Treatment of myasthenia with alkaline phosphatase

Abstract

The present disclosure features methods of treating or alleviating at least one symptom in a subject suffering from or susceptible to a myasthenia disease by administering to the subject a therapeutically effective amount of at least one recombinant polypeptide having alkaline phosphatase activity. The subject is characterized by not carrying a loss of function mutation in the ALPL gene.

Description

Cross Reference to Related Applications

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 463,539, filed May 2, 2023, the entirety of which is incorporated by reference. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and which is hereby incorporated by reference in its entirety. The XML copy, created on March 22, 2024, is named 0467WO_SL.xml and is 31,813 bytes in size. BACKGROUND

[0003] Hypophosphatasia (HPP)-like disease is caused by unknown etiology and results in a wide range of symptoms and severity, from rickets (osteomalacia) to almost complete absence of bone mineralization in utero. Most subjects exhibit features of skeletal changes, short stature, lower limb pain, gait disturbance, and premature tooth loss. In addition, these subjects exhibit muscle weakness and hypotonia, which severely reduce their quality of life due to the physical impairment caused by these types of disease every day. SUMMARY

[0004] The present invention features a method of treating muscle weakness in a subject having, or at risk of having, a muscle weakness disease, or reducing the risk of developing muscle weakness in the subject, by administering to the subject a therapeutically effective amount of at least one recombinant polypeptide having alkaline phosphatase activity, wherein the subject exhibits a reduced tissue non-specific alkaline phosphatase (TNSALP) concentration, and wherein the subject does not carry a loss-of-function mutation in the ALPL gene.

[0005] The reduced TNSALP concentration can be caused by reduced ALPL transcription, reduced ALPL mRNA translation, elevated or reduced TNSALP post-translational modification, or reduced TNSALP enzyme activity. The reduced ALPL transcription, reduced ALPL mRNA translation, elevated or reduced TNSALP post-translational modification, or reduced TNSALP enzyme activity can be measured relative to a normal subject.

[0006] Subjects may carry mutations in the 3' untranslated region (UTR), 5' UTR, or intron regions of the ALPL gene. Subjects may carry mutations 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. Subjects may carry the ATP1A3 variant c.357+1G>A. Subjects may carry the ADCK3 variant m.1665G>A. Subjects may carry the MTRNR2 variant m.1836A>G.

[0007] The muscles of the subjects (e.g., leg muscles, such as soleus or extensor digitorum longus (EDL)) did not differ significantly from those of normal subjects without myasthenia gravis in at least one property selected from the proportion of muscle fiber types and fiber contractile properties.

[0008] Myasthenic disease may be caused by decreased alkaline phosphatase activity. Subjects may have elevated serum pyrophosphate (PPi) concentrations, and / or myasthenic disease may be caused by elevated PPI concentrations. Elevated pyrophosphate (PPi) concentrations can exacerbate myasthenia in said subjects. Administration of the recombinant peptide reduced PPI concentrations in subjects (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) compared to untreated subjects.

[0009] The recombinant peptide can be administered to subjects daily for at least one week, one month, three months, six months, or one year. The recombinant peptide can be administered subcutaneously, intravenously, intramuscularly, sublingually, intrathecally, or intradermally.

[0010] The recombinant polypeptide may include at least one of the following: tissue nonspecific alkaline phosphatase (TNALP), placental alkaline phosphatase (PALP), germ cell alkaline phosphatase (GCALP), intestinal alkaline phosphatase (IALP), and their biologically functional fragments, fusions, or chimeric constructs. The recombinant polypeptide may include at least one soluble fragment of TNALP, PALP, GCALP, and IALP. Tissue nonspecific alkaline phosphatase (TNALP) may contain amino acids 1-485 of SEQ ID NO: 1.

[0011] The recombinant polypeptide can be a fusion protein. The recombinant polypeptide can include an immunoglobulin molecule, such as a fragment crystallizable (Fc) region. The Fc has the amino acid sequence of SEQ ID NO: 20. The recombinant polypeptide can include a negatively charged peptide. The negatively charged polypeptide can include one to fifty negatively charged amino acids, such as aspartic acid or glutamic acid. The negatively charged peptide can include at least one of D 10 , D 16 , E 10 , and E 16 .

[0012] The recombinant polypeptide can include a bone-targeting alkaline phosphatase having the structure:

[0013] Z-sALP-Y-spacer-X-W n -V,

[0014] where sALP is an extracellular domain of alkaline phosphatase;

[0015] V is absent or an amino acid sequence of at least one amino acid;

[0016] X is absent or an amino acid sequence of at least one amino acid;

[0017] Y is absent or an amino acid sequence of at least one amino acid;

[0018] Z is absent or an amino acid sequence of at least one amino acid; and

[0019] W n is a polyaspartate or polyglutamate, where n = 10 to 16.

[0020] The spacer can comprise an Fc region (e.g., FC having the amino acid sequence of SEQ ID NO: 20). The recombinant polypeptide can include the structure of sALP-Fc-D 10 .

[0021] The recombinant polypeptide can 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). The recombinant polypeptide can be administered at a dose of about 1 mg / kg six times per week, about 2 mg / kg three times per week, or 3 mg / kg two times per week.

[0022] The subject can be characterized by having a six-minute walk distance of about 350 meters or less prior to administration of the recombinant polypeptide. Administration of the recombinant polypeptide can result in an increase in the subject's six-minute walk distance of at least 100 meters or more. The subject can exhibit a six-minute walk distance of about 500 meters or more after administration of the recombinant polypeptide. The subject can exhibit a reduced dependence on assistive mobility devices (e.g., walkers, wheelchairs, braces, canes, and orthotics) after administration of the recombinant polypeptide.

[0023] The subject can be characterized by having a plasma PPi concentration of about 4.5 μΜ or more prior to administration of the recombinant polypeptide. Administration of the recombinant polypeptide can result in a median decrease in PPi concentration in the subject's plasma sample of at least about 1 μΜ. The subject can exhibit a plasma PPi concentration of about 2 μΜ to about 5 μΜ after administration of the recombinant polypeptide.

[0024] In some embodiments,

[0025] i) the subject is 0 days to 14 days of age and is characterized by having a plasma ALP concentration of about 90 U / L or less prior to administration of the recombinant polypeptide;

[0026] ii) the subject is 15 days to less than 1 year of age and is characterized by having a plasma ALP concentration of about 134 U / L or less prior to administration of the recombinant polypeptide;

[0027] iii) the subject is about 1 year to less than 10 years of age and is characterized by having a plasma ALP concentration of about 156 U / L or less prior to administration of the recombinant polypeptide;

[0028] iv) the subject is about 10 years to about 13 years of age and is characterized by having a plasma ALP concentration of about 141 U / L or less prior to administration of the recombinant polypeptide;

[0029] v) the subject is a female about 13 years to about 15 years of age and is characterized by having a plasma ALP concentration of about 62 U / L or less prior to administration of the recombinant polypeptide;

[0030] vi) the subject is a male about 13 years to about 15 years of age and is characterized by having a plasma ALP concentration of about 127 U / L or less prior to administration of the recombinant polypeptide;

[0031] vii) the subject is a female about 15 years to about 17 years of age and is characterized by having a plasma ALP concentration of about 54 U / L or less prior to administration of the recombinant polypeptide;

[0032] viii) the subject is a male aged about 15 years to about 17 years, and prior to administration of the recombinant polypeptide, is characterized by having a plasma ALP concentration of about 89 U / L or less;

[0033] ix) the subject is about 17 years of age or older, and prior to administration of the recombinant polypeptide, is characterized by having a plasma ALP concentration of about 48 U / L or less; or

[0034] x) the subject is about 17 years of age or older, and prior to administration of the recombinant polypeptide, is characterized by having a plasma ALP concentration of about 59 U / L or less.

[0035] Administration of the recombinant polypeptide can result in an increase in the median plasma ALP concentration in the subject of at least about 100 U / L or more.

[0036] In some embodiments,

[0037] i) the subject is 0 days to 14 days of age, and after administration of the recombinant polypeptide, is characterized by having a plasma ALP concentration of about 273 U / L or more;

[0038] ii) the subject is 15 days to less than 1 year of age, and after administration of the recombinant polypeptide, is characterized by having a plasma ALP concentration of about 518 U / L or more;

[0039] iii) the subject is about 1 year to less than about 10 years of age, and after administration of the recombinant polypeptide, is characterized by having a plasma ALP concentration of about 369 U / L or more;

[0040] iv) the subject is about 10 years to about 13 years of age, and after administration of the recombinant polypeptide, is characterized by having a plasma ALP concentration of about 460 U / L or more;

[0041] v) the subject is a female aged about 13 years to about 15 years, and after administration of the recombinant polypeptide, is characterized by having a plasma ALP concentration of about 280 U / L or more;

[0042] vi) the subject is a male aged about 13 years to about 15 years, and after administration of the recombinant polypeptide, is characterized by having a plasma ALP concentration of about 517 U / L or more;

[0043] vii) the subject is a female aged about 15 years to about 17 years, and after administration of the recombinant polypeptide, is characterized by having a plasma ALP concentration of about 128 U / L or more;

[0044] viii) the subject is a male aged about 15 years to about 17 years, and after administration of the recombinant polypeptide, is characterized as having a plasma ALP concentration of about 365 U / L or more;

[0045] ix) the subject is a female aged about 17 years or more, and after administration of the recombinant polypeptide, is characterized as having a plasma ALP concentration of about 95 U / L or more; or

[0046] x) the subject is a male aged about 17 years or more, and after administration of the recombinant polypeptide, is characterized as having a plasma ALP concentration of about 164 U / L or more.

[0047] The subject can be characterized as having a mean Bruininks-Oseretsky Test of Motor Proficiency 2nd Edition (BOT-2) strength score of about 10 or less prior to administration of the recombinant polypeptide. The subject can be characterized as having a mean BOT-2 speed of run and agility score of about 5 or less prior to administration of the recombinant polypeptide. Administration of the recombinant polypeptide can result in a mean BOT-2 strength score of about 10 or more for the subject. Administration of the recombinant polypeptide can result in a mean BOT-2 speed of run and agility score of about 5 or more for the subject.

[0048] The subject can be characterized as having a mean Childhood Health Assessment Questionnaire (CHAQ) Index score of about 0.8 or more prior to administration of the recombinant polypeptide. Administration of the recombinant polypeptide can result in a mean CHAQ Index score of about 0.5 or less for the subject.

[0049] The subject can be characterized as having a mean Pediatric Outcomes Data Collection Instrument (PODCI) score of about 40 or less prior to administration of the recombinant polypeptide. Administration of the recombinant polypeptide can result in a mean PODCI score of about 40 or more for the subject.

[0050] The subject can be characterized as having a mean muscle strength rating of less than about 5 prior to administration of the recombinant polypeptide. Administration of the recombinant polypeptide can result in an average increase in muscle strength rating of about 1 or more for the subject.

[0051] The subject can be characterized as having a mean hand-held dynamometry (HHD) value of less than about 80% of the predicted HHD value prior to administration of the recombinant polypeptide. Administration of the recombinant polypeptide can result in a mean HHD value of about 80% or more of the predicted HHD value for the subject. The HHD value can represent grip strength, knee flexion, knee extension, hip flexion, hip extension, or hip abduction for the subject.

[0052] The subject has a muscle weakness disease selected from the group consisting of muscular dystrophy, myasthenia gravis and calcium pyrophosphate deposition disease (CPPD), amyotrophic lateral sclerosis (ALS), myositis, myotonic dystrophy, myotonia, Guillain-Barre syndrome, Duchenne muscular dystrophy (DMD), and Lambert-Eaton myasthenic syndrome.

[0053] In some embodiments of any of the treatment methods 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 disease (CPPD) are expressly excluded.

[0054] Definitions

[0055] As used herein, “a” or “an” means “at least one” or “one or more” unless otherwise indicated. Furthermore, the singular forms “a”, “one”, and “the” include plural referents unless the context clearly dictates otherwise.

[0056] As used herein, “about” refers to a floating range of the quantity of ±10% of the recited value, and preferably ±5% of the recited value, or more preferably ±2% of the recited value.

[0057] “asfotase alfa” means a soluble glycoprotein human TNALP (hTNALP) fusion protein comprising two identical polypeptide chains, wherein each polypeptide chain comprises amino acid residues 1-726 of SEQ ID NO: 1. The structure of each polypeptide chain comprises the catalytic domain of hTNALP, a human immunoglobulin G1 Fc domain, and a ten aspartate peptide that serves as a bone targeting domain (structure hTNALP-Fc-D 10 ). The two polypeptide chains are covalently linked by two disulfide bonds. Asfotase alfa has been approved in the United States, Europe, Japan, Canada, Israel, Australia, Korea under the trade name STRENSIQ ® (Alexion Pharmaceuticals, Inc., Boston, MA) for the treatment of subjects with HPP.

[0058] The terms“individual,”“subject,” and“patient” are used interchangeably and refer to any subject for whom diagnosis, treatment, or therapy is desired, particularly a human. As used herein, a subject“at risk” or a subject“predisposed” to a disease is a subject identified as being at risk of developing a disease, disorder, or symptom associated with a muscle weakness disease.

[0059] As used herein,“mean” refers to a numerical value that represents the average or median of a data set. The mean of a data set is calculated by dividing the sum of the values in the data set by the number thereof. The median of a data set is calculated by determining the middle value in an odd numbered list of data or by determining the average of the middle two data values in an even numbered list.

[0060] The term“wild type” or“wild type sequence” for a TNALP or other gene or protein used in the present disclosure refers to the typical form of such gene or protein found in nature in a normal human, non-human mammal, or other living organism. A wild type sequence can refer to the standard“normal” allele at a locus or the standard“normal” primary amino acid sequence of a polypeptide or protein (optionally with standard“normal” post-translational modifications and / or interchain bonds and / or interactions between amino acid residues), as opposed to a sequence resulting from a non-standard“mutant” allele or amino acid sequence / modification / interaction. A“mutant” allele can vary to a large extent, and even become wild type in the case of genetic drift within a population. It is now recognized that most or all loci (and, although less frequently, most polypeptide sequences) exist in multiple allelic forms that have different frequencies of occurrence throughout the geographic range of a species, and that there can not necessarily be a uniform wild type. In general, however, the most prevalent allele or amino acid sequence (i.e., the allele or amino acid sequence having the highest frequency in normal human individuals or other organisms) is considered the wild type allele or amino acid sequence in the present disclosure.

[0061] The term "normal subject" as used herein, unless otherwise indicated, refers to a subject, e.g., a human, who does not have any evidence of a muscle weakness disease and / or symptoms or physiological consequences thereof, e.g., caused by or associated with, e.g., an abnormal alkaline phosphatase activity (which can be due to, e.g., insufficient or lack of gene expression or protein levels and / or loss of function due to gene or protein mutation). An example of a normal subject is a human who does not have muscle weakness or symptoms of muscle weakness and who does not exhibit an alkaline phosphatase gene or protein (e.g., TNSALP) mutation or modification that can lead to HPP-related muscle weakness. A normal subject can also be a subject who does not exhibit an abnormal endogenous alkaline phosphatase activity (which can be tested by, e.g., assessing levels of PPi, PEA, and PLP relative to healthy controls).

[0062] As used herein, an "elevated" or "increased" concentration refers to a concentration of a molecule (e.g., a substrate of TNSALP, such as PPi) in a subject having or susceptible to a muscle weakness disease described herein that is higher than the concentration of that molecule in a normal subject (such as a subject who does not have a muscle weakness disease) or higher than the concentration of that molecule in the same subject at a time point when they do not have overt symptoms of a muscle weakness disease (e.g., after treatment). The "elevated concentration" of the molecule can be determined in a cell, tissue, or organ of the subject, such as in the blood or serum of the subject.

[0063] As used herein, the term "Bayley Scales of Infant and Toddler Development, 3 rdEdition)” or“BSID-III” refers to a series of standardized measures used to assess a subject’s motor skills (fine and gross motor), language skills (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. BSID-III measures include a series of developmental play tasks to be administered to the subject. 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 and age-matched subjects. BSID-III can also include a social-emotional adaptive behavior questionnaire completed by the parent / caregiver to determine the range of the subject’s adaptive behaviors. For example, measures used to determine BSID-III scores (e.g., BSID-III gross motor functioning scores) can include grasp, perceptual-motor integration, motor planning and speed, visual tracking, reach, object grasp, object manipulation, functional hand skills, response to tactile information, movement of limbs and trunk, static posture maintenance, dynamic movement, balance, and motor planning. These subject measurements are then converted to BSID-III scale scores (e.g., BSID-III gross motor functioning scale scores) ranging from 0 to 14, with scores of about 7 to about 13 considered to be in the normal range for healthy subjects.

[0064] As used herein, the term“bone targeting moiety” refers to an amino acid sequence of 1 to 50 amino acid residues in length that has sufficient affinity for bone matrix such that the bone targeting moiety alone has an in vivo binding affinity for bone matrix of about 10 -6 M to about 10 -15 M (e.g., 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M, or 10 -15 M) for bone matrix.

[0065] As used herein, the term "Bruininks-Oseretsky Test of Motor Proficiency, Second Edition" or "BOT-2" refers to the second edition of a standardized test for gross and fine motor performance of a subject (e.g., aged about 4 years to about 21 years). See Bruininks, R. H. (2005). Bruininks-Oseretsky Test of Motor Proficiency, (BOT-2). Minneapolis, MN: Pearson Assessment, which is hereby incorporated by reference in its entirety. The BOT-2 is administered individually to assess gross motor and fine motor skills in a range of subjects. Specifically, the BOT-2 can be used to evaluate physical impairments and mobility limitations in subjects having 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, hand dexterity, bilateral coordination, balance, and upper limb coordination. For example, BOT-2 strength scores can be determined by having the subject perform sit-ups, V-up, standing long jump, wall sit, and push-ups. Running speed and agility scores can be determined by having the subject cross a balance beam or perform a shuttle run, double-leg hop, or single-leg hop. Both BOT-2 strength and BOT-2 running speed and agility scores range from 0 to 25, with scores of about 10 to 20 considered to represent healthy subjects.

