendency toward muscle weakness disorders (e.g., HPP-like disorders) to provide, for example, a relative BOT-2 score for the subject.

[0104] The BOT-2 score (e.g., strength, running speed and agility, fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance, and upper limb coordination score) ranges from approximately 0 to approximately 25, where a score of approximately 10 to approximately 20 is considered to represent a healthy subject (e.g., a subject without muscle weakness disease (e.g., HPP-like disease)). Subjects with an average BOT-2 score of less than approximately 10 (e.g., strength, running speed and agility, fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance, and upper limb coordination score) may be treated with alkaline phosphatase, or a polypeptide having alkaline phosphatase activity, such as sALP such as TNALP, such as the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, such as asfotase alfa.

[0105] For example, subjects with a BOT-2 strength score of less than 10 (e.g., approximately 0, approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, or approximately 10) who have or are prone to muscle weakness may be treated with sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) for the duration of the subject's life. Similarly, subjects with a BOT-2 running speed and agility score of less than 10 (e.g., about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10) who have or are prone to muscle weakness may be treated with sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) for the duration of the subject's life.

[0106] The method may result in an improvement in the BOT-2 score (e.g., muscle strength, running speed and agility, fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance, and / or upper limb coordination score) in subjects with or prone to muscle weakness disorders (e.g., HPP-like disorders). For example, treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha), such as treatment with sALP for a certain period, may result in an average increase of approximately 10 to approximately 20 (e.g., approximately 10, approximately 11, approximately 12, approximately 13, approximately 14, approximately 15, approximately 16, approximately 17, approximately 18, approximately 19, or approximately 20) in the BOT-2 strength score. Furthermore, treatment with sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha) may result in an average increase of approximately 5 to approximately 20 (e.g., approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, approximately 10, approximately 11, approximately 12, approximately 13, approximately 14, approximately 15, approximately 16, approximately 17, approximately 18, approximately 19, or approximately 20) in the running speed and agility scores of BOT-2.

[0107] Increases in BOT-2 scores (e.g., strength, running speed and agility, fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance, and / or upper limb coordination scores) may persist for a certain period during administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha). Similarly, reductions in muscular physical impairment after administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity may persist during administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity.

[0108] For subjects with or prone to muscle weakness disorders (such as HPP), the BOT-2 score (strength, running speed and agility, fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance, and upper limb coordination score) can be used alone or in combination with other metrics to evaluate the effectiveness of treatment with alkaline phosphatase or polypeptides with alkaline phosphatase activity such as sALP (e.g., TNALP, e.g., sALP polypeptide of SEQ ID NO: 1, or polypeptide variants having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa). Improvements to specific test scores demonstrate that alkaline phosphatase or polypeptides with alkaline phosphatase activity are effective in treating muscle dysfunction associated with muscle weakness disorders. For example, if administration of sALP to a subject with or a tendency toward muscle weakness results in an average increase of approximately 5 or more in BOT-2 running speed and agility scores, and the subject previously had an average BOT-2 running speed and agility score of less than approximately 5, then sALP is considered effective, for example, in treating physical disabilities associated with muscle weakness.

[0109] Furthermore, for subjects who have or are prone to having a muscle weakness disorder (e.g., HPP-like disorder, CPPD, familial hypophosphatemia, etc.) within each BOT-2 domain (strength, running speed and agility, fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance, and upper limb coordination), one or more tests may be performed to determine the subject's BOT-2 score.

[0110] Alternatively, if administration of alkaline phosphatase, or an alkaline phosphatase-containing polypeptide, such as sALP, does not result in an average increase of more than approximately 5 in BOT-2 running speed and agility scores, the dose and / or frequency of administration may be modified to determine an effective amount of alkaline phosphatase, or an alkaline phosphatase-containing polypeptide, for subjects with or prone to muscle weakness disorders (e.g., HPP-like disorders). For example, the dose of sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) may be increased, for example, from about 3 mg / kg / week to about 6 mg / kg / week, or from about 6 mg / kg / week to about 9 mg / kg / week.

[0111] Child Health Assessment Questionnaire (CHAQ) The Child Health Assessment Questionnaire (CHAQ) may be administered to assess the health status of children with muscle weakness disorders (e.g., HPP-like disorders) and to generate a CHAQ index score for the child (as described by Bruce & Fries (J. Rheumatol. 30(1):167-178, 2003) and Klepper (Arthritis & Rheumatism, 49:S5-S14, 2003) (the entire questionnaire is incorporated herein by reference). The CHAQ consists of eight categories of questions about grooming, getting up, eating, walking, hygiene, reaching, grasping, and activity, in which a parent or guardian records the degree of difficulty the child with a muscle weakness disorder (e.g., HPP-like disorder) has in performing each activity. The score ranges from 0 to 3 within each category, with a score of 0 indicating no problem; a score of 1 indicating some difficulty; a score of 2 indicating very difficulty; and a score of 3 indicating the child is unable to perform the activity.

[0112] Children with or prone to muscle weakness who have a mean CHAQ index score greater than approximately 0.8 (e.g., approximately 0.8, approximately 1, approximately 1.2, approximately 1.4, approximately 1.6, approximately 1.8, approximately 2.0, approximately 2.2, approximately 2.4, approximately 2.6, approximately 2.8, or approximately 3.0) (e.g., indicating activity of daily living (ADL) and / or pain) may be treated by administering alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa). For example, children with a mean CHAQ index score greater than approximately 0.8 may be treated by administering alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa), for the duration of the subject's life. Furthermore, children with or prone to muscle weakness disorders as disclosed herein may be asked one or more questions in one or more of the eight categories (grooming, getting up, eating, walking, hygiene, reaching, grasping, and activity) to reach a mean CHAQ index score, and if the mean CHAQ index score is greater than approximately 0.8, the child may be treated by administering alkaline phosphatase or a polypeptide having alkaline phosphatase activity, e.g., sALP.

[0113] The CHAQ index scores of children with or prone to muscle weakness disorders disclosed herein may be compared, for example, to the CHAQ index scores of children without such muscle weakness disorders in order to determine the standard deviation of the CHAQ index scores. Furthermore, the CHAQ index scores of children with or prone to muscle weakness disorders disclosed herein may be compared to the CHAQ index scores of other children with or prone to muscle weakness disorders disclosed herein to determine, for example, the standard deviation of the CHAQ index scores.

[0114] The present method may result in improvement of the CHAQ index score (e.g., indicating impairment in ADL and / or pain) in children with or prone to muscle weakness disorders as disclosed herein. For example, treatment with sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa), e.g., treatment with sALP for the lifetime of the child may result in an average reduction of the CHAQ index score to about 0 to about 0.5 or less (e.g., about 0, about 0.1, about 0.2, about 0.4, or about 0.5) in children with HPP-like disorders.

[0115] A decrease in the CHAQ index score in children with or prone to muscle weakness disorders (e.g., HPP-like disorders) may persist throughout the administration of alkaline phosphatase or polypeptides with alkaline phosphatase activity such as sALP (e.g., TNALP, e.g., sALP polypeptide of SEQ ID NO: 1, or polypeptide variants having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa), for example, up to the child's lifetime. Similarly, an increase in activities of daily living (ADL) and / or a decrease in pain in children may persist throughout the administration of sALP (e.g., TNALP, e.g., sALP polypeptide of SEQ ID NO: 1, or polypeptide variants having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa), for example, up to the child's lifetime.

[0116] The effectiveness of treatment with alkaline phosphatase or polypeptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., sALP polypeptide of SEQ ID NO: 1, or polypeptide variants having at least 95% sequence identity with SEQ ID NO: 1, e.g., asfotase alfa), can be evaluated using the CHAQ index score of children with or a tendency toward muscle weakness disorders (e.g., HPP-like disorders). Improvements to specific test scores demonstrate that alkaline phosphatase or polypeptides with alkaline phosphatase activity are effective in treating, for example, activities of daily living (ADL) and pain associated with muscle weakness disorders. In particular, children with or a tendency toward muscle weakness disorders may be asked one or more questions in one or more of the eight categories (grooming, getting up, eating, walking, hygiene, reaching, grasping, and activity) to reach a mean CHAQ index score and evaluate the effectiveness of treatment with sALP administration. For example, if administration of sALP to a child with or prone to muscle weakness results in an average reduction of approximately 0.5 or less in the CHAQ index score, and the child previously had an average CHAQ index score greater than approximately 0.8, then sALP is effective in treating, for example, activities of daily living (ADL) and pain associated with muscle weakness. Alternatively, if administration of sALP does not result in an average reduction of approximately 0.5 or less in the CHAQ index score, the dose and / or frequency of sALP administration may be modified to determine an effective dose of sALP for a child with or prone to muscle weakness. For example, the dose of sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) may be increased, for example, from approximately 3 mg / kg / week to approximately 6 mg / kg / week, or from approximately 6 mg / kg / week to approximately 9 mg / kg / week.

[0117] Pediatric Outcome Data Acquisition System (PODCI) Certain subjects with or prone to muscle weakness disorders (e.g., HPP-like disorders) may be identified for treatment with alkaline phosphatase or an alkaline phosphatase-containing polypeptide such as sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) using a Pediatric Outcome Data Acquisition Device (PODCI). PODCI may be performed to assess the subject's health status and generate a PODCI score (as described in Plint et al. (J. Pediatr. Orthop. 23(6):788-790, 2003)). PODCI consists of eight categories of questions that may be completed by the subject with or prone to muscle weakness disorders (e.g., HPP-like disorders) or by the subject's parent / guardian. Categories that may be used to determine PODCI for individuals with or prone to muscle weakness disorders include: 1) Upper limb and physical function scales to measure the difficulties encountered in performing daily personal care and student activities; 2) Mobility and basic mobility scales to measure the difficulties experienced in performing daily movements and motor activities in daily activities; 3) Sport / physical function scales to measure the difficulties or limitations encountered in participating in more active activities or sports; 4) Pain / comfort scales to measure the level of pain experienced in the past week; 5) Treatment expectancy scales to measure the long-term expectation of treatment; 6) Well-being scales to measure overall satisfaction with the sense of similarity to friends and others of the same age, and with the individual's appearance; 7) Symptom satisfaction scales to measure the individual's acceptance of current limitations if this is a lifelong condition; and 8) Overall function scales, a general combined scale calculated from the first four scales above. For each category, a standardized score was determined for subjects who have or are prone to muscle weakness disorders, and then converted to a 0-100 scale, where 0 represents significant impairment and 100 represents less significant impairment.

