WO2024092238A1 - Klotho polypeptide or polynucleotide for improving cognition - Google Patents

Klotho polypeptide or polynucleotide for improving cognition Download PDF

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Publication number
WO2024092238A1
WO2024092238A1 PCT/US2023/078094 US2023078094W WO2024092238A1 WO 2024092238 A1 WO2024092238 A1 WO 2024092238A1 US 2023078094 W US2023078094 W US 2023078094W WO 2024092238 A1 WO2024092238 A1 WO 2024092238A1
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dose
concentrations
polypeptide
fold
administered
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PCT/US2023/078094
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French (fr)
Inventor
Yan Poon
Kenneth Greenberg
Nathaniel David
Dena DUBAL
Stacy CASTNER
Graham Williams
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Unity Biotechnology, Inc.
The Regents Of The University Of California
Yale University
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Publication of WO2024092238A1 publication Critical patent/WO2024092238A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01031—Beta-glucuronidase (3.2.1.31)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered

Definitions

  • Brain health is one of the biggest biomedical challenges with few if any effective medical treatments. Cognition is a highly valued and central manifestation of brain health that is impaired or becomes disrupted in normal aging, numerous neurodegenerative, neurologic, and psychiatric diseases, childhood developmental syndromes, traumatic brain injury, and stress. Cognition is also disrupted by jet lag, medication side effects, and certain medical treatments, such as those for cancer. Thus, the potential to enhance cognition or counter cognitive dysfunction is of enormous relevance across the human lifespan in health and disease and unmet need remains.
  • the method comprises improving cognition in an individual comprising administering to the individual an effective amount of a protein comprising a Klotho polypeptide or a functional variant or fragment thereof, thereby improving cognitive function in the individual.
  • this disclosure features a method of increasing cognitive function in a human subject, comprising: parenterally administering to said subject at least one dose of a polypeptide, or a polynucleotide that is capable of expressing a polypeptide, that includes at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations by at least 2.5 fold, and no more than 25-fold, compared to pre-administration (baseline) concentrations.
  • KL1 domain of human klotho KL
  • this disclosure features a method of increasing cognitive function in a human subject, comprising: parenterally administering to said subject at least one dose of a polypeptide, or a polynucleotide that is capable of expressing a polypeptide, that includes at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations to at least 1000 pg/mL and no more than 5000 pg/mL.
  • KL human klotho
  • this disclosure features a method of increasing cognitive function in a human subject, comprising: parenterally administering to said subject a plurality of doses of a polypeptide, or a polynucleotide that is capable of expressing a polypeptide, that includes at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations by at least 2.5 fold, and no more than 25-fold, compared to pre-administration (baseline) concentrations, and wherein the plurality of doses is administered on an intermittent dosing schedule.
  • KL1 domain of human klotho KL
  • this disclosure features a method of increasing cognitive function in a human subject, comprising: parenterally administering to said subject a plurality of doses of a polypeptide, or a polynucleotide that is capable of expressing a polypeptide, that includes at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations to at least 1000 pg/mL and no more than 5000 pg/mL, and wherein the plurality of doses is administered on an intermittent dosing schedule.
  • KL1 domain of human klotho KL
  • FIG. 1A illustrates the experimental protocol of hippocampal LTP recordings from young male mice treated with either Vehicle (Veh) or Rhesus KL (10 ⁇ g/kg, s.c.).
  • FIG. 1C shows average fEPSP slope of the last 10 minutes of each recording for mice treated with Veh or Rhesus KL. **p ⁇ 0.01 (two-tailed t-test).
  • FIG. 1H illustrates the experimental protocol for measuring KL levels in the serum of aged rhesus macaques 4 hrs following treatment with Veh or varying doses of Rhesus KL (0.4 ⁇ g/kg, 2 ⁇ g/kg, 10 ⁇ g/kg, 20 ⁇ g/kg, or 30 ⁇ g/kg, s.c.).
  • FIG. II shows the relative KL serum levels of aged rhesus macaques at baseline and 4 hrs following injection with Veh or varying doses of Rhesus KL.
  • FIG. 2B is a diagram of the spatial delayed task.
  • (Top) Aged rhesus macaques were shown a food reward, a visible cue. A screen was lowered between the monkey and the wells and the wells were covered. (Bottom) Following a delay, the screen was raised and each monkey attempted to remember the spatial location of the hidden food reward. The task tested normal memory load (NML) with 4-7 wells present and a high memory load (HML) with more wells (6-9) present.
  • NML normal memory load
  • HML high memory load
  • FIG. 2D presents the percent correct choices made by monkeys in the HML task 4 hrs and 14 days after a single injection with Veh or Rhesus KL (10 ⁇ g/kg).
  • the dashed line represents mean performance and shaded gray represents range of performance in Veh-treated monkeys.
  • *p ⁇ 0.05 vs Veh linear mixed model ANOVA and Satterthwaite’s t-tests).
  • FIG. 2F shows the percent correct choices made by monkeys in the NML between 4h and 14 days after a single injection with Veh or Rhesus KL (10 ⁇ g/kg).
  • the dashed line represents mean performance and shaded gray represents range of performance in Veh-treated monkeys.
  • FIG. 2H graphs the percent increase in cognition in the NML task, compared to Veh- treatment, at varying doses of Rhesus KL treatment.
  • *p ⁇ 0.05 vs Veh; # 0.057 vs Veh (linear mixed model ANOVA and Satterthwaite’s t-tests).
  • the term “cognitive function” refers to at least one observable mental task or function.
  • the mental task or function is selected from a memory task or function (e.g., a semantic, episodic, procedural, priming, or working memory task or function); an orientation task or function; a language task or function; a problem solving task or function; a visual perception task or function; a construction task or function; an integration task or function; a planning task or function; an organizational task or function; selective attention; inhibitory control; or ability to mentally manipulate information.
  • cognitive function is measured by a clinical test (also known as a clinical instrument).
  • a given condition e.g., treatment with klotho
  • cognition absent the condition e.g., absent treatment with klotho
  • an improvement in cognition might be a reduction in the rate of cognitive decline (i.e., an improvement compared to the absence of treatment), but not an actual improvement in cognitive ability.
  • An increase in cognitive function can also be an increase in brain activity in a specified area, e.g., as determined by MRI, or an inhibition of brain activity that results in better overall cognitive function.
  • An increase in cognitive function can also be improvement in a cognitive performance test as described in more detail herein.
  • An improvement or increase in cognitive function can be in any one cognitive function, or any combination of individual cognitive functions.
  • the terms “therapy,” “treatment,” and “amelioration” refer to any reduction in the severity of symptoms (cognitive decline), or improvement in cognitive function, or where motor function is affected, an improvement in motor function.
  • the terms “treat” and “prevent” are not intended to be absolute terms.
  • Treatment and prevention can refer to any delay in cognitive decline, amelioration of symptoms (e.g., confusion, delirium), etc.
  • Treatment and prevention can be complete or partial, such that cognition is better than would have been expected without treatment (e.g., compared to cognition in the same individual before treatment or compared to cognition in similar non-treated individuals).
  • the effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment.
  • the terms “effective amount,” “effective dose,” “therapeutically effective amount,” etc. refer to that amount of the therapeutic agent sufficient to ameliorate a disorder, as described above.
  • a therapeutically effective amount will show a reduction in the severity of symptoms of at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.
  • Therapeutic efficacy can also be expressed as “-fold” increase or decrease.
  • a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.
  • Subject “Subject,” “patient,” “individual” and like terms are used interchangeably and refer to, except where indicated, mammals such as humans and non-human primates, as well as dogs, horses, pigs, mice, rats, and other mammalian species.
  • mammals such as humans and non-human primates, as well as dogs, horses, pigs, mice, rats, and other mammalian species.
  • the term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical supervision.
  • a patient can be an individual who is seeking treatment, monitoring, adjustment or modification of an existing therapeutic regimen, etc.
  • a method for improving cognitive function in a human subject.
  • the method comprises: parenterally administering to said subject at least one dose of a polypeptide, or a polynucleotide that is capable of expressing a polypeptide, that includes at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations by at least 2.5 fold, and no more than 25-fold, compared to pre-administration (baseline) concentrations.
  • KL1 domain of human klotho KL
  • the dose is sufficient to raise serum KL concentrations by at least
  • the dose is sufficient to raise serum KL concentrations by at least
  • the dose is sufficient to raise serum KL concentrations by at least 7.5-fold compared to baseline concentrations. In some embodiments, the dose is sufficient to raise serum KL concentrations by at least 10-fold compared to baseline concentrations.
  • the dose is sufficient to raise serum KL concentrations by no more than 25-fold, no more than 24.5-fold, no more than 24-fold, no more than 23.5-fold, no more than 23-fold, no more than 22.5-fold, no more than 22-fold, 21.5-fold, no more than 21-fold, no more than 20.5-fold, no more than 20-fold, no more than 19.5-fold, no more than 19-fold, no more than 18.5-fold, no more than 18-fold, no more than 17.5-fold, no more than 17-fold, no more than 16.5-fold, no more than 16-fold, no more than 15.5-fold, no more than 15-fold, no more than 14.5-fold, no more than 14-fold, no more than 13.5-fold, no more than 13-fold, no more than 12.5-fold, no more than 12-fold, no more than 11.5-fold, no more than 11-fold, no more than 10.5-fold, no more than 10-fold, no more than 9.5-fold,
  • the dose is sufficient to raise serum KL concentrations by no more than 20-fold compared to baseline concentrations. In some embodiments, the dose is sufficient to raise serum KL concentrations by no more than 15-fold compared to baseline concentrations. In some embodiments, the dose is sufficient to raise serum KL concentrations by no more than 10- fold compared to baseline concentrations.
  • the dose is sufficient to raise serum KL concentrations by about 2.5-fold to about 10-fold. In some embodiments, the dose is sufficient to raise serum KL concentrations by about 5-fold to about 7.5-fold compared to baseline concentrations..
  • a method for improving cognitive function in a human subject comprises parenterally administering to said subject at least one dose of a polypeptide, or a polynucleotide that is capable of expressing a polypeptide, that includes at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations to at least 1000 pg/mL and no more than 5000 pg/mL.
  • KL human klotho
  • the dose is sufficient to raise serum KL concentrations to at least 1000 pg/mL, to at least 1500 pg/mL, to at least 2000 pg/mL, to at least 2500 pg/mL, to at least 3000 pg/mL, to at least 3500 pg/mL, to at least 4000 pg/mL, or to at least 4500 pg/mL.
  • the dose is sufficient to raise serum KL concentrations to at least 1000 pg/mL. In some embodiments, the dose is sufficient to raise serum KL concentrations to at least 1500 pg/mL. In some embodiments, the dose is sufficient to raise serum KL concentrations to at least 2000 pg/mL. In some embodiments, the dose is sufficient to raise serum KL concentrations to at least 2500 pg/mL. In some embodiments, the dose is sufficient to raise serum KL concentrations to at least 3000 pg/mL. In some embodiments, the dose is sufficient to raise serum KL concentrations to at least 3500 pg/mL. In some embodiments, the dose is sufficient to raise serum KL concentrations to no more than 4000 pg/mL.
  • the dose is sufficient to raise serum KL concentration to no more than 5000 pg/mL, to no more than 4500 pg/mL, to no more than, pg/mL, to no more than 4000 pg/mL, to no more than 3500 pg/mL, to no more than 3000 pg/mL, to no more than 2500 pg/mL, to no more than 2000 pg/mL, to no more than 1500 pg/mL, or to no more than 1000 pg/mL.
  • the dose is sufficient to raise serum KL concentrations to no more than 3000 pg/mL. In some embodiments, the dose is sufficient to raise serum KL concentrations to no more than 2500 pg/mL.
  • cognition is improved by at least 1%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some embodiments, cognition is improved by at least 2, 3, 5, 7, 10, 15, 20, 25%, 50%, 75%, 80%, or 90%, or more, determined using tests of cognition, molecular proxies, or structural changes associated with brain function.
  • motor function is improved by at least 1%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some embodiments, motor function is improved by at least 2, 3, 5, 7, 10, 15, 20, 25%, 50%, 75%, 80%, or 90%, or more, determined using tests of motor function. 7.3. Dose and Dose Schedule
  • a method for improving cognitive function in a human subject comprises: parenterally administering to said subject a plurality of doses of a polypeptide, or a polynucleotide that is capable of expressing a polypeptide, that includes at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations by at least 2.5 fold, and no more than 25-fold, compared to preadministration (baseline) concentrations, and wherein the plurality of doses is administered on an intermittent dosing schedule.
  • KL1 domain of human klotho KL
  • this disclosure features a method of increasing cognitive function in a human subject, comprising: parenterally administering to said subject a plurality of doses of a polypeptide, or a polynucleotide that is capable of expressing a polypeptide, that includes at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations to at least 1000 pg/mL and no more than 5000 pg/mL, and wherein the plurality of doses is administered on an intermittent dosing schedule.
  • KL1 domain of human klotho KL
  • successive doses are administered no less frequently than once every 6 months and no more frequently than once per day.
  • successive doses are administered no less frequently than once every 3 months. In some embodiments, successive doses are administered no less frequently than once a month.
  • successive doses are administered no less frequently than once every four weeks. In some embodiments, successive doses are administered no less frequently than once every other week. In some embodiments, successive doses are administered no less frequently than once a week.
  • successive doses are administered no more frequently than once a week. In some embodiments, successive doses are administered no more frequently than one every two weeks. In some embodiments, successive doses are administered no more frequently than once every four weeks. [0053] In some embodiments, successive doses are administered no more frequently than once a month. In some embodiments, successive doses are administered once a day. In some embodiments, successive doses are administered once a week. In some embodiments, successive doses are administered once every two weeks. In some embodiments, successive doses are administered once every four weeks.
  • the dose is a polypeptide administered subcutaneously (s.c.).
  • the dose is at least 5 ⁇ g/kg s.c. and no more than 50 ⁇ g/kg s.c (e.g., at least 5 ⁇ g/kg s.c. and no more than 45 ⁇ g/kg s.c., at least 5 ⁇ g/kg s.c. and no more than 40 ⁇ g/kg s.c., at least 5 ⁇ g/kg s.c. and no more than 35 ⁇ g/kg s.c., at least 5 ⁇ g/kg s.c. and no more than 30 ⁇ g/kg s.c., at least 5 ⁇ g/kg s.c.
  • ⁇ g/kg s.c. at least 10 ⁇ g/kg s.c. and no more than 35 ⁇ g/kg s.c., at least 10 ⁇ g/kg s.c. and no more than 30 ⁇ g/kg s.c., at least 10 ⁇ g/kg s.c. and no more than 25 ⁇ g/kg s.c., at least 10 ⁇ g/kg s.c. and no more than 20 ⁇ g/kg s.c., at least 10 ⁇ g/kg s.c. and no more than 15 ⁇ g/kg s.c., at least 15 ⁇ g/kg s.c.
  • the dose is at least 5 gg/kg s.c. (e.g., at least 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, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 gg/kg s.c.)
  • the dose is at least 7.5 gg/kg s.c.
  • the dose is at least 10 gg/kg s.c.
  • the dose is at least 15 gg/kg s.c.
  • the dose is at least 20 gg/kg s.c.
  • the dose is at least 25 gg/kg s.c.
  • the dose is no more than 40 gg/kg s.c. (e.g., no more than 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 gg/kg s.c.). In some embodiments, the dose is no more than 30 gg/kg s.c. In some embodiments, the dose is no more than 25 gg/kg s.c. In some embodiments, the dose is no more than 20 gg/kg s.c. In some embodiments, the dose is no more than 15 gg/kg s.c. In some embodiments, the dose is no more than 10 gg/kg s.c. In some embodiments, the dose is no more than 5 gg/kg s.c.
  • the Klotho polypeptide can be administered at the initial dosage of at least 5 gg/kg s.c. and no more than 50 gg/kg s.c. daily (or other intermittent period (e.g., every other day, every third day, every fourth day, every fifth day, every sixth day, once per week, once every two weeks, once every three weeks, and once a month)) and adjusted over time.
  • a daily dose range of about 10 gg/kg to about 40 gg/kg, or about 20 gg/kg to about 30 gg/kg can be used.
  • the dosage is varied depending upon the requirements of the patient, the severity of the condition being treated, and the route of administration.
  • the effective dose is typically in the range of 5-50 gg/kg, while for direct delivery to the central nervous system (CNS), the effective dosage is lower, e.g., 5-30 gg/kg.