[0066] As used herein, the term "Child Health Assessment Questionnaire" or "CHAQ" refers to a questionnaire for assessing the health status (e.g., ability to perform activities of daily living (ADLs) and incidence of pain) of a subject aged 1 year to 19 years, such as a subject having a HPP-like disease. For a description of the CHAQ score, see Bruce & Fries (J. Rheumatol. 30(1): 167-178, 2003), which is hereby incorporated by reference in its entirety. For children aged greater than 8 years, the CHAQ can be administered by interview or self-report. The CHAQ includes eight subscales for dressing / grooming, getting-up, eating, walking, hygiene, reaching, gripping, and activities. Scores within each category range from 0 to 3, with a score of 0 indicating no difficulty at all; a score of 1 indicating some difficulty; a score of 2 indicating greater difficulty; and a score of 3 indicating the subject is unable to perform the activity. The CHAQ index can also be used to determine the presence and severity of pain.

[0067] "Extracellular domain" means the extracellular portion of a native protein, such as alkaline phosphatase. Specifically, the extracellular domain lacks the signal peptide.

[0068] “Fc” means the region of an immunoglobulin (e.g., IgG-1, IgG-2, IgG-3, IgG-3, or IgG-4) that is capable of crystallization, including the CH2 domain and the CH3 domain of an immunoglobulin heavy chain. Fc can also include any portion of the hinge region connecting the Fab region and the Fc region. Fc can be from any mammal, including a human, and can be post-translationally modified (e.g., by glycosylation). In one non-limiting example, Fc can be the region of a human IgG-1 that is capable of crystallization having the amino acid sequence of SEQ ID NO: 20.

[0069] “Fragment” means a portion of a polypeptide or nucleic acid molecule that preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the entire length of the referenced nucleic acid molecule or polypeptide. A fragment can 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 entire length of the polypeptide. An exemplary soluble alkaline phosphatase (sALP) fragment has 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 NO: 2-6) and can include additional C-terminal and / or N-terminal portions.

[0070] The terms “hand-held dynamometer” and “HHD” as used interchangeably herein refer to a method for measuring grip strength and muscle strength of a subject, particularly a subject having or susceptible to a muscle weakness disease. The dynamometer can be used to assess grip strength, knee flexion, knee extension, hip flexion, hip extension, and hip abduction of a subject, e.g., a subject having or susceptible to a muscle weakness disease. For example, knee flexion and extension and hip flexion, extension, and abduction of a subject having or susceptible to a muscle weakness disease can be measured using, e.g., the MICROFET2 ™Grip strength can be measured using, for example, a Jamar grip dynamometer. Specifically, an administrator holds the dynamometer stationary and the subject exerts maximum force against the dynamometer. Peak force data is collected in pounds, which is then converted to Newtons (N). Torque values are then calculated according to limb length in units of N-meters. Torque values can then be compared to, for example, torque values of normal subjects of approximately the same age, the same gender, and / or the same height and expressed as a percentage value to generate a HHD value for the subject.

[0071] As used herein, the term “hypophosphatasia” or “HPP” refers to a rare, inheritable bone disorder caused by one or more loss-of-function mutations in the ALPL (alkaline phosphatase, liver / bone / kidney) gene that encodes tissue-nonspecific alkaline phosphatase (TNALP). HPP can be further characterized as infantile HPP, childhood HPP, perinatal HPP (e.g., benign perinatal HPP or lethal perinatal HPP), or dental HPP.

[0072] As used herein, the term “hypophosphatasia-like disease” or “HPP-like disease” refers to a disease that exhibits similar symptoms to HPP but is not caused by a loss-of-function mutation in the ALPL gene.

[0073] “Untreated patient” or “untreated subject” means a patient or subject having a myasthenic disease described herein who has never received treatment with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., an 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).

[0074] “Pain” as used herein refers to physical distress or discomfort caused by a myasthenic disease described herein, such as muscle pain. For example, pain symptoms can include, for example, aching, tightness, or stiffness. The severity of pain can vary from subject to subject (e.g., chronic pain or acute pain). Specifically, chronic pain refers to pain that lasts more than three to six months, or pain that exceeds the expected healing period of time. In contrast, acute pain refers to pain that typically lasts less than three to six months. As described herein, a therapeutic composition (e.g., comprising sALP, such as asfotase alfa) can be administered to a subject having pain (e.g., muscle pain) in an amount sufficient to reduce or at least partially reduce pain symptoms (e.g., discomfort, aching, tightness, or stiffness) and complications thereof (e.g., fatigue, insomnia, weakened immune system, depression, anxiety, stress, irritability, or disability).

[0075] 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-translationally modified (e.g., glycosylated or phosphorylated), that constitutes all or part of a naturally occurring or non-naturally occurring polypeptide or peptide, as described herein.

[0076] "Pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" means a carrier or excipient that is physiologically tolerable to a subject (e.g., a human) and that does not significantly interfere with the therapeutic properties of a molecule with which it is administered. An exemplary pharmaceutically acceptable carrier substance is physiological saline. For example, a pharmaceutically acceptable carrier can include sodium chloride (e.g., 150 mM sodium chloride) and sodium phosphate (e.g., 25 mM sodium phosphate). Other physiologically tolerable carriers and their formulations are known to those of skill 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).

[0077] "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. The pharmaceutical composition can be approved by a government regulatory agency for manufacture or sale as part of a therapeutic regimen to treat or prevent a disease or event in a subject. The pharmaceutical composition can be formulated, for example, for subcutaneous administration, intravenous administration (e.g., as a sterile solution free of particulate emboli and dissolved in a solvent system suitable for intravenous use), for oral administration (e.g., a tablet, capsule, caplet, capletlet, or syrup), or any other formulation described herein, for example, in the form of a unit dosage form. In one embodiment, the pharmaceutical composition of the present disclosure is administered subcutaneously or formulated for subcutaneous administration.

[0078] As used herein, the term "physical impairment" refers to a physiological condition, such as the bone and muscle weakness diseases described herein, that can limit or cause a loss of the subject's ability to, for example, walk, functional endurance, and perform activities of daily living (ADLs). In particular, the physical impairment can limit or cause a loss of the subject's ability to perform ADLs, which are routine activities that a healthy subject performs every day without assistance, such as functional ambulation or transfers (e.g., walking), bathing and showering, dressing, self-feeding, and personal hygiene and grooming. As described herein, a therapeutic composition (e.g., a composition comprising sALP, such as asfotase alfa) can be administered to a subject to reduce the severity and / or frequency of physical impairment associated with muscle weakness.

[0079] As used herein, the term“pediatric outcome data collection instrument” or“PODCI” refers to a questionnaire used to assess the overall health status, incidence of pain, and ability to perform ADLs in subjects under the age of 19 years, particularly subjects with chronic health conditions, such as subjects with HPP-like diseases. For a description of the PODCI, see Plint et al. (J. Pediatr. Orthop. 23(6): 788-790, 2003), which is hereby incorporated by reference in its entirety. The questionnaire can be completed by the subject, or by the subject’s parent / guardian with knowledge of the subject’s condition. Eight scales generated from the PODCI include the following: 1) an upper extremity and body function scale to measure difficulties encountered in performing daily personal care and school activities; 2) a transfer and basic mobility scale to measure difficulties experienced in performing routine actions and motor activities in daily activities; 3) a sports / physical function scale to measure difficulties or limitations encountered in participating in more active activities or sports; 4) a pain / comfort scale to measure the level of pain experienced over the past week; 5) a treatment expectations scale to measure long-term expectations for treatment; 6) a well-being scale to measure overall satisfaction with one’s appearance and perception of similarity to peers; 7) a symptom satisfaction scale to measure the subject’s acceptance of current limitations if those limitations were to be a lifelong condition; and 8) a global function scale, which is a general composite scale calculated from the first four scales listed above. Standardized scores are generated from a series of questions from the PODCI and converted to a scale of 0 to 100, where 0 represents significant disability and 100 represents less disability.

[0080] As used herein, the term “Peabody Developmental Motor Scales, 2nd Edition” or “PDMS-2” refers to a program for early motor development in children that provides an assessment of gross and fine motor skills in subjects (e.g., infants and children) from birth through childhood. For a description of the PDMS-2 scales, see van Hartingsveldt et al. (Occup. Ther. Int. 12(1): 1-13, 2005), which is hereby incorporated by reference in its entirety. The PDMS-2 consists of six subtests that measure interrelated motor abilities of early development. The six subtests include the following: 1) Motor subtest, which measures the subject’s ability to move from one place to another (measuring items include crawling, walking, running, hopping on one foot, and jumping forward); 2) Reflexes subtest, which measures the subject’s ability to react automatically to environmental events; 3) Stationary Subtest, which measures the subject’s ability to maintain his or her body’s center of gravity stable and balanced; 4) Object Manipulation subtest, which measures the subject’s ability to manipulate objects, such as catching, throwing, and kicking a ball; 5) Grasp subtest, which measures the subject’s ability to use his or her hands, such as the ability to hold an object with one hand and actions involving the use of both hands’ fingers; and 6) Visual-Motor Integration subtest, which measures the subject’s ability to use his or her visual perceptual skills for complex eye-hand coordination tasks, such as reaching and grasping objects, block building, and pattern tracing. The PDMS-2 measurements for each subtest are converted to a PDMS-2 score, such as a PDMS-2 motor standard score ranging from 0 to 13, where healthy subjects range from about 7 to about 13.

[0081] The terms“sALP,”“soluble alkaline phosphatase,” and“extracellular domain of alkaline phosphatase” are used interchangeably and refer to a soluble, non-membrane bound alkaline phosphatase or a domain, biologically active fragment, or biologically active variant thereof. sALP includes, for example, an alkaline phosphatase lacking the C-terminal glycolipid anchor (GPI signal sequence, e.g., a polypeptide comprising or consisting of amino acid residues 18-502 of human TNALP (SEQ ID NO: 2, 3, 4, 5, or 6)). In particular, TNALP can include, for example, a polypeptide comprising or consisting of amino acid residues 1-485 of SEQ ID NO: 1 (such as asfotase alfa), or a polypeptide variant 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 SQ ID NO: 1 and SEQ ID NO: 22 has 96.08% sequence identity to SEQ ID NO: 1). sALP also includes, for example, mammalian orthologs of human TNALP, such as Rhesus TNALP (SEQ ID NO: 7), rat TNALP (SEQ ID NO: 8), canine TNALP (SEQ ID NO: 9), pig TNALP (SEQ ID NO: 10), murine TNALP (SEQ ID NO: 11), bovine TNALP (SEQ ID NO: 12-14), or feline TNALP (SEQ ID NO: 15). sALP also includes soluble, non-membrane bound forms of human PALP (e.g., a polypeptide comprising or consisting of amino acid residues 18-502 of SEQ ID NO: 16 or 17), GCALP (e.g., a polypeptide comprising or consisting of amino acid residues 18-502 of SEQ ID NO: 18), and IALP (e.g., a polypeptide comprising or consisting of amino acid residues 18-502 of SEQ ID NO: 19), as well as additional variants and analogs thereof that retain alkaline phosphatase activity (e.g., the ability to hydrolyze PPi). In particular, sALP lacks the N-terminal signal peptide (e.g., aa 1-17 of SEQ ID NO: 2-6, 8, 11-13, or 15 or aa 1-25 of SEQ ID NO: 7).

[0082] "sALP polypeptide" means a polypeptide having the structure A-sALP-B, where sALP is as defined herein, and each of A and B is either absent or an amino acid sequence of at least one amino acid. An exemplary sALP polypeptide has an amino acid sequence comprising 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., an 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 alfa).

[0083] "signal peptide" means a short peptide (5 to 30 amino acids long) located at the N-terminus of a polypeptide that directs the polypeptide to a secretory pathway (e.g., the extracellular space). The signal peptide is typically cleaved during secretion of the polypeptide. A signal sequence can direct a polypeptide to an intracellular compartment or organelle, e.g., the Golgi apparatus. A signal sequence can be identified by homology or bioactivity comparisons with peptides having known functions of targeting polypeptides to specific regions of a cell. One of ordinary skill in the art can identify signal peptides by using readily available software (e.g., the Sequence Analysis Software Package of the University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, or PILEUP / PRETTYBOX programs). A signal peptide can be, for example, a signal peptide substantially identical to amino acid residues 1-17 of SEQ ID NOs: 2-6 or amino acid residues 1-25 of SEQ ID NO: 7.

[0084] As used herein, when a polypeptide or nucleic acid sequence is referred to as having "at least X% sequence identity" to a reference sequence, where "X" is a real number, this means that at least X% of the amino acid residues or nucleotides in the polypeptide or nucleic acid are identical with those of the reference sequence, when the sequences are optimally aligned. Optimal alignment of sequences can be determined in various ways within the skill in the art, for example, 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 alignment algorithms and other alignment algorithms can be computed 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, M.O., 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. Moreover, one of skill can determine appropriate parameters for measuring alignment, including any algorithm needed to achieve optimal alignment over the length of the sequences being compared.

[0085] "Therapeutically effective amount" means an amount of a polypeptide or nucleic acid molecule described herein sufficient to significantly improve, treat, prevent, delay, inhibit or stop at least one symptom of muscle weakness. A therapeutically effective amount of a composition described herein can depend on the severity of the condition being treated and the subject's condition, weight, and overall state, and can be determined by one of ordinary skill in the art after considering these factors. A therapeutically effective amount of a composition described herein can be administered to a subject in a single dose or multiple doses over a period of time.

[0086] "Treat" means to apply medical management to a subject with the intent to cure, alleviate, stabilize, prevent, delay, inhibit or stop a muscle weakness disease (e.g., to a subject having low muscle tone) or reduce the likelihood of having the muscle weakness disease, and / or to manage a subject exhibiting or likely to have a muscle weakness disease (e.g., to a subject having low muscle tone), for example, by administering a pharmaceutical composition (e.g., sALP, such as asfotase alfa).

[0087] The term includes curative treatment, i.e. treatment specifically aimed at the improvement or cure associated with a disease, pathological condition, disorder or event, and also causal treatment, i.e. treatment aimed at eliminating the cause of the relevant disease, pathological condition, disorder or event. In addition, the term includes palliative treatment, i.e. treatment aimed at reducing or improving at least one symptom rather than curing the disease, pathological condition, disorder or event; symptomatic treatment, i.e. treatment aimed at the general symptoms of the relevant disease, pathological condition, disorder or event; preventive treatment, i.e. treatment aimed at reducing as much as possible or partially or totally inhibiting the development of the relevant disease, pathological condition, disorder or event, for example in a subject who is not yet ill but who is predisposed to a particular disease, pathological condition, disorder or event or is in another way at risk of this particular disease, pathological condition, disorder or event; and supportive treatment, i.e. treatment used to complement another specific therapy aimed at the improvement of the relevant disease, pathological condition, disorder or event.

[0088] As used herein, “ambulatory ability” refers to the ability of a subject (e.g., a subject having a myasthenic disease described herein) to lift and lower each foot in turn. Ambulatory ability can be assessed by a test, specifically, the six-minute walk test (6MWT). See American Journal of Respiratory and Critical Care Medicine, 166(1): 111-7, 2002, which is hereby incorporated by reference in its entirety. BRIEF DESCRIPTION OF DRAWINGS

[0089] Figure 1 FIG. 1 is a graph showing the RNA sequencing results for ATP1A3 transcripts. The x-axis represents the position in the transcript and the y-axis is the number of reads. Results for Patient A, mother, father, and three siblings are shown.

[0090] Figure 2A FIG. 2 is a graph showing the ALPL transcriptional profile for Patient A (proband), mother, father, and three siblings. The transcriptional profile is measured in Transcripts per Million (TPM).

[0091] Figure 2B FIG. 3 is a Western blot showing the TNAP translational profile for Patient A (proband), mother, father, and three siblings. Lanzoparine was used as a standard.

[0092] Figure 3 FIG. 4 illustrates the Seahorse mitochondrial respiratory pressure test profile of the extensor digitorum longus (EDL) muscle fibers of age- and sex-matched HPP mice and WT mice.

[0093] Figure 4The reserve respiratory capacity in muscle of HPP mice after in vivo treatment with vehicle or SEQ ID NO: 21 is illustrated.

[0094] Figure 5 The branched chain amino acids (BCAAs) are involved in the pathway of adenosine triphosphate (ATP) generation via the electron transport chain (ETC) and oxidative phosphorylation (Ox-Phos) is illustrated.

[0095] Figure 6 , Figure 7 and Figure 8 The levels of the BCAAs valine, leucine, and isoleucine in the tibialis muscle of tissue non-specific alkaline phosphatase (TNSALP) knockout mice (Akp2- / - (KO) mice) treated with SEQ ID NO: 1 for 35 days followed by treatment with vehicle for 12 days (cessation of SEQ ID NO: 1), or wild-type untreated mice, are illustrated. DETAILED DESCRIPTION

[0096] Muscle weakness is a prominent symptom of several diseases and conditions. One such disease is hypophosphatasia (HPP), a congenital deficiency caused by loss-of-function mutations in the ALPL gene that encodes tissue non-specific alkaline phosphatase (TNSALP). asfotase alfa (STRENSIQ® ® , Alexo Therapeutics) is a TNSALP fusion protein and is the first and only treatment available for subjects diagnosed with HPP. Surprisingly, we have found that subjects who do not carry loss-of-function mutations in the ALPL gene but also exhibit symptoms similar to HPP, including muscle weakness, such as low muscle tone, also respond to asfotase alfa treatment. Accordingly, the present invention features methods of treating muscle weakness in subjects who do not carry loss-of-function mutations in ALPL by administering a recombinant polypeptide having alkaline phosphatase activity (e.g., asfotase alfa) in subjects who are characterized as having reduced TNSALP concentrations (e.g., in their serum).

[0097] Subjects that can be treated using the compositions and methods described herein

[0098] The present invention features methods of treatment for treating muscle weakness in a subject having reduced levels of TNSALP by administering a polypeptide having alkaline phosphatase activity. The subject is one who does not carry a loss-of-function mutation in ALPL but can carry a mutation in the non-coding region of the ALPL gene. The reduced levels of TNSALP can be caused by one or more factors such as reduced ALPL transcription (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), reduced ALPL mRNA translation (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), increased (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) or reduced (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) post-translational modification of TNSALP, and / or reduced TNSALP enzymatic activity (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). The reduced ALPL transcription, reduced ALPL mRNA translation, increased or reduced post-translational modification of TNSALP, and / or reduced TNSALP enzymatic activity can be measured relative to an average, a normalized value, a baseline, a subject value prior to onset of disease, or a healthy subject.