[0118] Subjects with or prone to having a muscle weakness disorder (e.g., HPP-like disorder) with an average PODCI score of less than approximately 40 (e.g., approximately 5, 10, 15, 20, 25, 30, 35, or 39) (e.g., indicating impairment in ADL and / or pain) may be treated by administering alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa). For example, subjects with an average PODCI score of less than 40 may be treated by administering sALP for the duration of their lifetime. Furthermore, subjects with or prone to muscle weakness disorders may be asked one or more questions on one or more of the eight scales mentioned above (e.g., mobility and basic mobility, sports / physical function, and pain / comfort scales) in order to reach an average PODCI score, and if the average PODCI score is greater than or less than 40, the subject may be treated by administering sALP.

[0119] The methods described herein may result in an increase in the PODCI score (e.g., indicating impairment in activities of daily living and / or pain) in subjects with or prone to muscle weakness disorders. For example, treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa), for example, treatment with sALP over the lifetime of the subject, may result in an average increase in the PODCI score of about 40 to about 50 (e.g., about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50).

[0120] An increase in the PODCI score may persist throughout the entire duration of administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha), for the lifetime of the subject, for example, a subject with or prone to muscle weakness. Similarly, an increase in ADL and / or a decrease in pain may persist throughout the duration of administration of sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha), for the lifetime of the subject.

[0121] The effectiveness of treatment with alkaline phosphatase or polypeptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., sALP polypeptide of SEQ ID NO: 1, or polypeptide variants with at least 95% sequence identity to SEQ ID NO: 1, e.g., asfotase alfa), can be evaluated using the PODCI score of subjects with or a tendency toward muscle weakness disorders (e.g., HPP-like disorders). Improvements to specific test scores demonstrate that alkaline phosphatase or polypeptides with alkaline phosphatase activity are effective in treating, for example, activities of daily living (ADL) and pain associated with muscle weakness disorders. In particular, subjects with or a tendency toward muscle weakness disorders can be evaluated for treatment effectiveness with sALP administration by taking one or more questions on one or more of the eight scales (upper limb and physical function scale, mobility and basic mobility scale, sports / physical function scale, pain / comfort scale, treatment expectation scale, well-being scale, symptom satisfaction scale, and overall function scale) to reach a mean PODCI score.

[0122] For example, if administration of sALP to a subject with or a tendency toward muscle weakness results in an average increase of approximately 40 or greater than approximately 40 in the PODCI score, and the subject previously had an average PODCI score of less than approximately 40, then sALP is effective in addressing, for example, activities of daily living (ADL) and pain associated with muscle weakness. Alternatively, if administration of sALP does not result in an average increase of approximately 40 or greater than approximately 40 in the PODCI score, the dose and frequency of sALP administration may be modified to determine an effective dose of sALP for subjects with or a tendency toward muscle weakness. For example, the dose of sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) may be increased, for example, from approximately 3 mg / kg / week to approximately 6 mg / kg / week, or from approximately 6 mg / kg / week to approximately 9 mg / kg / week.

[0123] Bailey Scale for Infant and Toddler Development, 3rd Edition (BSID-III) Another endpoint, the Bayley Scales of Infant and Toddler Development, Third Edition (BSID-III), as described in Bayley (2006), Bayley scales of infant and toddler development: administration manual, San Antonio, TX: Harcourt Assessment, may be applied to assess the health status of subjects who have or are prone to having a muscle weakness disorder (e.g., HPP-like disorder) from birth, and to generate a BSID-III score for the subject. The BSID-III includes a set of developmental competition tasks that may be administered to the subject to determine a raw BSID-III score. For example, categories for determining the BSID-III score for a subject with or prone to a muscle weakness disorder (e.g., an infant under approximately 3 years of age with HPP) may include grasping, perceptual-motor integration, motor planning and speed, visual tracking, reaching, object grasping, object manipulation, functional hand skills, response to tactile information, limb and torso movement, static positioning, dynamic movement, balance, and motor planning. Next, the BSID-III measurements can be converted to a scaled BSID-III score, which can then be used to determine the subject's capacity compared to a healthy, age-adjusted subject. The BSID-III scale score for subjects with or prone to muscle weakness disorders (e.g., subjects with HPP-like disorders) may range from 0 to 14, where a score of approximately 7 to 13 is considered the normal range for a healthy subject.

[0124] Subjects with or prone to muscle weakness disorders may be tested as infants (e.g., approximately 3 years of age or under 3 years of age) in one or more of the listed categories (grasp, perceptual-motor integration, motor planning and speed, visual tracking, reaching, object grasping, object manipulation, functional hand skills, response to tactile information, limb and torso movement, static positioning, dynamic movement, balance, and motor planning) to generate a BSID-III score indicating delayed motor development. Infants with or prone to muscle weakness who have a mean BSID-III score of less than approximately 2 in one or more of the listed categories (grasping, perceptual-motor integration, motor planning and speed, visual tracking, reaching, object grasping, object manipulation, functional hand skills, response to tactile information, limb and torso movement, static positioning, dynamic movement, balance, and motor planning) may be treated by administering sALP (TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa). In particular, infants with or prone to muscle weakness who have a mean BSID-III score of less than approximately 2 may be treated by administering sALP for the lifetime of the subject.

[0125] The method may result in an improvement in the mean BSID-III score (e.g., indicating delayed motor development) in subjects with or prone to muscle weakness disorders. For example, treatment with sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa), e.g., sALP treatment over the lifetime of the subject may result in an mean increase of approximately 5 in the BSID-III score (e.g., approximately 5, 6, 7, 8, 9, 10, 11, 12, or 13).

[0126] An increase in BSID-III score may persist for the lifetime of a subject with or a tendency toward muscle weakness during administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity, such as alkaline phosphatase or sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha). Similarly, an increase in motor development may persist for the lifetime of a subject during administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity, such as alkaline phosphatase or sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha).

[0127] The effectiveness of treatment with alkaline phosphatase or polypeptides with alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or polypeptide variants with at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha), can be evaluated using the BSID-III score of subjects with muscle weakness disorders (e.g., HPP-like disorders). Improvements in specific test scores demonstrate that alkaline phosphatase or polypeptides with alkaline phosphatase activity are effective in treating, for example, delayed motor development associated with muscle weakness disorders. In particular, subjects with or prone to muscle weakness disorders may be tested as infants (e.g., approximately 3 years or younger with HPP) in one or more of the listed categories (grasp, perceptual-motor integration, motor planning and speed, visual tracking, reaching, object grasping, object manipulation, functional hand skills, response to tactile information, limb and torso movement, static positioning, dynamic movement, balance, and motor planning) to evaluate the therapeutic effectiveness of sALP administration once they reach a mean BSID-III score.

[0128] For example, if administration of sALP to a child with or prone to muscle weakness disorder results in an average increase of approximately 5 in the BSID-III scale score, and the child previously had an average BSID-III scale score of less than approximately 2 as an infant (e.g., at approximately 3 years of age or under 3 years of age), then sALP is effective in treating, for example, delayed motor development associated with HPP-like disorders. Alternatively, if administration of sALP does not result in an average increase of approximately 5 in the BSID-III scale score, the dose and / or frequency of sALP administration may be modified to determine an effective dose of sALP for children with or prone to muscle weakness disorder. For example, the dose of sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) may be increased, for example, from approximately 3 mg / kg / week to approximately 6 mg / kg / week, or from approximately 6 mg / kg / week to approximately 9 mg / kg / week.

[0129] Peabody Developmental Motor Scale, 2nd Edition (PDMS-2) Another endpoint, the Peabody Developmental Motor Scale, Second Edition (PDMS-2), may be administered to assess the health status of subjects who have or are prone to having a muscle weakness disorder (e.g., HPP-like disorder) from birth, and generate a PDMS-2 score for the subject (as described by van Hartingsveldt et al. (Occup.Ther.Int. 12(1):1-13, 2005)). The PDMS-2 includes six sub-tests to measure the motor skills of subjects, such as those with HPP.

[0130] In particular, PDMS-2 measurements can be determined from the following subtests: 1) Spontaneous movement subtest, which measures the subject's ability to move from one place to another (measurements include crawling, walking, running, hopping, and jumping forward); 2) Reflex subtest, which measures the subject's ability to react automatically to environmental events; 3) Static subtest, which measures the subject's ability to maintain body control within the center of gravity and maintain balance; 4) Object manipulation subtest, which measures the subject's ability to manipulate objects, such as catching, throwing, and kicking a ball; 5) Grasping subtest, which measures the subject's ability to use their hands, such as the ability to hold an object with one hand and actions involving controlled use of the fingers of both hands; and 6) Visual-motor integration subtest, which measures the subject's ability to use their visual perception skills to perform complex eye-hand coordination tasks, such as picking up and grasping objects, building with blocks, and copying designs. PDMS-2 measurements can be determined for subjects who have or are prone to muscle weakness disorders (e.g., HPP-like disorders) in one or more of these categories, and then converted to a PDMS-2 score, such as a PDMS-2 exercise standard score ranging from 0 to 13, while the range for healthy subjects (e.g., subjects without muscle weakness disorders) is approximately 7 to 13.

[0131] Subjects with a mean PDMS score (e.g., indicating delayed motor development) and who have or are prone to muscle weakness disorders may be treated by administering sALP (TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa).

[0132] The methods described herein may result in an improvement in the PDMS-2 score (e.g., an indication of delayed motor development) in subjects with or prone to muscle weakness disorders. For example, treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha) may result in an average increase of about 7 to about 13 (e.g., about 7, about 8, about 9, about 10, about 11, about 12, or about 13) in the PDMS-2 score.

[0133] An increase in PDMS-2 score may persist for a long period, for example, up to the lifetime of a subject with muscle weakness disorder, during the administration of alkaline phosphatase or a polypeptide with alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha). Similarly, an increase in motor development may persist for a lifetime of a subject with muscle weakness disorder, or a subject with muscle weakness disorder, during the administration of sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha).

[0134] The effectiveness of treatment with alkaline phosphatase or a polypeptide with alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa), can be evaluated using the PDMS-2 score of subjects with muscle weakness disorders (e.g., HPP-like disorders). Improvements to specific test scores demonstrate that alkaline phosphatase or a polypeptide with alkaline phosphatase activity is effective in treating, for example, delayed motor development associated with muscle weakness disorders. For example, children with muscle weakness disorders or those with a tendency towards them may be tested at approximately 5 years of age or under 5 years of age in one or more of the described categories (spontaneous movement, reflexes, stillness, object manipulation, grasping, and visuomotor function) to reach a mean PDMS-2 score and to evaluate the effectiveness of treatment with sALP.