  • the effective dose is higher, e.g., in the range of 50-10,000 gg/kg (e.g., 100 gg/kg-2 mg/kg). The dose is chosen in order to provide effective therapy for the patient.
  • the dose may be repeated at an appropriate frequency which may be in the range of once or twice per day, once or twice per week to once every three months, depending on the pharmacokinetics of the Klotho polypeptide composition (e.g., half-life in the circulation) and the pharmacodynamic response (e.g., the duration of the therapeutic effect).
  • an appropriate frequency may be in the range of once or twice per day, once or twice per week to once every three months, depending on the pharmacokinetics of the Klotho polypeptide composition (e.g., half-life in the circulation) and the pharmacodynamic response (e.g., the duration of the therapeutic effect).
  • the dose is at least 350 ⁇ g and no more than 3500 ⁇ g (e.g., at least 350 ⁇ g and no more than 3000 ⁇ g, at least 350 ⁇ g and no more than 2500 ⁇ g, at least 350 ⁇ g and no more than 2000 ⁇ g, at least 350 ⁇ g and no more than 1500 ⁇ g, at least 350 ⁇ g and no more than 1000 ⁇ g, at least 350 ⁇ g and no more than 750 ⁇ g, at least 350 ⁇ g and no more than 500, at least 500 ⁇ g and no more than 3500 ⁇ g, at least 500 ⁇ g and no more than 3000 ⁇ g, at least 500 ⁇ g and no more than 2500 ⁇ g, at least 500 ⁇ g and no more than 2000 ⁇ g, at least 500 ⁇ g and no more than 1500 ⁇ g, at least 500 ⁇ g and no more than 1000 ⁇ g, at least 500 ⁇ g and no more than 750 ⁇ g, at least 750 ⁇ g and
  • the dose is at least 500 ⁇ g (e.g., at least 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, or 3400 ⁇ g).
  • the dose is at least 750 ⁇ g.
  • the dose is at least 1000 ⁇ g.
  • the dose is at least 1500 ⁇ g.
  • the dose is at least 2000 ⁇ g.
  • the dose is at least 2500 ⁇ g.
  • the dose is at least 3000 ⁇ g.
  • the dose is no more than 3000 ⁇ g (e.g., no more than 2900, 2800, 2700, 2600, 2500, 2400, 2300, 2200. 2100, 2000, 1900, 1800, 1700, 1600, 1500. 1400, 1300. 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 ⁇ g).
  • the dose is no more than 2500 ⁇ g.
  • the dose is no more than 2000 ⁇ g.
  • the dose is no more than 1500 ⁇ g.
  • the dose is no more than 1000 ⁇ g.
  • the Klotho polypeptide can be administered at the initial dosage of at least 350 ⁇ g and no more than 3500 ⁇ g daily (or other intermittent period (e.g., every other day, every third day, every fourth day, every fifth day, every sixth day, once per week, once every two weeks, once every three weeks, and once a month)) and adjusted over time.
  • the dosage is varied depending upon the requirements of the patient, the severity of the condition being treated, and the route of administration.
  • the effective dose is typically in the range of 350-3500 ⁇ g, while for direct delivery to the central nervous system (CNS). the effective dosage is lower, e.g., 350-2000 ⁇ g/kg.
  • the effective dose is higher, e.g., in the range of 50-10,000 ⁇ g.
  • the dose is chosen in order to provide effective therapy for the patient.
  • the dose may be repeated at an appropriate frequency which may be in the range of once or twice per day, once or twice per week to once every three months, depending on the pharmacokinetics of the Klotho polypeptide composition (e.g., half-life in the circulation) and the pharmacodynamic response (e.g., the duration of the therapeutic effect).
  • Dosages can be empirically determined considering the type and severity of cognitive condition diagnosed in a particular patient.
  • the dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time.
  • the size of the dose will also be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of any particular composition in a particular patient, as will be recognized by the skilled practitioner.
  • additional administration is dependent on patient progress, e.g., the patient is monitored between administrations.
  • the patient can be monitored for cognitive ability or for side effects, e.g., weakness, dizziness, nausea, etc.
  • the individual has a chronic condition, so that klotho is administered over an indefinite period, e.g., for the lifetime of the patient. In such cases, administration is typically periodic.
  • Diseases that are considered long-term or chronic include, but are not limited to Alzheimer's disease, Parkinson's disease, Huntington's disease, and cognitive decline associated with hypertension and heart disease.
  • the subject is a human.
  • the subject, prior to first administration has normal cognitive function.
  • the human subject has at least normal cognitive function and the administering results in improved cognitive function compared to before the administering.
  • the subject, prior to first administration has cognitive impairment.
  • the subject, prior to first administration has mild cognitive impairment (MCI).
  • MCI mild cognitive impairment
  • the human subject has impaired cognitive function and the administering results in improved cognitive function compared to before the administering.
  • Klotho polypeptides are used to prevent or reduce cognitive decline associated with aging, for example, in individuals 45 years of age or older, or upon initial signs of cognitive decline.
  • the patient is older than 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 years of age.
  • the patient is older than 76, 77, 78, 79, 80, 81.
  • the patient is 40-50 years old, 50-60 years old, 52-62 years old, 63-70 years old, 60-70 years old, 70-80 years old, or 80-90 years old.
  • the patient is 51, 52. 53. 54. 55. 56. 57. 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69. 70. 71. 72. 73.
  • the subject is at least 21 years old. In some embodiments, the subject is at least 45 years old. In some embodiments, the subject is at least 60 year's old. In some embodiments, the subject is at least 65 years old. In some embodiments, the subject is at least 80 years old.
  • the subject is under 21 years old. [0071] In some embodiments, the subject has at least one ApoE4 allele.
  • the subject prior to first administration, has dementia.
  • the subject has Alzheimer’s disease, frontotemporal dementia, Lewy Body Dementia, Parkinson's disease, Huntington's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasalar degeneration, vascular cognitive impairment, vascular dementia, amyotropic lateral sclerosis, prion disorder, posterior cortical atrophy, primary progressive aphasia HIV-related dementia, global aphasia, mixed transcortical aphasia, Broca’s aphasia, transcortical motor aphasia, Wernicke’s aphasia, transcortical sensory aphasia, conduction aphasia, anomic aphasia, multiple system atrophy; mental or mood disorders: depression, schizophrenia, attention deficit/ hyperactivity disorder, autism spectrum disorder, intellectual disability, mood disorder, psychotic disorder, psychosis; childhood neurodevelopment al syndromes and brain tumors: X-linked mental disability or
  • klotho polypeptides can also be administered to provide increased cognition for individuals desiring improved cognition (e.g., individuals exposed to stress, or sleep deprivation), or for individuals requiring superior cognitive function.
  • the klotho polypeptide composition can be administered 2-24 hours before the desired effect, which can last about 3-5 days for working memory and about 2 weeks for spatial memory.
  • Klotho polypeptides are be used to treat individuals with age-related, non-age related, or disease related conditions including, but not limited to: neurodegenerative diseases, mental or mood disorders, childhood neurodevelopmental syndromes and brain tumors, genetic syndromes affecting learning, metabolic disorders affecting cognition, cognitive decline associated with chemotherapy and/or radiation therapy, and conditions with impaired motor functions.
  • Non-limiting examples of neurodegenerative diseases and dementia that can be treated by administering to a subject a klotho polypeptide (and functional variants and fragments thereof) include: Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasalar degeneration, mild cognitive impairment, vascular- dementia, Lewy body dementia, amyotropic lateral sclerosis, prion disorder, and HIV-related dementia.
  • Non-limiting examples of mental or mood disorders that can be treated by administering to a subject a klotho polypeptide (and functional variants and fragments thereof) include: depression, schizophrenia, attention deficit/hyperactivity disorder, autism spectrum disorder, intellectual disability, a mood disorder, and a psychotic disorder.
  • Non-limiting examples of childhood neurodevelopmental syndromes and brain tumors that can be treated by administering to a subject a klotho polypeptide (and functional variants and fragments thereof) include: X-linked mental disability or retardation, astrocytoma, ependymoma, medulloblastoma, and oligodendroglioma.
  • Non-limiting examples of genetic syndromes affecting learning that can be treated by administering to a subject a klotho polypeptide (and functional variants and fragments thereof) include: Down's syndrome, Angelman's syndrome, and Rett's syndrome.
  • Non-limiting examples of metabolic disorders affecting cognition include: phenylketonuria, Lesch-Nyhan, galactosemia, and adrenoleukodystrophy.
  • cognitive decline associated with chemotherapy and/or radiation therapy can be treated by administering to a subject a klotho polypeptide (and functional variants and fragments thereof).
  • the Klotho polypeptide composition is administered to an (e.g., human) individual having impaired motor function.
  • the individual has stroke to the brain or spinal cord (ischemic or hemorrhagic), neurodegenerative disease (Parkinson's disease, Lewy body dementia, multiple system atrophy, amyotropic lateral sclerosis, prion disorder, Huntington's disease, supranuclear palsy), Parkinsonism, traumatic brain injury, neuroinfectious brain lesions, multiple sclerosis and related autoimmune and demyelinating disease, spinal cord lesions (compressive, infectious, toxic or metabolic, autoimmune, oncologic), brain tumor, epilepsy, paraneoplastic disorder, neurodevelopmental disorder (mitochondrial, autosomal genetic), muscle disease (polymyositis, dermatomyositis, inclusion body myositis, infectious, endocrine, metabolic, toxic, congenital myopathy, congential muscular dystrophy, her
  • Non-limiting examples of additional conditions and disorders that can be treated by administering to a subject a klotho polypeptide (and functional variants and fragments thereof) include::: pain-associated cognitive effects, traumatic brain injury, stroke, multiple sclerosis, neuroautoimmune disease, epilepsy, delirium, paraneoplastic disorder, developmental delay, and leukodystrophies.
  • Cognitive ability can be measured using any method known in the art, e.g., for testing memory, language ability, executive functions, visuospatial function, dementia, or multiparameter neuropsychological abilities.
  • Klotho administration results in at least a 1%, 2%, 5%, 7%, 10%, 15%, 20%. 30%. 40%, 50%, 60%, 70%. 80%. 90%, 100% or greater improvement in score on a standard cognitive ability test (e.g., measured 1-3 days after administration).
  • the testing is carried out more than once for an individual, e.g., one or more time over the course of treatment with Klotho.
  • Standard tests for memory and learning can be applied, e.g., to determine semantic, episodic, procedural, priming, and/or working (i.e., short term) memory.
  • Common tests include Cambridge prospective memory test (CAMPROMPT), memory assessment scales (MAS), Rey auditory verbal learning test, Rivermead behavioral memory test, Test of memory and learning (TOMAL), Wechsler memory scale (WMS), and Test of memory malingering (TOMM).
  • Tests for language functions include, e.g., Boston Diagnostic Aphasia Examination (BDAE), Comprehensive aphasia test (CAT), and Multilingual aphasia examination (MAE).
  • Executive function e.g., problem solving, planning, organization, inhibitory control
  • BADS Behavioral assessment of dysexecutive syndrome
  • CNS vital signs Brief Core Battery
  • COW AT Controlled oral word association test
  • D-KEFS Delis-Kaplan Executive Function System
  • Digit vigilance test Kaplan Baycrest neurocognitive assessment (KBNA), Hayling and Brixton tests
  • TOVA Tests of variables of attention
  • WST Wisconsin card sorting test
  • TAA Test of everyday attention
  • Visuospatial ability e.g., visual perception, construction and integration
  • VOT Hooper visual organization task
  • Rey-Osterrieth complex figure tests Dementia can be quantified using the clinical dementia rating or dementia rating scale.
  • Multi-parameter tests for neuropsychological function include but are not limited to the Barcelona neuropsychological test (BNT), Cambridge neuropsychological test automated battery (CANTAB), Cognistat, Cognitive assessment screening instrument (CAST), Cognitive function scanner (CFS), Dean-Woodcock neuropsychology assessment system (DWNAS), General practitional assessment of cognition (GPCOG) Mini mental state examination (MMSE), NEPSY, or the CDR computerized assessment system.
  • BNT Barcelona neuropsychological test
  • CANTAB Cambridge neuropsychological test automated battery
  • Cognistat Cognitive assessment screening instrument
  • CAST Cognitive assessment screening instrument
  • CFS Cognitive function scanner
  • DWNAS Dean-Woodcock neuropsychology assessment system
  • GPCG General practitional assessment of cognition
  • MMSE Mini mental state examination
  • NEPSY or the CDR computerized assessment system.
  • cognition can be determined using structural or molecular proxies for cognitive activity, e.g., compared over time to detect changes.
  • Cognitive changes can be detected, e.g., by observing changes to brain structure, connectivity, activation, inhibition, or synaptic plasticity, e.g., by MRI, fMRI, EEG, TMS and TES, and/or any combination of these.
  • brain activity is observed.
  • Klotho administration results in a 1.5-fold, 2-fold, 5-fold, 7-fold, 10-fold, or greater increase in brain activity (e.g., measured 1-3 days after administration).
  • Molecular proxies for improved cognition include, but arc not limited to: increased levels of GluN2B, increased GluN2B synaptic localization, increased NMDA receptor activation, and/or increased c-fos activation in the brain. These measures are particularly relevant to cognition.
  • Such method can include, e.g., obtaining a sample of neuronal tissue or CSF from an individual and using standard assays to determine gene expression or activation.
  • the method further comprises monitoring the individual for cognitive ability, either through a molecular proxy (e.g., changes NMDA receptor or c-fos activation, or GluN2B levels in the brain), changes in MRI brain scans (e.g., functional MRI), changes in EEG, changes in TMS and TES, changes in neuropsychologic test scores, or tests of cognitive ability (e.g., for learning, short or long term memory, executive functions, language ability, and visuospatial function).
  • the individual is monitored using more than one of the above tests in any combination.
  • the dose of the Klotho polypeptide for each administration is determined based on the therapeutic progress of the individual, e.g., where a higher dose is administered if the individual is not responding sufficiently to therapy.
  • administration results in improved motor function.
  • Changes in motor function can be assayed as known in the art.
  • Exemplary motor function assays include but are not limited to electromyogram and nerve conduction studies, direct or device-assisted clinical testing of strength, tone, and muscle bulk, reflex examination, coordination examination, and gait analysis.
  • Assays for testing etiologies causing deficits of motor function include but are not limited to magnetic resonance imaging of the central nervous system, muscle biopsy, nerve biopsy, and laboratory studies.
  • Klotho is a single-pass type 1 membrane protein.
  • Residues 1-33 (underlined) constitute the signal sequence; residues 34-981 are extracellular; 982-1002 are transmembrane, and 1003- 1012 are cytoplasmic.
  • the signal sequence is cleaved and the mature full-length Klotho protein has the following sequence: [0095] Mature full length soluble Klotho includes two conserved domains (KL1 and KL2) with homology to beta glycosidase proteins. The KL1 domain consists of residues 34-549 (numbering relative to full length protein inclusive of the signal sequence):
  • glycosyl hydrolase-1 domain 1 comprises residues 57-506 (numbering relative to full length protein inclusive of the signal sequence): and glycosyl hydrolase-1 domain 2 comprises residues 515-953 (numbering relative to the full length protein inclusive of the signal sequence):
  • klotho polypeptides that can be used for administration to a subject in the methods described herein include species homologs (e.g., non-human primate, mouse, rat), allelic variants, functional fragments, and functional variants of the wild type sequence that retain cognition improving activity.
  • Examples include secreted Klotho, fragments comprising the KL1 domain, fragments comprising the KL2 domain, fragments comprising the KL1 and KL2 domains, variants comprising the KL1 domain with at least one (e.g., 1-20, 5-50, 25-100) non-conserved amino acid in the KL1 domain substituted with a different amino acid or deleted, variants comprising the KL2 domain with at least one non-conserved amino acid in the KL2 domain substituted with a different amino acid.
  • variants comprising the KL1 domain with at least one (e.g., 1-20, 5-50, 25-100) non-conserved amino acid in the KL1 domain substituted with a different amino acid or deleted variants comprising the KL2 domain with at least one non-conserved amino acid in the KL2 domain substituted with a different amino acid.