[0099] Loss-of-function mutations in ALPL are known in the art and disclosed, for example, in PCT publication WO2011134084, the disclosure of which is hereby incorporated by reference in its entirety. Thus, the present invention features methods for treating a subject having muscle weakness who does not carry one of the mutations described herein or otherwise known. Non-loss-of-function mutations (e.g., mutations in the regulatory region, 3' or 5' UTR, enhancer, promoter, or intronic region of ALPL) can result in reduced levels of TNSALP. Alternatively or additionally, the subject can carry a mutation (e.g., a single nucleotide polymorphism (SNP)) anywhere in their genome, such as in the regulatory region, 3' or 5' UTR, enhancer, promoter, or intronic region of a non-ALPL gene.

[0100] A subject can carry one or more structural variants in their genome that result in reduced TNSALP activity, which in turn causes muscle weakness. Examples of structural variants include copy number variants, insertions, duplications, inversions, translocations, and breakpoint ends. A subject can carry a mutation or structural variant in the coding region of a protein, enzyme, or regulatory RNA (e.g., non-coding RNA) other than TNSALP. For example, other proteins or RNAs that interact with the TNSALP or ALPL gene to directly or indirectly modulate the function, activity, and / or expression of TNSALP can have mutations or structural variants that produce a downstream effect of reducing TNSALP levels. For example, a subject can carry a mutation or structural variant 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. The mutation or structural variant can be in the coding region, 5' UTR, 3' UTR, or intronic sequence. A subject can carry ATP1A3 variant c.357+1G>A. A subject can carry ADCK3 variant m.1665G>A. A subject can carry MTRNR2 variant m.1836A>G.

[0101] The ATP1A3 gene encodes the a-subunit of the Na+ / K+ ATPase pump, which uses energy from ATP to transport ions into and out of cells to maintain electrochemical gradients. It also plays a role in neuronal function, muscle contraction, and reuptake of neurotransmitters in the central nervous system. All reported deleterious ATP1A3 mutations exhibit a dominant mode of inheritance with variable penetrance. In addition, deleterious mutations are associated with a rapidly onset dystonia-parkinsonism phenotype characterized by bradykinesia and ataxia, as well as alternating hemiplegia in children, as well as cognitive problems. The phenotype can vary in children and can include hypotonia, ataxia, difficulty speaking, and slurred speech. As described herein, a subject carrying a mutation in ATP1A3 can exhibit reduced ALP levels or symptoms associated therewith. A subject carrying a mutation in ATP1A3 who has muscle weakness can benefit from treatment with a polypeptide having alkaline phosphatase activity according to the methods described herein.

[0102] The ADCK3 gene encodes a mitochondrial protein that functions in the electron transfer membrane protein complex in the respiratory chain. Mutations in this gene impair the production of coenzyme Q10. PLP is also an essential cofactor for enzymes in the Q10 biosynthetic pathway. Mutations in the ADCK3 gene are associated with autosomal recessive cerebellar ataxia. Subjects carrying mutations in ADCK3 can exhibit reduced ALP levels or symptoms associated therewith, as described herein. Subjects carrying mutations in ATP1A3 who have muscle weakness can benefit from treatment with a polypeptide having alkaline phosphatase activity according to the methods described herein.

[0103] A subject treated according to the methods described herein can be a subject characterized as having one or more of the following: elevated PPi concentration, reduced alkaline phosphatase concentration and / or activity, an average BOT-2 strength score of, e.g., less than 10, an average BOT-2 run speed and agility score of, e.g., less than 5, an average CHAQ index score of, e.g., greater than about 0.8, or an average PODCI score of, e.g., less than about 40, an average 6MWT of, e.g., less than about 80% of the predicted 6MWT value (e.g., where the predicted 6MWT value is the 6MWT value of an age-matched and / or sex-matched normal subject), a muscle strength grade of, e.g., less than 5, and / or an average HHD value (e.g., average HHD muscle strength or grip value) of, e.g., less than about 50% of the predicted HHD value (e.g., where the predicted HHD value is the HHD value of an age-matched and / or sex-matched normal subject). In particular, the subject can be a subject who has been identified as having or being susceptible to muscle weakness.

[0104] A subject can be identified as in need of treatment. For example, a subject identified as having or being susceptible to a muscle weakness disease (e.g., a disease not caused by a loss-of-function mutation in ALPL, but which is characterized by, e.g., reduced ALP levels or activity or increased ALP substrate levels). Such a subject can be treated by administration of a recombinant polypeptide having alkaline phosphatase activity, such as a TNALP, e.g., a 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. A subject can be characterized as having elevated PPi concentration, reduced ALP concentration, e.g., an average BOT-2 strength score of less than 10, e.g., an average BOT-2 speed and agility score of less than 5, e.g., an average CHAQ index score of greater than about 0.8, e.g., an average PODCI score of less than about 40, e.g., an average 6MWT of less than about 80% of the predicted 6MWT value, e.g., a muscle strength grade of less than 5, and / or e.g., an average HHD value (e.g., average HHD muscle strength or grip strength value) of less than about 80% of the predicted HHD value. For example, an elevated PPi concentration in a sample (e.g., a plasma sample) from an infant or child (e.g., a subject less than about 12 years of age) of about 5.71 µM or greater, an elevated PPi concentration in a sample (e.g., a plasma sample) from an adolescent (e.g., a subject about 13 to about 18 years of age) of about 4.78 µM or greater; and an elevated PPi concentration in a sample (e.g., a plasma sample) from an adult (e.g., a subject greater than about 18 years of age) of about 5.82 µM or greater can be used to identify these subjects as in need of treatment. In particular, a reduced ALP concentration in a sample (e.g., a plasma sample) from a subject, e.g., about 90 U / L or less for a subject 0 days to 14 days of age; about 134 U / L or less for a subject 15 days to less than 1 year of age; about 156 U / L or less for a subject about 1 year to less than 10 years of age; about 141 U / L or less for a subject about 10 years to less than about 13 years of age; about 62 U / L or less for a female subject about 13 years to less than about 15 years of age; about 127 U / L or less for a male subject about 13 years to less than about 15 years of age; about 54 U / L or less for a female subject about 15 years to less than about 17 years of age; about 89 U / L or less for a male subject about 15 years to less than about 17 years of age; about 48 U / L or less for a female subject about 17 years of age or older; about 59 U / L or less for a male subject about 17 years of age or older can be used to identify these subjects as in need of treatment.

[0105] A subject having or susceptible to a muscle weakness disease (e.g., a subject not carrying a loss-of-function mutation in ALPL) can be identified by elevated PPi concentration, reduced alkaline phosphatase concentration, and / or reduced grip or muscle strength (e.g., as assessed using BOT-2, 6MWT, CHAQ, PODCI, muscle strength grade, and / or HHD). The subject can have reduced ALPL transcription, reduced ALPL mRNA translation, elevated or reduced TNSALP post-translational modification, and / or reduced TNSALP enzymatic activity, e.g., relative to a mean, normalized value, baseline, pre-disease onset subject value, or healthy subject.

[0106] The application can also feature a method of identifying a subject having or susceptible to a muscle weakness disease, wherein the subject has elevated PPi concentration, reduced ALP concentration, and / or reduced grip or muscle strength; and then treating, ameliorating, or reducing the risk of at least one symptom of the muscle weakness disease in the subject, e.g., by administering an ALP (e.g., asfotase alfa).

[0107] A subject can be identified regardless of whether the subject has been previously diagnosed with hypophosphatasia (HPP), calcium pyrophosphate deposition disease (CPPD), or familial hypophosphatemia (such as autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets, X-linked hypophosphatemic rickets, X-linked hypophosphatemia (XLH), etc.). For example, a subject can be identified based on elevated PPi concentration. Causes of elevated PPi concentration include, e.g., insufficient signaling molecules, or mutations in genes encoding such signaling molecules, that regulate the production, degradation, or stability of PPi. For example, insufficient or mutated signaling molecules can result in overexpression of PPi or reduced degradation or hydrolysis of PPi. Insufficient signaling molecules also include insufficient co-factors or other molecules that facilitate the function of signaling molecules. For example, in CPPD, Mg deficiency, which acts as a co-factor for various phosphatases, can result in elevated PPi levels.

[0108] Targeting muscle weakness diseases

[0109] Muscle weakness, myopathy, and myasthenia gravis that can be treated by the methods described herein include any disease or condition that results in, is attributed to, or is associated with at least one symptom of muscle weakness. The terms “muscle weakness,” “myopathy,” and “myasthenia gravis” or other similar expressions in the present disclosure refer to a condition associated with an impaired state of muscle function, such as a lack or deficiency in muscle strength, as compared to other subjects not suffering from the condition or the same subject at a time point prior to suffering from the condition. Muscle weakness can be classified as a condition with true muscle weakness or perceived muscle weakness. True muscle weakness can include a condition in which less force is exerted by the muscle than expected. For example, true muscle weakness includes various skeletal muscle diseases, including muscular dystrophy and inflammatory myopathy. Other examples include neuromuscular junction disorders, such as myasthenia gravis. Muscle weakness can also result from low levels of potassium and other electrolytes within muscle cells, at which time less force is exerted by the muscle than expected. Perceived muscle weakness (or non-neuromuscular weakness) describes a condition in which a subject feels that more effort is required (i.e., as compared to other subjects not suffering from the condition or the same subject at a time point prior to suffering from the condition) to exert a certain amount of force, but the actual muscle strength is normal, such as chronic fatigue syndrome.

[0110] In some cases, such as myasthenia gravis, muscle strength is normal at rest, but true weakness occurs after the muscle is subjected to exercise. This is also true for some cases of chronic fatigue syndrome, in which objective exercise has been measured to result in muscle weakness and delayed recovery time, which is a feature of the partial published definition. These diseases or conditions are also examples of “muscle weakness diseases” within the scope of the present disclosure.

[0111] Muscle weakness can also be classified as “proximal” or “distal” based on the location of the muscle it affects. Proximal muscle weakness affects muscles closest to the midline of the body, while distal muscle weakness affects muscles further from the limb. Proximal muscle weakness can be seen in Cushing’s Syndrome and hyperthyroidism.

[0112] There are also other categories of muscle weakness that are clinically recognized. For example, neuromuscular fatigue can be classified as “central” or “peripheral” based on its cause. Central muscle fatigue manifests as a global feeling of energy deficiency, while peripheral muscle fatigue manifests as a situation in which a specific muscle cannot complete a task

[0113] The severity of muscle weakness can be classified into different “grades” based on the following exemplary criteria:

[0114] Grade 0: No contraction or muscle movement.

[0115] Grade 1: Slight contraction is visible, but there is no movement at the joint.

[0116] Grade 2: movement at the joint in the absence of gravity.

[0117] Grade 3: able to move against gravity, but not against additional resistance.

[0118] Grade 4: able to move against external resistance, but with less strength than normal.

[0119] Grade 5: normal strength.

[0120] A subject classified as having any of grades 0-4 of a muscle weakness disorder can be treated according to the methods disclosed herein.

[0121] Hypophosphatasia (HPP) and muscle weakness

[0122] Hypophosphatasia (HPP) is a rare, inherited metabolic disorder caused by loss-of-function mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. We have identified a subset of subjects who were diagnosed with HPP but did not exhibit loss-of-function mutations in ALPL, which encodes TNSALP, upon sequencing analysis. Thus, some other mechanism contributes to the onset of HPP-like symptoms. The biochemical hallmark in these subjects is low ALP activity in serum (hypophosphatasemia), which leads to elevated levels of three phosphorous-containing compound substrates in the blood and / or urine: inorganic pyrophosphate (PPi), phosphoethanolamine (PEA), and pyridoxal 5'-phosphate (PLP). TNSALP deficiency can lead to a range of sequelae, including premature loss of primary teeth, rickets, stunted growth, muscle weakness, impaired physical functioning, and pain. Asfotase alfa treatment significantly improves bone mineralization in HPP patients, but it is not yet clear whether it is effective in treating muscle weakness. As described herein, we found that asfotase alfa has a therapeutic effect on muscle in subjects (e.g., subjects with a muscle weakness disease, such as subjects with a HPP-like disease), even in subjects who do not carry loss-of-function mutations in ALPL.

[0123] Calcium pyrophosphate deposition disease (CPPD or CPDD) and muscle weakness

[0124] Calcium pyrophosphate deposition disease (CPPD or CPDD) or calcium pyrophosphate dihydrate crystal deposition disease is a metabolic arthropathy caused by deposition of calcium pyrophosphate dihydrate crystals within and around joints, particularly in articular cartilage and fibrocartilage. These diseases can exist in subjects who do not carry loss-of-function mutations in ALPL and exhibit reduced levels of TNSALP. While CPPD is often asymptomatic, with only radiographic changes observed (i.e., chondrocalcinosis), various clinical symptoms can arise, including acute arthritis (pseudogout) and chronic arthritis. Crystal deposition causes inflammation of the joint, which can lead to breakdown of the articular cartilage. The disease can present in several different arthritic-related forms: osteoarthritis, chronic rheumatoid arthritis (RA)-like inflammatory arthritis, or an acute painful inflammatory condition known as pseudogout. The name pseudogout derives from its similarity to another acute painful state known as gout. The main difference is the type of crystal involved in the inflammation and damage. While the knee, wrist, and hip are most commonly affected by CPPD, almost any joint can be affected. This condition is the most common cause of secondary metabolic osteoarthritis. Subjects with CPPD can present with severe morbidity due to the pain of acute-onset pseudogout or symptoms of chronic arthropathy. Treatment of CPPD on a symptomatic basis is important to prevent further end-organ damage, but the treatment does not reverse the joint disease.

[0125] The exact mechanism of development of CPPD is not known. Due to aging, genetic factors, or both, subjects have increased breakdown of adenosine triphosphate, which leads to increased concentrations of inorganic pyrophosphate in the joint. Changes in the cartilage matrix can play an important role in promoting deposition of calcium pyrophosphate dihydrate crystals. Overactivity of enzymes that break down triphosphates, such as triphosphonucleoside pyrophosphohydrolase, has been observed in the cartilage of subjects with CPPD. Thus, inorganic pyrophosphate can bind calcium, leading to deposition in the cartilage and synovium. Hyaline cartilage is most often affected, but fibrocartilage, such as the meniscal cartilage of the knee, can also be affected (Pritzker et al., 1988 J Rheumatol. 15(5):828-835).

[0126] Other diseases and muscle weakness

[0127] Similar to HPP and CPPD (or CPDD), other diseases or conditions can include at least one symptom of muscle weakness. Among them, certain types of muscle weakness diseases have characteristic elevated inorganic pyrophosphate (PPi) concentrations. These muscle weakness diseases with elevated PPi concentrations are also targets for treatment with asfotase alfa in the present disclosure.

[0128] For example, subjects diagnosed with familial hypophosphatemia (such as autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets, X-linked hypophosphatemic rickets, and X-linked hypophosphatemia (XLH), among others) are often characterized as having a myasthenic phenotype. Hypophosphatemia or hypophosphatemic rickets is a form of rickets characterized by low serum phosphate levels and resistance to treatment with ultraviolet radiation or vitamin D intake. X-linked hypophosphatemia (XLH) is a dominant disorder that accounts for more than 80% of all familial hypophosphatemia. XLH is considered a systemic disorder that results from mutations in the phosphatonin-regulating gene (PHEX) that is homologous to an endopeptidase on the X chromosome. XLH subjects show normal or low serum 1,25-dihydroxyvitamin D3concentrations, indicating insufficient formation of this vitamin D metabolite. The remaining 20% of subjects with familial hypophosphatemia have autosomal dominant hypophosphatemic rickets, which is caused by gain-of-function autosomal recessive hypophosphatemic rickets and hereditary hypophosphatemic rickets with hypercalciuria.

[0129] Other diseases characterized by a myasthenic disease that can be treated according to the methods described herein include, for example, myasthenia gravis, amyotrophic lateral sclerosis (ALS), myositis, myotonic dystrophy, myotonia, Guillain-Barre syndrome, Duchenne muscular dystrophy (DMD), and Lambert-Eaton myasthenic syndrome.

[0130] Methods of treatment

[0131] Provided herein are methods for treating or ameliorating at least one symptom in a subject (e.g., a child, adolescent, or adult, e.g., a subject who does not carry a loss-of-function mutation in ALPL and has reduced levels of TNSALP) having or susceptible to a myasthenic disease. Such treatment can include administering an alkaline phosphatase or a polypeptide having alkaline phosphatase activity to reduce elevated PPi concentrations in such a subject. For example, a soluble alkaline phosphatase (sALP, such as a TNALP, e.g., a 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 administered to a child, adolescent, or adult.

[0132] The subject is diagnosed with a muscle weakness disease (such as HPP or HPP-like disease, CPPD, familial hypophosphatemia, etc. as described herein) prior to administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, such as TNALP, e.g., a 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). Additionally, the subject with or susceptible to a muscle weakness disease can be a treatment-naive subject who has not previously been treated with sALP (such as TNALP, e.g., a 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). The subject can have a muscle weakness disease other than HPP, CPPD, or familial hypophosphatemia.

[0133] The method includes administering an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, such as TNALP, e.g., a 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) to a subject having or susceptible to a muscle weakness disease in a single dose or multiple doses over a period of time. In particular, sALP (such as asfotase alfa) can be administered to a subject previously determined to have elevated inorganic pyrophosphate (PPi) concentration or at least one predetermined biomarker / score of muscle weakness, such as a mean BOT-2 strength score of less than 10, a mean BOT-2 run speed and agility score of less than 5, a mean CHAQ Index score of greater than about 0.8, and / or a mean PODCI score of less than about 40, a mean 6MWT of less than about 80% of the predicted 6MWT value, a muscle strength grade of less than 5, and / or, for example, a mean HHD value (e.g., mean HHD muscle strength or grip strength value) of less than about 80% of the predicted HHD value. For example, sALP can be administered to an infant or child (e.g., a subject less than about 12 years of age) having a PPi concentration greater than about 5.71 mM in a previously determined sample (e.g., a plasma sample); a teenager (e.g., a subject about 13 to about 18 years of age) having a PPi concentration greater than about 4.78 mM in a sample; or an adult (e.g., a subject greater than about 18 years of age) having a PPi concentration greater than about 5.82 mM in a sample. In other embodiments, the muscle weakness disease described herein is caused by an elevated concentration of at least one alkaline phosphatase substrate (e.g., PPi, PLP, PEA, etc.). Alternatively, an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, such as TNALP, e.g., a 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 administered to a subject having or susceptible to a muscle weakness disease prior to determining a muscle weakness score (e.g., using a BOT-2 strength score, a BOT-2 run speed and agility score, a CHAQ Index score, a BSID-III scale score, a PDMS-2 criterion score, a muscle strength score, a 6MWT value, and / or a HHD value). Treatment with ALP according to the methods described herein can promote, for example, an increase in ADL activity, a reduction in pain, and / or an improvement in motor development.