[0135] For example, if administration of sALP to a child with or prone to muscle weakness disorder results in an average increase of approximately 7 in the PDMS-2 standard score, and the child previously had an average PDMS-2 standard score of approximately 5, then sALP is effective in treating, for example, delayed motor development associated with HPP-like disorders. Alternatively, if administration of sALP does not result in an average increase of approximately 7 in the PDMS-2 standard score, the dose and / or frequency of sALP administration may be modified to determine an effective dose of sALP for the child. For example, the dose of sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) may be increased, for example, from approximately 3 mg / kg / week to approximately 6 mg / kg / week, or from approximately 6 mg / kg / week to approximately 9 mg / kg / week.

[0136] 6-minute walk test (6MWT) Subjects with muscle weakness disorders may be identified using the 6MWT for treatment with alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa). In particular, the 6MWT may be used to assess walking ability in adults with muscle weakness disorders and to generate adult 6MWT values. The 6MWT may be conducted indoors or outdoors using a flat, straight, enclosed walkway with a hard surface (e.g., approximately 30 meters in length). Time may be tracked using a stopwatch or other timer, and the distance walked by the subject with muscle weakness disorders (e.g., in meters) may be determined using a mechanical counter or other device. For example, the length of the walkway may be marked every 3 meters, with a 30-meter turning point and a starting line also marked, to determine the number of meters walked by the subject with muscle weakness disorders. Next, the distance walked by a subject with muscle weakness in 6 minutes can be compared to the predicted number of meters walked by, for example, a normal subject of approximately the same age, sex, and / or height, and expressed as a percentage to generate the subject's 6MWT value. The 6MWT value of a subject with muscle weakness can be compared to the subject's baseline 6MWT value. Furthermore, the 6MWT value of a subject with muscle weakness can be compared to the 6MWT value of a normal subject.

[0137] Subjects with muscle weakness disorders whose mean 6MWT is less than approximately 80% of the predicted 6MWT value (e.g., compared to normal subjects of similar age, same sex, and / or same height) should be given alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alpha) for at least two weeks (e.g., Treatment may be provided by administering the drug for a treatment period of at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years, or over the lifetime of the subject, in particular for at least 6 weeks. For example, subjects with muscle weakness disease having an average 6MWT of less than approximately 80% of the predicted 6MWT value (e.g., approximately 50%, 55%, 60%, 65%, 70%, or 75% of the predicted 6MWT value) may be treated with alkaline phosphatase or a polypeptide having alkaline phosphatase activity for at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years, or over the lifetime of the subject, in particular, at least six weeks).

[0138] This method may improve 6MWT values ​​in subjects with muscle weakness disorders. For example, treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa), for example, for at least 2 weeks (e.g., at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, or less) Treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity for a treatment period of at least 8 months, at least 9 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years, or throughout the lifetime of the subject, in particular at least 6 weeks, may result in an average increase in the 6MWT value of the subject of about 80% or more of the predicted 6MWT value (e.g., about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or more of the predicted 6MWT value).

[0139] An increase in 6MWT values ​​in subjects with muscle weakness disorders may persist for at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least nine months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least seven years, at least eight years, at least nine years, or at least ten years of treatment, or throughout the lifetime of the subject, in particular at least six weeks) during the administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, at least TNALP, at least SEQ ID NO: 1 sALP polypeptide, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) for a treatment period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years). For example, 6MWT values ​​increased to over 80% of the predicted 6MWT value in subjects with muscle weakness during treatment with alkaline phosphatase or polypeptides with alkaline phosphatase activity, and remained within ±10% of the increased 6MWT value.

[0140] Similarly, improvements in walking ability in subjects with muscle weakness may persist during the administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity, for example, for at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years of treatment, or throughout the subject's lifetime, in particular, at least six weeks). For example, subjects with muscle weakness may show reduced reliance on assistive mobility devices such as walkers, wheelchairs, braces, crutches, or orthopedic devices during treatment with sALP.

[0141] Alternatively, if administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa) does not result in a mean increase in 6MWT values ​​to more than 80% of the predicted 6MWT value (e.g., for normal subjects of approximately the same age, sex, and / or height), the dose and / or frequency of administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity may be modified to determine an effective dose of alkaline phosphatase or a polypeptide having alkaline phosphatase activity for subjects with muscle weakness disorders. For example, the dose of alkaline phosphatase or a polypeptide having alkaline phosphatase activity may be increased, for example, from about 2.1 mg / kg / week or about 3.5 mg / kg / week to about 6 mg / kg / week or about 9 mg / kg / week.

[0142] Handheld Dynamometer (HHD) The grip strength and muscle strength of subjects with or prone to muscle weakness disorders can be assessed using a handheld dynamometer (HHD). For example, knee flexion and extension, as well as hip flexion, extension, and abduction, can be measured using, for example, a MICROFET2® dynamometer, while the subject's grip strength can be measured using, for example, a Jamar Grip dynamometer. Specifically, the administrator holds the dynamometer stationary, and the subject applies maximum force to the dynamometer. Peak force data is collected in pounds and then converted to Newtons (N). The torque value is then calculated using the limb length in N meters. The torque value can then be compared to, for example, the torque value of a normal subject of approximately the same age, sex, and / or height, and expressed as a percentage to generate the subject's HHD value.

[0143] Subjects with muscle weakness disorders whose mean HHD value is less than approximately 80% of their predicted HHD value (e.g., compared to normal subjects of similar age, sex, and / or height) should be given alkaline phosphatase or a polypeptide with alkaline phosphatase activity (e.g., sALP, e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alpha) for at least two weeks. (For example, treatment may be administered for a treatment period of at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years, or over the lifetime of the subject, in particular for at least 6 weeks). For example, subjects with muscle weakness disease having an average HHD of less than approximately 80% of the predicted HHD value (e.g., approximately 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the predicted HHD value) may be treated with alkaline phosphatase or a polypeptide having alkaline phosphatase activity for at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, or at least ten years, or over the lifetime of the subject, in particular, at least six weeks).

[0144] This method may improve HHD levels in subjects with muscle weakness disorders. For example, treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha), for example, for at least 2 weeks (e.g., at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months) Treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity for a treatment period of months, at least 8 months, at least 9 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years, or throughout the lifetime of the subject, in particular at least 6 weeks, may result in an average increase in HHD value of up to about 80% or more of the subject's predicted HHD value (e.g., about 83%, 85%, 87%, 90%, 93%, 95%, 97%, or about 100%, or about 100% of the predicted HHD value).

[0145] An increase in HHD levels in subjects with muscle weakness disorders may persist for at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least nine months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least seven years, at least eight years, at least nine years, or at least ten years of treatment, or throughout the lifetime of the subject, in particular at least six weeks) during the administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, at least TNALP, at least SEQ ID NO: 1 sALP polypeptide, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) for a treatment period of at least two weeks (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven months, at least eight months, at least nine months, at least one year, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years). For example, during treatment with alkaline phosphatase or a polypeptide with alkaline phosphatase activity, HHD levels in subjects with muscle weakness disorders increased to over 80% of their predicted HHD levels, and remained within ±10% of the increased HHD level.

[0146] Alternatively, if administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) does not result in an average increase in HHD values ​​of more than 80% of the predicted HHD value (e.g., in subjects with muscle weakness disease of approximately the same age, sex, and / or height), the dose and / or frequency of administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity may be modified to determine an effective dose of alkaline phosphatase or a polypeptide having alkaline phosphatase activity for subjects with muscle weakness disease. For example, the dose of alkaline phosphatase or a polypeptide having alkaline phosphatase activity may be increased, for example, from about 2.1 mg / kg / week or about 3.5 mg / kg / week to about 6 mg / kg / week or about 9 mg / kg / week.

[0147] Alkaline phosphatase Asfotase alfa is a human TNALP (hTNALP; SEQ ID NO: 1) fusion protein formulated for the treatment of HPP. In particular, asfotase alfa (SEQ ID NO: 1) can be effectively used to treat hypophosphatasia (HPP), its symptoms, and associated physical impairments in subjects who have or are prone to having muscle weakness disorders over a long period of time (e.g., at least 1 day, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, or more than 10 years (e.g., the lifetime of the subject)).

[0148] In light of the results described herein, this disclosure is not limited to specific alkaline phosphatases (ALPs) or nucleic acid sequences encoding ALPs. Alkaline phosphatases encompass a group of enzymes that catalyze the cleavage of phosphate moieties (e.g., pyrophosphate, PP). i (Hydrolysis of) There are four known mammalian alkaline phosphatase (ALP) isozymes: tissue-nonspecific alkaline phosphatase (TNALP; further described below), placental alkaline phosphatase (PLALP) (e.g., accession numbers P05187, NP_112603, and NP_001623), germ cell alkaline phosphatase (GALP) (e.g., accession number P10696), and intestinal alkaline phosphatase (IALP) (e.g., accession numbers P09923, and NP_001622). In addition to the exemplary ALPs discussed above, the disclosure also provides any polypeptide having the same or similar catalytic site structure and / or enzymatic activity as ALP for treating subjects having or prone to muscle weakness disorders. Bone delivery conjugates, including sALP, are further described in PCT Publication WO2005 / 103263 and WO2008 / 138131.

[0149] Examples of TNALPs that may be used in accordance with the methods described herein include human TNALP (accession numbers NP_000469, AAI10910, AAH90861, AAH66116, AAH21289, and AAI26166); rhesus monkey TNALP (accession number XP_01109717); rat TNALP (accession number NP_037191); dog TNALP (accession number AAF64516); pig TNALP (accession number AAN64273), mouse (accession number NP_031457), bovine TNALP (accession numbers NP_789828, NP_776412, AAM8209, and AAC33858), and cat TNALP (accession number NP_001036028). In particular, TNALP may be recombinant human TNALP (e.g., SEQ ID NO: 1, asfotase alfa, see U.S. Patents 7,763,712 and 7,960,529, which are incorporated herein by reference in their entirety) used to treat subjects with or prone to muscle weakness disorders. TNALP may also exhibit at least about 95% sequence identity to the TNALP polypeptide or nucleic acid sequence described above.