  • functional fragments of the Klotho polypeptide that can be used for administration to a subject as described herein include the extracellular domain (e.g., corresponding to or substantially identical or similar to amino acids 34-981 of human Klotho (e.g., corresponding to amino acid residues 34-981 or SEQ ID NO: 1)), secreted Klotho (e.g., corresponding to or substantially identical or similar to 549 amino acid form (e.g., corresponding to amino acids residues of 1-549 of SEQ ID NO: 1)), a KL1 domain (e.g., corresponding to or substantially identical or similar to amino acids 34-549 of human Klotho (e.g., corresponding to amino acid residues 34-549 of SEQ ID NO: 1)), a glycosyl hydrolase consensus sequence (e.g., corresponding to or substantially identical or similar to amino acids 57-506 of human Klotho (e.g., corresponding to amino acid residues 57-506 of SEQ ID NO: 1)),
  • the klotho polypeptide has at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 1. In some embodiments, the klotho polypeptide has at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 2.
  • the klotho polypeptide has at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 3. In some embodiments, the klotho polypeptide has at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 4.
  • the Klotho polypeptide has at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to amino acids 34-981 of SEQ ID NO: 1.
  • the polypeptide, that includes at least the klotho (KL) protein has at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to:
  • Residues 1-19 constitute the signal sequence.
  • the human klotho polypeptide has the sequence of SEQ ID NO: 6.
  • the klotho polypeptide is as described in U.S. Patent No.
  • the Klotho polypeptide is part of a larger fusion protein.
  • the fusion protein comprises the Klotho polypeptide as described herein and further comprises no more than 100, 75, 50, or 30 additional amino acids.
  • the Klotho polypeptide e.g., any of the human klotho polypeptides described herein
  • FGF Fibroblast growth factor
  • the Klotho polypeptide is not a fusion protein that also contains a fibroblast growth factor (FGF).
  • the Klotho polypeptide comprises (e.g., is fused to) an affinity tag (e.g., a histidine tag) or a conjugate to increase stability or half-life in vivo.
  • an affinity tag e.g., a histidine tag
  • the Klotho polypeptide is linked to a stabilizing moiety such as PEG, glycosylation, or a liposome or other nanocarrier.
  • a stabilizing moiety such as PEG, glycosylation, or a liposome or other nanocarrier.
  • the Klotho polypeptide is linked to a liposome.
  • liposomes containing phosphatidyl-ethanolamine (PE) can be prepared by established procedures as described herein. The inclusion of PE provides an active functional site on the liposomal surface for attachment.
  • the Klotho polypeptide is linked to an affinity tag, e.g., a histidine tag (e.g., 4-16 histidine residues), streptavidin, or an antibody target.
  • an affinity tag e.g., a histidine tag (e.g., 4-16 histidine residues), streptavidin, or an antibody target.
  • the Klotho polypeptide is glycosylated. In some embodiments, the polypeptide is N-glycosylated. In some embodiments, the polypeptide is O-glycosylated. In some embodiments, the polypeptide is both N-glycosylated and O-glycosylated.
  • a functional variant or fragment of a Klotho polypeptide is a variant or fragment that retains any klotho activity, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%. or 100% of the level of any activity of soluble klotho.
  • Soluble klotho activities include those described above, and include binding FGF-23, binding to FGFRlc, betaglucuronidase activity, suppression of wnt signaling, suppression of insulin signaling, suppression of TFG-beta 1 activity, increasing GluN2B expression and/or synaptic localization, c-fos induction.
  • Additional Klotho activities include causing changes in magnetic resonance imaging (MRI) brain scans, e.g., functional MRI, electroencephalograph (EEG), and transcranial magnetic and electrical stimulation (TMS and TES); and improved performance in neuropsychologic testing and cognitive ability. 7.7. Formulations and administration of Klotho
  • MRI magnetic resonance imaging
  • EEG electroencephalograph
  • TMS and TES transcranial magnetic and electrical stimulation
  • the klotho polypeptide is administered as described in U.S. Patent No. 10,300,117, which is herein incorporated by reference in its entirety.
  • the Klotho polypeptide is administered in a pharmaceutical composition with a physiologically (i.e., pharmaceutically) acceptable carrier.
  • a physiologically (i.e., pharmaceutically) acceptable carrier refers to a typically inert substance used as a diluent or vehicle for a diagnostic or therapeutic agent. The term also encompasses a typically inert substance that imparts cohesive qualities to the composition.
  • Physiologically acceptable carriers can be liquid, e.g., physiological saline, phosphate buffer, normal buffered saline (135-150 mM NaCl), water, buffered water, 0.4% saline, 0.3% glycine, glycoproteins to provide enhanced stability (e.g., albumin, lipoprotein, globulin, etc.), and the like. Since physiologically acceptable carriers are determined in part by the particular composition being administered as well as by the particular method used to administer the composition, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (See, e.g.. Remington's Pharmaceutical Sciences, 17th ed., 1989).
  • the compositions can be sterilized by conventional, well-known sterilization techniques or may be produced under sterile conditions.
  • Aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • the compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.
  • Sugars can also be included for stabilizing the compositions, such as a stabilizer for lyophilized antibody compositions.
  • the Klotho polypeptide is formulated as a dosage forms prepared for mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous, intravenous, intramuscular-, or intraarterial injection, either bolus or infusion), oral, or transdermal administration to a patient.
  • mucosal e.g., nasal, sublingual, vaginal, buccal, or rectal
  • parenteral e.g., subcutaneous, intravenous, intramuscular-, or intraarterial injection, either bolus or infusion
  • oral or transdermal administration to a patient.
  • dosage forms include, but are not limited to: dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.
  • suspensions e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or a water-in-
  • the Klotho polypeptide is formulated for injection.
  • injectable compositions can comprise a solution of the Klotho polypeptide suspended in an acceptable carrier, such as an aqueous carrier.
  • an acceptable carrier such as an aqueous carrier.
  • aqueous carriers e.g., water, buffered water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, 5% dextrose, and the like, and may include glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc.
  • normal buffered saline (135-150 mM NaCl) is used.
  • compositions can contain pharmaceutically acceptable auxiliary substances to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Injection solutions and suspensions can also be prepared from sterile powders, granules, and tablets.
  • the composition is administered by intravenous infusion, topically, intraperitoneally, intravesically, or intrathecally.
  • the Klotho polypeptide formulation can be provided in unit-dose or multi-dose scaled containers, such as ampoules and vials.
  • the Klotho polypeptide composition alone or in combination with other suitable components, can be made into aerosol formulations (“nebulized”) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, and nitrogen.
  • the pharmaceutical composition (e.g., the klotho polypeptide and a carrier) can be packaged or prepared in unit dosage form.
  • the preparation is subdivided into unit doses containing appropriate quantities of the active component, e.g., according to the dose of Klotho polypeptide.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation.
  • the composition can, if desired, also contain other compatible therapeutic agents.
  • the Klotho polypeptide composition can be formulated in a kit for administration.
  • a pharmaceutical composition comprising a klotho polypeptide is administered orally. In some embodiments, a pharmaceutical composition comprising a klotho polypeptide is administered mucosally, e.g., nasally. In some embodiments, a pharmaceutical composition comprising a klotho polypeptide is administered by injection, e.g., subcutaneous, intraperitoneal, intravenous, or intramuscular. In some embodiments, a pharmaceutical composition comprising a klotho polypeptide is administered by infusion, e.g., using a reservoir or osmotic minipump.
  • An example of administration of a pharmaceutical composition includes storing the Klotho polypeptide at 10 mg/ml in sterile isotonic aqueous saline solution at 4° C., and diluting it in an appropriate solution for injection prior to administration to the patient.
  • the Klotho polypeptide composition can be administered by intravenous infusion over the course of 0.25-2 hours.
  • the administration procedure is via bolus injection.
  • the Klotho polypeptide composition is administered to an (e.g., human) individual having at least normal cognitive function.
  • an individual receiving the Klotho polypeptide composition begins initially with at least normal cognition and following administration of the Klotho polypeptide composition attains improved cognition compared to the initial level of cognition.
  • the level of cognition of an individual can be determined as is known in the art. Normal cognitive functions are determined by scores from sets of cognitive tests that are compiled into global cognitive scores, as described in Dubai D B et al.
  • cognition tests include tests of executive function and working memory such as Trails A and Trails B (Dubai D B et al. (2014) Cell Reports 7: 1065-1076).
  • administration of Klotho results in an improvement of cognition (whether initially at least normal or impaired), by at least 5%, 10%, 20% or more.
  • the Klotho polypeptide can also be formulated as a sustained-release preparation (e.g., in a semi-permeable matrices of solid hydrophobic polymers (e.g., polyesters, hydrogels (for example, poly (2-hydroxyethyl-methacrylate), or poly (vinylalcohol)), polylactides.
  • solid hydrophobic polymers e.g., polyesters, hydrogels (for example, poly (2-hydroxyethyl-methacrylate), or poly (vinylalcohol)
  • the Klotho polypeptide can be entrapped in a nanoparticle prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxy methylcellulose or gelatin microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • this disclosure features a method of increasing cognitive function in a human subject, comprising: parenterally administering to said subject at least one dose of a polynucleotide that is capable of expressing at least the KL1 domain of human klotho (KL) (, wherein each dose is sufficient to raise serum KL concentrations by at least 2.5 fold, and no more than 25-fold, compared to pre-administration (baseline) concentrations.
  • a polynucleotide that is capable of expressing at least the KL1 domain of human klotho (KL) (, wherein each dose is sufficient to raise serum KL concentrations by at least 2.5 fold, and no more than 25-fold, compared to pre-administration (baseline) concentrations.
  • this disclosure features a method of increasing cognitive function in a human subject, comprising: parenterally administering to said subject at least one dose of a polynucleotide that is capable of expressing at least the KL1 domain of human klotho (KL) (, wherein each dose is sufficient to raise serum KL concentrations to at least 1000 ⁇ g/mL and no more than 5000 pg/mL.
  • KL human klotho
  • this disclosure features a method of increasing cognitive function in a human subject, comprising: parenterally administering to said subject a plurality of doses of a polynucleotide that is capable of expressing at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations by at least 2.5 fold, and no more than 25-fold, compared to pre-administration (baseline) concentrations, and wherein the plurality of doses is administered on an intermittent dosing schedule.
  • KL1 domain of human klotho KL
  • this disclosure features a method of increasing cognitive function in a human subject, comprising: parenterally administering to said subject a plurality of doses of a polynucleotide that is capable of expressing at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations to at least 1000 pg/mL and no more than 5000 pg/mL, and wherein the plurality of doses is administered on an intermittent dosing schedule.
  • KL1 domain of human klotho KL
  • the polynucleotide is administered subcutaneously.
  • the polynucleotide is an mRNA.
  • the mRNA comprises: a) at least one 5’cap structure; b) a 5’UTR (untranslated region); c) an mRNA open reading frame encoding the polypeptide (e.g., any of the klotho polypeptides or klotho domains described herein); d) a 3’UTR (untranslated region); and e) a poly-A region of at least 100 nucleotides in length.
  • the dose is administered in a pharmaceutical formulation that further comprises a pharmaceutically acceptable carrier for the polynucleotide.
  • the formulation comprises a lipid nanoparticle, polymer, liposome, dendrimer, exosomes, or a lipid.
  • the polynucleotide is an expression vector.
  • the expression vector is a viral expression vector.
  • the viral expression vector is a retroviral vector, a lentiviral vector, an AAV vector, or an adenoviral vector.
  • the viral expression vector is an AAV vector.
  • the viral expression vector is a retroviral vector.
  • the viral expression vector is a lentiviral vector.
  • the viral expression vector is engineered to preferentially transduce at least one target tissue following parenteral administration.
  • the at least one target tissue is liver, muscle, or central nervous system (CNS) cells.
  • the at least one target tissue is CNS cells.
  • the viral expression vector crosses the blood brain barrier following parenteral administration.
  • Rhesus macaque was selected as a model system because of their phylogenetic similarity to humans, demonstrated complex higher-order cognitive functions, and the propensity to undergo targeted functional decline of similar brain regions such, as the prefrontal cortex, as do aged humans.
  • the primary goal was to test if a Klotho (KL) dose could be found that could enhance cognition in rhesus macaque monkeys.
  • KL a Klotho
  • the secondary goal was to explore higher KL doses in rhesus macaques to test whether KL-mediated benefits on cognition could be dose-dependent.
  • Klotho 20 ⁇ g/kg and 30 ⁇ g/kg
  • klotho-mediated cognitive enhancement demonstrate klotho-mediated cognitive enhancement in NHPs who have a complex genetic, anatomic, and functional brain similar to humans, and that the dose response is complex, with a previously unknown effective dose window.
  • mice were on a congenic C57BL/6J background and kept on a 12 hour light/dark cycle with ad libitum access to food and water.
  • the standard housing group was five mice per cage. Behavior tasks were carried out during the light cycle. All studies were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco, and conducted in compliance with NIH guidelines.
  • Adult aged male and female rhesus macaque monkeys (Macaca mulatta) were maintained in accordance with the Yale University Institutional Animal Care and Use Committee and federal guidelines for the care and use of nonhuman primates. Monkeys were maintained in a 12 hour light/dark cycle, fed standard monkey chow and fruit, and were tested during the light cycle.
  • Mouse KL, and rhesus KL were diluted in PBS (pH 7.5) and injected subcutaneous (s.c.) at specified doses prior to behavior testing or sample collection for serum KL measurements.
  • Recombinant proteins were used within one week of thawing from -80°C stock solutions and stored at 4°C. Proteins were coded to keep experimenters blind to the identity of treatment used in experiments for all mouse studies and for the majority of monkey studies performed.
  • Enzyme-linked immunosorbent assay was performed to measure KL from monkey serum samples, according to the manufacturer’s directions as described in Dubai et al. (Cell Rep 7, 1065-1076, doi: 10.1016/j.celrep.2014.03.076 (2014)) and Yokoyama et al. (Ann. Clin. Transl. Neurol. 2, 215-230, doi: 10.1002/acn3.161 (2015)), each of which are herein incorporated by reference in their entireties. Briefly, each monkey serum sample was diluted with ELISA buffer (1:2) and analyzed for KL by ELISA. One serum value, several standard deviation above the cohort mean was excluded from analysis.
  • IP-IB immunoprecipitation-immunoblot
  • mice were anesthetized and the brain was immediately placed in ice-cold artificial cerebrospinal fluid (aCSF) and sliced on a vibratome (Leica VT1200). Slices were incubated at 32°C for 30 minutes and then incubated at RT (room temperature) for 1 hour prior to testing.
  • aCSF artificial cerebrospinal fluid
  • RT room temperature
  • acute hippocampal slices were placed on a Med64-Quad II multielectrode array (Alpha MED Scientific).
  • Field Excitatory Post Synaptic Potentials fEPSP
  • LTP Long term potentiation
  • mice were tested in the small Y maze, also known as spontaneous alternation Y maze with spatial cues, as described in Dubai et al. (Cell Rep., 7, 1065-1076, doi: 10.1016/j.celrep.2014.03.076 (2014)); Dubai et al. (J. Neurosci. 35, 2358-2371, doi:10.1523/JNEUROSCI.5791-12.2015 (2015)); and Leon et al. ⁇ Cell Rep., 20, 1360-1371, doi: 10.1016/j.celrep.2017.07.024 (2017)), each of which are herein incorporated by reference in their entireties. Briefly, a mouse was placed inside one of the three identical arms of the Y-maze and allowed to explore the apparatus for 6 minutes. The recorded spontaneous alterations were scored manually, and percent alternations were calculated.
  • Stable performance was first achieved for each monkey by titrating task difficulty by increasing the number of spatial locations or lengthening the delays (a variable multiplier between 1 and 10 applied to a set of baseline delays of 0, 1, 2, 3, 4 sec). Once stable, the number of wells and delay multiplier was kept fixed for that monkey for the duration of the testing for the current study. For testing, monkeys watched the spatially distanced wells while the investigator put a preferred treat in a single well. The wells were covered, an opaque screen between the monkey and wells was lowered, and after a predetermined delay, the screen was raised. The monkey was able to retrieve the treat if the choice of well was correct. Based on the number of wells and the time between cue and task the behavior is classified as either high memory load (HML) (6-9 wells;0-15 second delay) or normal memory load (NML) (2-7 wells; 0-32 seconds).
  • HML high memory load
  • NML normal memory load
  • FIG. 2A Cognitive testing of aged rhesus macaques was performed using the spatial delayed response task (FIG. 2A), assessing frontal-temporal circuits and regions of the brain including the hippocampus and frontal cortex.