[0134] Additionally, each of the scores described herein (e.g., BOT-2 strength scores, BOT-2 running speed and agility scores, CHAQ index scores, BSID-III scale scores, PDMS-2 normative scores, 6MWT, 12-POMA-G, modified performance-oriented mobility assessment (mPOMA-G, such as the scoring described in Phillips et al., 2015 Bone Abstracts 4:P136, or HHD values) in a subject having or susceptible to a muscle weakness disease can be used alone or in any combination to assess the efficacy of treatment with a sALP (such as a TNALP, e.g., a 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), where an improvement in a certain test score indicates that the sALP is effective in treating the muscle weakness disease.

[0135] For example, when a subject having or susceptible to a muscle weakness disease is administered an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., a sALP, such as a TNALP, e.g., a 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) such that the BOT-2 strength score is increased on average to about 10 or greater than about 10, where the subject previously had an average BOT-2 strength score of less than about 10, then the treatment with the alkaline phosphatase or the polypeptide having alkaline phosphatase activity is effective in treating, for example, physical impairment associated with the muscle weakness disease. Alternatively, when the administration of the sALP does not increase the BOT-2 strength score on average to about 10 or greater than about 10, the dose and / or frequency of administration of the alkaline phosphatase or the polypeptide having alkaline phosphatase activity can be changed in order to determine an effective amount of the alkaline phosphatase or the polypeptide having alkaline phosphatase activity for the subject. For example, the dose of the sALP (such as a TNALP, e.g., a 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 increased from, for example, about 3 mg / kg / week to about 6 mg / kg / week, or from about 6 mg / kg / week to about 9 mg / kg / week.

[0136] Additionally, when a subject having or susceptible to a muscle weakness disease is administered an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, such as a TNALP, e.g., a 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) such that the subject’s muscle strength grade classification is improved in one or more aspects (e.g., improved from a prior lower muscle strength grade to a muscle strength grade of 1, 2, 3, 4, or 5), where the subject previously had an average muscle strength grade of less than about 5, then the alkaline phosphatase or polypeptide having alkaline phosphatase activity treatment is effective to treat, for example, physical impairment associated with the muscle weakness disease. Alternatively, where administration of the sALP does not result in an improvement in the subject’s muscle strength grade classification from one or more of the prior lower muscle strength grades, the dose and / or frequency of the alkaline phosphatase or polypeptide having alkaline phosphatase activity administration can be changed (e.g., increased) in order to determine an effective amount of the alkaline phosphatase or polypeptide having alkaline phosphatase activity for the subject. For example, the dose of sALP (such as a TNALP, e.g., a 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 increased from, for example, about 3 mg / kg / week to about 6 mg / kg / week, or from about 6 mg / kg / week to about 9 mg / kg / week.

[0137] Biomarkers / endpoints for diagnosis and / or treatment of muscle weakness diseases

[0138] Muscle weakness disorders (such as HPP, including, for example, perinatal HPP, infantile HPP, childhood HPP, and dentinogenesis imperfecta, HPP-like disorders, CPPD, and familial hypophosphatasia, as described herein) can be treated with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, such as TNALP, e.g., a 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). The methods described herein can also be used to diagnose a subject as having or being susceptible to a muscle weakness disorder, identify a subject as having or being susceptible to a muscle weakness disorder, or test the efficacy of a treatment for a muscle weakness disorder. For example, a subject can be diagnosed as having or being susceptible to a muscle weakness disorder if the subject can be characterized as having certain characteristic biomarkers or index scores. The subject can be treated with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, such as TNALP, e.g., a 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 the treatment efficacy or effect can be analyzed using the characteristic biomarkers or index scores. Such biomarkers can include, for example, elevated inorganic pyrophosphate (PPi) concentration and / or reduced alkaline phosphatase (ALP) in the serum, bone, or muscle tissue, or urine of the subject. Exemplary indices that can be used in the methods described herein to determine the efficacy of a treatment for muscle weakness can include: (1) Bruiniel s Test of Motor Proficiency 2nd Edition (BOT-2), (2) Childhood Health Assessment Questionnaire (CHAQ), (3) Pediatric Outcomes Data Collection Instrument (PODCI), (4) Bayley Scales of Infant and Toddler Development 3rd Edition (BSID-III), (5) Peabody Developmental Motor Scales 2nd Edition (PDMS-2), (6) Six Minute Walk Test (6MWT), (7) Muscle Strength Rating, and (8) Hand-Held Dynamometer (HHD), which are described in further detail below.

[0139] Plasma inorganic pyrophosphate (PPi) and alkaline phosphatase (ALP) concentrations

[0140] By measuring inorganic pyrophosphate (PPi) and / or alkaline phosphatase (ALP) concentrations in a sample from a subject, such as a plasma or urine sample, a subject having or susceptible to a muscle weakness disease can be identified for treatment with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, such as a TNALP, e.g., a 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). Any method known to one of skill in the art can be used to quantify PPi and / or ALP concentrations in a plasma sample or, alternatively, a urine sample, as detailed in Whyte et al. 1995 (J. Clin. Invest. 95(4): 1440-1445), which is hereby incorporated by reference in its entirety. Methods of quantifying PPi concentrations in plasma or urine samples are also 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 hereby incorporated by reference in its entirety.

[0141] Specifically, an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, such as TNALP, e.g., a 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 administered to a subject (e.g., a subject not carrying a loss-of-function mutation in ALPL) having or susceptible to a muscle weakness disease, the subject having been previously determined to have a plasma PPi concentration of up to about 6 µM (e.g., a plasma PPi concentration of about 4.5 µM, about 5 µM, or about 5.5 µM, or a plasma PPi concentration ranging from about 4.5 µM to about 6 µM). For example, an alkaline phosphatase or a polypeptide having alkaline phosphatase activity is administered to an infant or child (e.g., a subject less than about 12 years of age) having a plasma PPi concentration of about 5.71 µM or greater; an adolescent (e.g., a subject about 13 years of age to about 18 years of age) having a plasma PPi concentration of about 4.78 µM or greater; or an adult (e.g., a subject greater than about 18 years of age) having a plasma PPi concentration of about 5.82 µM or greater. Additionally, an alkaline phosphatase or a polypeptide having alkaline phosphatase activity can be administered to a subject (e.g., a human) having or susceptible to a muscle weakness disease, the subject having been previously determined to have a plasma ALP concentration of: e.g., about 90 U / L or less for a subject 0 days to 14 days of age; about 134 U / L or less for a subject 15 days to less than 1 year of age; about 156 U / L or less for a subject about 1 year to less than 10 years of age; about 141 U / L or less for a subject about 10 years to less than about 13 years of age; about 62 U / L or less for a female subject about 13 years to less than about 15 years of age; about 127 U / L or less for a male subject about 13 years to less than about 15 years of age; about 54 U / L or less for a female subject about 15 years to less than about 17 years of age; about 89 U / L or less for a male subject about 15 years to less than about 17 years of age; about 48 U / L or less for a female subject about 17 years of age or older; or about 59 U / L or less for a male subject about 17 years of age or older.

[0142] The plasma PPi concentration and / or plasma ALP concentration of a subject (e.g., a human) having or susceptible to a muscle weakness disease can be compared to the plasma PPi concentration and / or plasma ALP of a normal subject to determine the therapeutic effect of administering an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, such as TNALP, e.g., a 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) to the subject. In particular, the alkaline phosphatase or polypeptide having alkaline phosphatase activity can be administered for 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, such as the life of the subject). Alternatively, the methods can include determining the plasma PPi concentration and / or plasma ALP concentration prior to administering the alkaline phosphatase or polypeptide having alkaline phosphatase activity to assess the effect of treatment with the alkaline phosphatase or polypeptide having alkaline phosphatase activity on the subject.

[0143] The methods result in a decrease in PPi and / or an increase in ALP concentration in a sample (e.g., a plasma sample) from a subject (e.g., a human subject, e.g., a human subject not carrying a loss-of-function mutation in ALPL) having or susceptible to a muscle weakness disease. For example, treatment with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, such as TNALP, e.g., a 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) results in a decrease in PPi concentration in a sample (e.g., a plasma sample) from the subject of about 1 µM, about 1.5 µM, about 2 µM, about 2.5 µM, or about 3 µM, or 25% or more (e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more than 60%). Thus, the subject exhibits a plasma PPi concentration of, e.g., about 2 µM to about 5 µM, about 3 µM to about 5 µM, about 2 µM to about 4 µM, or about 2 µM to about 3 µM, after administration of the alkaline phosphatase or polypeptide having alkaline phosphatase activity.

[0144] Likewise, treatment with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity results in an increase in ALP concentration in a sample (e.g., a plasma sample) from a subject (e.g., a human) having or susceptible to a muscle weakness disease by 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more than 60% relative to the subject prior to administration of the alkaline phosphatase or the polypeptide having alkaline phosphatase activity. For example, administration of the alkaline phosphatase or the polypeptide having alkaline phosphatase activity results in an increase in ALP concentration in a sample (e.g., a plasma sample) from a subject of: e.g., about 273 U / L or more for a subject aged 0 days to 14 days; about 518 U / L or more for a subject aged 15 days to less than 1 year; about 369 U / L or more for a subject aged about 1 year to less than 10 years; about 460 U / L or more for a subject aged about 10 years to less than about 13 years; about 280 U / L or more for a female subject aged about 13 years to less than about 15 years; about 517 U / L or more for a male subject aged about 13 years to less than about 15 years; about 128 U / L or more for a female subject aged about 15 years to less than about 17 years; about 365 U / L or more for a male subject aged about 15 years to less than about 17 years; about 95 U / L or more for a female subject aged about 17 years or more; or about 164 U / L or more for a male subject aged about 17 years or more.

[0145] Decreases in plasma PPi and / or increases in ALP concentration in a subject (e.g., a human) having or susceptible to a muscle weakness disease can be sustained throughout administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, such as TNALP, e.g., a 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, during treatment with sALP, plasma PPi concentration is decreased by about 25% and remains at ±10% of the decreased plasma PPi concentration, and / or during treatment with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, plasma ALP concentration is increased by about 50% and remains at ±10% of the increased plasma ALP concentration.

[0146] Alternatively, when administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, such as a TNALP, e.g., a 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 decrease the PPi concentration in a plasma sample from a subject (e.g., a human) having or susceptible to a myasthenia disease by an average of about 25% or more, the dose and / or frequency of administration of the sALP can be altered in order to determine an effective amount of the sALP for the subject. Likewise, when administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity does not increase the ALP concentration in a plasma sample from a subject by an average of about 50% or more, the dose and / or frequency of administration of the alkaline phosphatase or a polypeptide having alkaline phosphatase activity can be altered in order to determine an effective amount of the alkaline phosphatase or a polypeptide having alkaline phosphatase activity for the subject. For example, the dose of the alkaline phosphatase or a polypeptide having alkaline phosphatase activity can be increased from, e.g., 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] Assays for identifying subjects having reduced alkaline phosphatase (ALP) concentration

[0148] Biochemical and diagnostic assays can be used to identify subjects having reduced ALP concentration, which can be caused by reduced ALPL transcription, reduced ALPL mRNA translation, elevated or reduced post-translational modification of TNSALP, and / or reduced TNSALP enzyme activity. The subject can carry a mutation (e.g., a single nucleotide polymorphism (SNP)) in their genome, e.g., in a regulatory region of ALPL or another gene associated with (e.g., directly or indirectly interacting with) ALPL or TNSALP, or not associated with ALPL or TNSALP, such as in a promoter, an upstream open reading frame (uORF), a downstream ORF, a 3’ or 5’ untranslated region. The subject can carry a mutation in the coding region of a protein or enzyme other than TNSALP. For example, another protein that interacts with TNSALP or directly or indirectly modulates the function, activity, and / or expression of TNSALP can have a mutation, resulting in a downstream effect that impacts TNSALP concentration. The genome of a subject having myasthenia can be sequenced (e.g., using whole genome sequencing (WGS), intron sequencing, or exon sequencing) to identify relevant mutations outside of the coding region of the ALPL gene that cause or contribute to the symptoms of myasthenia described herein.

[0149] Assays for measuring ALPL transcription, ALPL mRNA translation, TNSALP post-translational modification, and / or reduced TNSALP enzyme activity are known to those of skill in the art. For example, assays for measuring transcriptional status include, for example, quantitative PCR (qPCR), RT-PCR, RNA-Seq, and next generation sequencing (NGS). These assays can yield information about transcriptional dysregulation of ALPL or other genes. Assays for measuring translational status include, for example, Western blot analysis, ELISA, Bradford assay, and polysome analysis. These assays can be used to quantify protein levels. Assays for measuring post-translational modification of TNSALP (e.g., phosphorylation or glycosylation, e.g., N-linked or O-linked) include, for example, Western blot analysis or ELISA assays, e.g., using antibodies that bind to the post-translationally modified TNSALP. Assays for measuring TNSALP activity are well known in the art and described, for example, in PCT Publication No. WO2005103263, which is hereby incorporated by reference in its entirety. Assays for measuring TNSALP activity include fluorescence-based enzymatic assays as well as hydroxyapatite binding. In addition, TNSALP localization assays can be used to reveal whether TNSALP is present at the cell membrane. Further, biochemical assays measuring ALP serum substrate levels (e.g., PPi, PLP, PEA) can be used as an output of the assay for TNSALP activity.

[0150] Bunsey's motor proficiency test 2nd edition (BOT-2)

[0151] An exemplary Bruininks-Oseretsky Test of Motor Proficiency, 2nd Edition (BOT-2) is described in Bruininks, R. H. (2005). Bruininks-Oseretsky Test of Motor Proficiency, (BOT-2), Minneapolis, MN: Pearson Assessment, which is hereby incorporated by reference in its entirety. In particular, the BOT-2 can be used to assess physical impairments and mobility limitations of a subject having or susceptible to a muscle weakness disease (e.g., a HPP-like disease) to generate a BOT-2 score for the subject.

[0152] The BOT-2 includes a series of tests to assess a subject's physical impairments, which can be performed using, for example, a kit including these tests. The BOT-2 provides a composite BOT-2 score in the following areas: strength, speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination. For example, a subject having or susceptible to a muscle weakness disease can determine a BOT-2 strength score by performing sit-ups, V-up, standing long jump, static lunge against wall, and / or push-ups. A subject having or susceptible to a muscle weakness disease can determine a BOT-2 speed and agility score by crossing a balance beam or performing shuttle run, double-leg hop, and / or single-leg hop. A subject having or susceptible to a muscle weakness disease can determine a BOT-2 fine motor precision score by cutting out a circle and / or connecting dots. A subject having or susceptible to a muscle weakness disease can determine a BOT-2 fine motor integration score by copying a star and / or copying a square. A subject having or susceptible to a muscle weakness disease can determine a hand dexterity score by transferring pennies, sorting cards, and / or stacking blocks. A subject having or susceptible to a muscle weakness disease can determine a BOT-2 bilateral coordination score by tapping his or her foot and fingers and / or performing jumping jacks. A subject having or susceptible to a muscle weakness disease can determine a BOT-2 balance score by walking along a line and / or standing on a balance beam on one leg. A subject having or susceptible to a muscle weakness disease can determine a BOT-2 upper limb coordination score by throwing a ball to a target and / or catching a thrown ball.

[0153] A subject having or susceptible to a muscle weakness disease (e.g., an HPP-like disease) can be tested for one or more of the aspects described (strength, speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination) to produce a BOT-2 score indicative of the subject's physical impairments. Within each BOT-2 aspect (strength, speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination), such a subject can determine the subject's BOT-2 score by performing one or more tests, for example, the subject can determine a BOT-2 strength score by performing one or more of sit-ups, V-up, standing long jump, static lunge against wall, and push-ups. Thus, a BOT-2 score (e.g., a BOT-2 strength score) for a subject having or susceptible to a muscle weakness disease (e.g., an HPP-like disease) can be determined by performing only one test (e.g., a test selected from sit-ups, V-up, standing long jump, static lunge against wall, and push-ups).

[0154] The BOT-2 score (e.g., strength, speed and agility run, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination) of a subject having or susceptible to a muscle weakness disease (e.g., HPP-like disease) can be compared to the BOT-2 score of a subject not having a muscle weakness disease (e.g., HPP-like disease), for example, to determine a baseline comparison of the BOT-2 score. The BOT-2 score (e.g., strength, speed and agility run, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination) of a subject having or susceptible to a muscle weakness disease (e.g., HPP-like disease) can be compared to the BOT-2 score of another subject having or susceptible to a muscle weakness disease (e.g., HPP-like disease), for example, to provide a relative BOT-2 score of the subject.

[0155] The BOT-2 score (e.g., strength, speed and agility run, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination score) ranges from about 0 to about 25 or less, with a score of about 10 to about 20 considered to represent a healthy subject (e.g., a subject not having a muscle weakness disease (e.g., HPP-like disease)). A subject having an average BOT-2 score (e.g., strength, speed and agility run, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination score) of less than about 10 can be treated with a basic phosphatase or a polypeptide having basic phosphatase activity (e.g., sALP, such as TNALP, e.g., a 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).

[0156] For example, a subject having or susceptible to a muscle weakness disease with a BOT-2 strength 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) can be treated with a sALP (such as a TNALP, e.g., a 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 a period of time, even throughout the subject’s life. Likewise, a subject having or susceptible to a muscle weakness disease with a BOT-2 run 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) can then be treated with a sALP (such as a TNALP, e.g., a 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 a period of time, even throughout the subject’s life.

[0157] These methods can result in an improvement in BOT-2 scores (e.g., strength, run speed and agility, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and / or upper limb coordination scores) in subjects having or susceptible to a muscle weakness disease (e.g., a HPP-like disease). For example, treatment with a basic phosphatase or a polypeptide having basic phosphatase activity, such as a sALP (e.g., a TNALP, e.g., a 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), such as treatment with a sALP for a period of time, can result in an average increase in BOT-2 strength scores to about 10 to about 20 (e.g., about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20). Additionally, treatment with a sALP (such as a TNALP, e.g., a 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 result in an average increase in BOT-2 run speed and agility scores to about 5 to about 20 (e.g., about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20).

[0158] Increases in BOT-2 scores (e.g., strength, speed and agility run, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and / or upper limb coordination scores) can be sustained throughout the administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., a 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 a period of time. Likewise, reductions in physical impairments in muscle following administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity can be sustained throughout the administration of the alkaline phosphatase or the polypeptide having alkaline phosphatase activity.