[0150] Soluble alkaline phosphatase The ALP of the present invention comprises a soluble (e.g., extracellular or non-membrane-bound) form of any of the alkaline phosphatases described herein. sALP may be, for example, a soluble form of human tissue-nonspecific alkaline phosphatase (human TNALP (hTNALP)). This disclosure is not limited to specific sALPs and may include any sALP polypeptide that is physiologically active against, for example, phosphoethanolamine (PEA), inorganic pyrophosphate (PPi), and pyridoxal 5'-phosphate (PLP). In particular, the sALPs of the present invention are catalytically suitable for improving skeletal calcification in bone. This disclosure further includes nucleic acids encoding the sALPs described herein that may be used to treat the muscle weakness conditions described herein, including, for example, HPP-like diseases, CPPD, and familial hypophosphatemia (autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets, X-linked hypophosphatemic rickets, X-linked hypophosphatemia (XLH), etc.).

[0151] TNALP is a membrane-bound protein (TNALP) fixed at its C-terminus by a glycolipid moiety (Swiss-Prot, P05186). This glycolipid anchor (GPI) is added post-translation after removal of the hydrophobic C-terminus and serves both as a transient membrane anchor and as a signal for GPI addition. The GPI anchor is located at the cell membrane, while the rest of TNALP is extracellular. In particular, TNALP (e.g., human TNALP (hTNALP)) can be engineered to replace the first amino acid (alanine) of the hydrophobic C-terminal sequence with a stop codon, thereby producing engineered hTNALP that contains all the amino acid residues of the native anchor type of TNALP but lacks the GPI membrane anchor. Those skilled in the art will understand that the position of the GPI membrane anchor varies in different ALPs, and may include, for example, the last 10, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 45, 50, or more amino acid residues on the C-terminus of the polypeptide. Recombinant sTNALP may include, for example, amino acids 1-502 (18-502 if secreted), amino acids 1-501 (18-501 if secreted), amino acids 1-504 (18-504 if secreted), amino acids 1-505 (18-505 if secreted), or amino acids 1-502. Thus, the C-terminus of natural ALPs may be cleaved by specific amino acids without affecting ALP activity.

[0152] In addition to the C-terminal GPI anchor, TNALP also possesses an N-terminal signal peptide sequence. The N-terminal signal peptide is present on the synthetic protein when synthesized but is cleaved from TNALP after translocation to the ER. sALP includes both secreted (i.e., lacking an N-terminal signal) and non-secreted (i.e., possessing an N-terminal signal) forms. Those skilled in the art will understand that the position of the N-terminal signal peptide varies in different alkaline phosphatases and may include, for example, the first 5, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 30 or more amino acid residues on the N-terminus of the polypeptide. Those skilled in the art will know that the location of signal sequence cleavage sites can be predicted by a suitable computer algorithm, such as that described in Bendtsen et al. (J.Mol.Biol.340(4):783-795,2004), which is available on the web at www.cbs.dtu.dk / services / SignalP / .

[0153] The present invention also includes sALP consensus sequences derived from the extracellular domains of ALP isozymes (e.g., TNALP, PALP, GCALP, IALP, etc.). Therefore, similar to sTNALP discussed above, the disclosure also provides other soluble human ALP isozymes, i.e., those that do not have a peptide signal and preferably include the extracellular domain of ALP. sALP also includes polypeptide sequences that satisfy consensus sequences derived from human ALP isozymes and the extracellular domains of mammalian TNALP orthologs (human, mouse, rat, bovine, cat, and dog), or consensus sequences derived from the extracellular domains of mammalian TNALP orthologs (human, mouse, rat, bovine, cat, and dog). sALP also includes sequences that satisfy similar consensus sequences derived from various combinations of these TNALP orthologs or human ALP isozymes. Such consensus sequences are shown, for example, in International Publication No. 2008 / 138131.

[0154] The sALP of the present invention may include not only the wild-type sequence of sALP described above, but also any polypeptide having at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with these alkaline phosphatases (e.g., SEQ ID NOs: 1-24, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alpha). Examples of mutations that can be introduced into the ALP sequence are described in U.S. Patent Application Publication No. 2013 / 0323244, which is incorporated herein by reference in its entirety. sALP can be optionally glycosylated at any one or more suitable amino acid residues. Furthermore, sALP may have at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with any of the sALPs described herein (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alpha). sALP may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additions, deletions, or substitutions to any of the sALPs described herein (TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha).

[0155] sALP-fusion polypeptide Either the sALP and the linker described herein may be combined in a sALP polypeptide, e.g., an A-sALP-B sALP polypeptide, where A and B are either absent or an amino acid sequence of at least one amino acid (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha). If present, A and / or B may be any linker described herein. In some sALP polypeptides, A is absent, B is absent, or both A and B are absent. The sALP polypeptide may optionally contain an Fc region to provide an sALP fusion polypeptide as described herein. The sALP polypeptide may optionally contain a bone-targeting moiety as described herein. In some sALP polypeptides, a linker, such as a flexible linker, may be present between the sALP and a bone-targeting moiety, such as a dipeptide sequence (e.g., leucine-lysine or aspartate-isoleucine). Further exemplary Fc regions, linkers, and bone-targeting moieties are described below.

[0156] Any of the sALP, linker, and Fc regions described herein are fusion polypeptides, for example, structure Z-sALP-Y-spacer-XW n -V, ZW n -X-Spacer-Y-sALP-V, Z-sALP-YW n -X-Spacer-V, and ZW n -X-sALP-Y-spacer-V can be combined in recombinant fusion polypeptides (TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or polypeptide variants having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha). In particular, the structure Z-sALP-Y-spacer-XW n -V, or ZW n-X-spacer-Y-sALP-V may be the full length of ALP, such as the soluble extracellular domain of ALP, or a functional fragment of ALP, as described herein (e.g., TNALP, PALP, GCALP, and IALP). One of X, Y, Z, and V and / or the spacer may be absent or an amino acid sequence of at least one amino acid. n The bone-targeting moiety, if present, can be, for example, a series of consecutive Asp or Glu residues, where n=1 to 50, for example, n=3 to 30, for example, 5 to 15, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. The bone-targeting moiety, if present, can be located anywhere in the fusion polypeptide, for example, at the N-terminus or C-terminus, or in its vicinity, and / or in the linker region. For example, the bone-targeting moiety is at the C-terminus. sALP polypeptides and fusion polypeptides may also lack a bone-targeting moiety.

[0157] The sALP fusion polypeptide has the structure hTNALP-Fc-D 10 It may have the following characteristics. In particular, the sALP fusion polypeptide may contain the amino acid sequence of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as asfotase alpha.

[0158] sALP fusion polypeptides may have a negatively charged polypeptide containing 1 to 50 (e.g., 6 to 10, 10 to 15, 10 to 16, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 19, 20, 25, 30, 35, 40, 45, or 50) negatively charged amino acids, such as aspartic acid or glutamic acid residues. The negatively charged peptide is D 10 , D 16 , E 10 , and E 16It consists of or may include at least one of the following.

[0159] Useful spacers include, but are not limited to, polypeptides containing Fc, as well as terminally highly negatively charged peptides (e.g., W nExamples include hydrophilic and flexible polypeptides that can mitigate the repulsive force caused by the presence of ). For example, sALP may be a fusion polypeptide having an Fc region of immunoglobulin in its N-terminal or C-terminal domain. Immunoglobulin molecules have a structure well known in the art. It consists of two light chains (each about 23 kD) and two heavy chains (each about 50-70 kD) linked by interchain disulfide bonds. Immunoglobulins are readily cleaved proteolytically (e.g., by papain cleavage) to obtain Fab (containing the light chain and the VH and CH1 domains of the heavy chain) and Fc (containing the CH2 and CH3 domains of the heavy chain, along with adjacent sequences). The useful Fc fragments described herein include Fc fragments of any immunoglobulin molecule, comprising IgG, IgM, IgA, IgD, or IgE, and their various subclasses (e.g., IgG-1, IgG-2, IgG-3, IgG-4, IgA-1, IgA-2), derived from any mammal (e.g., human). For example, the Fc fragment is human IgG-1. The Fc fragment may comprise, for example, the CH2 and CH3 domains of the heavy chain, as well as any portion of the hinge region. The Fc region may be optionally glycosylated with one or more suitable amino acid residues known to those skilled in the art. In particular, the Fc fragment of the fusion polypeptide has the amino acid sequence of SEQ ID NO: 20 or has at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 20. Manipulated, for example, non-naturally occurring Fc regions may also be used (see, for example, International Patent Application Publication WO2005 / 007809, incorporated herein by reference). The Fc fragments described herein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, or more additions, deletions, or substitutions compared to any of the Fc fragments described herein.

[0160] The sALP fusion polypeptides described herein (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or polypeptide variants having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha) may include a peptide linker region between the Fc fragments. Furthermore, the peptide linker region may be located between the Fc fragments and an optional bone targeting moiety. The linker region may have any sequence and length that allows sALP to remain biologically active, for example, without steric hindrance. Exemplary linker lengths are 1 to 200 amino acid residues, for example, 1 to 5, 6 to 10, 11 to 15, 16 to 20, 21 to 25, 26 to 30, 31 to 35, 36 to 40, 41 to 45, 46 to 50, 51 to 55, 56 to 60, 61 to 65, 66 to 70, 71 to 75, 76 to 80, 81 to 85, 86 to 90, 91 to 95, 96 to 100, 101 to 110, 111 to 120, 121 to 130, 131 to 140, 141 to 150, 151 to 160, 161 to 170, 171 to 180, 181 to 190, or 191 to 200 amino acid residues. For example, a linker may include or consist of a mobile portion, such as a region that does not have a significant fixed secondary or tertiary structure. An exemplary flexible linker is a glycine-rich linker containing, for example, at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% glycine residues. The linker may also contain, for example, serine residues. In some cases, the amino acid sequence of the linker consists only of glycine and serine residues. The linker may be optionally glycosylated with any one or more suitable amino acid residues. Furthermore, the linkers described herein may include any other sequences or portions linked by covalent or non-covalent bonds. The linker may also be absent, in which case the Fc fragment and sALP are fused together directly without intervening residues. Certain Fc-sALPs, or sALP-Fc fusion polypeptides, may be considered, according to this disclosure, to either 1) lack a linker, or 2) have a linker corresponding to a portion of the sALP.For example, an Fc directly fused to hsTNALP(1-502) may be considered to have no linker if hsTNALP is amino acid 1-502, or it may be considered to have a 17-amino acid linker if hsTNALP(18-502).