  • This task assesses working and spatial memory for both a normal memory load (NML, easier task) and high memory load (HML, harder task) (see Castner, S. A. & Goldman-Rakic, P. S., J. Neurosci.. 24. 1446-1450, doi:10.1523/JNEUROSCI.3987- 03.2004 (2004)) (FIG. 2B).
  • monkeys were trained to achieve a stable baseline response of 65-75% correct for the NML task in choosing the spatial location of a food reward, among 2-7 wells covered with identical plaques, after variable delays of up to 28 seconds from the time an opaque shutter was lowered in front of them.
  • Well number and duration were titrated previously for each animal in order to achieve the desired stable baseline performance. All macaques were then treated with vehicle (s.c.) to habituate to effects of the procedure and stress of an injection on cognitive performance.
  • monkeys were treated with either vehicle or rhesus KL; 4 hours later, monkeys underwent the HML task (with up to 9 wells) followed by a series of NML tasks (up to 7 wells) over two weeks (FIG. 2A), ending with another HML task.
  • KL (10 ⁇ g/kg, s.c.) enhanced cognition in aged rhesus macaques in the short-term and in the long-term during both NML and HML testing. As expected, performance between baseline or vehicle treatment did not differ. KL (10 ⁇ g/kg, s.c.) increased HML performance by 4 hours after treatment (FIG. 2C), within the same rapid time frame it increased cognition in mice (see FIG. IE). KL-mediated cognitive enhancement of HML, a harder memory task (given the increased number of wells from which to choose and the increase in delays) persisted at 2 weeks (FIG. 2D). KL (10 ⁇ g/kg, s.c.) also enhanced the average NML performance (FIG. 2E), an effect that largely persisted across multiple tests over two weeks (FIG. 2F).
  • klotho-mediated cognitive enhancement extends to NHPs in a complex genetic, anatomic, and functional brain similar to humans.
  • the data also suggest lower, more “physiological” levels of the hormone in the body may be required for a therapeutic window of cognitive enhancement in humans.
  • KL has pleiotropic actions including on insulin (see Wolf et al.. Oncogene 27, 7094-7105, doi: 10.1038/onc.2008.292 (2008) and FGF signaling (Urakawa et al., Nature 444, 770-774, doi:10.1038/nature05315 (2006), Wnt (Liu et al., Science 317, 803-806, doi: 10.1126/science.l 143578 (2007), and NMD AR functions (Dubai et al. (Cell Rep. 7, 1065- 1076, doi: 10.1016/j.celrep.2014.03.076 (2014)); Dubai et al. (J. Neurosci.
  • SEQ ID NO: 1 full length Klotho (amino acid 1-1012):
  • SEQ ID NO: 2 full length without signal sequence (amino acids 34-1012 of full length):
  • SEQ ID NO: 3 (KL1 domain comprising amino acids 34-549 of full length KL):
  • SEQ ID NO: 4 (Glycosyl hydrolase- 1 domain 1 (as identified by Uniprot) comprising amino acids 57-506 of full length):
  • SEQ ID NO: 5 (glycosyl hydrolase- 1 domain 2 (as identified by Uniprot) comprising amino acid 515-953):
  • SEQ ID NO: 6 (signal sequence + 34-981 of SEQ ID NO: 1):

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Abstract

Methods of improving cognitive function in a human subject, comprising parenterally administering to said subject at least one dose of a polypeptide, or a polynucleotide that is capable of expressing a polypeptide, that includes at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations, are provided herein.

Description

KLOTHO POLYPEPTIDE OR POLYNUCLEOTIDE FOR IMPROVING COGNITION
1. CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 63/381,539, filed October 28, 2022, which is hereby incorporated by reference in its entirety.
2. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under R01 NS092918 awarded by the National Institutes of Health. The government has certain rights in the invention.
3. BACKGROUND OF THE INVENTION
[0003] Brain health is one of the biggest biomedical challenges with few if any effective medical treatments. Cognition is a highly valued and central manifestation of brain health that is impaired or becomes disrupted in normal aging, numerous neurodegenerative, neurologic, and psychiatric diseases, childhood developmental syndromes, traumatic brain injury, and stress. Cognition is also disrupted by jet lag, medication side effects, and certain medical treatments, such as those for cancer. Thus, the potential to enhance cognition or counter cognitive dysfunction is of enormous relevance across the human lifespan in health and disease and unmet need remains.
4. SEQUENCE LISTING
[0004] The instant application contains a Sequence Listing which has been submitted electronically via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on October XX, 2023, is named 51758WO_sequencelisting.xml, and is XXXX bytes in size.
5. SUMMARY OF THE INVENTION
[0005] Provided herein are methods and compositions for improving cognition through systemic administration of klotho or a protein comprising klotho or a functional fragment thereof. In some embodiments, the method comprises improving cognition in an individual comprising administering to the individual an effective amount of a protein comprising a Klotho polypeptide or a functional variant or fragment thereof, thereby improving cognitive function in the individual.
[0006] In first aspect, this disclosure features a method of increasing cognitive function in a human subject, comprising: parenterally administering to said subject at least one dose of a polypeptide, or a polynucleotide that is capable of expressing a polypeptide, that includes at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations by at least 2.5 fold, and no more than 25-fold, compared to pre-administration (baseline) concentrations.
[0007] In another aspect, this disclosure features a method of increasing cognitive function in a human subject, comprising: parenterally administering to said subject at least one dose of a polypeptide, or a polynucleotide that is capable of expressing a polypeptide, that includes at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations to at least 1000 pg/mL and no more than 5000 pg/mL.
[0008] In another aspect, this disclosure features a method of increasing cognitive function in a human subject, comprising: parenterally administering to said subject a plurality of doses of a polypeptide, or a polynucleotide that is capable of expressing a polypeptide, that includes at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations by at least 2.5 fold, and no more than 25-fold, compared to pre-administration (baseline) concentrations, and wherein the plurality of doses is administered on an intermittent dosing schedule.
[0009] In another aspect, this disclosure features a method of increasing cognitive function in a human subject, comprising: parenterally administering to said subject a plurality of doses of a polypeptide, or a polynucleotide that is capable of expressing a polypeptide, that includes at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations to at least 1000 pg/mL and no more than 5000 pg/mL, and wherein the plurality of doses is administered on an intermittent dosing schedule.
6. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS [0010] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:
[0011] FIG. 1A illustrates the experimental protocol of hippocampal LTP recordings from young male mice treated with either Vehicle (Veh) or Rhesus KL (10 μg/kg, s.c.).
[0012] FIG. IB shows fEPSP recordings from acute hippocampal slices of mice (age = 3 months) treated with Veh or Rhesus KL (n=8 slices from 3 mice for Veh; 10 slices from 3 mice for Rhesus KL).
[0013] FIG. 1C shows average fEPSP slope of the last 10 minutes of each recording for mice treated with Veh or Rhesus KL. **p<0.01 (two-tailed t-test).
[0014] FIG. ID illustrates the experimental protocol for testing male mice (age=4 months) in the small Y maze following treatment with Veh or Rhesus KL (10 μg/kg, s.c.).
[0015] FIG. IE shows the percent alternations in the small Y maze of mice treated with Veh or Rhesus KL (n=28-29 mice per experimental group). *p<0.05 (two-tailed t-test).
[0016] FIG. IF illustrates the experimental protocol for measuring KL levels in the serum of young male mice (age = 4 mo.s) 4 hrs following treatment with Veh or mouse KL (10 μg/kg, s.c.).
[0017] FIG. 1G shows relative KL serum levels from nontransgenic (NTG) and KL transgenic overexpressing (KL TG) mice alongside serum from young mice (age = 4 months) 4 hrs following treatment with Veh or mouse KL (10 μg/kg, s.c.) (n = 5-7 mice per experimental group). **p<0.01 vs NTG (two-tailed t-test), ***p<0.001 vs Veh (two-tailed t-test).
[0018] FIG. 1H illustrates the experimental protocol for measuring KL levels in the serum of aged rhesus macaques 4 hrs following treatment with Veh or varying doses of Rhesus KL (0.4 μg/kg, 2 μg/kg, 10 μg/kg, 20 μg/kg, or 30 μg/kg, s.c.).
[0019] FIG. II shows the relative KL serum levels of aged rhesus macaques at baseline and 4 hrs following injection with Veh or varying doses of Rhesus KL. (n=21 monkeys at baseline: n=16 female, n=5 male monkeys; n=l-3 monkeys at each KL dose, all female except one male at 10 μg/kg). Values are relative to baseline. ***p<0.001 vs Baseline (Bonferroni-corrected).
[0020] FIG. 2A illustrates the experimental paradigm for testing aged rhesus macaques (age = 18-27 year's) in a spatial delayed memory task following treatment with vehicle or Rhesus KL (10 μg/kg, s.c.).
[0021] FIG. 2B is a diagram of the spatial delayed task. (Top) Aged rhesus macaques were shown a food reward, a visible cue. A screen was lowered between the monkey and the wells and the wells were covered. (Bottom) Following a delay, the screen was raised and each monkey attempted to remember the spatial location of the hidden food reward. The task tested normal memory load (NML) with 4-7 wells present and a high memory load (HML) with more wells (6-9) present.
[0022] FIG. 2C shows the percent correct choices made by monkeys in the HML task, representing spatial and working memory, 4 hrs following treatment with either Veh or Rhesus KL (10 μg/kg) (n=l 1-18 monkeys per experimental group: n=6-13 female, n= 3-5 male monkeys per experimental group). *p<0.05 vs Veh (linear mixed model ANOVA and Satterthwaite’s t-tests).
[0023] FIG. 2D presents the percent correct choices made by monkeys in the HML task 4 hrs and 14 days after a single injection with Veh or Rhesus KL (10 μg/kg). The dashed line represents mean performance and shaded gray represents range of performance in Veh-treated monkeys. (n=ll monkeys per experimental group: n=6-8 female; 3-4 male monkeys per experimental group). *p<0.05 vs Veh (linear mixed model ANOVA and Satterthwaite’s t-tests).
[0024] FIG. 2E Percent correct choices made by monkeys in the NML task averaged over two weeks, following a single treatment with either Veh or Rhesus KL (10 μg/kg). (n=l 1-18 monkeys per experimental group: n=6-13 female, n= 3-5 male monkeys per experimental group). *p<0.05 vs Veh (linear mixed model ANOVA and Satterthwaite’s t-tests).
[0025] FIG. 2F shows the percent correct choices made by monkeys in the NML between 4h and 14 days after a single injection with Veh or Rhesus KL (10 μg/kg). The dashed line represents mean performance and shaded gray represents range of performance in Veh-treated monkeys. (n=l l monkeys per experimental group: n=6-8 female; 3-4 male monkeys per experimental group). *p<0.05 vs Veh (linear mixed model ANOVA and Satterthwaite’s t-tests).
[0026] FIG. 2G graphs the percent increase in cognition in the HML task, compared to Veh- treatment, at varying doses of Rhesus KL treatment. Dashed line represents mean performance and shaded gray represents range of performance in Veh-treated monkeys. (n=7-13 monkeys per experimental group: n=6 female, 3 male monkeys for 10 μg/kg; n = 5 female, 2 male for 20 μg/kg; n = 9 female, 4 male for 30 μg/kg group). *p<0.05 vs Veh (linear mixed model ANOVA and Satterthwaite’s t-tests).
[0027] FIG. 2H graphs the percent increase in cognition in the NML task, compared to Veh- treatment, at varying doses of Rhesus KL treatment. (n=7-13 monkeys per experimental group: n=6 female, 3 male monkeys for 10 μg/kg; n = 5 female, 2 male for 20 μg/kg; n = 9 female, 4 male for 30 μg/kg group). *p<0.05 vs Veh; # = 0.057 vs Veh (linear mixed model ANOVA and Satterthwaite’s t-tests).
7. DETAILED DESCRIPTION OF THE INVENTION
7.1. Definitions
[0028] As used herein, the term “cognitive function” refers to at least one observable mental task or function. In various embodiments, the mental task or function is selected from a memory task or function (e.g., a semantic, episodic, procedural, priming, or working memory task or function); an orientation task or function; a language task or function; a problem solving task or function; a visual perception task or function; a construction task or function; an integration task or function; a planning task or function; an organizational task or function; selective attention; inhibitory control; or ability to mentally manipulate information. In some embodiments, cognitive function is measured by a clinical test (also known as a clinical instrument).
[0029] The terms “cognitive ability” and “cognition” are used synonymously herein with “cognitive function.”
[0030] As used herein, the terms “improved cognitive function,” or “increased cognitive function”, and synonymously, “improved (or increased) cognition” and “improved (or increased) cognitive ability”, and like terms, refer to an improvement in cognition function under a given condition (e.g., treatment with klotho) compared to cognition absent the condition (e.g., absent treatment with klotho). For an individual experiencing cognitive decline, an improvement in cognition might be a reduction in the rate of cognitive decline (i.e., an improvement compared to the absence of treatment), but not an actual improvement in cognitive ability. An increase in cognitive function can also be an increase in brain activity in a specified area, e.g., as determined by MRI, or an inhibition of brain activity that results in better overall cognitive function. An increase in cognitive function can also be improvement in a cognitive performance test as described in more detail herein. An improvement or increase in cognitive function can be in any one cognitive function, or any combination of individual cognitive functions.
[0031] As used herein, the terms “therapy,” “treatment,” and “amelioration” refer to any reduction in the severity of symptoms (cognitive decline), or improvement in cognitive function, or where motor function is affected, an improvement in motor function. As used herein, the terms “treat” and “prevent” are not intended to be absolute terms. Treatment and prevention can refer to any delay in cognitive decline, amelioration of symptoms (e.g., confusion, delirium), etc. Treatment and prevention can be complete or partial, such that cognition is better than would have been expected without treatment (e.g., compared to cognition in the same individual before treatment or compared to cognition in similar non-treated individuals). The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment.
[0032] As used herein, the terms “effective amount,” “effective dose,” “therapeutically effective amount,” etc. refer to that amount of the therapeutic agent sufficient to ameliorate a disorder, as described above. For example, for the given parameter, a therapeutically effective amount will show a reduction in the severity of symptoms of at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.
[0033] “Subject,” “patient,” “individual” and like terms are used interchangeably and refer to, except where indicated, mammals such as humans and non-human primates, as well as dogs, horses, pigs, mice, rats, and other mammalian species. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical supervision. A patient can be an individual who is seeking treatment, monitoring, adjustment or modification of an existing therapeutic regimen, etc.
7.2. Method of improving cognition
7.2.1. Relative fold changes in serum KL compared to baseline
[0034] In one aspect, a method is presented for improving cognitive function in a human subject. The method comprises: parenterally administering to said subject at least one dose of a polypeptide, or a polynucleotide that is capable of expressing a polypeptide, that includes at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations by at least 2.5 fold, and no more than 25-fold, compared to pre-administration (baseline) concentrations.
[0035] In some embodiments, the dose is sufficient to raise serum KL concentrations by at least
1.5-fold, by at least 2-fold, by at least 2.5-fold, by at least 3-fold, by at least 3.5-fold, by at least
4-fold, by at least 4.5-fold, by at least 5.0-fold, by at least 5.5-fold, by at least 6.0-fold, by at least
6.5-fold, by at least 7-fold, by at least 7.5-fold, by at least 8-fold, by at least 8.5-fold, by at least 9-fold, by at least 9.5-fold, by at least 10-fold, by at least 10.5-fold, by at least 11-fold, by at least
11.5-fold, by at least 12-fold, by at least 12.5-fold, by at least 13-fold, by at least 13.5-fold, by at least 14-fold, by at least 14.5-fold, by at least 15-fold, by at least 15.5-fold, by at least 16-fold, by at least 16.5-fold, by at least 17-fold, by at least 17.5-fold, by at least 18-fold, by at least 18.5- fold, by at least 19-fold, by at least 19.5-fold, by at least 20-fold, by at least 20.5-fold, by at least 21-fold, by at least 21.5-fold, by at least 22-fold, by at least 22.5-fold, by at least 23-fold, by at least 23.5-fold, by at least 24-fold, or by at least 24.5-fold compared to baseline concentrations.