[0159] BOT-2 scores (strength, speed and agility run, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination scores) of subjects having or susceptible to a muscle weakness disease (e.g., HPP) can be used alone or in combination with other indicators to assess the efficacy of treatment with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., a 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), where improvements in certain test scores indicate that the alkaline phosphatase or the polypeptide having alkaline phosphatase activity is effective in treating muscle impairments associated with the muscle weakness disease. For example, sALP is considered effective, e.g., in treating physical impairments associated with a muscle weakness disease, when administration of sALP to a subject having or susceptible to a muscle weakness disease results in an average increase in BOT-2 speed and agility run scores of about 5 or greater, where the subject previously had an average BOT-2 speed and agility run score of less than about 5.

[0160] Additionally, within each BOT-2 aspect (strength, speed and agility run, fine motor precision, fine motor integration, hand dexterity, bilateral coordination, balance, and upper limb coordination), a subject having or susceptible to a muscle weakness disease (e.g., a HPP-like disease, CPPD, familial hypophosphatasia as described herein, etc.) can determine the subject’s BOT-2 score by performing one or more tests.

[0161] Alternatively, when administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP, does not result in an average increase in BOT-2 run speed and agility score of greater than about 5, the dose and / or frequency of administration can be changed in order to determine an effective amount of alkaline phosphatase or a polypeptide having alkaline phosphatase activity for a subject having or susceptible to a muscle weakness disease, e.g., a HPP-like disease. For example, the dose of sALP, such as TNALP, e.g., an 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 increased from, e.g., about 3 mg / kg / week to about 6 mg / kg / week, or from about 6 mg / kg / week to about 9 mg / kg / week.

[0162] Child health assessment questionnaire (CHAQ)

[0163] The health status of a child having a muscle weakness disease, e.g., a HPP-like disease, can be evaluated by administering the Childhood Health Assessment Questionnaire (CHAQ) to generate a CHAQ index score for the child, as described in Bruce and Fries (J. Rheumatol. 30(1): 167-178, 2003) and Klepper (Arthritis & Rheumatism, 49: S5-S14, 2003), which are hereby incorporated by reference in their entireties. The CHAQ includes eight categories of questions for dressing / grooming, getting-up, eating, walking, hygiene, reaching, gripping, and activities, in which a parent or guardian records the degree of difficulty a child having a muscle weakness disease, e.g., a HPP-like disease, has in performing the respective activity. The scoring range within each category is 0 to 3, where a score of 0 indicates no difficulty at all; a score of 1 indicates some difficulty; a score of 2 indicates greater difficulty; and a score of 3 indicates that the child is unable to perform the activity.

[0164] A child having or susceptible to a muscle weakness disease with an average CHAQ Index score (e.g., indicative of activities of daily living (ADL) disability and / or pain) greater than about 0.8 (e.g., about 0.8, about 1, about 1.2, about 1.4, about 1.6, about 1.8, about 2.0, about 2.2, about 2.4, about 2.6, about 2.8, or about 3.0) can be treated by administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., a 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, a child with an average CHAQ Index score greater than about 0.8 can be treated by administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., a 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 a period of time (even throughout the life of the subject). Moreover, a child having or susceptible to a muscle weakness disease disclosed herein can be asked one or more questions for one or more of the eight categories (dressing / grooming, getting-up, eating, walking, hygiene, reaching, gripping, and moving about) to arrive at an average CHAQ Index score, and if the average CHAQ Index score is greater than about 0.8, the child can be treated by administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP.

[0165] A CHAQ Index score of a child having or susceptible to a muscle weakness disease disclosed herein can be compared to a CHAQ Index score of a child not having such a muscle weakness disease, e.g., to determine a standard deviation of CHAQ Index scores. Additionally, a CHAQ Index score of a child having or susceptible to a muscle weakness disease disclosed herein can be compared to a CHAQ Index score of another child having or susceptible to a muscle weakness disease disclosed herein, e.g., to determine a standard deviation of CHAQ Index scores.

[0166] These methods can result in an improvement in a CHAQ Index score (e.g., indicative of ADL disability and / or pain) of a child having or susceptible to a muscle weakness disease disclosed herein. For example, treatment with sALP (such as TNALP, e.g., a 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), such as treatment with sALP for a period of time, even throughout the life of the child, can result in an average decrease in a CHAQ Index score of a child having a HPP-like disease to about 0 to equal to or less than about 0.5 (e.g., about 0, about 0.1, about 0.2, about 0.4, or about 0.5).

[0167] A reduction in the CHAQ Index score of a child having or susceptible to a muscle weakness disease (e.g., a HPP-like disease) can be sustained throughout the entire administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., a 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 a period of time, even throughout the life of the child. Likewise, an increase in the child’s ADL and / or a reduction in pain can be sustained throughout the entire administration of sALP (such as TNALP, e.g., a 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 a period of time, even throughout the life of the child.

[0168] The CHAQ Index Score of a child having or susceptible to a muscle weakness disease (e.g., a HPP-like disease) can be used to assess the efficacy of treatment with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., an 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), wherein an improvement in the score relative to a certain test score indicates that the alkaline phosphatase or polypeptide having alkaline phosphatase activity is effective in treating, e.g., activities of daily living (ADL) disability and pain associated with a muscle weakness disease. Specifically, one or more questions can be asked of a child having or susceptible to a muscle weakness disease for one or more of the eight categories (dressing / grooming, getting-up, eating, walking, hygiene, reaching, gripping, and ambulation) to derive an average CHAQ Index Score and assess the efficacy of sALP administration. For example, when sALP is administered to a child having or susceptible to a muscle weakness disease such that the average CHAQ Index Score is reduced to equal or less than about 0.5, wherein the child previously had an average CHAQ Index Score greater than about 0.8, then sALP is considered effective in treating, e.g., activities of daily living (ADL) disability and pain associated with a muscle weakness disease. Alternatively, when administration of sALP does not reduce the average CHAQ Index Score to equal or less than about 0.5, the dose and / or frequency of sALP administration can be changed in order to determine an effective amount of sALP for a child having or susceptible to a muscle weakness disease. For example, the dose of sALP (such as TNALP, e.g., an 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 increased from, e.g., about 3 mg / kg / week to about 6 mg / kg / week, or from about 6 mg / kg / week to about 9 mg / kg / week.

[0169] Pediatric outcomes data collection instrument (PODCI)

[0170] Certain subjects having or susceptible to a muscle weakness disease (e.g., HPP-like disease) can be identified using the Pediatric Outcomes Data Collection Instrument (PODCI) for treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., a 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). The PODCI can be administered to assess the health status of a child to generate a PODCI score for the subject, as described in Plint et al. (J. Pediatr. Orthop. 23(6): 788-790, 2003). The PODCI includes eight categories of questions that can be completed by a subject having or susceptible to a muscle weakness disease (e.g., HPP-like disease) or the subject’s parent / guardian. Categories of the PODCI that can be used to determine a subject having or susceptible to a muscle weakness disease include the following: 1) Upper Extremity and Body Function Scales to measure difficulties encountered in performing daily personal care and school activities; 2) Transfer and Basic Mobility Scales to measure difficulties experienced in performing routine actions and motor activities in daily activities; 3) Sports / Physical Function Scales to measure difficulties or limitations encountered in participating in more active activities or sports; 4) Pain / Comfort Scales to measure the level of pain experienced over the past week; 5) Therapy Expectation Scales to measure long-term expectations for treatment; 6) Well-Being Scales to measure overall satisfaction with personal appearance and perception of similarity to peers; 7) Symptom Satisfaction Scales to measure the subject’s acceptance of current limitations if those limitations were to be a lifelong condition; and 8) Global Function Scale, which is a general composite scale calculated from the first four scales listed above. In each of these categories, a standardized score for a subject having or susceptible to a muscle weakness disease is determined and then converted to a scale of 0 to 100, where 0 indicates significant disability and 100 indicates less disability.

[0171] A subject having or susceptible to a muscle weakness disease (e.g., a HPP-like disease) having an average PODCI score (e.g., indicative of ADL disability and / or pain) of less than about 40 (e.g., about 5, about 10, about 15, about 20, about 25, about 30, about 35, or about 39) can be treated by administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., a 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, a subject having an average PODCI score of less than 40 can be treated by administration of sALP for a period of time (even throughout the subject’s life). Further, a subject having or susceptible to a muscle weakness disease can be asked one or more questions for one or more of the eight scales (e.g., the Transfers and Basic Mobility scale, the Sports / Physical Function scale, and the Pain / Comfort scale) described above to arrive at an average PODCI score, and if the average PODCI score is greater than less than 40, the subject can be treated by administration of sALP.

[0172] The methods described herein can increase the PODCI score (e.g., indicative of ADL disability and / or pain) of a subject having or susceptible to a muscle weakness disease. For example, treatment with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., a 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), such as treatment with sALP for a period of time, even throughout the subject’s life, can increase the average PODCI score to 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).

[0173] The increase in the PODCI score can persist throughout the administration of the alkaline phosphatase or the polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., a 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 a period of time, even throughout the subject’s life having or susceptible to a muscle weakness disease. Likewise, the increase in ADL and / or the decrease in pain can persist for a period of time, even throughout the subject’s life, throughout the administration of sALP (such as TNALP, e.g., a 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).

[0174] PODCI scores of subjects having or susceptible to a muscle weakness disease (e.g., a HPP-like disease) can be used to assess the efficacy of treatment with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., a 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), where an improvement in the score relative to a certain test score indicates that the alkaline phosphatase or polypeptide having alkaline phosphatase activity is effective in treating, for example, activities of daily living (ADL) disability and pain associated with a muscle weakness disease. Specifically, one or more questions can be asked of a subject having or susceptible to a muscle weakness disease with respect to one or more of the eight scales (upper extremity and body function scale, mobility and basic movement scale, sports / recreation physical function scale, pain / comfort scale, treatment expectations scale, well-being scale, symptom satisfaction scale, and overall function scale) to derive an average PODCI score and assess the efficacy of treatment with sALP.

[0175] For example, sALP is considered effective in treating, for example, activities of daily living (ADL) disability and pain associated with a muscle weakness disease when administration of sALP to a subject having or susceptible to a muscle weakness disease results in an average increase in the PODCI score to about 40 or greater, where the subject previously had an average PODCI score of less than about 40. Alternatively, when administration of sALP does not result in an average increase in the PODCI score to about 40 or greater, the dose and frequency of administration of sALP can be changed in order to determine an effective amount of sALP for a subject having or susceptible to a muscle weakness disease. For example, the dose of sALP (such as TNALP, e.g., a 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 increased from, for example, about 3 mg / kg / week to about 6 mg / kg / week, or from about 6 mg / kg / week to about 9 mg / kg / week.

[0176] Bayley Scales of Infant and Toddler Development 3rd edition (BSID-III)

[0177] Another endpoint can be administered, the Bayley Scales of Infant and Toddler Development, Third Edition (BSID-III), to assess the health status of a subject having or susceptible to a muscle weakness disease (e.g., HPP-like disease) from birth to generate a BSID-III score for the subject, as described in Bayley. (2006). Bayley scales of infant and toddler development: administration manual. San Antonio, TX: Harcourt Assessment. The BSID-III includes a series of developmental play tasks that can be administered to a subject to determine a raw BSID-III score. For example, categories for determining a BSID-III score for a subject having or susceptible to a muscle weakness disease (e.g., an infant having HPP who is about three years of age or younger) can include grasp, perceptual-motor integration, motor planning and speed, visual tracking, reach, object grasp, object manipulation, functional hand skills, response to tactile information, movement of limbs and trunk, static posture maintenance, dynamic movement, balance, and motor planning. The BSID-III measurements are then converted to a scale BSID-III score, which can be used to determine the performance of a subject compared to a healthy and age-matched subject. The BSID-III scale score for a subject having or susceptible to a muscle weakness disease (e.g., a subject having an HPP-like disease) can range from 0 to 14, with a score of about 7 to about 13 considered to be within the normal range for a healthy subject.

[0178] A subject having or susceptible to a muscle weakness disease can be tested in infancy (e.g., at an age of about 3 years or less than 3 years) for one or more of the categories (grasp, perceptual-motor integration, motor planning and speed, visual tracking, reach, object grasp, object manipulation, functional hand skills, reaction to tactile information, movement of limbs and trunk, static posture maintenance, dynamic movement, balance, and motor planning) to generate a BSID-III score indicative of motor developmental delay. A subject having or susceptible to a muscle weakness disease having an average BSID-III score for one or more of the described categories (grasp, perceptual-motor integration, motor planning and speed, visual tracking, reach, object grasp, object manipulation, functional hand skills, reaction to tactile information, movement of limbs and trunk, static posture maintenance, dynamic movement, balance, and motor planning) of less than about 2 can be treated in infancy by administration of a sALP (such as a TNALP, e.g., a 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). In particular, a subject having or susceptible to a muscle weakness disease having an average BSID-III score of less than about 2 can be treated in infancy by administration of a sALP for a period of time (even throughout the subject’s life).

[0179] These methods can result in an improvement in the average BSID-III score (e.g., indicative of motor developmental delay) of a subject having or susceptible to a muscle weakness disease. For example, treatment with a sALP (such as a TNALP, e.g., a 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), such as treatment with a sALP for a period of time, even throughout the subject’s life, can result in an average increase in the BSID-III score to greater than about 5 (e.g., about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or about 13).

[0180] Increases in BSID-III scores can persist for a period of time, even throughout the life of a subject having or susceptible to a muscle weakness disease, throughout the entire administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., a 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). Likewise, increases in motor development can persist throughout the entire administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., a 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 a period of time, even throughout the life of a subject.

[0181] BSID-III scores of subjects having or susceptible to a muscle weakness disease (e.g., a HPP-like disease) can be used to assess the efficacy of treatment with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., a 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), where improvements in certain test scores indicate that the alkaline phosphatase or polypeptide having alkaline phosphatase activity is effective in treating, for example, motor developmental delays associated with a muscle weakness disease. In particular, subjects having or susceptible to a muscle weakness disease can be tested in one or more of the described categories (grasp, perceptual-motor integration, motor planning and speed, visual tracking, reach, object grasp, object manipulation, functional hand skills, response to tactile information, movement of limbs and trunk, static posture maintenance, dynamic movement, balance, and motor planning) in infancy (e.g., having HPP at about age three or younger) to derive an average BSID-III score and assess the efficacy of treatment with sALP.

[0182] For example, when administration of sALP to a child with or susceptible to myasthenia gravis results in an average increase in BSID-III score to greater than about 5, where the child previously had an average BSID-III score of less than about 2 in infancy (e.g., at an age of about 3 years or less), sALP is considered effective in treating, for example, motor developmental delay associated with HPP-like disorders. Alternatively, when administration of sALP does not result in an average increase in BSID-III score to greater than about 5, the dosage and / or frequency of sALP administration may be varied to determine the effective amount of sALP for a child with or susceptible to myasthenia gravis. For example, the dosage of sALP (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as asfotase alfa) may be increased from, for example, about 3 mg / kg / week to about 6 mg / kg / week, or from about 6 mg / kg / week to about 9 mg / kg / week.

[0183] Peabody Developmental Motor Scales 2nd edition (PDMS-2)

[0184] Another endpoint, the Peabody Developmental Motor Scale, Version 2 (PDMS-2), can be applied to assess the health status of subjects with or predisposed to myasthenic disorders (such as HPP-like disorders) from birth to generate a PDMS-2 score for the subject, as described by van Hartingsveldt et al. (Occup. Ther. Int. 12(1): 1-13, 2005). PDMS-2 includes six subtests for measuring the motor skills of subjects, such as those with HPP.

[0185] Specifically, PDMS-2 measurements can be determined from the following subtests: 1) a motor subtest to measure the subject’s ability to move from one place to another (measured items include crawling, walking, running, hopping on one foot, and jumping forward); 2) a reflexes subtest to measure the subject’s ability to automatically react to environmental events; 3) a statics subtest to measure the subject’s ability to maintain a stable body center of gravity and balance; 4) an object manipulation subtest to measure the subject’s ability to manipulate objects, such as catching, throwing, and kicking a ball; 5) a grasping subtest to measure the subject’s ability to use his or her hands, such as the ability to hold an object with one hand and actions involving the use of both hands’ fingers; and 6) a visual-motor integration subtest to measure the subject’s ability to use his or her visual perceptual skills for complex eye-hand coordination tasks, such as reaching and grasping objects, block building, and pattern copying. PDMS-2 measurements can be determined for one or more of these categories for subjects having or susceptible to a muscle weakness disease (e.g., a HPP-like disease), and then converted to a PDMS-2 score, such as a PDMS-2 motor composite score ranging from 0 to 13, where healthy subjects (e.g., subjects not having a muscle weakness disease) range from about 7 to about 13.

[0186] Subjects having or susceptible to a muscle weakness disease with an average PDMS score (e.g., indicative of motor developmental delay) can be treated by administration of a sALP, such as a TNALP, e.g., a 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.

[0187] The methods described herein can result in an improvement in the PDMS-2 score (e.g., indicative of motor developmental delay) of a subject having or susceptible to a muscle weakness disease. For example, treatment with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as a sALP (e.g., a TNALP, e.g., a 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 result in an average increase in the PDMS-2 score to about 7 to about 13 (e.g., about 7, about 8, about 9, about 10, about 11, about 12, or about 13).

[0188] The increase in PDMS-2 score can continue for an extended period of time, for example, for a period of time, even throughout the life of a subject having or susceptible to a muscle weakness disease, throughout the administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., a 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). Likewise, the increase in motor development can continue for a period of time, even throughout the life of a subject having or susceptible to a muscle weakness disease, throughout the administration of sALP (such as TNALP, e.g., a 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).

[0189] The PDMS-2 score of a subject having or susceptible to a muscle weakness disease (e.g., a HPP-like disease) can be used to assess the efficacy of treatment with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity, such as sALP (e.g., TNALP, e.g., a 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), where an improvement in a certain test score indicates that the alkaline phosphatase or a polypeptide having alkaline phosphatase activity is effective in treating, for example, motor developmental delay associated with a muscle weakness disease. For example, a child having or susceptible to a muscle weakness disease can be tested for one or more of the categories described (motor, reflex, still, object manipulation, grasp, and visual-motor) at an age of about 5 years or less than 5 years to derive an average PDMS-2 score and assess the efficacy of treatment with sALP.