[0161] sALP and sALP fusion polypeptides (TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or polypeptide variants having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha) can associate into dimers or tetramers. For example, two sALP-Fc monomers can be covalently bonded via two disulfide bonds located in the hinge region of the Fc fragment. Furthermore, polypeptides or fusion polypeptides (e.g., sALP polypeptides or fusion polypeptides) can be glycosylated or PEGylated.

[0162] Production of nucleic acids and polypeptides Nucleic acids encoding sALP and sALP fusion polypeptides (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha) can be produced by any method known in the art. Typically, the nucleic acid encoding the desired fusion polypeptide is produced using molecular cloning methods and generally placed in a vector, such as a plasmid or virus. The vector is used to transform the nucleic acid into a host cell suitable for the expression of the fusion polypeptide. A typical method is disclosed, for example, by Maniatis et al. (Cold Springs Harbor Laboratory, 1989). Many cell types can be used as suitable host cells, but mammalian cells are preferred because they can confer appropriate post-translational modifications. Host cells of the present invention may include, for example, Chinese hamster ovary (CHO) cells, L cells, C127 cells, 3T3 cells, BHK cells, COS-7 cells, or any other suitable host cells known in the art. For example, the host cells are Chinese hamster ovary (CHO) cells (e.g., CHO-DG44 cells).

[0163] sALP and sALP fusion polypeptides (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or polypeptide variants having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha) can be produced under any conditions suitable for inducing sALP polypeptide expression in host cells. Such conditions include a suitable selection of a medium prepared with components such as buffers, bicarbonates and / or HEPES, chlorides, phosphates, ions such as calcium, sodium, potassium, magnesium, iron, carbon sources such as monosaccharides, amino acids, potentially lipids, nucleotides, vitamins, and growth factors such as insulin, supplemented with 2–4 mM L-glutamine and 5% fetal bovine serum, such as standard commercial media like alpha-MEM, DMEM, Ham's-F12, and IMDM; and 2–4 mM L-glutamine supplemented with Hyclone® SFM4CHO, Sigma CHO DHFR. - There are commercially available animal protein-free culture media, such as Cambrex POWER® CHO CD. These media are preferably prepared without thymidine, hypoxanthine, and L-glycine to maintain selective pressure and enable stable protein product expression.

[0164] Pharmaceutical compositions, formulations, and medications Compositions of the present disclosure containing sALP or sALP fusion polypeptides (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or polypeptide variants having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) can be formulated by various methods known in the art. As will be understood by those skilled in the art, the formulation can be determined by the route of administration and / or form of administration. The route of administration may depend on various factors, such as the environment and the therapeutic purpose. In particular, the polypeptides and fusion polypeptides described herein may be formulated for administration by any route known in the art, e.g., subcutaneous (e.g., by subcutaneous injection), intravenous, oral, nasal, intramuscular, sublingual, intrathecal, or intradermal. For example, pharmaceutical compositions may be in the form of liquids, solutions, suspensions, pills, capsules, tablets, gel caps, powders, gels, ointments, creams, nebulas, mists, atomized vapors, aerosols, or phytosomes.

[0165] formulation Compositions comprising sALP and sALP fusion polypeptides (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) can be formulated by standard methods. Pharmaceutical formulations are an established technology, for example, Gennaro (2000) "Remington: The Science and Practice of Pharmacy" 20 th Edition, Lippincott, Williams & Wilkins (ISBN:0683306472); Ansel et al. (1999) “Pharmaceutical Dosage Forms and Drug Delivery Systems” 7 thEdition, Lippincott Williams & Wilkins Publishers (ISBN:0683305727); and Kibbe (2000) “Handbook of Pharmaceutical Excipients American Pharmaceutical Association”, 3 rd Further details are provided in Edition (ISBN: 091733096X). For example, an sALP composition (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha) may be formulated, for example, as a buffer solution suitable for storage at 2-8°C (e.g., 4°C) at a suitable concentration. The composition may also be formulated for storage at temperatures below 0°C (e.g., -20°C or -80°C). In some embodiments, the composition may be further formulated for storage at 2-8°C (e.g., 4°C) for up to 2 years (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, or 2 years). Thus, in some embodiments, the compositions described herein may be stable for storage at 2-8°C (e.g., 4°C) for at least 1 year.

[0166] Compositions comprising sALP and sALP-fusion polypeptides (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or polypeptide variants having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha) can be in various forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and injectable solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends in part on the intended mode of administration and therapeutic application.

[0167] Compositions containing a composition intended for systemic or local delivery may, for example, be in the form of an injectable or injectable solution. Therefore, a composition (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa) may be formulated for parenteral administration (e.g., subcutaneous, intravenous, intraperitoneal, or intramuscular injection). As used herein, “parenteral administration,” “administered parenterally,” and other grammatically equivalent terms refer to, but are not limited to, methods of administration other than intra-intestinal and local administration, usually by injection, including, but are not limited to, intravenous, intranasal, intraocular, pulmonary, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, carotid, and intrasternal injections and infusions.

[0168] Compositions comprising sALP and sALP-fusion polypeptides (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or polypeptide variants having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha) can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for stable storage at high concentrations. Sterile injection solutions can be prepared by incorporating the required amount of the compositions described herein into a suitable solvent, along with one or a combination of the components listed above, as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the compositions described herein into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. For sterile powders for the preparation of sterile injection solutions, the preparation method includes vacuum drying and lyophilization, obtaining the powder of the compositions described herein, along with any additional desired components (see below), from its pre-sterilized filtered solution. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of a surfactant. Sustained adsorption of the injectable composition can be achieved by including absorption-delaying agents, such as monostearate and gelatin, in the composition.

[0169] The compositions described herein can also be formulated in immunoliposome compositions. Such formulations can be prepared by methods known to those skilled in the art, such as those described in Epstein et al. (1985) Proc Natl Acad Sci USA 82:3688; Hwang et al. (1980) Proc Natl Acad Sci USA 77:4030, and U.S. Patents 4,485,045 and 4,544,545. Liposomes with extended circulation time are disclosed, for example, in U.S. Patent 5,013,556.

[0170] Compositions comprising sALP and sALP fusion polypeptides (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or polypeptide variants having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha) can also be formulated with a carrier that protects the composition (e.g., sALP polypeptide or sALP fusion polypeptide) from rapid release, such as in controlled-release formulations including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Various methods for preparing such formulations are well known in the art. See, for example, JR Robinson (1978) "Sustained and Controlled Release Drug Delivery Systems," Marcel Dekker, Inc., New York.

[0171] A composition comprising sALP or sALP fusion polypeptide (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha) can be formulated as a clear, colorless to slightly yellow aqueous solution for injection, pH 7.4. sALP or sALP polypeptide (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha) can be formulated at concentrations of 12 mg / 0.3 mL, 18 mg / 0.45 mL, 28 mg / 0.7 mL, 40 mg / 1 mL, or 80 mg / 0.8 mL. sALP, or sALP polypeptide (formulated at concentrations of, for example, 12 mg / 0.3 mL, 18 mg / 0.45 mL, 28 mg / 0.7 mL, 40 mg / 1 mL, or 80 mg / 0.8 mL), may be formulated with a carrier such as sodium chloride and / or sodium phosphate (e.g., particularly about 150 mM NaCl and / or about 25 mM sodium phosphate at pH about 7.4). In particular, the composition can be formulated as a 40 mg / mL solution for injection containing 40 mg of sALP or sALP polypeptide per 1 mL of solution (for example, each vial may contain 0.3 mL of solution and 12 mg of sALP (40 mg / mL), each vial may contain 0.45 mL of solution and 18 mg of sALP (40 mg / mL), each vial may contain 0.7 mL of solution and 28 mg of sALP (40 mg / mL), or each vial may contain 1.0 mL of solution and 40 mg of asfotase alfa (40 mg / mL)). sALP, or sALP polypeptide (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa), may be formulated as an injectable solution at a concentration of 100 mg / mL, with each 1 mL solution containing 100 mg of sALP, or sALP polypeptide (e.g., each vial contains 0.8 mL of solution and 80 mg of asfotase alfa (100 mg / mL)).

[0172] When a composition is used in combination with a second active agent, the composition may be formulated together with the second agent, or it may be formulated separately from the second drug formulation. For example, each pharmaceutical composition may be mixed and administered together, for example, immediately before administration, or administered separately, for example, simultaneously or at different times.

[0173] Compositions comprising sALP and sALP fusion polypeptides (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) may be formulated for administration to a subject in conjunction with intravenous gamma globulin therapy (IVIG), plasma exchange therapy, plasma replacement, or plasma exchange, or, when administered to a fetus, for administration to a woman pregnant with such a fetus.

[0174] Carrier / vehicle Preparations containing sALP or sALP-fusion polypeptides (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or polypeptide variants having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha) may be provided to subjects with or prone to muscle weakness disorders in combination with pharmaceutically acceptable sterile aqueous or non-aqueous solvents, suspensions, or emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils, fish oils, and injectable organic esters. Aqueous carriers include water, water-alcohol solutions, emulsions, or suspensions, and include physiological saline and buffered parenteral medical vehicles (including sodium chloride solution, Ringer's dextrose solution, dextrose + sodium chloride solution, Ringer's solution containing lactose, or non-volatile oils). For example, a pharmaceutically acceptable carrier may include sodium chloride and / or sodium phosphate, and the composition may include, for example, about 150 mM sodium chloride and / or about 25 mM sodium phosphate, pH 7.4.