[0036] In some embodiments, the dose is sufficient to raise serum KL concentrations by at least
5-fold compared to baseline concentrations. In some embodiments, the dose is sufficient to raise serum KL concentrations by at least 7.5-fold compared to baseline concentrations. In some embodiments, the dose is sufficient to raise serum KL concentrations by at least 10-fold compared to baseline concentrations. [0037] In some embodiments, the dose is sufficient to raise serum KL concentrations by no more than 25-fold, no more than 24.5-fold, no more than 24-fold, no more than 23.5-fold, no more than 23-fold, no more than 22.5-fold, no more than 22-fold, 21.5-fold, no more than 21-fold, no more than 20.5-fold, no more than 20-fold, no more than 19.5-fold, no more than 19-fold, no more than 18.5-fold, no more than 18-fold, no more than 17.5-fold, no more than 17-fold, no more than 16.5-fold, no more than 16-fold, no more than 15.5-fold, no more than 15-fold, no more than 14.5-fold, no more than 14-fold, no more than 13.5-fold, no more than 13-fold, no more than 12.5-fold, no more than 12-fold, no more than 11.5-fold, no more than 11-fold, no more than 10.5-fold, no more than 10-fold, no more than 9.5-fold, no more than 9-fold, no more than 8.5-fold, no more than 8-fold, no more than 7.5-fold, no more than 7-fold, no more than 6.5- fold, no more than 6-fold, no more than 5.5-fold, no more than 5-fold, no more than 4.5-fold, no more than 4-fold, no more than 3.5-fold, no more than 3-fold, no more than 2.5-fold, no more than 2-fold, or no more than 1.5-fold compared to baseline concentrations.
[0038] In some embodiments, the dose is sufficient to raise serum KL concentrations by no more than 20-fold compared to baseline concentrations. In some embodiments, the dose is sufficient to raise serum KL concentrations by no more than 15-fold compared to baseline concentrations. In some embodiments, the dose is sufficient to raise serum KL concentrations by no more than 10- fold compared to baseline concentrations.
[0039] In some embodiments, the dose is sufficient to raise serum KL concentrations by about 2.5-fold to about 10-fold. In some embodiments, the dose is sufficient to raise serum KL concentrations by about 5-fold to about 7.5-fold compared to baseline concentrations..
7.2.2. KL Cmax
[0040] In another aspect, a method is presented for improving cognitive function in a human subject, where ethe method comprises parenterally administering to said subject at least one dose of a polypeptide, or a polynucleotide that is capable of expressing a polypeptide, that includes at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations to at least 1000 pg/mL and no more than 5000 pg/mL. [0041] In some embodiments, the dose is sufficient to raise serum KL concentrations to at least 1000 pg/mL, to at least 1500 pg/mL, to at least 2000 pg/mL, to at least 2500 pg/mL, to at least 3000 pg/mL, to at least 3500 pg/mL, to at least 4000 pg/mL, or to at least 4500 pg/mL.
[0042] In some embodiments, the dose is sufficient to raise serum KL concentrations to at least 1000 pg/mL. In some embodiments, the dose is sufficient to raise serum KL concentrations to at least 1500 pg/mL. In some embodiments, the dose is sufficient to raise serum KL concentrations to at least 2000 pg/mL. In some embodiments, the dose is sufficient to raise serum KL concentrations to at least 2500 pg/mL. In some embodiments, the dose is sufficient to raise serum KL concentrations to at least 3000 pg/mL. In some embodiments, the dose is sufficient to raise serum KL concentrations to at least 3500 pg/mL. In some embodiments, the dose is sufficient to raise serum KL concentrations to no more than 4000 pg/mL.
[0043] In some embodiments, the dose is sufficient to raise serum KL concentration to no more than 5000 pg/mL, to no more than 4500 pg/mL, to no more than, pg/mL, to no more than 4000 pg/mL, to no more than 3500 pg/mL, to no more than 3000 pg/mL, to no more than 2500 pg/mL, to no more than 2000 pg/mL, to no more than 1500 pg/mL, or to no more than 1000 pg/mL.
[0044] In some embodiments, the dose is sufficient to raise serum KL concentrations to no more than 3000 pg/mL. In some embodiments, the dose is sufficient to raise serum KL concentrations to no more than 2500 pg/mL.
[0045] In some embodiments, cognition is improved by at least 1%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some embodiments, cognition is improved by at least 2, 3, 5, 7, 10, 15, 20, 25%, 50%, 75%, 80%, or 90%, or more, determined using tests of cognition, molecular proxies, or structural changes associated with brain function.
[0046] In some embodiments, motor function is improved by at least 1%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some embodiments, motor function is improved by at least 2, 3, 5, 7, 10, 15, 20, 25%, 50%, 75%, 80%, or 90%, or more, determined using tests of motor function. 7.3. Dose and Dose Schedule
[0047] In another aspect, a method is presented for improving cognitive function in a human subject, where the method comprises: parenterally administering to said subject a plurality of doses of a polypeptide, or a polynucleotide that is capable of expressing a polypeptide, that includes at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations by at least 2.5 fold, and no more than 25-fold, compared to preadministration (baseline) concentrations, and wherein the plurality of doses is administered on an intermittent dosing schedule.
[0048] In another aspect, this disclosure features a method of increasing cognitive function in a human subject, comprising: parenterally administering to said subject a plurality of doses of a polypeptide, or a polynucleotide that is capable of expressing a polypeptide, that includes at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations to at least 1000 pg/mL and no more than 5000 pg/mL, and wherein the plurality of doses is administered on an intermittent dosing schedule.
[0049] In some embodiments, successive doses are administered no less frequently than once every 6 months and no more frequently than once per day.
[0050] In some embodiments, successive doses are administered no less frequently than once every 3 months. In some embodiments, successive doses are administered no less frequently than once a month.
[0051] In some embodiments, successive doses are administered no less frequently than once every four weeks. In some embodiments, successive doses are administered no less frequently than once every other week. In some embodiments, successive doses are administered no less frequently than once a week.
[0052] In some embodiments, successive doses are administered no more frequently than once a week. In some embodiments, successive doses are administered no more frequently than one every two weeks. In some embodiments, successive doses are administered no more frequently than once every four weeks. [0053] In some embodiments, successive doses are administered no more frequently than once a month. In some embodiments, successive doses are administered once a day. In some embodiments, successive doses are administered once a week. In some embodiments, successive doses are administered once every two weeks. In some embodiments, successive doses are administered once every four weeks.
[0054] In some embodiments, the dose is a polypeptide administered subcutaneously (s.c.).
[0055] In some embodiments, the dose is at least 5 μg/kg s.c. and no more than 50 μg/kg s.c (e.g., at least 5 μg/kg s.c. and no more than 45 μg/kg s.c., at least 5 μg/kg s.c. and no more than 40 μg/kg s.c., at least 5 μg/kg s.c. and no more than 35 μg/kg s.c., at least 5 μg/kg s.c. and no more than 30 μg/kg s.c., at least 5 μg/kg s.c. and no more than 25 μg/kg s.c., at least 5 μg/kg s.c. and no more than 20 μg/kg s.c., at least 5 μg/kg s.c. and no more than 15 μg/kg s.c., at least 5 μg/kg s.c. and no more than 10 μg/kg s.c., at least 10 μg/kg s.c. and no more than 50 μg/kg s.c., at least 10 μg/kg s.c. and no more than 45 μg/kg s.c., at least 10 μg/kg s.c. and no more than 40 μg/kg s.c., at least 10 μg/kg s.c. and no more than 35 μg/kg s.c., at least 10 μg/kg s.c. and no more than 30 μg/kg s.c., at least 10 μg/kg s.c. and no more than 25 μg/kg s.c., at least 10 μg/kg s.c. and no more than 20 μg/kg s.c., at least 10 μg/kg s.c. and no more than 15 μg/kg s.c., at least 15 μg/kg s.c. and no more than 50 μg/kg s.c., at least 15 μg/kg s.c. and no more than 45 μg/kg s.c., at least 15 μg/kg s.c. and no more than 40 μg/kg s.c., at least 15 μg/kg s.c. and no more than 35 μg/kg s.c., at least 15 μg/kg s.c. and no more than 30 μg/kg s.c., at least 15 μg/kg s.c. and no more than 25 μg/kg s.c., at least 15 μg/kg s.c. and no more than 20 μg/kg s.c., at least 20 μg/kg s.c. and no more than 50 μg/kg s.c., at least 20 μg/kg s.c. and no more than 45 μg/kg s.c., at least 20 μg/kg s.c. and no more than 40 μg/kg s.c., at least 20 μg/kg s.c. and no more than 35 μg/kg s.c., at least 20 μg/kg s.c. and no more than 30 μg/kg s.c., at least 20 μg/kg s.c. and no more than 25 μg/kg s.c., at least 25 μg/kg s.c. and no more than 50 μg/kg s.c., at least 25 μg/kg s.c. and no more than 45 μg/kg s.c., at least 25 μg/kg s.c. and no more than 40 μg/kg s.c., at least 25 μg/kg s.c. and no more than 35 μg/kg s.c., at least 25 μg/kg s.c. and no more than 30 μg/kg s.c., at least 30 μg/kg s.c. and no more than 50 μg/kg s.c., at least 30 μg/kg s.c. and no more than 45 μg/kg s.c., at least 30 μg/kg s.c. and no more than 40 μg/kg s.c., at least 30 μg/kg s.c. and no more than 35 μg/kg s.c., at least 35 μg/kg s.c. and no more than 50 μg/kg s.c., at least 35 μg/kg s.c. and no more than 45 μg/kg s.c., at least 35 μg/kg s.c. and no more than 40 μg/kg s.c., at least 40 μg/kg s.c. and no more than 50 gg/kg s.c., at least 40 gg/kg s.c. and no more than 45 gg/kg s.c., or at least 45 gg/kg s.c. and no more than 50 gg/kg s.c.)
[0056] In some embodiments, the dose is at least 5 gg/kg s.c. (e.g., at least 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, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 gg/kg s.c.) In some embodiments, the dose is at least 7.5 gg/kg s.c. In some embodiments, the dose is at least 10 gg/kg s.c. In some embodiments, the dose is at least 15 gg/kg s.c. In some embodiments, the dose is at least 20 gg/kg s.c. In some embodiments, the dose is at least 25 gg/kg s.c.
[0057] In some embodiments, the dose is no more than 40 gg/kg s.c. (e.g., no more than 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 gg/kg s.c.). In some embodiments, the dose is no more than 30 gg/kg s.c. In some embodiments, the dose is no more than 25 gg/kg s.c. In some embodiments, the dose is no more than 20 gg/kg s.c. In some embodiments, the dose is no more than 15 gg/kg s.c. In some embodiments, the dose is no more than 10 gg/kg s.c. In some embodiments, the dose is no more than 5 gg/kg s.c.
[0058] In some embodiments, the Klotho polypeptide can be administered at the initial dosage of at least 5 gg/kg s.c. and no more than 50 gg/kg s.c. daily (or other intermittent period (e.g., every other day, every third day, every fourth day, every fifth day, every sixth day, once per week, once every two weeks, once every three weeks, and once a month)) and adjusted over time. For example, a daily dose range of about 10 gg/kg to about 40 gg/kg, or about 20 gg/kg to about 30 gg/kg can be used. In some embodiments, the dosage is varied depending upon the requirements of the patient, the severity of the condition being treated, and the route of administration. For example, for injection of Klotho polypeptide, the effective dose is typically in the range of 5-50 gg/kg, while for direct delivery to the central nervous system (CNS), the effective dosage is lower, e.g., 5-30 gg/kg. For oral administration, the effective dose is higher, e.g., in the range of 50-10,000 gg/kg (e.g., 100 gg/kg-2 mg/kg). The dose is chosen in order to provide effective therapy for the patient. The dose may be repeated at an appropriate frequency which may be in the range of once or twice per day, once or twice per week to once every three months, depending on the pharmacokinetics of the Klotho polypeptide composition (e.g., half-life in the circulation) and the pharmacodynamic response (e.g., the duration of the therapeutic effect).
[0059] In some embodiments, the dose is at least 350 μg and no more than 3500 μg (e.g., at least 350 μg and no more than 3000 μg, at least 350 μg and no more than 2500 μg, at least 350 μg and no more than 2000 μg, at least 350 μg and no more than 1500 μg, at least 350 μg and no more than 1000 μg, at least 350 μg and no more than 750 μg, at least 350 μg and no more than 500, at least 500 μg and no more than 3500 μg, at least 500 μg and no more than 3000 μg, at least 500 μg and no more than 2500 μg, at least 500 μg and no more than 2000 μg, at least 500 μg and no more than 1500 μg, at least 500 μg and no more than 1000 μg, at least 500 μg and no more than 750 μg, at least 750 μg and no more than 3500 μg, at least 750 μg and no more than 3000 μg, at least 750 μg and no more than 2500 μg, at least 750 μg and no more than 2000 μg, at least 750 μg and no more than 1500 μg, at least 750 μg and no more than 1000 μg, at least 1000 μg and no more than 3500 μg, at least 1000 μg and no more than 3000 μg, at least 1000 μg and no more than 2500 μg, at least 1000 μg and no more than 2000 μg, at least 1000 μg and no more than 1500 μg, at least 1500 μg and no more than 3500 μg, at least 1500 μg and no more than 3000 μg, at least 1500 μg and no more than 2500 μg, at least 1500 μg and no more than 2000 μg, at least 2000 μg and no more than 3500 μg, at least 2000 μg and no more than 3000 μg, at least 2000 μg and no more than 2500 μg, at least 2500 μg and no more than 3500 μg, at least 2500 μg and no more than 3000 μg, or at least 3000 μg and no more than 3500 μg).
[0060] In some embodiments, the dose is at least 500 μg (e.g., at least 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, or 3400 μg). In some embodiments, the dose is at least 750 μg. In some embodiments, the dose is at least 1000 μg. In some embodiments, the dose is at least 1500 μg. In some embodiments, the dose is at least 2000 μg. In some embodiments, the dose is at least 2500 μg. In some embodiments, the dose is at least 3000 μg.
[0061] In some embodiments, the dose is no more than 3000 μg (e.g., no more than 2900, 2800, 2700, 2600, 2500, 2400, 2300, 2200. 2100, 2000, 1900, 1800, 1700, 1600, 1500. 1400, 1300. 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 μg). In some embodiments, the dose is no more than 2500 μg. In some embodiments, the dose is no more than 2000 μg. In some embodiments, the dose is no more than 1500 μg. In some embodiments, the dose is no more than 1000 μg.
[0062] In some embodiments, the Klotho polypeptide can be administered at the initial dosage of at least 350 μg and no more than 3500 μg daily (or other intermittent period (e.g., every other day, every third day, every fourth day, every fifth day, every sixth day, once per week, once every two weeks, once every three weeks, and once a month)) and adjusted over time. In some embodiments, the dosage is varied depending upon the requirements of the patient, the severity of the condition being treated, and the route of administration. For example, for injection of Klotho polypeptide, the effective dose is typically in the range of 350-3500 μg, while for direct delivery to the central nervous system (CNS). the effective dosage is lower, e.g., 350-2000 μg/kg. For oral administration, the effective dose is higher, e.g., in the range of 50-10,000 μg. The dose is chosen in order to provide effective therapy for the patient. The dose may be repeated at an appropriate frequency which may be in the range of once or twice per day, once or twice per week to once every three months, depending on the pharmacokinetics of the Klotho polypeptide composition (e.g., half-life in the circulation) and the pharmacodynamic response (e.g., the duration of the therapeutic effect).
[0063] Dosages can be empirically determined considering the type and severity of cognitive condition diagnosed in a particular patient. The dose administered to a patient, in the context of the present disclosure, should be sufficient to effect a beneficial therapeutic response in the patient over time. The size of the dose will also be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of any particular composition in a particular patient, as will be recognized by the skilled practitioner.
[0064] In some embodiments, additional administration is dependent on patient progress, e.g., the patient is monitored between administrations. For example, after the first administration or round of administrations, the patient can be monitored for cognitive ability or for side effects, e.g., weakness, dizziness, nausea, etc.
[0065] In some embodiments, the individual has a chronic condition, so that klotho is administered over an indefinite period, e.g., for the lifetime of the patient. In such cases, administration is typically periodic. Diseases that are considered long-term or chronic include, but are not limited to Alzheimer's disease, Parkinson's disease, Huntington's disease, and cognitive decline associated with hypertension and heart disease.
7.4. Subjects to be treated
[0066] In typical embodiments, the subject is a human.
[0067] In some embodiments, the subject, prior to first administration, has normal cognitive function. In some embodiments, the human subject has at least normal cognitive function and the administering results in improved cognitive function compared to before the administering. In some embodiments, the subject, prior to first administration, has cognitive impairment. In some embodiments, the subject, prior to first administration, has mild cognitive impairment (MCI). In some embodiments, the human subject has impaired cognitive function and the administering results in improved cognitive function compared to before the administering.