[0190] For example, when administration of sALP to a child having or susceptible to a muscle weakness disease results in an average increase in PDMS-2 standard score to about 7, where the child previously had an average PDMS-2 standard score of about 5, then sALP is considered effective in treating, for example, motor developmental delay associated with a HPP-like disease. Alternatively, when administration of sALP does not result in an average increase in PDMS-2 standard score to about 7, the dose and / or frequency of sALP administration can be altered in order to determine an effective amount of sALP for the child. For example, the dose of sALP (such as TNALP, e.g., a 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 increased from, for example, about 3 mg / kg / week to about 6 mg / kg / week, or from about 6 mg / kg / week to about 9 mg / kg / week.

[0191] Six minute walk test (6MWT)

[0192] A subject having a muscle weakness disease can be identified using a 6MWT for treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, such as TNALP, e.g., a 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). In particular, the 6MWT can be used to assess the walking ability of an adult having a muscle weakness disease to generate a 6MWT value for the adult. The 6MWT can be performed indoors or outdoors using a flat, straight, and enclosed corridor (e.g., about 30 meters in length) having a hard surface. A stopwatch or other timer can be used to record the time, and a mechanical counter or other device can be used to determine the distance (e.g., in meters) walked by the subject having a muscle weakness disease. For example, the length of the corridor can be marked every three meters to determine the number of meters walked by the subject having a muscle weakness disease, with a turnaround point set at 30 meters and a starting line also marked. The distance walked by the subject having a muscle weakness disease in six minutes can then be compared to a predicted number of meters walked by a normal subject having approximately the same age, the same gender, and / or the same height, for example, and expressed as a percentage value to generate a 6MWT value for the subject. The 6MWT value for the subject having a muscle weakness disease can be compared to the 6MWT value for that subject at baseline. In addition, the 6MWT value for the subject having a muscle weakness disease can be compared to the 6MWT value for a normal subject.

[0193] A subject having a muscle weakness disease with an average 6MWT value that is less than about 80% of a predicted 6MWT value (e.g., relative to a normal subject with about the same age, same gender, and / or same height) can be treated with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, such as TNALP, e.g., a 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), such as by administering the alkaline phosphatase or polypeptide having alkaline phosphatase activity 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 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 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 for the life of the subject; particularly at least six weeks). For example, a subject having a muscle weakness disease with an average 6MWT value that is less than about 80% of a predicted 6MWT value (e.g., about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of a predicted 6MWT value) can be treated with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity 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 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 for the life of the subject; particularly at least six weeks).

[0194] These methods can result in an improvement in the 6MWT value of a subject having a muscle weakness disease. For example, treatment with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, such as a TNALP, e.g., a 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), such as 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 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 the life of the subject; particularly at least six weeks), can result in an average increase in the 6MWT value of the subject to about 80% or more of the predicted 6MWT value (e.g., about 82%, about 84%, about 86%, about 88%, about 90%, about 92%, about 94%, about 96%, about 98%, or more of the predicted 6MWT value).

[0195] The increase in the 6MWT value of a subject having a muscle weakness disease can be sustained throughout the administration of the alkaline phosphatase or the polypeptide having alkaline phosphatase activity (e.g., sALP, such as a TNALP, e.g., a 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), such as 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 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 the life of the subject; particularly at least six weeks). For example, the 6MWT value of a subject having a muscle weakness disease is increased to greater than about 80% of the predicted 6MWT value during treatment with the alkaline phosphatase or the polypeptide having alkaline phosphatase activity and remains within ±10% of the increased 6MWT value.

[0196] Similarly, improvements in walking ability in subjects having a muscle weakness disease can persist throughout the entire administration of the alkaline phosphatase or polypeptide having alkaline phosphatase activity, such as 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 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 for the life of the subject; particularly, at least six weeks). For example, subjects having a muscle weakness disease exhibit reduced dependence on assistive mobility devices (such as a walker, a wheelchair, a brace, a cane, or an orthosis) during treatment with sALP.

[0197] Alternatively, when administration of the alkaline phosphatase or polypeptide having alkaline phosphatase activity (e.g., sALP, such as TNALP, e.g., a 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 increase the 6MWT value, on average, to greater than 80% of the predicted 6MWT value (e.g., of a normal subject having approximately the same age, the same gender, and / or height), the dose and / or frequency of administration of the alkaline phosphatase or polypeptide having alkaline phosphatase activity can be changed in order to determine an effective amount of the alkaline phosphatase or polypeptide having alkaline phosphatase activity for a subject having a muscle weakness disease. For example, the dose of the alkaline phosphatase or polypeptide having alkaline phosphatase activity can be increased from, e.g., 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.

[0198] Hand-held dynamometry (HHD)

[0199] Grip strength and muscle strength in a subject having or susceptible to a muscle weakness disease can be assessed using a handheld dynamometer (HHD). For example, knee flexion and extension and hip flexion, extension, and abduction in a subject having or susceptible to a muscle weakness disease can be measured using, e.g., a MICROFET2 ™ dynamometer, while grip strength in the subject can be measured using, e.g., a Jamar grip dynamometer. Specifically, an administrator holds the dynamometer still and the subject exerts a maximum force on the dynamometer. Peak force data is collected in pounds, which is then converted to Newtons (N). Torque values are then calculated according to limb length in units of N-meters. Torque values can then be compared to, and expressed as a percentage value of, torque values of normal subjects having approximately the same age, the same gender, and / or the same height, to generate HHD values for the subject.

[0200] A subject having a muscle weakness disease with an average HHD value less than about 80% of a predicted HHD value (e.g., relative to normal subjects with about the same age, same gender, and / or same height) can be treated with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, such as TNALP, e.g., a 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), such as by administering the alkaline phosphatase or polypeptide having alkaline phosphatase activity 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 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 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 for the life of the subject; particularly at least six weeks). For example, a subject having a muscle weakness disease with an average HHD value less than about 80% of a predicted HHD value (e.g., about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of a predicted HHD value) can be treated with an alkaline phosphatase or a polypeptide having alkaline phosphatase activity 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 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 for the life of the subject; particularly at least six weeks).

[0201] These methods can result in an improvement in the HHD value of a subject having a muscle weakness disease. For example, treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, such as a TNALP, e.g., a 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), such as 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 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 the life of the subject; particularly at least six weeks), can result in an average increase in the HHD value of the subject to about 80% or more of the predicted HHD value (e.g., about 83%, about 85%, about 87%, about 90%, about 93%, about 95%, about 97%, or about 100% or about 100% of the predicted HHD value).

[0202] The increase in the HHD value of a subject having a muscle weakness disease can be sustained throughout the administration of alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, such as a TNALP, e.g., a 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), such as 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 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 the life of the subject; particularly at least six weeks). For example, during treatment with alkaline phosphatase or a polypeptide having alkaline phosphatase activity, the HHD value of a subject having a muscle weakness disease increases to greater than about 80% of the predicted HHD value and remains within ±10% of the increased HHD value.

[0203] Alternatively, when administration of an alkaline phosphatase or a polypeptide having alkaline phosphatase activity (e.g., sALP, such as TNALP, e.g., a 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 increase the HHD value, on average, to more than 80% of the predicted HHD value (e.g., of a subject having a muscle weakness disease having approximately the same age, the same gender, and / or height), the dose and / or frequency of administration of the alkaline phosphatase or a polypeptide having alkaline phosphatase activity can be changed in order to determine an effective amount of the alkaline phosphatase or a polypeptide having alkaline phosphatase activity for a subject having a muscle weakness disease. For example, the dose of the alkaline phosphatase or a polypeptide having alkaline phosphatase activity can be increased from, e.g., 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.

[0204] Alkaline phosphatase

[0205] 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) is effective for the treatment of hypophosphatasia (HPP), symptoms thereof, and physical impairments associated therewith in a subject having or susceptible to a muscle weakness disease for an extended period of time (e.g., at least one day, at least one week, at least two weeks, at least three weeks, at least one month, at least three months, at least six 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, at least ten years, or more than ten years (e.g., the subject’s lifetime)).

[0206] In view of the results described herein, the present disclosure is not limited to a particular alkaline phosphatase (ALP) or nucleic acid sequence encoding an ALP. Alkaline phosphatases encompass a group of enzymes that catalyze the cleavage (e.g., hydrolysis) of a phospho moiety from a variety of substrates, including, but not limited to, pyrophosphates (PPi), phosphonates, and phosphates (e.g., phosphodiester bonds, phosphoramidates, and phosphonamidates). In particular, the present disclosure is not limited to a particular ALP or nucleic acid sequence encoding an ALP, and encompasses any ALP or nucleic acid sequence encoding an ALP that is effective for the treatment of hypophosphatasia (HPP), symptoms thereof, and physical impairments associated therewith in a subject having or susceptible to a muscle weakness disease for an extended period of time (e.g., at least one day, at least one week, at least two weeks, at least three weeks, at least one month, at least three months, at least six 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, at least ten years, or more than ten years (e.g., the subject’s lifetime)). iThe enzyme. There are four known mammalian alkaline phosphatase (ALP) isozymes: tissue nonspecific alkaline phosphatase (TNALP; described further below), placental alkaline phosphatase (PLALP) (e.g., Accession Nos. P05187, NP_112603, and NP_001623), germ cell alkaline phosphatase (GALP) (e.g., Accession No. P10696), and intestinal alkaline phosphatase (IALP) (e.g., Accession Nos. P09923 and NP_001622). In addition to the exemplary ALPs discussed above, the present disclosure provides any polypeptide having the same or similar catalytic site structure and / or enzymatic activity as ALP for use in treating a subject having or susceptible to a muscle weakness disorder. Bone delivery conjugates comprising sALP are further described in PCT Publication Nos. WO 2005 / 103263 and WO 2008 / 138131.

[0207] TNALPs that can be used according to the methods described herein include, for example, human TNALP (Accession Nos. NP_000469, AAI10910, AAH90861, AAH66116, AAH21289, and AAI26166); rhesus TNALP (Accession No. XP_01109717); rat TNALP (Accession No. NP_037191); dog TNALP (Accession No. AAF64516); pig TNALP (Accession No. AAN64273), mouse (Accession No. NP_031457), bovine TNALP (Accession Nos. NP_789828, NP_776412, AAM8209, and AAC33858), and cat TNALP (Accession No. NP_001036028). In particular, the TNALP can be recombinant human TNALP (e.g., SEQ ID NO: 1, asfotase alfa; see U.S. Patents Nos. 7,763,712 and 7,960,529, which are incorporated by reference in their entireties) for use in treating a subject having or susceptible to a muscle weakness disorder. The TNALP can also be a TNALP exhibiting at least about 95% sequence identity to the polypeptide or nucleic acid sequence of the TNALPs described above.

[0208] Soluble alkaline phosphatase

[0209] The ALP of the present invention includes a soluble (e.g., extracellular or non-membrane bound) form of any of the alkaline phosphatases described herein. The sALP can be, for example, a soluble form of human tissue nonspecific alkaline phosphatase (human TNALP (hTNALP)). The present disclosure is not limited to a particular sALP, and can include any sALP polypeptide that has physiological activity toward, for example, phosphoethanolamine (PEA), inorganic pyrophosphate (PPi), and 5'-phosphopyridoxal (PLP). In particular, the sALP of the present invention has catalytic ability to improve bone mineralization in bone. The present disclosure also includes nucleic acids encoding the sALP described herein that can be used to treat the muscle weakness conditions described herein, including, for example, HPP-like diseases, CPPD, familial hypophosphatemia (such as autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets, X-linked hypophosphatemic rickets, X-linked hypophosphatemia (XLH), and the like), and the like.

[0210] TNALP is a membrane-bound protein anchored at the C-terminus by a glycolipid moiety (Swiss-Prot, P05186). The glycolipid anchor (GPI) is added post-translationally after removal of the hydrophobic C-terminus, which serves as both a temporary membrane anchor and a signal for addition of the GPI. While the GPI anchor is located in the cell membrane, the remainder of the TNALP is extracellular. In particular, TNALP (e.g., human TNALP (hTNALP)) can be engineered to replace the first amino acid residue (alanine) of the hydrophobic C-terminal sequence with a stop codon, resulting in an engineered hTNALP that contains all of the amino acid residues of the native anchored form of TNALP and is absent of the GPI membrane anchor. Those skilled in the art will appreciate that the location of the GPI membrane anchor will vary among different ALPs, and can 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. The recombinant sTNALP can include, for example, amino acids 1 to 502 (18 to 502 when secreted), amino acids 1 to 501 (18 to 501 when secreted), amino acids 1 to 504 (18 to 504 when secreted), amino acids 1 to 505 (18 to 505 when secreted), or amino acids 1 to 502. Thus, the C-terminus of the native ALP can be truncated by certain amino acids without affecting the activity of the ALP.

[0211] In addition to the C-terminal GPI anchor, TNALP has an N-terminal signal peptide sequence. The N-terminal signal peptide is present on the synthesized protein at the time of synthesis, but is cleaved from the TNALP after translocation into the ER. sALP includes both its secreted form (i.e., lacking the N-terminal signal) and non-secreted form (i.e., having the N-terminal signal). Those skilled in the art will appreciate that the location of the N-terminal signal peptide will vary among different alkaline phosphatases, and can 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 can predict the location of the signal sequence cleavage site, for example, by appropriate computer algorithms such as described in Bendtsen et al. (J. Mol. Biol. 340(4):783-795, 2004) and available from the website www.cbs.dtu.dk / services / SignalP / .

[0212] The present disclosure also provides sALP derived from the extracellular domain of ALP isozymes (e.g., TNALP, PALP, GCALP, IALP, etc.). Thus, similar to sTNALP discussed above, the present disclosure also provides other soluble human ALP isozymes, i.e., lacking the peptide signal, preferably comprising the extracellular domain of ALP. sALP also includes polypeptide sequences that satisfy a consensus sequence derived from the ALP extracellular domain of human ALP isozymes and mammalian TNALP orthologs (human, mouse, rat, bovine, cat, and dog), or a consensus sequence derived from the ALP extracellular domain of mammalian TNALP orthologs (human, mouse, rat, bovine, cat, and dog) only. sALP also includes those polypeptide sequences that satisfy similar consensus sequences derived from various combinations of these TNALP orthologs or human ALP isozymes. Such consensus sequences are given, for example, in WO 2008 / 138131.

[0213] The sALP of the present application can include not only the wild-type sequence of the sALPs 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 to 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 to the sequence of SEQ ID NO: 1, e.g., asfotase alfa). Examples of mutations that can be introduced into the ALP sequence are described in U.S. Pub. No. 2013 / 0323244, which is hereby incorporated by reference in its entirety. The sALP can optionally be glycosylated at any appropriate amino acid residue(s). Further, the sALP can 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 to any of the sALPs described herein, such as a 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. The sALP can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additions, deletions, or substitutions relative to any of the sALPs described herein, such as a 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.

[0214] sALP fusion polypeptides

[0215] Any sALP and linker described herein can be combined in a sALP polypeptide, such as a sALP polypeptide of the structure A-sALP-B, where each of A and B is absent, or is an amino acid sequence of at least one amino acid (such as a TNALP, e.g., a 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). When present, A and / or B can be any linker described herein. In some sALP polypeptides, A is absent, B is absent, or both A and B are absent. A sALP polypeptide can optionally comprise an Fc region to provide a sALP fusion polypeptide as described herein. A sALP polypeptide can optionally comprise a bone targeting moiety as described herein. In some sALP polypeptides, a linker, e.g., a flexible linker such as a dipeptide sequence (e.g., leucine-lysine or aspartate-isoleucine), can be included between the bone targeting moiety and the sALP. Additional exemplary Fc regions, linkers, and bone targeting moieties are described below.

[0216] Any sALP, linker, and Fc region described herein can be combined in a fusion polypeptide (e.g., a recombinant fusion polypeptide) of the structure Z-sALP-Y-spacer-X-W n -V, Z-W n -X-spacer-Y-sALP-V, Z-sALP-Y-W n -X-spacer-V and Z-W n -X-sALP-Y-spacer-V (such as a TNALP, e.g., a 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). In particular, the structure can be Z-sALP-Y-spacer-X-W n -V or Z-W n -X-spacer-Y-sALP-V. The sALP can be a full-length or functional fragment of an ALP, such as a soluble extracellular domain of an ALP as described herein (e.g., TNALP, PALP, GCALP, and IALP). Any of X, Y, Z, and V and / or the spacer can be absent, or is an amino acid sequence of at least one amino acid. W nmay be a bone-targeting moiety, e.g., having a series of consecutive Asp or Glu residues, where n = 1 to 50, e.g., n = 3 to 30, e.g., 5 to 15, e.g., 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, e.g., at or near the N- or C-terminus, and / or in the linker region. For example, the bone-targeting moiety is located at the C-terminus. The sALP polypeptide and fusion polypeptide can also lack a bone-targeting moiety.

[0217] The sALP fusion polypeptide can have the structure hTNALP-Fc-D 10 . In particular, the sALP fusion polypeptide can comprise the amino acid sequence 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.

[0218] The sALP fusion polypeptide can have a negatively charged polypeptide comprising 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 aspartate or glutamate residues. The negatively charged peptide can consist of or include at least one of D 10 , D 16 , E 10 , and E 16 .

[0219] Useful spacers include, but are not limited to, polypeptides comprising Fc, and are capable of mitigating the effects of having a highly negatively charged peptide at the terminus (e.g., W n) and flexible polypeptides that cause repulsion. For example, the sALP can be a fusion polypeptide comprising an Fc region of an immunoglobulin in either the N-terminal or C-terminal domain. Immunoglobulin molecules have a structure well known in the art. They comprise two light chains (each about 23 kD) and two heavy chains (each about 50 to 70 kD) linked by interchain disulfide bonds. Immunoglobulins are readily cleaved by proteolysis (e.g., by papain cleavage) into Fab (containing the light chain and the VH and CHI domains of the heavy chain) and Fc (containing the CH2 and CH3 domains of the heavy chain, and the adjoining sequences). Useful Fc fragments as described herein include Fc fragments from any immunoglobulin molecule of any mammal (e.g., human), including IgG, IgM, IgA, IgD, or IgE, and the various subclasses thereof (e.g., IgG-1, IgG-2, IgG-3, IgG-4, IgA-1, IgA-2). For example, the Fc fragment is human IgG-1. The Fc fragment can include, for example, any portion of the CH2 and CH3 domains of the heavy chain and the hinge region. The Fc region can optionally be glycosylated at any appropriate amino acid residue or residues known to those of skill 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 to SEQ ID NO: 20. Engineered (e.g., non-naturally occurring) Fc regions can also be used (see, e.g., International Application Publication No. WO 2005 / 007809, which is hereby incorporated by reference). The Fc fragment as described herein can 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 relative to any Fc fragment described herein.