[0175] Intravenous vehicles may contain liquids and nutritional and electrolyte supplements, such as those based on ringer's dextrose. They may also contain pharmaceutically acceptable salts, such as mineral salts like hydrochloride, hydrobromide, phosphate, and sulfate; and salts of organic acids such as acetate, propionate, malonate, and benzoate. Furthermore, auxiliary substances, such as wetting agents or emulsifiers and pH buffers, may be present in such vehicles. A detailed discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0176] dose The sALP polypeptides described herein (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or polypeptide variants having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alpha) and / or the fusion proteins of SEQ ID NO: 21 or SEQ ID NO: 22, or variants having at least 95% sequence identity to the sequence of SEQ ID NO: 21 or SEQ ID NO: 22, are, for example, 0.01 mg / kg to 500 mg / kg (e.g., 0.05 mg / kg to 500 mg / kg, 0.1 mg / kg to 20 mg / kg, 5 mg / kg to 500 mg / kg, 0.1 mg / kg to 100 mg / kg, 10 mg / kg to 100 mg / kg, 0.1 mg / kg to 50 mg / kg, 0.5 mg / kg to 25 mg) g / kg, 1.0 mg / kg to 10 mg / kg, 1.5 mg / kg to 5 mg / kg, or 2.0 mg / kg to 3.0 mg / kg), or 1 μg / kg to 1,000 μg / kg (e.g., 5 μg / kg~1,000μg / kg, 1μg / kg~750μg / kg, 5μg / kg~750μg / kg, 10μg / kg~750μg / kg, 1μg / kg~500μg / kg, 5μg Individual doses within the range of 1 / kg~500μg / kg, 10μg / kg~500μg / kg, 1μg / kg~100μg / kg, 5μg / kg~100μg / kg, 10μg / kg~100μg / kg, 1μg / kg~50μg / kg, 5μg / kg~50μg / kg, or 10μg / kg~50μg / kg may be administered to subjects with or prone to muscle weakness disorders.

[0177] Examples of sALP doses include, for example, 0.01, 0.05, 0.1, 0.5, 1, 2, 2.5, 5, 10, 20, 25, 50, 100, 125, 150, 200, 250, or 500 mg / kg; or 1, 2, 2.5, 5, 10, 20, 25, 50, 100, 125, 150, 200, 250, 500, 750, 900, or 1,000 μg / kg. For all doses or ranges described herein, the term "about" may be used to change these doses by ±10% of the stated value or the endpoint of the range. In particular, the compositions according to this disclosure (e.g., including sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa)) may be administered to subjects in doses ranging from about 0.001 mg / kg / day to about 500 mg / kg / day, about 0.01 mg / kg / day to about 100 mg / kg / day, or about 0.01 mg / kg / day to about 20 mg / kg / day. For example, a sALP composition (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) may be administered to a subject in weekly doses ranging from, for example, about 0.5 mg / kg / week to about 140 mg / kg / week, e.g., about 0.8 mg / kg / week to about 50 mg / kg / week, or about 1 mg / kg / week to about 10 mg / kg / week (e.g., about 6 or about 9 mg / kg / week). In particular, sALP (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) may be administered in doses of 2 mg / kg three times per week (total dose 6 mg / kg / week), 1 mg / kg six times per week (total dose 6 mg / kg / week), 3 mg / kg three times per week (total dose 9 mg / kg / week), 0.5 mg / kg three times per week (total dose 1.5 mg / kg / week), or 9.3 mg / kg three times per week (total dose 28 mg / kg / week). The dose is adapted by the clinician according to conventional factors such as the severity of the disease and different parameters from subjects who have or are prone to muscle weakness.

[0178] The dosage of a composition comprising sALP and sALP fusion polypeptide (TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa), and / or the fusion protein of SEQ ID NO: 21 or SEQ ID NO: 22, or a variant having at least 95% sequence identity to the sequence of SEQ ID NO: 21 or SEQ ID NO: 22, may be provided in a single-dose or multi-dose regimen. The dosage may be administered, for example, hourly, every other hour, daily, every other day, twice a week, three times a week, four times a week, five times a week, six times a week, weekly, every other week, monthly, every other month, or annually. Alternatively, the dosage may be administered, for example, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve times per day. In particular, the dosage regimen is once a week. The duration of the administration regimen may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, weeks, or months, or even the remainder of the life of the subject who has or is prone to muscle weakness. The dosage, frequency, and duration of the medication are adapted by the clinician according to conventional factors such as different parameters from the subject who has or is prone to muscle weakness, and the severity of the disease.

[0179] For example, the recommended dose of sALP or sALP-fusion polypeptide (e.g., TNALP, e.g., the sALP polypeptide of SEQ ID NO: 1, or a polypeptide variant having at least 95% sequence identity to the sequence of SEQ ID NO: 1, e.g., asfotase alfa) is a regimen of 2 mg / kg body weight administered subcutaneously three times a week, or 1 mg / kg body weight administered subcutaneously six times a week. Further dose information is provided below (Table 1).

[0180] [Table 1]

[0181] The following embodiments are intended to be illustrative rather than limiting to the present disclosure. [Examples]

[0182] Example 1. Decreased muscle tone in subjects with HPP-like disease without loss-of-function mutations in ALPL. Patient A was diagnosed with HPP at 5 years and 9 months of age, at which time she underwent evaluation for decreased bone mineral density associated with severe hypotonia. She was found to have a low alkaline phosphatase level of 149 U / L (150–420), a high PLP level of 172.4 (20–125), and a high urinary PEA level of 190 mg / dl (0–106). Skeletal imaging showed a normal X-ray of the wrist, but X-rays of the knee showed decreased bone mineral density, thin bone, and abnormal tibial epiphysis. DXA scans showed low bone mineral density (Z-scores of -4.6 and -3.3 for the whole body except the lumbar spine and head, respectively). She had no fractures. She had ALPL gene sequencing, including deletion and duplication studies, and no mutations in the ALPL gene were identified.

[0183] Prior to the HPP diagnosis, Patient A underwent extensive molecular testing to identify the cause of severe hypotonia that began in infancy. During her first year of life, evaluations of spinal muscular atrophy, muscle biopsy, and neuro- and electromyographic (EMG) studies did not provide a specific explanation for her persistent hypotonia and weakness. SMA gene testing did not reveal any disease-causing mutations. Muscle biopsy revealed chronic, progressive myopathy exhibiting characteristics of fibrous disequilibrium. Interpretation of EMG revealed possible mild abnormalities in neuromuscular transmission. Brain MRI revealed volume loss / dysplasia, including the lower part of the cerebellar vermis with mild bilateral cerebellar volume loss.

[0184] Subsequent extensive genetic testing failed to provide a definitive explanation for Patient A's complex clinical presentation. A comprehensive mitochondrial DNA (mtDNA) analysis panel was performed at Baylor College of Medicine and reported an unknown, rare variant of significant significance in the MTRNR2 gene (m.1836A>G). A coenzyme Q10 deficiency panel was also performed at Baylor College of Medicine and reported an unknown, rare variant of significant significance in the ADCK3 gene (c.1665G>A). Whole exome sequencing (WES) was performed using GeneDx, which revealed two variants, neither of which explained her phenotype. One variant was a known ADCK3 variant previously identified by the Baylor coenzyme Q10 deficiency panel. This variant is likely pathogenic but is associated with autosomal recessive coenzyme Q10 deficiency. This is not a significant explanation, as Patient A has only maternal mutations. The second variant was a paternally inherited variant in the ATP1A3 gene. This variant (c.357+1G>A) is classified as highly pathogenic, while her father, who has the same variant, is unaffected. Therefore, the father's non-affected status in patient A appears to be evidence that having the c.357+1G>A variant in the ATP1A3 gene is not the cause of her clinical phenotype. The variant c.357+1G>A is located between exons 4 and 5 and can affect the splicing of these two exons. The mother does not carry this variant and showed a distinct splicing site between exons 4 and 5 compared to other family members. Other family members showed varying degrees of “leak” between exons 4 and 5, while patient A showed the largest number of reads mapped to the intron between exons 4 and 5, suggesting less efficient splicing of the intron (Figure 1). However, ATP1A3 was expressed at very low levels in PBMCs, and the number of reads mapped to the affected introns was low.

[0185] Example 2. Quantification of ALPL transcription and translation Patient A was subsequently examined for impaired ALPL gene expression at the RNA and / or protein levels, due to ALPL transcriptional dysregulation, by RNA sequencing of ALPL transcripts and Western blotting of ALPL protein. RNA sequencing showed that ALPL transcript levels were normal in patient A, as well as in patient A's mother, father, and three siblings (Figure 2A). ALPL translation was identified using Western blotting. The analysis confirmed that TNAP protein was produced in patient A, as well as in patient A's mother, father, and three siblings (Figure 2B). Confirmatory and quantitative analyses using positive controls were performed.

[0186] Example 3. Quantification of ALPL translation Western blot analysis of ALPL is performed using a sample from patient A to determine whether the ALPL protein is produced at normal levels and with the correct molecular weight. A determination that the protein is not present at normal concentrations, despite patient A showing normal transcription levels, suggests a possible defect in ALPL translation.

[0187] Patient A may then be analyzed together with her parents and three siblings to obtain a complete genetic profile. This study includes the collection of 20 mL of blood samples from Patient A, her parents, and her three siblings for informed consent, Western blot analysis, RT-PCR analysis, or RNA sequencing of ALPL transcripts, and Western blot analysis of ALPL proteins. In addition, 7 mL of blood will be collected from the parents and siblings for determination of PLP, vitamin D2+D3, a comprehensive metabolic panel, and urinary PEA measurement. A medical history questionnaire and a review of anthropometric medical records will also be conducted.

[0188] Example 4. Treatment of muscle hypotonia with asfotase alpha A 12-year-old female with chromosome duplication 22q11.21q11.22, developmental delay, Asperger's syndrome (autism spectrum disorder), extreme fatigue, nocturnal pain (shoulder, back, and leg), and findings consistent with HPP (low ALP, elevated PLP, and PEA, and decreased bone mineralization) was initially diagnosed with HPP. Later, it was discovered that the patient did not have the ALPL mutation. Within months of starting the STRENSIQ®, the female patient's physical strength, agility, and endurance improved. Her 6MWT improved from 320 meters to 597 meters (age / sex criterion is 672 ± 55). Overall, she showed less pain (2 out of 10 instead of 5 out of 10 reported during previous visits) and improved mobility.

[0189] Example 5. The treatment can address muscle weakness, such as that measured by a decrease in SRC, which is unrelated to the state of bone mineralization. Experiments were conducted to analyze the effects of fusion proteins on muscle weakness, as measured by a decrease in SRC, independent of bone mineralization status. In this context, fusion proteins from two variants of SEQ ID NO: 1, including SEQ ID NO: 21 and SEQ ID NO: 22, were considered. A comparison of the three fusion protein structures is shown in Table 2, and the exact sequences are shown in Tables 3, 4, and 5. Further data on treatment with the SEQ ID NO: 21 fusion protein are included below.