[0068] In some embodiments, Klotho polypeptides (and functional variants and fragments thereof) are used to prevent or reduce cognitive decline associated with aging, for example, in individuals 45 years of age or older, or upon initial signs of cognitive decline. In some embodiments, the patient is older than 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 years of age. In certain embodiments, the patient is older than 76, 77, 78, 79, 80, 81. 82, 82, 84, 85, 86, 87, 88, 89 or 90 years of age. In various embodiments, the patient is 40-50 years old, 50-60 years old, 52-62 years old, 63-70 years old, 60-70 years old, 70-80 years old, or 80-90 years old. In certain embodiments, the patient is 51, 52. 53. 54. 55. 56. 57. 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69. 70. 71. 72. 73. 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 years old.
[0069] In some embodiments, the subject is at least 21 years old. In some embodiments, the subject is at least 45 years old. In some embodiments, the subject is at least 60 year's old. In some embodiments, the subject is at least 65 years old. In some embodiments, the subject is at least 80 years old.
[0070] In some embodiments, the subject is under 21 years old. [0071] In some embodiments, the subject has at least one ApoE4 allele.
[0072] In some embodiments, the subject, prior to first administration, has dementia.
[0073] In some embodiments, the subject has Alzheimer’s disease, frontotemporal dementia, Lewy Body Dementia, Parkinson's disease, Huntington's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasalar degeneration, vascular cognitive impairment, vascular dementia, amyotropic lateral sclerosis, prion disorder, posterior cortical atrophy, primary progressive aphasia HIV-related dementia, global aphasia, mixed transcortical aphasia, Broca’s aphasia, transcortical motor aphasia, Wernicke’s aphasia, transcortical sensory aphasia, conduction aphasia, anomic aphasia, multiple system atrophy; mental or mood disorders: depression, schizophrenia, attention deficit/ hyperactivity disorder, autism spectrum disorder, intellectual disability, mood disorder, psychotic disorder, psychosis; childhood neurodevelopment al syndromes and brain tumors: X-linked mental disability or retardation, astrocytoma, ependymoma, medulloblastoma, oligodendroglioma; genetic syndromes affecting learning: Down's syndrome, Angelman's syndrome, Rett’ s syndrome; Metabolic disorders affecting cognition: phenylketonuria, Lesch-Nyhan, galactosemia, and adrenoleukodystrophy; Cognitive decline associated with chemotherapy and/or radiation therapy or “chemo brain”; and additional conditions and disorders: pain-associated cognitive effects, traumatic brain injury, stroke, multiple sclerosis, neuroautoimmune disease, epilepsy, delirium, paraneoplastic disorder, developmental delay, hydrocephalus, leukodystrophies sleep deprivation, jet lag.
[0074] In some embodiments, klotho polypeptides (and functional variants and fragments thereof) can also be administered to provide increased cognition for individuals desiring improved cognition (e.g., individuals exposed to stress, or sleep deprivation), or for individuals requiring superior cognitive function. In such cases, the klotho polypeptide composition can be administered 2-24 hours before the desired effect, which can last about 3-5 days for working memory and about 2 weeks for spatial memory.
[0075] In some embodiments, Klotho polypeptides (and functional variants and fragments thereof) are be used to treat individuals with age-related, non-age related, or disease related conditions including, but not limited to: neurodegenerative diseases, mental or mood disorders, childhood neurodevelopmental syndromes and brain tumors, genetic syndromes affecting learning, metabolic disorders affecting cognition, cognitive decline associated with chemotherapy and/or radiation therapy, and conditions with impaired motor functions.
[0076] Non-limiting examples of neurodegenerative diseases and dementia that can be treated by administering to a subject a klotho polypeptide (and functional variants and fragments thereof) include: Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasalar degeneration, mild cognitive impairment, vascular- dementia, Lewy body dementia, amyotropic lateral sclerosis, prion disorder, and HIV-related dementia.
[0077] Non-limiting examples of mental or mood disorders that can be treated by administering to a subject a klotho polypeptide (and functional variants and fragments thereof) include: depression, schizophrenia, attention deficit/hyperactivity disorder, autism spectrum disorder, intellectual disability, a mood disorder, and a psychotic disorder.
[0078] Non-limiting examples of childhood neurodevelopmental syndromes and brain tumors that can be treated by administering to a subject a klotho polypeptide (and functional variants and fragments thereof) include: X-linked mental disability or retardation, astrocytoma, ependymoma, medulloblastoma, and oligodendroglioma.
[0079] Non-limiting examples of genetic syndromes affecting learning that can be treated by administering to a subject a klotho polypeptide (and functional variants and fragments thereof) include: Down's syndrome, Angelman's syndrome, and Rett's syndrome.
[0080] Non-limiting examples of metabolic disorders affecting cognition that can be treated by administering to a subject a klotho polypeptide (and functional variants and fragments thereof) include: phenylketonuria, Lesch-Nyhan, galactosemia, and adrenoleukodystrophy.
[0081] In some embodiments, cognitive decline associated with chemotherapy and/or radiation therapy can be treated by administering to a subject a klotho polypeptide (and functional variants and fragments thereof).
[0082] In some embodiments, the Klotho polypeptide composition is administered to an (e.g., human) individual having impaired motor function. For example, in some embodiments, the individual has stroke to the brain or spinal cord (ischemic or hemorrhagic), neurodegenerative disease (Parkinson's disease, Lewy body dementia, multiple system atrophy, amyotropic lateral sclerosis, prion disorder, Huntington's disease, supranuclear palsy), Parkinsonism, traumatic brain injury, neuroinfectious brain lesions, multiple sclerosis and related autoimmune and demyelinating disease, spinal cord lesions (compressive, infectious, toxic or metabolic, autoimmune, oncologic), brain tumor, epilepsy, paraneoplastic disorder, neurodevelopmental disorder (mitochondrial, autosomal genetic), muscle disease (polymyositis, dermatomyositis, inclusion body myositis, infectious, endocrine, metabolic, toxic, congenital myopathy, congential muscular dystrophy, hereditary), neuropathies (Guillain-Barre syndrome, axonal and demyelinating, diabetic, toxic, metabolic, infectious, critical illness, entrapment), tick paralysis, myasthenia gravis, and spinal muscular- atrophy.
[0083] Non-limiting examples of additional conditions and disorders that can be treated by administering to a subject a klotho polypeptide (and functional variants and fragments thereof) include:: pain-associated cognitive effects, traumatic brain injury, stroke, multiple sclerosis, neuroautoimmune disease, epilepsy, delirium, paraneoplastic disorder, developmental delay, and leukodystrophies.
7.5. Measuring Cognitive Ability/Function
[0084] Cognitive ability can be measured using any method known in the art, e.g., for testing memory, language ability, executive functions, visuospatial function, dementia, or multiparameter neuropsychological abilities. In some embodiments, Klotho administration results in at least a 1%, 2%, 5%, 7%, 10%, 15%, 20%. 30%. 40%, 50%, 60%, 70%. 80%. 90%, 100% or greater improvement in score on a standard cognitive ability test (e.g., measured 1-3 days after administration). In some embodiments, the testing is carried out more than once for an individual, e.g., one or more time over the course of treatment with Klotho.
[0085] For example, standard tests for memory and learning can be applied, e.g., to determine semantic, episodic, procedural, priming, and/or working (i.e., short term) memory. Common tests include Cambridge prospective memory test (CAMPROMPT), memory assessment scales (MAS), Rey auditory verbal learning test, Rivermead behavioral memory test, Test of memory and learning (TOMAL), Wechsler memory scale (WMS), and Test of memory malingering (TOMM). Tests for language functions include, e.g., Boston Diagnostic Aphasia Examination (BDAE), Comprehensive aphasia test (CAT), and Multilingual aphasia examination (MAE).
[0086] Executive function (e.g., problem solving, planning, organization, inhibitory control) can be tested using Behavioral assessment of dysexecutive syndrome (BADS), CNS vital signs (Brief Core Battery), Controlled oral word association test (COW AT), Delis-Kaplan Executive Function System (D-KEFS), Digit vigilance test, Kaplan Baycrest neurocognitive assessment (KBNA), Hayling and Brixton tests, Tests of variables of attention (TOVA), Wisconsin card sorting test (WCST), or Test of everyday attention (TEA). Visuospatial ability (e.g., visual perception, construction and integration) can be tested using the Clock Test, Hooper visual organization task (VOT), or Rey-Osterrieth complex figure tests. Dementia can be quantified using the clinical dementia rating or dementia rating scale.
[0087] Multi-parameter tests for neuropsychological function (e.g., cognitive function) include but are not limited to the Barcelona neuropsychological test (BNT), Cambridge neuropsychological test automated battery (CANTAB), Cognistat, Cognitive assessment screening instrument (CAST), Cognitive function scanner (CFS), Dean-Woodcock neuropsychology assessment system (DWNAS), General practitional assessment of cognition (GPCOG) Mini mental state examination (MMSE), NEPSY, or the CDR computerized assessment system.
[0088] Alternatively, cognition can be determined using structural or molecular proxies for cognitive activity, e.g., compared over time to detect changes. Cognitive changes can be detected, e.g., by observing changes to brain structure, connectivity, activation, inhibition, or synaptic plasticity, e.g., by MRI, fMRI, EEG, TMS and TES, and/or any combination of these. In some embodiments, brain activity is observed. In some embodiments, Klotho administration results in a 1.5-fold, 2-fold, 5-fold, 7-fold, 10-fold, or greater increase in brain activity (e.g., measured 1-3 days after administration). Molecular proxies for improved cognition include, but arc not limited to: increased levels of GluN2B, increased GluN2B synaptic localization, increased NMDA receptor activation, and/or increased c-fos activation in the brain. These measures are particularly relevant to cognition. Such method can include, e.g., obtaining a sample of neuronal tissue or CSF from an individual and using standard assays to determine gene expression or activation.
[0089] In some embodiments, the method further comprises monitoring the individual for cognitive ability, either through a molecular proxy (e.g., changes NMDA receptor or c-fos activation, or GluN2B levels in the brain), changes in MRI brain scans (e.g., functional MRI), changes in EEG, changes in TMS and TES, changes in neuropsychologic test scores, or tests of cognitive ability (e.g., for learning, short or long term memory, executive functions, language ability, and visuospatial function). In some embodiments, the individual is monitored using more than one of the above tests in any combination. In some embodiments, the dose of the Klotho polypeptide for each administration is determined based on the therapeutic progress of the individual, e.g., where a higher dose is administered if the individual is not responding sufficiently to therapy.
[0090] In some embodiments, administration results in improved motor function. Changes in motor function can be assayed as known in the art. Exemplary motor function assays include but are not limited to electromyogram and nerve conduction studies, direct or device-assisted clinical testing of strength, tone, and muscle bulk, reflex examination, coordination examination, and gait analysis. Assays for testing etiologies causing deficits of motor function include but are not limited to magnetic resonance imaging of the central nervous system, muscle biopsy, nerve biopsy, and laboratory studies.
[0091] Similarly, in mice and other non-human animals, cognitive ability can be tested with measures of executive function (working memory, attention, processing speed, set shifting), visiospatial learning and memory, object memory, pattern recognition, fear memory, passive avoidance memory, habituation, and novel object recognition, for example. Common tests include but are not limited to the Moms water maze, Barnes maze, radial arm water maze, y- maze, T-maze, and open field habituation. Brain imaging techniques are similarly applicable.
7.6. Klotho Protein
[0092] Human Klotho (canonical sequence) is described in Uniprot Accession No. Q9UEF7:
Figure imgf000022_0001
[0093] Klotho is a single-pass type 1 membrane protein. Residues 1-33 (underlined) constitute the signal sequence; residues 34-981 are extracellular; 982-1002 are transmembrane, and 1003- 1012 are cytoplasmic.
[0094] When produced in production host cells for use as a therapeutic agent, the signal sequence is cleaved and the mature full-length Klotho protein has the following sequence:
Figure imgf000022_0002
[0095] Mature full length soluble Klotho includes two conserved domains (KL1 and KL2) with homology to beta glycosidase proteins. The KL1 domain consists of residues 34-549 (numbering relative to full length protein inclusive of the signal sequence):
Figure imgf000023_0001
[0096] The extracellular region of the protein has two glycosyl hydrolase- 1 domains. As annotated in Uniprot, Glycosyl hydrolase-1 domain 1 comprises residues 57-506 (numbering relative to full length protein inclusive of the signal sequence):
Figure imgf000023_0002
and glycosyl hydrolase-1 domain 2 comprises residues 515-953 (numbering relative to the full length protein inclusive of the signal sequence):
Figure imgf000023_0003
[0097] Orthologs exist in other species. Mouse and rat Klotho respectively share 86% and 85% identity with the human Klotho polypeptide, which is shown as SEQ ID NO: 1. [0098] In some embodiments, klotho polypeptides that can be used for administration to a subject in the methods described herein include species homologs (e.g., non-human primate, mouse, rat), allelic variants, functional fragments, and functional variants of the wild type sequence that retain cognition improving activity. Examples include secreted Klotho, fragments comprising the KL1 domain, fragments comprising the KL2 domain, fragments comprising the KL1 and KL2 domains, variants comprising the KL1 domain with at least one (e.g., 1-20, 5-50, 25-100) non-conserved amino acid in the KL1 domain substituted with a different amino acid or deleted, variants comprising the KL2 domain with at least one non-conserved amino acid in the KL2 domain substituted with a different amino acid.
[0099] In some embodiments, functional fragments of the Klotho polypeptide that can be used for administration to a subject as described herein include the extracellular domain (e.g., corresponding to or substantially identical or similar to amino acids 34-981 of human Klotho (e.g., corresponding to amino acid residues 34-981 or SEQ ID NO: 1)), secreted Klotho (e.g., corresponding to or substantially identical or similar to 549 amino acid form (e.g., corresponding to amino acids residues of 1-549 of SEQ ID NO: 1)), a KL1 domain (e.g., corresponding to or substantially identical or similar to amino acids 34-549 of human Klotho (e.g., corresponding to amino acid residues 34-549 of SEQ ID NO: 1)), a glycosyl hydrolase consensus sequence (e.g., corresponding to or substantially identical or similar to amino acids 57-506 of human Klotho (e.g., corresponding to amino acid residues 57-506 of SEQ ID NO: 1)), or a beta-glucosidase/6- phospho-beta-glucosidase/beta-galactosidase consensus sequence (e.g., corresponding to or substantially identical or similar to amino acids 62-497 of human Klotho (numbering relative to full length protein inclusive of the signal sequence)). In some embodiments, the Klotho polypeptide comprises or is substantially identical or similar to amino acids 34-549 of human Klotho (e.g., corresponding to amino acid residues of 34-549 of SEQ ID NO: 1).
[0100] In some embodiments, the klotho polypeptide has at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 1. In some embodiments, the klotho polypeptide has at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 2. In some embodiments, the klotho polypeptide has at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 3. In some embodiments, the klotho polypeptide has at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 4. In some embodiments, the Klotho polypeptide has at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to amino acids 34-981 of SEQ ID NO: 1.
[0101] In some embodiments, the polypeptide, that includes at least the klotho (KL) protein has at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to:
Figure imgf000025_0001
Residues 1-19 (underlined) constitute the signal sequence.
[0102] In some embodiments, the human klotho polypeptide has the sequence of SEQ ID NO: 6.
[0103] In some embodiments, the klotho polypeptide is as described in U.S. Patent No.
10,300,117, which is herein incorporated by reference in its entirety.
[0104] In some embodiments, the Klotho polypeptide is part of a larger fusion protein. In some embodiments, the fusion protein comprises the Klotho polypeptide as described herein and further comprises no more than 100, 75, 50, or 30 additional amino acids. In some embodiments, the Klotho polypeptide (e.g., any of the human klotho polypeptides described herein) is not fused to a Fibroblast growth factor (FGF). In some embodiments, the Klotho polypeptide (e.g., any of the human klotho polypeptides described herein) is not a fusion protein that also contains a fibroblast growth factor (FGF).
[0105] In some embodiments, the Klotho polypeptide comprises (e.g., is fused to) an affinity tag (e.g., a histidine tag) or a conjugate to increase stability or half-life in vivo.