[0220] The sALP fusion polypeptides described herein, 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, can comprise a peptide linker region between the Fc fragments. In addition, a peptide linker region can be comprised between the Fc fragments and the optional bone targeting moiety. The linker region can have any sequence and length that allows the sALP to retain biological activity, e.g., without steric hindrance. Exemplary linker lengths are 1 to 200 amino acid residues, e.g., 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, the linker comprises or consists of a flexible portion, e.g., a region that does not have a significant fixed secondary or tertiary structure. An exemplary flexible linker is a glycine-rich linker, e.g., containing at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% glycine residues. The linker can also contain, e.g., serine residues. In some cases, the amino acid sequence of the linker consists only of glycine and serine residues. The linker can optionally be glycosylated at any appropriate amino acid residue(s). In addition, the linker as described herein can comprise any other sequence or moiety attached covalently or non-covalently. The linker can also be absent, where the Fc fragments are fused directly together with the sALP without intervening residues. According to the present disclosure, certain Fc-sALP or sALP-Fc fusion polypeptides can be considered 1) to have no linker, or 2) to have a linker corresponding to a portion of the sALP. For example, an Fc fused directly to hsTNALP (1-502), e.g., where hsTNALP is amino acids 1-502, can be considered to have no linker, or to have a linker of 17 amino acids, e.g., where hsTNALP (18-502).

[0221] sALP and sALP fusion polypeptides, 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, can associate into dimers or tetramers. For example, two sALP-Fc monomers can be covalently linked by two disulfide bonds located in the hinge region of the Fc fragment. Additionally, the polypeptides or fusion polypeptides, e.g., sALP polypeptides or fusion polypeptides, can be glycosylated or pegylated.

[0222] Production of nucleic acids and polypeptides

[0223] Nucleic acids encoding sALP and sALP fusion polypeptides, 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, can be produced by any method known in the art. Typically, a nucleic acid encoding the desired fusion polypeptide is generated using molecular cloning methods, and the nucleic acid is typically placed within a vector, such as a plasmid or virus. The vector is used to transform the nucleic acid into a host cell suitable for expression of the fusion polypeptide. Representative methods are disclosed, for example, in Maniatis et al. (Cold Springs Harbor Laboratory, 1989). Although mammalian cells are preferred (because they are capable of imparting appropriate post-translational modifications), many cell types can also be used as suitable host cells. Host cells of the present application can include, for example, Chinese hamster ovary (CHO) cells, L cells, C127 cells, 3T3 cells, BHK cells, COS-7 cells, or any other suitable host cell known in the art. For example, the host cell is a Chinese hamster ovary (CHO) cell (e.g., a CHO-DG44 cell).

[0224] sALP and sALP fusion polypeptides, 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, can be produced under any conditions suitable for effecting expression of the sALP polypeptide in a host cell. Such conditions include a medium prepared with appropriate selected components such as buffers, bicarbonate and / or HEPES; ions such as chloride, phosphate, calcium, sodium, potassium, magnesium, iron; carbon sources such as monosaccharides; amino acids, lipids, nucleotides, vitamins, and growth factors such as insulin, as can be present; conventional, commercially available media such as a-MEM, DMEM, Ham's-F12, and IMDM, supplemented with 2 mM to 4 mM L-glutamine and 5% fetal calf serum; conventional, commercially available animal protein-free media such as Hyclone F-12, IMDM, and RPMI-1640, supplemented with 2 mM to 4 mM L-glutamine and 5% fetal calf serum; and the like.™ SFM4CHO, Sigma CHO DHFR - Cambrex POWER ™ CHO CD, supplemented with 2 mM to 4 mM L-glutamine. These media are best prepared in the absence of thymidine, hypoxanthine, and L-glycine to maintain selective pressure and thus achieve stable expression of protein products.

[0225] Pharmaceutical compositions, formulations, and administration

[0226] Compositions of this disclosure containing sALP or sALP fusion peptides (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa) may be formulated by a variety of methods known in the art. As those skilled in the art will understand, routes of administration and / or modes may be used to prescribe formulations. Routes of administration may depend on a variety of factors, such as environment and therapeutic goals. Specifically, the peptides and fusion peptides described herein may be formulations for administration via any route known in the art, such as 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, capsule tablets, powders, gels, ointments, creams, mists, sprays, atomized vapors, aerosols, or phospholipid complexes.

[0227] Formulations

[0228] Compositions containing sALP and sALP fusion peptides (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa) can be formulated according to standard methods. Drug preparation is a well-established technique and is further described in, for example, the following literature: 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 Edition (ISBN: 091733096X). For example, sALP compositions (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as asfotase alfa) can be formulated, for example, as a buffer solution with a suitable concentration and are suitable for storage at 2°C to 8°C (e.g., 4°C). The compositions can also be formulated for storage at temperatures below 0°C (e.g., -20°C or -80°C). The compositions can also be formulated for storage at 2°C to 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, 1½ years or 2 years). Therefore, the compositions described herein can be stored stably at 2°C to 8°C (e.g., 4°C) for at least 1 year.

[0229] Compositions containing sALP and sALP fusion peptides (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1, or peptide variants having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa) can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. Preferred forms depend in part on the intended method of administration and therapeutic application.

[0230] For example, compositions intended for systemic or local delivery may be in the form of injectable or infusionable solutions. Thus, compositions such as TNALP, for example, the sALP polypeptide of SEQ ID NO: 1, or polypeptide variants having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as asfotase alfa, may be formulated for administration via parenteral routes (e.g., subcutaneous, intravenous, intraperitoneal, or intramuscular injection). As used herein, “parenteral administration,” “parenteral,” and other grammatically equivalent phrases refer to methods of administration other than enteral and local administration, typically by injection, and including but not limited to subcutaneous, intradermal, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, tracheal, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, intracerebral, intracranial, carotid, and intrasternal injections and infusions.

[0231] Compositions containing sALP and sALP fusion peptides (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1, or peptide variants having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa) can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for stable storage at high concentrations. Sterile injectable solutions can be prepared by incorporating the composition described herein in the desired amount with one or a combination of the ingredients listed above (as needed) into a suitable solvent, followed by sterilization by filtration. Dispersions are typically prepared by incorporating the composition described herein into a sterile medium containing a base dispersion medium and desired other ingredients from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preparation methods include vacuum drying and freeze-drying, which yield powders of the composition described herein with any additional desired ingredients (see below) from a previously sterile filtered solution. This can be achieved, for example, by using coatings (such as lecithin), by maintaining the desired particle size in the case of dispersions, and by maintaining the appropriate fluidity of the solution by using surfactants. Extended absorption of injectable compositions can be achieved by including agents that delay absorption (such as monostearate and gelatin) in the composition.

[0232] The compositions described herein can also be formulated into immunoliposome compositions. Such formulations can be prepared by methods known in the art, such as those described in the following literature: 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. Patent Nos. 4,485,045 and 4,544,545. Liposomes with increased cycle times are disclosed, for example, in U.S. Patent No. 5,013,556.

[0233] Compositions containing sALP and sALP fusion peptides (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1, or peptide variants having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa) may also be formulated with a carrier that protects the composition (e.g., the sALP peptide or sALP fusion peptide) from rapid release, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene-vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Many methods for preparing such formulations are known in the art. See, for example, JR Robinson (1978), “Sustained and Controlled Release Drug Delivery Systems,” Marcel Dekker, Inc., New York.

[0234] Compositions containing sALP or sALP fusion peptides (such as TNALP, for example the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as asfotase alfa) can be formulated as injection solutions that are clear, colorless to pale yellow aqueous solutions at pH 7.4. sALP or sALP peptides (such as TNALP, for example the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as asfotase alfa) 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 peptides (e.g., 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) can be formulated with a carrier such as sodium chloride and / or sodium phosphate (e.g., about 150 mM NaCl and / or about 25 mM sodium phosphate, particularly at a pH of about 7.4). Specifically, the composition can be formulated as a 40 mg / mL solution for injection, wherein each mL of solution contains 40 mg of sALP or sALP peptide (e.g., 0.3 mL solution and 12 mg sALP (40 mg / mL) per vial, 0.45 mL solution and 18 mg sALP (40 mg / mL) per vial, 0.7 mL solution and 28 mg sALP (40 mg / mL) per vial, or 1.0 mL solution and 40 mg asfotase alfa (40 mg / mL) per vial). sALP or sALP peptides (such as TNALP, for example the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as asfotase alfa) can be formulated as an injectable solution with a concentration of 100 mg / ml, wherein each 1 mL of solution contains 100 mg of sALP or sALP peptide (e.g., each vial contains 0.8 mL of solution and 80 mg of asfotase alfa (100 mg / ml)).

[0235] When a composition is used in combination with a second active agent, the composition may be formulated together with the second active agent, or the composition may be formulated separately from the second active agent formulation. For example, the respective pharmaceutical compositions may be mixed and administered together, for example, just before administration, or may be administered separately, for example, at the same or different times.

[0236] Compositions containing sALP and sALP fusion peptides (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa) may be formulated for administration to a subject, or, in the case of administration to a fetus, to a woman carrying the fetus, while performing intravenous immunoglobulin therapy (IVIG), plasma ablation, plasma exchange, or plasma exchange.

[0237] Carriers / vehicles

[0238] Formulations containing sALP or sALP fusion peptides (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa) can be combined with pharmaceutically acceptable sterile aqueous or non-aqueous solvents, suspensions, or emulsions and administered to subjects with or susceptible to myasthenia gravis. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, fish oils, and injectable organic esters. Aqueous carriers include water, water-alcohol solutions, emulsions, or suspensions, including saline and buffered medical parenteral media, including sodium chloride solutions, Ringer's dextran solutions, dextran plus sodium chloride solutions, lactose-containing Ringer's solutions, or non-volatile oils. For example, pharmaceutically acceptable carriers may include sodium chloride and / or sodium phosphate, wherein the composition comprises, for example, about 150 mM sodium chloride and / or about 25 mM sodium phosphate, pH 7.4.

[0239] Intravenous carriers may include fluids and nutritional supplements, electrolyte supplements, such as those based on Ringer's dextran, etc. Pharmaceutically acceptable salts may be included, such as inorganic acid salts, such as hydrochlorides, hydrobroms, phosphates, sulfates, etc.; and salts of organic acids, such as acetates, propionates, malonates, benzoates, etc. Additionally, adjunctive substances, such as wetting agents or emulsifiers, pH buffers, etc., may be present in such carriers. A detailed discussion of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0240] Dosage

[0241] The sALP peptides described herein (such as TNALP, for example, the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as asfotase alfa), and / or fusion proteins of SEQ ID NO: 21 or SEQ ID NO: 22, or fusion proteins of SEQ ID NO: 21 or SEQ ID NO: 22, are described herein. Variants of sequence 22 with at least 95% sequence identity may be administered, for example, in the range of 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 / 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 from 1 μg / kg to 1,000 μg / kg. Individual doses within the range (e.g., 5 μg / kg to 1,000 μg / kg, 1 μg / kg to 750 μg / kg, 5 μg / kg to 750 μg / kg, 10 μg / kg to 750 μg / kg, 1 μg / kg to 500 μg / kg, 5 μg / kg to 500 μg / kg, 10 μg / kg to 500 μg / kg, 1 μg / kg to 100 μg / kg, 5 μg / kg to 100 μg / kg, 10 μg / kg to 100 μg / kg, 1 μg / kg to 50 μg / kg, 5 μg / kg to 50 μg / kg, or 10 μg / kg to 50 μg / kg) are administered to subjects who have or are susceptible to myasthenia gravis.

[0242] Exemplary doses of sALP include, for example, 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 25 mg / kg, 50 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 200 mg / kg, and 250 mg / kg. g or 500 mg / kg; or 1 μg / kg, 2 μg / kg, 2.5 μg / kg, 5 μg / kg, 10 μg / kg, 20 μg / kg, 25 μg / kg, 50 μg / kg, 100 μg / kg, 125 μg / kg, 150 μg / kg, 200 μg / kg, 250 μg / kg, 500 μg / kg, 750 μg / kg, 900 μg / kg, or 1,000 μg / kg. For all doses or ranges described herein, the term “about” may be used to adjust these doses to ±10% of the endpoints of the stated values ​​or ranges. Specifically, the compositions according to this disclosure (e.g., those comprising sALP (such as TNALP, such as 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, such as asfotase alfa)) may be administered to subjects at doses ranging from about 0.001 mg / kg / day to about 500 mg / kg / day, from about 0.01 mg / kg / day to about 100 mg / kg / day, or from about 0.01 mg / kg / day to about 20 mg / kg / day). For example, a sALP composition (such as TNALP, such as the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as asfotase alfa) may be administered to a subject at a weekly dose, for example, from about 0.5 mg / kg / week to about 140 mg / kg / week, for example, from about 0.8 mg / kg / week to about 50 mg / kg / week, or from about 1 mg / kg / week to about 10 mg / kg / week (for example, from about 6 mg / kg / week or about 9 mg / kg / week). Specifically, sALP (such as TNALP, for example the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, such as asfotase alfa) can be administered at doses of 2 mg / kg three times a week (total dose of 6 mg / kg / week), 1 mg / kg six times a week (total dose of 6 mg / kg / week), 3 mg / kg three times a week (total dose of 9 mg / kg / week), 0.5 mg / kg three times a week (total dose of 1.5 mg / kg / week), or 9.3 mg / kg three times a week (total dose of 28 mg / kg / week).The dosage will be adjusted by the clinician based on routine factors such as the severity of the disease and different parameters from subjects who have or are susceptible to myasthenia gravis.

[0243] Compositions comprising sALP and sALP fusion peptides (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa), and / or fusion proteins of SEQ ID NO: 21 or SEQ ID NO: 22, or variants having at least 95% sequence identity with the sequence of SEQ ID NO: 21 or SEQ ID NO: 22) may be provided in single-dose or multi-dose regimens. Dosage may be administered at frequencies such as once per hour, once every two hours, once daily, once every two days, twice weekly, three times weekly, four times weekly, five times weekly, six times weekly, once weekly, once every two weeks, once monthly, once every two months, or once a year. Alternatively, dosage may be administered daily, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times. Specifically, the dosing regimen is once weekly. The duration of the dosing regimen can 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 throughout the remaining life of a subject with or susceptible to myasthenia gravis. The dosage, frequency, and duration will be adjusted by the clinician based on routine factors such as the severity of the disease and different parameters from subjects with or susceptible to myasthenia gravis.

[0244] For example, the recommended dose of sALP or sALP fusion peptides (such as TNALP, e.g., the sALP peptide of SEQ ID NO: 1 or a peptide variant having at least 95% sequence identity with the sequence of SEQ ID NO: 1, e.g., asfotase alfa) is 2 mg / kg body weight subcutaneously three times a week, or 1 mg / kg body weight subcutaneously six times a week. Additional dosage information is provided below (Table 1).

[0245] Table 1. Dosing of asfotase alfa

[0246]

[0247]

[0248] The following examples are intended to illustrate, not limit, this disclosure.

[0249] Examples

[0250] Example 1. Muscle hypotonia in subjects with HPP-like disease and not carrying loss-of- function mutations in ALPL Figure 1

[0251] Patient A was diagnosed with HPP at 5 years and 9 months of age and presented for evaluation due to decreased bone mineral density associated with severe hypotonia. She was found to have low alkaline phosphatase levels of 149 U / L (150-420), elevated PLP levels of 172.4 (20-125), and elevated urinary PEA levels of 190 mg / dL (0-106). Skeletal imaging showed normal wrist X-rays, but knee X-rays revealed decreased bone mineral density, thin bone, and tibial epiphyseal abnormalities. DXA scans showed low bone mineral density (Z-scores for the lumbar spine and the whole body excluding the head were -4.6 and -3.3, respectively). She had no fractures. ALPL gene sequencing, including deletion and duplicate studies, was performed, and no mutations were found in the ALPL gene.

[0252] Prior to the diagnosis of HPP, Patient A underwent extensive molecular testing to determine the cause of her severe hypotonia that had been present since infancy. During her first year of life, evaluations for spinal muscular atrophy, muscle biopsies, and neurological and electromyographic (EMG) studies failed to provide a specific explanation for her persistent hypotonia and muscle weakness. SMA gene testing did not identify any disease-causing mutations. Muscle biopsy revealed chronic progressive myopathy with a characteristic feature of uneven fiber types. EMG interpretation suggested a possible mild abnormality in neuromuscular transmission. Brain MRI showed a reduction / hypoplasia in the lower cerebellar vermis, accompanied by mild bilateral cerebellar hypovolemia.

[0253] Extensive genetic testing was subsequently conducted, but none provided a clear explanation for patient A's complex clinical presentation. A comprehensive mitochondrial DNA (mtDNA) analysis was performed at Baylor College of Medicine, identifying a rare variant of undetermined significance (m.1836A>G) in the MTRNR2 gene. A coenzyme Q10 deficiency analysis was also performed at Baylor College of Medicine, identifying a rare variant of undetermined significance (c.1665G>A) in the ADCK3 gene. Whole-exome sequencing (WES) on GeneDx identified two variants, but neither could explain her phenotype. One variant was a known ADCK3 variant previously identified by Baylor's coenzyme Q10 deficiency analysis. Although this variant could be pathogenic, it is associated with autosomal recessive CoQ10 deficiency. This was not a significant explanation, as patient A only carried the maternal mutation. The second variant was a paternally inherited variant in the ATP1A3 gene. This variant (c.357+1G>A) was classified as potentially pathogenic, but her father carried the same variant and was unaffected. Therefore, it can be seen that the father's unaffected state proves that carrying the c.357+1G>A variant in the ATP1A3 gene is not the cause of her clinical phenotype. The c.357+1G>A variant is located between exons 4 and 5 and may affect the splicing of these two exons. The mother does not carry this variant and shows clear splicing connections between exons 4 and 5 relative to other family members. Other family members show varying degrees of "penetration" between exons 4 and 5, with patient A showing the highest number of reads mapped to the intron between exons 4 and 5, indicating lower splicing efficiency of that intron. Example 2. Quantification of ALPL transcription and translation However, ATP1A3 is expressed at very low levels in PBMCs and maps to fewer reads of the affected intron.