[0190] [Table 2]

[0191] [Table 3]

[0192] [Table 4]

[0193] [Table 5]

[0194] Figure 3 shows representative oxygen consumption rate (OCR) profiles normalized to the number of extensor digitorum longus (EDL) skeletal muscle fibers from 20-day-old female HPP mice and wild-type female littermates, demonstrating that HPP mouse EDL muscle fibers may have a reduced respiratory reserve compared to age- and sex-matched WT mice. These data suggest that a decrease in mitochondrial energy production rate contributes to muscle weakness in HPP. Abbreviations in Figure 3: ATP = adenosine triphosphate; EDL = extensor digitorum longus; FCCP = carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; HPP = hypophosphatasia; OCR = oxygen consumption rate; WT = wild-type.

[0195] Reserve respiratory volume (SRC), expressed as a percentage of the wild type, was comparatively evaluated in PBS-treated mice and HPP mice treated with the fusion protein of Sequence ID No. 21 (Figure 4 and Table 6). The abbreviations in Table 6 are as follows: HPP = hypophosphatasia; PBS = phosphate-buffered saline; SRC = reserve respiratory volume; WT = wild type.

[0196] [Table 6]

[0197] SRC in HPP mice treated with the SEQ ID NO: 21 fusion protein, expressed as a percentage of WT, was significantly increased compared to HPP mice treated with PBS alone (p=0.0008 using a one-sided Mann-Whitney test). These data demonstrate the ability of treatment with the SEQ ID NO: 21 fusion protein to improve mitochondrial bioenergy in skeletal muscle fibers of HPP mice. Bone phenotyping of PBS control mice revealed unaffected bone in 2 out of 9 HPP mice (22%), despite these mice having less than 50% of the corresponding WT SRC. HPP mice may have muscle weakness as measured by decreased SRC, independent of bone mineralization status. These data suggest that patients with HPP suffering from muscle weakness without skeletal signs may benefit from treatment with the SEQ ID NO: 21 fusion protein. Treatment with the SEQ ID NO: 21 fusion protein rescued the bone phenotype in all but 3 out of 19 HPP mice (84%). These three mice received doses of 8, 9, or 13, which is less than the 18 dose of 2 mg / kg of the fusion protein of SEQ ID NO: 21, which normalized the bone mineralization phenotype in 94% of HPP mice.

[0198] The data from Example 5 support a method for treating muscle weakness or reducing the risk of developing muscle weakness in subjects with or at risk of developing muscle weakness, comprising administering a therapeutically effective dose of at least one recombinant polypeptide having alkaline phosphatase activity to the subject, wherein the subject shows a decrease in tissue-nonspecific alkaline phosphatase (TNSALP) concentration and the subject does not have a loss-of-function mutation in the ALPL gene. In certain embodiments, the method comprises a fusion protein containing an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 21 or SEQ ID NO: 22 (or the recombinant polypeptide comprises a fusion protein containing amino acids of SEQ ID NO: 21 or SEQ ID NO: 22). In certain embodiments, the recombinant polypeptide of the method comprises an immunoglobulin molecule, the immunoglobulin molecule being an IgG2 / 4 fragment crystallizable (Fc) region, where Fc contains the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the therapeutically effective dose is administered every other week and may consist of approximately 0.1 mg / kg to approximately 20 mg / kg. In a particular embodiment, the method according to claim 66, wherein the muscle weakness disorder includes muscular dystrophy, myasthenia gravis, and calcium pyrophosphate deposition disease (CPPD), amyotrophic lateral sclerosis (ALS), myositis, myotonic dystrophy, myosrigerant, Guillain-Barré syndrome, Duchenne muscular dystrophy (DMD), and Lambert-Eaton myasthenic syndrome. Administration of the method may improve mitochondrial bioenergy in skeletal muscle fibers.

[0199] Example 6. Sequence ID No. 1 restores pyridoxal phosphate (PLP)-dependent BCAT activity, enabling adenosine triphosphate production from branched-chain amino acid transferases. In the absence of sequential SEQ ID NO: 1 treatment, branched-chain amino acids (BCAAs) (valine, leucine, and isoleucine) are elevated in the skeletal muscle of HPP mice (TNSALP knockouts) due to insufficient intracellular pyridoxal phosphate (PLP) for PLP-dependent branched-chain amino acid transferase (BCAT) catabolism of BCAAs to branched-chain keto acids (BCKAs). (BCKAs are catabolized (in a series of sequential steps) to produce acetyl-CoA or succinyl-CoA, which can enter the mitochondrial tricarboxylic acid (TCA) cycle and produce adenosine triphosphate (ATP) via oxidative phosphorylation of adenosine diphosphate (ADP).) This pathway is shown in Figure 5.

[0200] Treatment with Sequence ID No. 1 in HPP mice restores PLP-dependent BCAT activity and normalizes BCAA levels in skeletal muscle (making BCAAs available for energy production). This is shown in Figures 6, 7, and 8, and provides indirect evidence that sequential treatment with Sequence ID No. 1 normalizes BCAA levels in HPP mouse skeletal muscle (without significant difference compared to wild-type mice), restoring PLP-dependent BCAT activity and enabling ATP production from BCAAs. Discontinuation of treatment with Sequence ID No. 1 in TNSALP knockout mice significantly increases valine, leucine, and isoleucine levels in tibial muscle compared to wild-type mice, providing indirect evidence that PLP-dependent BCAT activity decreases and thereby restricts ATP production from BCAAs.

[0201] Other Embodiments All publications, patents, and patent applications are incorporated herein by reference in whole to the same extent as each independent publication, patent, or patent application is specifically and individually indicated to be incorporated by reference. Various modifications and variations of the methods, pharmaceutical compositions, and kits described herein will be apparent to those skilled in the art without departing from the scope and spirit of this disclosure. While this disclosure is described in relation to a particular aspect, it should be understood that this disclosure is subject to further modification and that the claimed invention should not be unduly limited to such specific embodiments.

Claims

1. A method for treating muscle weakness or reducing the risk of developing a muscle weakness disorder in a subject who has or is at risk of developing such a disorder, comprising administering to the subject a therapeutically effective amount of at least one recombinant polypeptide having alkaline phosphatase activity, wherein the subject shows a decrease in tissue-nonspecific alkaline phosphatase (TNSALP) concentration and the subject does not have a loss-of-function mutation in the ALPL gene.

2. The method according to claim 1, wherein the decrease in the concentration of TNSALP is caused by a decrease in ALPL transcription, a decrease in ALPL mRNA translation, an increase or decrease in post-translational modification of TNSALP, or a decrease in TNSALP enzyme activity.

3. The method according to claim 1 or 2, wherein a decrease in ALPL transcription, a decrease in ALPL mRNA translation, an increase or decrease in TNSALP post-translational modification, or a decrease in TNSALP enzyme activity is measured compared to a normal subject.

4. The method according to any one of claims 1 to 3, wherein the subject has a mutation in the 3' untranslated region (UTR), 5'UTR, or intron region of the ALPL gene.

5. The method according to any one of claims 1 to 4, wherein the subject has a mutation in one or more of the following: ATP1A3, ANKH, ENPP1, FGFR3, PHOSPHO1, PTH1R, PTH2R, SPP1, TNFRSF11A, TNFRSF11B, COL1A1, COL1A2, SOX9, PDXP, AOX1, PNPO, PDXK, ADCK3, MTRNR2, and S1PR1.

6. The method according to claim 5, wherein the subject has a mutation in ATP1A3.

7. The method according to claim 5 or 6, wherein the subject has a mutation in ADCK3.

8. The method according to any one of claims 1 to 7, wherein the target muscle is not significantly different from a normal target muscle without the muscle weakness disease in at least one characteristic selected from the proportion of muscle fiber types and fiber contraction characteristics.

9. The method according to any one of claims 1 to 8, wherein the muscle is at least one type of leg muscle, and optionally, the at least one type of muscle is selected from the soleus muscle and the extensor digitorum longus (EDL) muscle.

10. The method according to any one of claims 1 to 9, wherein the muscle weakness disorder is caused by a decrease in alkaline phosphatase activity.

11. The method according to any one of claims 1 to 10, wherein the subject has an elevated serum concentration of pyrophosphate (PPi), and optionally, the muscle weakness disorder is caused by the elevated concentration of PPi.

12. The method according to claim 11, wherein an increase in the concentration of pyrophosphate (PPi) enhances muscle weakness in the subject.

13. The method according to any one of claims 1 to 12, wherein the recombinant polypeptide reduces the concentration of PPi in the subject.

14. The method according to any one of claims 1 to 13, wherein the recombinant polypeptide is administered to the subject daily for at least one week, one month, three months, six months, or one year or longer.

15. The method according to any one of claims 1 to 14, wherein the recombinant polypeptide is administered subcutaneously, intravenously, intramuscularly, sublingually, intrathecally, or intradermally.

16. The method according to claim 15, wherein the recombinant polypeptide is administered subcutaneously.

17. The method according to any one of claims 1 to 16, wherein the recombinant polypeptide comprises at least one of tissue-nonspecific alkaline phosphatase (TNALP), placental alkaline phosphatase (PALP), germ cell alkaline phosphatase (GCALP), intestinal alkaline phosphatase (IALP), and biologically functional fragments, fusions, or chimeric constructs thereof.

18. The method according to claim 17, wherein the recombinant polypeptide comprises at least one soluble fragment of TNALP, PALP, GCALP, and IALP.

19. The method according to claim 17 or 18, wherein the tissue-nonspecific alkaline phosphatase (TNALP) comprises the amino acid sequence of amino acids 1 to 485 of SEQ ID NO:

1.

20. The method according to any one of claims 1 to 19, wherein the recombinant polypeptide is a fusion protein.

21. The method according to any one of claims 1 to 20, wherein the recombinant polypeptide comprises an immunoglobulin molecule.

22. The method according to claim 21, wherein the immunoglobulin molecule is a fragment crystallizable (Fc) region.

23. The method according to claim 22, wherein the Fc comprises the amino acid sequence of Sequence ID No.

20.

24. The method according to any one of claims 1 to 23, wherein the recombinant polypeptide comprises a negatively charged peptide.

25. The negatively charged peptide comprises 1 to 50 negatively charged amino acids, such as aspartic acid or glutamic acid, and optionally the negatively charged peptide is D １０ , D １６ , E １０ , and E １６ The method according to claim 24, wherein at least one of the above.

26. The method according to any one of claims 1 to 18, wherein the recombinant polypeptide comprises a bone-targeting alkaline phosphatase having the following structure: Z-sALP-Y-Spacer-X-W ｎ -V In the formula, sALP is the extracellular domain of alkaline phosphatase. V is either absent or is an amino acid sequence of at least one amino acid. X is either absent or an amino acid sequence of at least one amino acid. Y is either absent or is an amino acid sequence of at least one amino acid. Z is either absent or is an amino acid sequence of at least one amino acid. W ｎ However, the compound is polyaspartic acid or polyglutamic acid, and n = 10 to 16.