[0106] In some embodiments, the Klotho polypeptide is linked to a stabilizing moiety such as PEG, glycosylation, or a liposome or other nanocarrier. U.S. Pat. Nos. 4,732,863 and 7,892,554 and Chattopadhyay et al. (2010) Mol Pharm 7:2194 describe methods for attaching a polypeptide to PEG, PEG derivatives, and nanoparticles (e.g., liposomes).
[0107] In some embodiments, the Klotho polypeptide is linked to a liposome. For example, liposomes containing phosphatidyl-ethanolamine (PE) can be prepared by established procedures as described herein. The inclusion of PE provides an active functional site on the liposomal surface for attachment.
[0108] In some embodiments, the Klotho polypeptide is linked to an affinity tag, e.g., a histidine tag (e.g., 4-16 histidine residues), streptavidin, or an antibody target.
[0109] In some embodiments, the Klotho polypeptide is glycosylated. In some embodiments, the polypeptide is N-glycosylated. In some embodiments, the polypeptide is O-glycosylated. In some embodiments, the polypeptide is both N-glycosylated and O-glycosylated.
[0110] In some embodiments, a functional variant or fragment of a Klotho polypeptide is a variant or fragment that retains any klotho activity, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%. or 100% of the level of any activity of soluble klotho. Soluble klotho activities include those described above, and include binding FGF-23, binding to FGFRlc, betaglucuronidase activity, suppression of wnt signaling, suppression of insulin signaling, suppression of TFG-beta 1 activity, increasing GluN2B expression and/or synaptic localization, c-fos induction. Additional Klotho activities include causing changes in magnetic resonance imaging (MRI) brain scans, e.g., functional MRI, electroencephalograph (EEG), and transcranial magnetic and electrical stimulation (TMS and TES); and improved performance in neuropsychologic testing and cognitive ability. 7.7. Formulations and administration of Klotho
[0111] In some embodiments, the klotho polypeptide is administered as described in U.S. Patent No. 10,300,117, which is herein incorporated by reference in its entirety.
[0112] In some embodiments, the Klotho polypeptide is administered in a pharmaceutical composition with a physiologically (i.e., pharmaceutically) acceptable carrier. The term “earner” refers to a typically inert substance used as a diluent or vehicle for a diagnostic or therapeutic agent. The term also encompasses a typically inert substance that imparts cohesive qualities to the composition. Physiologically acceptable carriers can be liquid, e.g., physiological saline, phosphate buffer, normal buffered saline (135-150 mM NaCl), water, buffered water, 0.4% saline, 0.3% glycine, glycoproteins to provide enhanced stability (e.g., albumin, lipoprotein, globulin, etc.), and the like. Since physiologically acceptable carriers are determined in part by the particular composition being administered as well as by the particular method used to administer the composition, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (See, e.g.. Remington's Pharmaceutical Sciences, 17th ed., 1989).
[0113] In some embodiments, the compositions (e.g., klotho polypeptide and a carrier) can be sterilized by conventional, well-known sterilization techniques or may be produced under sterile conditions. Aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. Sugars can also be included for stabilizing the compositions, such as a stabilizer for lyophilized antibody compositions.
[0114] In some embodiments, the Klotho polypeptide is formulated as a dosage forms prepared for mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous, intravenous, intramuscular-, or intraarterial injection, either bolus or infusion), oral, or transdermal administration to a patient. Examples of dosage forms include, but are not limited to: dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.
[0115] In some embodiments, the Klotho polypeptide is formulated for injection. Injectable compositions can comprise a solution of the Klotho polypeptide suspended in an acceptable carrier, such as an aqueous carrier. Any of a variety of aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, 5% dextrose, and the like, and may include glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. In some embodiments, normal buffered saline (135-150 mM NaCl) is used. The compositions can contain pharmaceutically acceptable auxiliary substances to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
[0116] Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Injection solutions and suspensions can also be prepared from sterile powders, granules, and tablets. In some embodiments, the composition is administered by intravenous infusion, topically, intraperitoneally, intravesically, or intrathecally. The Klotho polypeptide formulation can be provided in unit-dose or multi-dose scaled containers, such as ampoules and vials. [0117] The Klotho polypeptide composition, alone or in combination with other suitable components, can be made into aerosol formulations (“nebulized”) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, and nitrogen.
[0118] In some embodiments, the pharmaceutical composition (e.g., the klotho polypeptide and a carrier) can be packaged or prepared in unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component, e.g., according to the dose of Klotho polypeptide. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation. The composition can, if desired, also contain other compatible therapeutic agents. In some embodiments, the Klotho polypeptide composition can be formulated in a kit for administration.
[0119] In some embodiments, a pharmaceutical composition comprising a klotho polypeptide is administered orally. In some embodiments, a pharmaceutical composition comprising a klotho polypeptide is administered mucosally, e.g., nasally. In some embodiments, a pharmaceutical composition comprising a klotho polypeptide is administered by injection, e.g., subcutaneous, intraperitoneal, intravenous, or intramuscular. In some embodiments, a pharmaceutical composition comprising a klotho polypeptide is administered by infusion, e.g., using a reservoir or osmotic minipump.
[0120] An example of administration of a pharmaceutical composition includes storing the Klotho polypeptide at 10 mg/ml in sterile isotonic aqueous saline solution at 4° C., and diluting it in an appropriate solution for injection prior to administration to the patient. In some embodiments, the Klotho polypeptide composition can be administered by intravenous infusion over the course of 0.25-2 hours. In some embodiments, the administration procedure is via bolus injection.
[0121] In some embodiments, the Klotho polypeptide composition is administered to an (e.g., human) individual having at least normal cognitive function. As described herein, it has been surprisingly discovered that not only can Klotho improve cognition in individuals with impaired cognition, Klotho can also improve cognition of individuals with at least normal cognition. Thus in some embodiments, the individual receiving the Klotho polypeptide composition begins initially with at least normal cognition and following administration of the Klotho polypeptide composition attains improved cognition compared to the initial level of cognition. The level of cognition of an individual can be determined as is known in the art. Normal cognitive functions are determined by scores from sets of cognitive tests that are compiled into global cognitive scores, as described in Dubai D B et al. (2014) Cell Reports 7:1065-1076. Such cognition tests include tests of executive function and working memory such as Trails A and Trails B (Dubai D B et al. (2014) Cell Reports 7: 1065-1076). In some embodiments, administration of Klotho results in an improvement of cognition (whether initially at least normal or impaired), by at least 5%, 10%, 20% or more.
[0122] The Klotho polypeptide can also be formulated as a sustained-release preparation (e.g., in a semi-permeable matrices of solid hydrophobic polymers (e.g., polyesters, hydrogels (for example, poly (2-hydroxyethyl-methacrylate), or poly (vinylalcohol)), polylactides. The Klotho polypeptide can be entrapped in a nanoparticle prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxy methylcellulose or gelatin microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.
7.8. Nucleic Acid Delivery of Polynucleotide Encoding Klotho
[0123] In another aspect, this disclosure features a method of increasing cognitive function in a human subject, comprising: parenterally administering to said subject at least one dose of a polynucleotide that is capable of expressing at least the KL1 domain of human klotho (KL) (, wherein each dose is sufficient to raise serum KL concentrations by at least 2.5 fold, and no more than 25-fold, compared to pre-administration (baseline) concentrations.
[0124] In another aspect, this disclosure features a method of increasing cognitive function in a human subject, comprising: parenterally administering to said subject at least one dose of a polynucleotide that is capable of expressing at least the KL1 domain of human klotho (KL) (, wherein each dose is sufficient to raise serum KL concentrations to at least 1000 μg/mL and no more than 5000 pg/mL. [0125] In another aspect, this disclosure features a method of increasing cognitive function in a human subject, comprising: parenterally administering to said subject a plurality of doses of a polynucleotide that is capable of expressing at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations by at least 2.5 fold, and no more than 25-fold, compared to pre-administration (baseline) concentrations, and wherein the plurality of doses is administered on an intermittent dosing schedule.
[0126] In another aspect, this disclosure features a method of increasing cognitive function in a human subject, comprising: parenterally administering to said subject a plurality of doses of a polynucleotide that is capable of expressing at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations to at least 1000 pg/mL and no more than 5000 pg/mL, and wherein the plurality of doses is administered on an intermittent dosing schedule.
[0127] In some embodiments, the polynucleotide is administered subcutaneously.
[0128] In some embodiments, the polynucleotide is an mRNA. In some embodiments, the mRNA comprises: a) at least one 5’cap structure; b) a 5’UTR (untranslated region); c) an mRNA open reading frame encoding the polypeptide (e.g., any of the klotho polypeptides or klotho domains described herein); d) a 3’UTR (untranslated region); and e) a poly-A region of at least 100 nucleotides in length.
[0129] In some embodiments, the dose is administered in a pharmaceutical formulation that further comprises a pharmaceutically acceptable carrier for the polynucleotide. In some embodiments, the formulation comprises a lipid nanoparticle, polymer, liposome, dendrimer, exosomes, or a lipid.
[0130] In some embodiments, the polynucleotide is an expression vector. In some embodiments, the expression vector is a viral expression vector. In some embodiments, the viral expression vector is a retroviral vector, a lentiviral vector, an AAV vector, or an adenoviral vector. In some embodiments, the viral expression vector is an AAV vector. In some embodiments, the viral expression vector is a retroviral vector. In some embodiments, the viral expression vector is a lentiviral vector. [0131] In some embodiments, the viral expression vector is engineered to preferentially transduce at least one target tissue following parenteral administration. In some embodiments, the at least one target tissue is liver, muscle, or central nervous system (CNS) cells. In some embodiments, the at least one target tissue is CNS cells.
[0132] In some embodiments, the viral expression vector crosses the blood brain barrier following parenteral administration.
8. EXAMPLES
8.1. Example 1: Klotho-mediated cognitive enhancement
[0133] We sought to test whether a low dose, subcutaneous administration of KL could boost cognition in aged rhesus macaques, a type of non-human primate (NHP). Rhesus macaque was selected as a model system because of their phylogenetic similarity to humans, demonstrated complex higher-order cognitive functions, and the propensity to undergo targeted functional decline of similar brain regions such, as the prefrontal cortex, as do aged humans.
[0134] The primary goal was to test if a Klotho (KL) dose could be found that could enhance cognition in rhesus macaque monkeys. For the primary goal, we demonstrated that that KL (10 μg/kg) enhanced cognition in aging rhesus macaques, an effect that persisted for at least two weeks in both the NML and HML measures of memory. The secondary goal was to explore higher KL doses in rhesus macaques to test whether KL-mediated benefits on cognition could be dose-dependent. For the secondary goal, we demonstrated higher doses of klotho (20 μg/kg and 30 μg/kg) induced dose-dependent attenuation of cognitive enhancement. Overall, these data demonstrate klotho-mediated cognitive enhancement in NHPs who have a complex genetic, anatomic, and functional brain similar to humans, and that the dose response is complex, with a previously unknown effective dose window.
8.1.1. Methods
8.1.1.1 Animals
[0135] All mice were on a congenic C57BL/6J background and kept on a 12 hour light/dark cycle with ad libitum access to food and water. The standard housing group was five mice per cage. Behavior tasks were carried out during the light cycle. All studies were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco, and conducted in compliance with NIH guidelines. Adult aged male and female rhesus macaque monkeys (Macaca mulatta) were maintained in accordance with the Yale University Institutional Animal Care and Use Committee and federal guidelines for the care and use of nonhuman primates. Monkeys were maintained in a 12 hour light/dark cycle, fed standard monkey chow and fruit, and were tested during the light cycle.
8.1.1.2 Drug Treatment
[0136] Mouse KL, and rhesus KL were diluted in PBS (pH 7.5) and injected subcutaneous (s.c.) at specified doses prior to behavior testing or sample collection for serum KL measurements. Recombinant proteins were used within one week of thawing from -80°C stock solutions and stored at 4°C. Proteins were coded to keep experimenters blind to the identity of treatment used in experiments for all mouse studies and for the majority of monkey studies performed.
8.1.1.3 Serum klotho measurements
[0137] Enzyme-linked immunosorbent assay (ELISAs) was performed to measure KL from monkey serum samples, according to the manufacturer’s directions as described in Dubai et al. (Cell Rep 7, 1065-1076, doi: 10.1016/j.celrep.2014.03.076 (2014)) and Yokoyama et al. (Ann. Clin. Transl. Neurol. 2, 215-230, doi: 10.1002/acn3.161 (2015)), each of which are herein incorporated by reference in their entireties. Briefly, each monkey serum sample was diluted with ELISA buffer (1:2) and analyzed for KL by ELISA. One serum value, several standard deviation above the cohort mean was excluded from analysis. For mouse serum, samples were assayed using immunoprecipitation-immunoblot (IP-IB) for mouse KL by the O’Brien Kidney Research Core at UT Southwestern as described in Barker (Nephrol. Dial. Transplant 30. 223- 233, doi: 10.1093/ndt/gfu291 (2015), which is herein incorporated by reference in its entirety).
8.1.1.4 Electrophysiology
[0138] Coronal mouse brain slices of 300 pm thickness were obtained as described with some modifications (see Dubai et al. (Cell Rep., 7, 1065-1076, doi: 10.1016/j.celrep.2014.03.076 (2014)); Dubai et al. (J. Neurosci. 35, 2358-2371, doi: 10.1523/JNEUROSCI.5791-12.2015 (2015); Leon et al. (Cell Rep., 20, 1360-1371, doi: 10.1016/j.celrep.2017.07.024 (2017)), each of which are herein incorporated by reference in their entireties) including that measurements were obtained from the hippocampal CAI region following stimulation of the Schaffer collateral path. Briefly, mice were anesthetized and the brain was immediately placed in ice-cold artificial cerebrospinal fluid (aCSF) and sliced on a vibratome (Leica VT1200). Slices were incubated at 32°C for 30 minutes and then incubated at RT (room temperature) for 1 hour prior to testing. For field potential recordings, acute hippocampal slices were placed on a Med64-Quad II multielectrode array (Alpha MED Scientific). Field Excitatory Post Synaptic Potentials (fEPSP) were elicited and recorded via planar electrodes of the Quad II 2x8 Probe AL-MED-PG501A by aligning the electrodes and the stratum radiatum region of hippocampal slices. Long term potentiation (LTP) was induced using a theta-burst protocol comprised of 2 trains delivered every 20 seconds, each train containing 10 bursts at 5 Hz, each burst containing four pulses at 100 Hz. Recordings and analysis were performed using Med64 Mobius Software (Alpha MED Scientific). Following stable LTP induction, the last 10 minutes of a usual 60 minute recording was averaged to compare the experimental groups.
8.1.1.5 Small Y maze
[0139] Mice were tested in the small Y maze, also known as spontaneous alternation Y maze with spatial cues, as described in Dubai et al. (Cell Rep., 7, 1065-1076, doi: 10.1016/j.celrep.2014.03.076 (2014)); Dubai et al. (J. Neurosci. 35, 2358-2371, doi:10.1523/JNEUROSCI.5791-12.2015 (2015)); and Leon et al. {Cell Rep., 20, 1360-1371, doi: 10.1016/j.celrep.2017.07.024 (2017)), each of which are herein incorporated by reference in their entireties. Briefly, a mouse was placed inside one of the three identical arms of the Y-maze and allowed to explore the apparatus for 6 minutes. The recorded spontaneous alterations were scored manually, and percent alternations were calculated.
8.1.1.6 Spatial delayed response task
[0140] The spatial delayed response task with normal memory load (NML) and high memory load (HML) was performed as described in Castner, S. A. & Goldman-Rakic, P. S. {J. Neurosci 24, 1446-1450, doi:10.1523/JNEUROSCI.3987-03.2004 (2004)) and Roberts, B. M. et al. {Neuroreport 21. 390-394, doi: 10.1097/WNR.0b013e3283381a4e (2010)). Briefly, following training to stability (65-75% correct) on the NML task, monkeys were tested at approximately the same time of day for highly palatable food rewards. Stable performance was first achieved for each monkey by titrating task difficulty by increasing the number of spatial locations or lengthening the delays (a variable multiplier between 1 and 10 applied to a set of baseline delays of 0, 1, 2, 3, 4 sec). Once stable, the number of wells and delay multiplier was kept fixed for that monkey for the duration of the testing for the current study. For testing, monkeys watched the spatially distanced wells while the investigator put a preferred treat in a single well. The wells were covered, an opaque screen between the monkey and wells was lowered, and after a predetermined delay, the screen was raised. The monkey was able to retrieve the treat if the choice of well was correct. Based on the number of wells and the time between cue and task the behavior is classified as either high memory load (HML) (6-9 wells;0-15 second delay) or normal memory load (NML) (2-7 wells; 0-32 seconds).