[0254] Figure 2A

[0255] Subsequently, RNA sequencing of ALPL transcripts and Western blot analysis of ALPL protein were performed to detect the ALPL gene expression impairment caused by ALPL transcriptional dysregulation in patient A at the RNA and / or protein levels. 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 2B Western blot analysis was used to identify the translation of ALPL. Analysis confirmed that TNAP protein was produced in patient A, as well as in patient A's mother, father, and three siblings. Example 3. Quantification of ALPL translation Confirmatory and quantitative analyses using positive controls will be performed.

[0256] Example 4. Treatment of muscle hypotonia with asfotase alfa

[0257] Western blot analysis of ALPL can be performed using a sample from patient A to determine whether the ALPL protein is being produced at normal levels and with the correct molecular weight. Even if patient A exhibits normal transcript levels, the presence of protein at abnormal concentrations suggests potential inadequate ALPL translation.

[0258] Subsequently, patient A, along with her parents and three siblings, can be analyzed to obtain a complete genetic profile. The study will include obtaining informed consent (for minors), collecting blood samples (20 mL) from patient A, her parents, and her three siblings for Western blot analysis, RT-PCR analysis, or RNA sequencing of ALPL transcripts and Western blot analysis of ALPL protein. Additionally, blood samples (7 mL) collected from the parents and siblings will be used to measure PLP, vitamin D2+D3, comprehensive metabolomics, and urinary PEA. A medical history questionnaire and anthropometry review will also be conducted.

[0259] Example 5. Treatment can resolve muscle weakness issues, as measured by reduced SRC, independent of bone mineralization status

[0260] A 12-year-old female with chromosomal duplication 22q11.21q11.22, developmental delay, autism spectrum disorder (Asperger's syndrome), extreme fatigue, and nocturnal awakenings due to pain (shoulder, back, and legs), along with clinical manifestations consistent with HPP (low ALP, elevated PLP and PEA, and decreased bone mineralization), was initially diagnosed with HPP. It was later discovered that the patient did not carry the ALPL mutation. She was started receiving STRESIQ. ® Over the course of several months, the female patient's strength, agility, and endurance improved. Her 6MWT score improved from 320 meters to 597 meters (the normal range for her age / sex is 672 ± 55). Overall, she showed reduced pain (a score of 2 out of 10 at this follow-up visit, instead of 5 out of 10 at the previous follow-up visit) and improved mobility.

[0261] Table 2: Fusion protein comparison results Table 3: SEQ ID NO: 1 (726 amino acids)

[0262] Experiments were conducted to analyze the effects of the fusion protein on myasthenia gravis, as measured by reduced SRC, independent of bone mineralization status. For the context, fusion proteins from two variants of SEQ ID NO: 1, including SEQ ID NO: 21 and SEQ ID NO: 22, were considered. Comparisons of the three fusion protein structures are shown in Table 2, and the exact sequences are described in Tables 3, 4, and 5. Additional data on treatment with the fusion protein of SEQ ID NO: 21 are included below.

[0263] Table 4: SEQ ID NO: 21 (724 amino acids) E108M, N213Q, N286Q

[0264]

[0265] Table 5: SEQ ID NO: 22 (721 amino acids) E108M, IgG2 / 4, D7

[0266]

[0267] Figure 3

[0268]

[0269] Figure 3

[0270]

[0271] Figure 4 Representative oxygen consumption rate (OCR) curves, normalized to the number of extensor digitorum longus (EDL) skeletal muscle fibers, were plotted in female HPP mice and WT female littermates at 20 days of age. The results indicate that HPP mice may lack reserve respiratory capacity in their EDL muscle fibers compared to age- and sex-matched WT mice. These data suggest that a reduced rate of mitochondrial energy production contributes to muscle weakness in HPP mice. Table 6: Percent of spare respiratory capacity (SRC) benchmarked to WT mice The abbreviations are: ATP = adenosine triphosphate; EDL = extensor digitorum longus; FCCP = carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; HPP = hypophosphatase; OCR = oxygen consumption rate; WT = wild type.

[0272] In control mice treated with PBS and HPP mice treated with the fusion protein of SEQ ID NO: 21, the percentage of standby respiratory capacity (SRC) based on WT mice was evaluated comparatively. Example 6. SEQ ID NO: 1 restores pyridoxal phosphate (PLP)-dependent BCAT activity, enabling production of adenosine triphosphate from branched chain amino acid transaminases (and Table 6). The abbreviations in Table 6 are: HPP = hypophosphatase; PBS = phosphate-buffered saline; SRC = reserve respiratory capacity; WT = wild type.

[0273] Figure 5

[0274]

[0275] Compared with HPP mice treated with PBS alone, HPP mice treated with the fusion protein of SEQ ID NO: 21 showed a significant increase in skeletal radiculoneuronic acid (SRC) as a percentage of WT mice (using a one-tailed Mann-Whitney test, p = 0.0008). These data suggest that treatment with the fusion protein of SEQ ID NO: 21 improves mitochondrial bioenergetics in skeletal muscle fibers of HPP mice. Bone phenotype analysis of PBS control mice showed that bone was unaffected in 2 out of 9 HPP mice (22%), although these mice had SRCs below 50% of matched WT mice. HPP mice may have myasthenia gravis, as measured by the reduced SRC, which is independent of bone mineralization status. These data suggest that HPP patients with myasthenia gravis but no skeletal symptoms may benefit from treatment with the fusion protein of SEQ ID NO: 21. Treatment with the fusion protein of SEQ ID NO: 21 improved the bone phenotype in all but 3 out of 19 HPP mice (84%). These three mice received 8, 9, or 13 doses, which is fewer than the 18 doses of the 2 mg / kg SEQ ID NO: 21 fusion protein required to restore 94% of the bone mineralization phenotype in HPP mice to normal.

[0276] Based on data from Example 5, a method for supporting the treatment of myasthenia gravis in subjects suffering from or at risk of developing myasthenia gravis, or for reducing the risk of such subjects developing myasthenia gravis, was discovered. This method comprises administering to the subject a therapeutically effective amount of at least one recombinant polypeptide with alkaline phosphatase activity, wherein the subject exhibits reduced tissue nonspecific alkaline phosphatase (TNSALP) concentrations and wherein the subject does not carry a loss-of-function mutation in the ALPL gene. In a specific embodiment, the method comprises a fusion protein containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 21 or SQ ID NO: 22 (or wherein the recombinant polypeptide comprises a fusion protein containing the amino acids of SEQ ID NO: 21 or SQ ID NO: 22). In a specific embodiment, the recombinant polypeptide of the method comprises an immunoglobulin molecule that is an IgG2 / 4 crystallizable fragment (Fc) region, and the Fc contains the amino acid sequence of SEQ ID NO: 21. In a specific embodiment, the therapeutically effective amount is administered once every two weeks and may comprise an amount from about 0.1 mg / kg to about 20 mg / kg. In a specific implementation, according to the method of claim 66, the myasthenic diseases include: muscular dystrophy, myasthenia gravis and calcium pyrophosphate deposition disease (CPPD), amyotrophic lateral sclerosis (ALS), myositis, myotonic dystrophy, myotonia, Guillain-Barré syndrome, Duchenne muscular dystrophy (DMD), and Lambert-Eaton myasthenic syndrome. By applying this method, mitochondrial bioenergy in skeletal muscle fibers can be improved.

[0277] Figure 6 Figure 7

[0278] In the absence of sequential treatment with SEQ ID NO: 1, insufficient intracellular pyridoxal phosphate (PLP) prevents the PLP-dependent branched-chain amino acid transferase (BCAT) from catabolizing BCAAs into branched-chain keto acids (BCKA), resulting in elevated levels of branched-chain amino acids (BCAAs) (valine, leucine, and isoleucine) in the skeletal muscle of HPP mice (TNSALP knockout). (BCKA is catabolized in a series of sequential steps to produce acetyl-CoA or succinyl-CoA, which can enter the mitochondrial tricarboxylic acid (TCA) cycle and be converted to adenosine triphosphate (ATP) via oxidative phosphorylation of adenosine diphosphate (ADP). This pathway is illustrated in...) Figure 8 middle.

[0279] Treatment of HPP mice with SEQ ID NO: 1 restored PLP-dependent BCAT activity, resulting in normal BCAA levels in skeletal muscle (making BCAA available for energy production). This result is shown in... Other embodiments, ​ and ​ In the study, continuous treatment with SEQ ID NO:1 restored BCAA levels in the skeletal muscle of HPP mice to normal (with no significant difference compared to wild-type mice), indirectly demonstrating the restoration of PLP-dependent BCAT activity, thus enabling ATP production from BCAA. In contrast, interruption (discontinuation) of SEQ ID NO:1 treatment in TNSALP knockout mice significantly increased valine, leucine, and isoleucine levels in tibial muscle compared to wild-type mice, indirectly demonstrating reduced PLP-dependent BCAT activity, thereby limiting ATP production from BCAA.

[0280] ​

[0281] All publications, patents, and patent applications mentioned in the foregoing description are incorporated herein by reference to the extent that each individual publication, patent, or patent application is specifically and individually cited in its entirety. Various modifications and variations to the described methods, pharmaceutical compositions, and kits will be apparent to those skilled in the art without departing from the scope and spirit of the claimed invention. Although this disclosure has been described in conjunction with specific embodiments, it should be understood that further modifications are possible and the claimed invention should not be unduly limited to such specific embodiments.

Claims

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

2. The method according to claim 1, wherein the decrease in TNSALP concentration is caused by reduced ALPL transcription, reduced ALPL mRNA translation, increased or decreased TNSALP post-translational modifications, or decreased TNSALP enzyme activity.

3. The method according to claim 1 or 2, wherein reduced ALPL transcription, reduced ALPL mRNA translation, increased or decreased TNSALP post-translational modification, or decreased TNSALP enzyme activity are measured relative to normal subjects.

4. The method according to any one of claims 1 to 3, wherein the subject carries 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 carries 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 of claim 5, wherein the subject carries a mutation in ATP1A3.

7. The method of claim 5 or 6, wherein the subject carries a mutation in ADCK3.

8. The method according to any one of claims 1 to 7, wherein the muscles of the subject are not significantly different from the muscles of a normal subject without the myasthenia gravis in terms of at least one property selected from the ratio of muscle fiber types and fiber contractile properties.

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

10. The method according to any one of claims 1 to 9, wherein the myasthenic disease 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 pyrophosphate (PPi) concentration, wherein optionally, the myasthenic disease is caused by the elevated PPi concentration.

12. The method of claim 11, wherein the elevated pyrophosphate concentration (PPi) exacerbates the subject's muscle weakness.

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

14. The method according to any one of claims 1 to 13, wherein the recombinant polypeptide may be 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 of 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 the following: tissue nonspecific alkaline phosphatase (TNALP), placental alkaline phosphatase (PALP), germ cell alkaline phosphatase (GCALP), intestinal alkaline phosphatase (IALP), and their biological functional fragments, fusions, or chimeric constructs.

18. The method of 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 1-485 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 of claim 21, wherein the immunoglobulin molecule is a crystallizable fragment region (Fc).

23. The method of claim 22, wherein the Fc comprises the amino acid sequence of SEQ 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 method of claim 24, wherein the negatively charged peptide comprises one to fifty negatively charged amino acids, such as aspartic acid or glutamic acid, wherein optionally, the negatively charged peptide is D. 10 D 16 E 10 and E 16 At least one of them.

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-interval region-XW n -V, sALP is the extracellular domain of the alkaline phosphatase. V is absent, or it is an amino acid sequence of at least one amino acid; X is absent, or it is an amino acid sequence of at least one amino acid; Y is absent, or it is an amino acid sequence of at least one amino acid; Z is absent, or it is an amino acid sequence of at least one amino acid; and W n It is polyaspartic acid or polyglutamic acid, where n = 10 to 16.

27. The method of claim 26, wherein the spacer region comprises a crystallizable fragment region (Fc).

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

20.

29. The method of claim 27 or 28, wherein the recombinant polypeptide comprises sALP-Fc-D 10 The structure.

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

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

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

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

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

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

36. The method according to any one of claims 1 to 35, wherein prior to administration of the recombinant polypeptide, the subject is characterized by having an average walking distance of about 350 meters or less in six minutes.

37. The method according to any one of claims 1 to 36, wherein administration of the recombinant polypeptide causes the subject to increase the average walking distance by at least 100 meters or more within six minutes.

38. The method according to any one of claims 1 to 37, wherein the subject exhibits an average walking distance of about 500 meters or longer within six minutes after administration of the recombinant peptide.

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

40. The method of claim 39, wherein the assistive mobility device is at least one of a walker, wheelchair, brace, cane, and orthosis.

41. The method according to any one of claims 1 to 40, wherein the subject is characterized by having a plasma PPi concentration of about 4.5 μM or higher prior to administration of the recombinant polypeptide.

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

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. The method according to any one of claims 1 to 43, wherein: i) The subjects are aged from 0 to 14 days and are characterized by having a plasma ALP concentration of about 90 U / L or lower prior to administration of the recombinant polypeptide; ii) The subject is 15 days to less than 1 year old and is characterized by having a plasma ALP concentration of about 134 U / L or less prior to administration of the recombinant polypeptide; iii) The subject is about 1 year old to less than 10 years old and is characterized by having a plasma ALP concentration of about 156 U / L or less prior to administration of the recombinant polypeptide; iv) The subjects are about 10 to about 13 years old and are characterized by having a plasma ALP concentration of about 141 U / L or less prior to administration of the recombinant polypeptide; v) The subjects are females aged about 13 to about 15 years, and are characterized by having a plasma ALP concentration of about 62 U / L or lower prior to administration of the recombinant polypeptide; vi) The subject is a male aged approximately 13 to approximately 15 years, and is characterized by having a plasma ALP concentration of approximately 127 U / L or lower prior to administration of the recombinant polypeptide; vii) The subjects were women aged about 15 to about 17 years, and were characterized by having a plasma ALP concentration of about 54 U / L or lower prior to administration of the recombinant polypeptide; viii) The subject is a male aged about 15 to about 17 years, and is characterized by having a plasma ALP concentration of about 89 U / L or less prior to administration of the recombinant polypeptide; (ix) The subject is approximately 17 years of age or older and, prior to administration of the recombinant peptide, is characterized by having a plasma ALP concentration of approximately 48 U / L or lower; or x) The subject is about 17 years of age or older and is characterized by having a plasma ALP concentration of about 59 U / L or lower prior to administration of the recombinant polypeptide.

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

46. ​​The method according to any one of claims 1 to 45, wherein: i) The subjects are aged from 0 to 14 days and, after administration of the recombinant polypeptide, are characterized by having a plasma ALP concentration of about 273 U / L or higher; ii) The subject is 15 days to less than 1 year old and is characterized by having a plasma ALP concentration of about 518 U / L or higher after administration of the recombinant polypeptide; iii) The subject is about 1 year old to less than about 10 years old, and is characterized by having a plasma ALP concentration of about 369 U / L or higher after administration of the recombinant polypeptide; iv) The subjects are about 10 to about 13 years old and, after administration of the recombinant polypeptide, are characterized by having a plasma ALP concentration of about 460 U / L or higher; v) The subjects are females aged about 13 to about 15 years, and are characterized by having a plasma ALP concentration of about 280 U / L or higher after administration of the recombinant polypeptide; vi) The subject is a male aged about 13 to about 15 years, and is characterized by having a plasma ALP concentration of about 517 U / L or higher after administration of the recombinant polypeptide; vii) The subjects were females aged about 15 to about 17 years, and were characterized by having a plasma ALP concentration of about 128 U / L or higher after administration of the recombinant polypeptide; viii) The subjects are males aged about 15 to about 17 years, and are characterized by having a plasma ALP concentration of about 365 U / L or higher after administration of the recombinant polypeptide; (ix) The subject is a woman aged approximately 17 years or older, and is characterized by having a plasma ALP concentration of approximately 95 U / L or higher after administration of the recombinant polypeptide; or x) The subject is a male aged about 17 years or older, and is characterized by having a plasma ALP concentration of about 164 U / L or higher after administration of the recombinant polypeptide.

47. The method according to any one of claims 1 to 46, wherein prior to administration of the recombinant polypeptide, the subject is characterized by having a mean Bunies Motor Skills Test Version 2 strength score of about 10 or lower.

48. The method of claim 47, wherein prior to administration of the recombinant polypeptide, the subject is characterized by having an average BOT-2 running speed and agility score of about 5 or lower.

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

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

51. The method according to any one of claims 1 to 50, wherein prior to administration of the recombinant polypeptide, the subject is characterized by having a mean Child Health Assessment Questionnaire (CHAQ) score of about 0.8 or higher.

52. The method according to any one of claims 1 to 51, wherein the administration of the recombinant polypeptide results in the subject having an average CHAQ score of about 0.5 or lower.

53. The method according to any one of claims 1 to 52, wherein prior to administration of the recombinant polypeptide, the subject is characterized by having a mean Pediatric Outcome Data Collection Tool (PODCI) score of about 40 or lower.

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

55. The method according to any one of claims 1 to 54, wherein prior to administration of the recombinant polypeptide, the subject is characterized by having an average muscle strength grade of less than about 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, wherein prior to administration of the recombinant polypeptide, the subject is characterized by having an average HHD value that is less than about 80% of the predicted handheld force gauge (HHD) value.

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

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

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

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

22.

62. The method of claim 61, wherein the recombinant polypeptide comprises a fusion protein comprising 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 contains an immunoglobulin molecule, which is the crystallizable fragment (Fc) region of IgG2 / 4, and the Fc contains the amino acid sequence of SEQ ID NO:

21.

64. The method of claim 61, wherein the therapeutically effective amount is administered once every two weeks.

65. The method of claim 61, wherein the therapeutically effective amount of the administration is from about 0.1 mg / kg to about 20 mg / kg.

66. The method of claim 65, wherein myasthenia gravis comprises: Muscular dystrophy, myasthenia gravis and calcium pyrophosphate deposition disease (CPPD), amyotrophic lateral sclerosis (ALS), myositis, myotonic dystrophy, myotonia, Guillain-Barré syndrome, Duchenne muscular dystrophy (DMD), and Lambert-Eaton myasthenic syndrome.

67. The method of claim 61, wherein the application improves mitochondrial bioenergy in skeletal muscle fibers.

68. The method of claim 1, wherein the treatment with the therapeutically effective amount of the at least one recombinant polypeptide having the alkaline phosphatase activity reduces the levels of valine, leucine, or isoleucine in the muscle of the subject.

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