27. The method according to claim 26, wherein the spacer includes a fragment crystallizable region (Fc).

28. The method according to claim 27, wherein the Fc comprises the amino acid sequence of SEQ ID NO:

20.

29. The recombinant polypeptide is sALP-Fc-D １０ The method according to claim 27 or 28, comprising the structure of

30. The method according to any one of claims 1 to 29, wherein the recombinant polypeptide is administered in a dose of about 0.1 mg / kg / day to about 20 mg / kg / day, or an equivalent weekly dose.

31. The method according to claim 30, wherein the recombinant polypeptide is administered in a dose of about 0.5 mg / kg / day to about 20 mg / kg / day, or an equivalent weekly dose.

32. The method according to claim 31, wherein the recombinant polypeptide is administered in a dose of about 0.5 mg / kg / day to about 10 mg / kg / day, or an equivalent weekly dose.

33. The method according to claim 32, wherein the recombinant polypeptide is administered in a dose of about 1 mg / kg / day to about 10 mg / kg / day, or an equivalent weekly dose.

34. The method according to claim 33, wherein the recombinant polypeptide is administered at a dose of about 6 mg / kg / week.

35. The method according to claim 34, wherein the recombinant polypeptide is administered at a dose of about 1 mg / kg six times a week, at a dose of about 2 mg / kg three times a week, or at a dose of 3 mg / kg twice a week.

36. The method according to any one of claims 1 to 35, characterized in that, prior to administration of the recombinant polypeptide, the subject has an average walking distance of approximately 350 meters or less in 6 minutes.

37. The method according to any one of claims 1 to 36, wherein the administration of the recombinant polypeptide promotes an increase in the average walking distance of the subject by at least 100 meters in 6 minutes.

38. The method according to any one of claims 1 to 37, wherein, after administration of the recombinant polypeptide, the subject exhibits an average walking distance of approximately 500 meters or more in 6 minutes.

39. The method according to any one of claims 1 to 38, wherein, after administration of the recombinant polypeptide, the subject exhibits a reduced dependence on an auxiliary transport device.

40. The method according to claim 39, wherein the assistive mobility device is at least one device selected from the group consisting of a walker, a wheelchair, a brace, crutches, and an orthopedic device.

41. The method according to any one of claims 1 to 40, characterized in that, prior to administration of the recombinant polypeptide, the subject has a plasma PPi concentration of about 4.5 μM or more.

42. The method according to any one of claims 1 to 41, wherein the administration of the recombinant polypeptide promotes a decrease in the median PPi concentration in a plasma sample of at least about 1 μM derived from the subject.

43. The method according to any one of claims 1 to 42, wherein, after administration of the recombinant polypeptide, the subject exhibits a plasma PPi concentration of about 2 μM to about 5 μM.

44. A method according to any one of claims 1 to 43, i) The subject is characterized in that it is 0 to 14 days old and has a plasma ALP concentration of about 90 U / L or less before administration of the recombinant polypeptide. ii) The subject is characterized in that it is between 15 days old and less than 1 year old, and has a plasma ALP concentration of approximately 134 U / L or less before administration of the recombinant polypeptide. iii) The subject is characterized in that it is approximately 1 year to less than 10 years old and has a plasma ALP concentration of approximately 156 U / L or less before administration of the recombinant polypeptide. iv) The subject is characterized in that it is approximately 10 to 13 years old and has a plasma ALP concentration of approximately 141 U / L or less before administration of the recombinant polypeptide. v) The subject is female, is about 13 to 15 years old, and has a plasma ALP concentration of about 62 U / L or less before administration of the recombinant polypeptide. vi) The subject is characterized in that he is male, is about 13 to about 15 years old, and has a plasma ALP concentration of about 127 U / L or less before administration of the recombinant polypeptide. vii) The subject is female, is about 15 to 17 years old, and has a plasma ALP concentration of about 54 U / L or less prior to administration of the recombinant polypeptide. viiii) The subject is characterized in that he is male, is about 15 to 17 years old, and has a plasma ALP concentration of about 89 U / L or less before administration of the recombinant polypeptide. ix) The subject is characterized in that he is about 17 years of age or older and has a plasma ALP concentration of about 48 U / L or less before administration of the recombinant polypeptide, or x) A method characterized in that the subject is approximately 17 years of age or older and has a plasma ALP concentration of approximately 59 U / L or less before administration of the recombinant polypeptide.

45. The method according to any one of claims 1 to 44, wherein the administration of the recombinant polypeptide promotes an increase in the median ALP concentration in a plasma sample derived from the subject, to at least about 100 U / L or more.

46. A method according to any one of claims 1 to 45, i) The subject is characterized in that it is 0 to 14 days old and has a plasma ALP concentration of approximately 273 U / L or higher after administration of the recombinant polypeptide. ii) The subject is characterized in that it is between 15 days old and less than 1 year old, and has a plasma ALP concentration of approximately 518 U / L or higher after administration of the recombinant polypeptide. iii) The subject is characterized in that it is approximately 1 year old to less than 10 years old and has a plasma ALP concentration of approximately 369 U / L or higher after administration of the recombinant polypeptide. iv) The subject is characterized in that it is approximately 10 to 13 years old and has a plasma ALP concentration of approximately 460 U / L or higher after administration of the recombinant polypeptide. v) The subject is female, is approximately 13 to 15 years old, and has a plasma ALP concentration of approximately 280 U / L or higher after administration of the recombinant polypeptide. vi) The subject is male, is approximately 13 to 15 years old, and has a plasma ALP concentration of approximately 517 U / L or higher after administration of the recombinant polypeptide. vii) The subject is female, is about 15 to 17 years old, and has a plasma ALP concentration of about 128 U / L or higher after administration of the recombinant polypeptide. viiii) The subject is male, is about 15 to 17 years old, and has a plasma ALP concentration of about 365 U / L or higher after administration of the recombinant polypeptide. ix) The subject is characterized in that she is female, is about 17 years of age or older, and has a plasma ALP concentration of about 95 U / L or higher after administration of the recombinant polypeptide, or x) A method characterized in that the subject is male, is about 17 years of age or older, and has a plasma ALP concentration of about 164 U / L or more after administration of the recombinant polypeptide.

47. The method according to any one of claims 1 to 46, characterized in that, prior to administration of the recombinant polypeptide, the subject has an average Blueinx-Oseletzky Test of Motor Skills, Second Edition (BOT-2) intensity score of approximately 10 or less.

48. The method according to claim 47, characterized in that, prior to administration of the recombinant polypeptide, the subject has an average BOT-2 running speed of about 5 or less and an agility score.

49. The method according to any one of claims 1 to 48, wherein administration of the recombinant polypeptide results in an average BOT-2 intensity score of about 10 or more for the subject.

50. The method according to any one of claims 1 to 49, wherein administration of the recombinant polypeptide results in the subject having an average BOT-2 running speed of about 5 or more and an agility score.

51. The method according to any one of claims 1 to 50, characterized in that, prior to administration of the recombinant polypeptide, the subject has an average Child Health Assessment Questionnaire (CHAQ) score of approximately 0.8 or higher.

52. The method according to any one of claims 1 to 51, wherein administration of the recombinant polypeptide results in an average CHAQ index score of approximately 0.5 or less for the subject.

53. The method according to any one of claims 1 to 52, characterized in that, prior to administration of the recombinant polypeptide, the subject has an average pediatric outcome data collection (PODCI) score of approximately 40 or less.

54. The method according to any one of claims 1 to 53, wherein administration of the recombinant polypeptide results in an average PODCI score of about 40 or more in the subject.

55. The method according to any one of claims 1 to 54, characterized in that, prior to administration of the recombinant polypeptide, the subject has an average muscle strength grade of less than approximately 5.

56. The method according to any one of claims 1 to 55, wherein administration of the recombinant polypeptide results in an average increase of about 1 or more in the muscle strength grade of the subject.

57. The method according to any one of claims 1 to 56, characterized in that, prior to administration of the recombinant polypeptide, the subject has an average HHD value of less than approximately 80% of the predicted handheld dynamometer (HHD) value.

58. The method according to any one of claims 1 to 57, wherein administration of the recombinant polypeptide results in an average HHD value of the subject that is approximately 80% or more of the predicted HHD value.

59. The method according to claim 57 or 58, wherein the HHD value represents the grip strength, knee flexion, knee extension, hip flexion, hip extension, or hip abduction of the subject.

60. The method according to any one of claims 1 to 59, wherein the subject has a muscle weakness disorder selected from muscular dystrophy, myasthenia gravis, calcium pyrophosphate deposition disease (CPPD), amyotrophic lateral sclerosis (ALS), myositis, myotonic dystrophy, myosrigerant, Guillain-Barré syndrome, Duchenne muscular dystrophy (DMD), and Lambert-Eaton myasthenic syndrome.

61. The method according to claim 1, wherein the recombinant polypeptide comprises a fusion protein comprising amino acids having at least 95% sequence identity with respect to SEQ ID NO: 21 or SEQ ID NO:

22.

62. The method according to claim 61, wherein the recombinant polypeptide comprises a fusion protein having the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO:

22.

63. The method according to claim 1, wherein the recombinant polypeptide comprises an immunoglobulin molecule, the immunoglobulin molecule is an IgG2 / 4 fragment crystallizable (Fc) region, and the Fc comprises the amino acid sequence of SEQ ID NO:

21.

64. The method according to claim 61, wherein the therapeutically effective dose is administered every other week.

65. The method according to claim 61, wherein the therapeutically effective dose administered is approximately 0.1 mg / kg to approximately 20 mg / kg.

66. The method according to claim 65, wherein the muscle weakness disorder includes muscular dystrophy, myasthenia gravis, calcium pyrophosphate deposition disease (CPPD), amyotrophic lateral sclerosis (ALS), myositis, myotonic dystrophy, myosrigerant, Guillain-Barré syndrome, Duchenne muscular dystrophy (DMD), and Lambert-Eaton myasthenic syndrome.

67. The method according to claim 61, wherein the administration improves mitochondrial bioenergy in skeletal muscle fibers.

68. The method according to claim 1, wherein treatment with a therapeutically effective amount of at least one recombinant polypeptide having alkaline phosphatase activity results in a decrease in the levels of valine, leucine, or isoleucine in the muscle of interest.

Images