8.1.1.7 Statistics
[0141] Statistical analyses were carried out with GraphPad Prism (version 7.0) for t-tests and analyses of variance (ANOVAs). Two-tailed t-tests were used to determine differences between two means and two-way ANOVA was used to determine differences between multiple means. Post-hoc corrections were performed for multiple comparisons as indicated. Linear mixed model analysis for determining effect of klotho on task performance was performed with R using the treatment concentration as a fixed effect and each rhesus macaque as a random effect. Error bars represent +SEM. Null hypotheses were rejected at or below a P value of 0.05.
8.1.2. Results
[0142] We first generated the rhesus macaque form of the KL protein (96% homologous to human klotho) and functionally validated its activity using assays in mice. We tested a KL dose (10 μg/kg) previously shown to increase synaptic and cognitive functions in mice5. As expected, 4 hrs after peripheral administration in mice (FIG. 1A), rhesus KL enhanced long term potentiation (LTP), a form of synaptic plasticity (FIGs. IB and 1C). In parallel to LTP, rhesus KL, 4 hours after peripheral administration in mice, enhanced working memory in the small Y maze (FIGs. ID and IE) without altering total movements. Collectively, these data show that the rhesus form of the KL protein was biologically active in the mouse brain, with a dose previously found to be effective using mouse KL (see Leon, et al. {Cell Rep., 20, 1360-1371, doi: 10.1016/j.celrep.2017.07.024 (2017)). [0143] We next assessed serum KL levels in mice with KL-mediated cognitive enhancement. We measured serum KL levels 4 hours after hormone administration since peripheral klotho treatment enhances cognition by that time point (FIG. IF). Mice were treated with either vehicle or mouse KL (10 μg/kg, s.c.) and 4 hours later their serum was collected and assayed for KL levels (FIG. IF). Peripheral KL treatment with 10 μg/kg increased serum KL levels by approximately 6-fold compared to vehicle-treated mice (FIG. 1G). Since transgenic overexpression of KL also enhances cognition in mice (see Dubai et al. (Cell Rep., 7, 1065-1076, doi: 10.1016/j.celrep.2014.03.076 (2014)) and Dubai et al. (J. Neurosci., 35, 2358-2371, doi: 10.1523/JNEUROSCI.5791-12.2015 (2015)), we measured serum KL in transgenic mice and found 3.5-fold increases compared to non-transgcnic mice (FIG. 1G). Thus, a range of 3.5-6 fold increases of serum KL were observed in klotho-mediated cognitive enhancement of mice.
[0144] We aimed to achieve similar- increases of serum klotho in aging rhesus macaques to those observed in cognitively-enhanced mice. Since metabolism of KL could differ between the species, we peripherally administered a range of KL doses in rhesus macaques (0.4-30 μg/kg, s.c.), collected their serum 4 hours later, and assayed KL levels. Compared to baseline levels of KL in aging rhesus macaques, treatment with 10 μg/kg was the lowest dose tested that significantly increased serum levels by 5-fold, within the desired therapeutic range observed in mice. Of note, 5-fold higher levels of serum KL are observed in human cord blood compared to adults (see, e.g., Ohata et al. (J. Clin. Endocrinol. Metab., 96, E943-947, doi:10.1210/jc.2010- 2357 (2011)), and decrease thereafter in aging (see, e.g., Semba et al. (J. Gerontol. A Biol. Sci. Med. Sci., 66, 794-800, doi: 10.1093/gerona/glr058 (2011)) and Dubai et al. (Cell Rep., 7, 1065- 1076, doi: 10.1016/j.celrep.2014.03.076 (2014)).
[0145] To execute the primary goal, we treated aged rhesus macaques with a single administration of vehicle or 10 μg/kg (s.c.) of KL and tested their cognition. This dose was chosen for primary analysis because it produced similar- increases of KL that are 1) effective in cognitive enhancement of mice and 2) present at birth in humans (see, e.g., Ohata et al. ( J. Clin. Endocrinol. Metab., 96, E943-947, doi: 10.1210/jc.2010-2357 (2011)).
[0146] Cognitive testing of aged rhesus macaques was performed using the spatial delayed response task (FIG. 2A), assessing frontal-temporal circuits and regions of the brain including the hippocampus and frontal cortex. This task assesses working and spatial memory for both a normal memory load (NML, easier task) and high memory load (HML, harder task) (see Castner, S. A. & Goldman-Rakic, P. S., J. Neurosci.. 24. 1446-1450, doi:10.1523/JNEUROSCI.3987- 03.2004 (2004)) (FIG. 2B). In brief, monkeys were trained to achieve a stable baseline response of 65-75% correct for the NML task in choosing the spatial location of a food reward, among 2-7 wells covered with identical plaques, after variable delays of up to 28 seconds from the time an opaque shutter was lowered in front of them. Well number and duration were titrated previously for each animal in order to achieve the desired stable baseline performance. All macaques were then treated with vehicle (s.c.) to habituate to effects of the procedure and stress of an injection on cognitive performance. Finally, monkeys were treated with either vehicle or rhesus KL; 4 hours later, monkeys underwent the HML task (with up to 9 wells) followed by a series of NML tasks (up to 7 wells) over two weeks (FIG. 2A), ending with another HML task.
[0147] In the primary analysis, KL (10 μg/kg, s.c.) enhanced cognition in aged rhesus macaques in the short-term and in the long-term during both NML and HML testing. As expected, performance between baseline or vehicle treatment did not differ. KL (10 μg/kg, s.c.) increased HML performance by 4 hours after treatment (FIG. 2C), within the same rapid time frame it increased cognition in mice (see FIG. IE). KL-mediated cognitive enhancement of HML, a harder memory task (given the increased number of wells from which to choose and the increase in delays) persisted at 2 weeks (FIG. 2D). KL (10 μg/kg, s.c.) also enhanced the average NML performance (FIG. 2E), an effect that largely persisted across multiple tests over two weeks (FIG. 2F).
[0148] In exploratory analysis of higher KL doses (20 and 30 μg/kg, s.c.), significant cognitive improvement was not observed in the HML tasks (FIG. 2G). In the NML task, there was a dose-dependent attenuation in cognitive improvement compared to the 10 μg/kg dose - with 20 μg/kg showing a trend in cognitive enhancement (p=0.057) and 30 μg/kg showing no cognitive improvement (FIG. 2H); this attenuation was not observed in mice treated with higher klotho doses. No klotho-induccd cognitive deficits were detected at any dose tested.
8.1.3. Discussion [0149] Collectively, the data show that KL (10 μg/kg) enhanced cognition in aging rhesus macaques, an effect that persisted for at least two weeks in both the NML and HML measures of memory. Klotho-mediated cognitive enhancement similarly persisted in mice for at least two weeks, suggesting organizational, longer-lasting, and beneficial effects on the synapse and brain. In both species, the cognitive effect outlasted the putative half-life of the hormone, estimated at 7 min in rodents and preliminarily around 27 hours in aging rhesus macaques (n=2). Higher doses of klotho (20 μg/kg and 30 μg/kg) induced dose-dependent attenuation of cognitive enhancement. Together, these data indicate klotho-mediated cognitive enhancement extends to NHPs in a complex genetic, anatomic, and functional brain similar to humans. The data also suggest lower, more “physiological” levels of the hormone in the body may be required for a therapeutic window of cognitive enhancement in humans.
[0150] Since KL has pleiotropic actions including on insulin (see Wolf et al.. Oncogene 27, 7094-7105, doi: 10.1038/onc.2008.292 (2008) and FGF signaling (Urakawa et al., Nature 444, 770-774, doi:10.1038/nature05315 (2006), Wnt (Liu et al., Science 317, 803-806, doi: 10.1126/science.l 143578 (2007), and NMD AR functions (Dubai et al. (Cell Rep. 7, 1065- 1076, doi: 10.1016/j.celrep.2014.03.076 (2014)); Dubai et al. (J. Neurosci. 35, 2358-2371 , doi:10.1523/JNEUROSCI.5791-12.2015 (2015)); and Leon et al. (Cell Rep., 20, 1360-1371, doi: 10.1016/j.celrep.2017.07.024 (2017)), each of which are herein incorporated by reference in their entireties, it is interesting to speculate that the specificity of its action at low doses, represents a balanced, multi-modal effect across signaling systems that inherently benefits biological substrates of cognition. Higher doses of KL, beyond what is experienced over the human lifespan, could differentially impact signaling systems to create imbalances that no longer enhance cognition. Whether even lower doses of KL than those tested could also enhance cognition remains to be determined.
[0151] Because KL levels decrease in human aging (see Semba et al., J. Gerontol. A Biol. Sci. Med. Sci., 66, 794-800, doi: 10.1093/gerona/glr058 (2011)); and Yamazaki et al., Biochem Biophys Res Commun 398, 513-518, doi: 10.1016/j.bbrc.2010.06.110 (2010)), our data show that a lower dose of KL can enhance cognition in aged rhesus macaques suggests that peripheral treatment, or replenishment, with this endogenous hormone may prove therapeutic in aging humans. 9. SEQUENCES
[0152] SEQ ID NO: 1 (full length Klotho (amino acid 1-1012):
Figure imgf000039_0001
[0153] SEQ ID NO: 2 (full length without signal sequence (amino acids 34-1012 of full length)):
Figure imgf000039_0002
[0154] SEQ ID NO: 3 (KL1 domain comprising amino acids 34-549 of full length KL):
Figure imgf000040_0001
[0155] SEQ ID NO: 4 (Glycosyl hydrolase- 1 domain 1 (as identified by Uniprot) comprising amino acids 57-506 of full length):
Figure imgf000040_0002
[0156] SEQ ID NO: 5 (glycosyl hydrolase- 1 domain 2 (as identified by Uniprot) comprising amino acid 515-953):
Figure imgf000040_0003
[0157] SEQ ID NO: 6 (signal sequence + 34-981 of SEQ ID NO: 1):
Figure imgf000040_0004
Figure imgf000041_0001
10. EQUIVALENTS AND INCORPORATION BY REFERENCE
[0158] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.
[0159] All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

Claims

WHAT IS CLAIMED IS:
1. A method of improving cognitive function in a human subject, comprising: parenterally administering to said subject at least one dose of a polypeptide, or a polynucleotide that is capable of expressing a polypeptide, that includes at least the KL1 domain of human klotho (KL), wherein each dose is sufficient to raise serum KL concentrations by at least 2.5-fold, and no more than 25-fold, compared to pre-administration (baseline) concentrations.
2. The method of claim 1, wherein the dose is sufficient to raise serum KL concentrations by at least 5-fold compared to baseline concentrations.
3. The method of any one of claims 1-2, wherein the dose is sufficient to raise serum KL concentrations by at least 7.5-fold compared to baseline concentrations.
4. The method of any one of claims 1-3, wherein the dose is sufficient to raise serum KL concentrations by at least 10-fold compared to baseline concentrations.
5. The method of any one of claims 1-4, wherein the dose is sufficient to raise serum KL concentrations by no more than 20-fold compared to baseline concentrations.
6. The method of any one of claims 1-5, wherein the dose is sufficient to raise serum KL concentrations by no more than 15-fold compared to baseline concentrations.
7. The method of any one of claims 1-6, wherein the dose is sufficient to raise serum KL concentrations by no more than 10-fold compared to baseline concentrations.
8. The method of any one of claims 1-7, wherein the dose is sufficient to raise serum KL concentrations by 2.5-fold to 10-fold compared to baseline concentrations.
9. The method of any one of claims 1-8, wherein the dose is sufficient to raise serum KL concentrations by 5-fold to 7.5-fold compared to baseline concentrations.
10. The method of any one of claims 1-9, wherein each dose is sufficient to raise serum KL concentrations to at least 1000 pg/mL and no more than 5000 pg/mL.
11. The method of claim 10, wherein the dose is sufficient to raise serum KL concentrations to at least 1500 pg/mL.
12. The method of claim 11, wherein the dose is sufficient to raise scrum KL concentrations to at least 2000 pg/mL.
13. The method of claim 12, wherein the dose is sufficient to raise serum KL concentrations to at least 2500 pg/mL.
14. The method of claim 13, wherein the dose is sufficient to raise serum KL concentrations to at least 3000 pg/mL.
15. The method of claim 14, wherein the dose is sufficient to raise serum KL concentrations to at least 3500 pg/mL.
16. The method of any one of claims 10-15, wherein the dose is sufficient to raise serum KL concentrations to no more than 4000 pg/mL.
17. The method of claim 16, wherein the dose is sufficient to raise serum KL concentrations to no more than 3000 pg/mL.
18. The method of claim 17, wherein the dose is sufficient to raise serum KL concentrations to no more than 2500 pg/mL.
19. The method of any one of claims 1-18, wherein a plurality of doses is administered on an intermittent dosing schedule.
20. The method of claim 19, wherein successive doses are administered no less frequently than once every 6 months and no more frequently than once per day.
21. The method of claim 20, wherein successive doses are administered no less frequently than once every 3 months.
22. The method of claim 21, wherein successive doses arc administered no less frequently than once a month.
23. The method of claim 22, wherein successive doses are administered no less frequently than once every four weeks.
24. The method of claim 23, wherein successive doses are administered no less frequently than once every other week.
25. The method of claim 24, wherein successive doses are administered no less frequently than once a week.
26. The method of any one of claims 19-25, wherein successive doses are administered no more frequently than once a week.
27. The method of claim 26, wherein successive doses are administered no more frequently than one every two weeks.
28. The method of claim 27, wherein successive doses are administered no more frequently than once every four weeks.
29. The method of claim 28, wherein successive doses are administered no more frequently than once a month.
30. The method of any one of claims 19-29, wherein successive doses are administered once a day.
31. The method of any one of claims 19-29, wherein successive doses are administered once a week.
32. The method of any one of claim 19-29, wherein successive doses arc administered once every two weeks.
33. The method of any one of claims 19-29, wherein successive doses are administered once every four weeks.
34. The method of any one of claims 1-33, wherein the subject is at least 21 years old.
35. The method of claim 34, wherein the subject is at least 45 years old.
36. The method of claim 35, wherein the subject is at least 60 years old.
37. The method of claim 36, wherein the subject is at least 65 years old.
38. The method of claim 37, wherein the subject is at least 80 years old.
39. The method of any one of claims 34-38, wherein the subject, prior to first administration, has normal cognitive function.
40. The method of claim 39, wherein the subject has at least one ApoE4 allele.
41. The method of any one of claims 34-38, wherein the subject, prior to first administration, has cognitive impairment.
42. The method of claim 41, wherein the subject, prior to first administration, has mild cognitive impairment (MCI).
43. The method of claim 41, wherein the subject, prior to first administration, has dementia.
44. The method of claim 42 or claim 43, wherein the subject has Alzheimer’s disease, frontotemporal dementia, Lewy Body Dementia, Parkinson's disease, Huntington's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasalar degeneration, vascular cognitive impairment, vascular dementia, amyotropic lateral sclerosis, prion disorder, posterior cortical atrophy, primary progressive aphasia HIV- related dementia, global aphasia, mixed transcortical aphasia, Broca’s aphasia, transcortical motor aphasia, Wernicke’s aphasia, transcortical sensory aphasia, conduction aphasia, anomic aphasia, multiple system atrophy; mental or mood disorders: depression, schizophrenia, attention deficit/ hyperactivity disorder, autism spectrum disorder, intellectual disability, mood disorder, psychotic disorder, psychosis; childhood neurodevelopmental syndromes and brain tumors: X-linked mental disability or retardation, astrocytoma, ependymoma, medulloblastoma, oligodendroglioma; genetic syndromes affecting learning: Down's syndrome, Angelman's syndrome, Rett' s syndrome;
Metabolic disorders affecting cognition: phenylketonuria, Lesch-Nyhan, galactosemia, and adrenoleukodystrophy;
Cognitive decline associated with chemotherapy and/or radiation therapy or “chemo brain”; and additional conditions and disorders: pain-associated cognitive effects, traumatic brain injury, stroke, multiple sclerosis, neuroautoimmune disease, epilepsy, delirium, paraneoplastic disorder, developmental delay, hydrocephalus, leukodystrophies sleep deprivation, jet lag.
45. The method of any one of claims 1-33, wherein the subject is under 21 years old.
46. The method of any one of claims 1-45, wherein the polypeptide has at least 85% sequence identity to SEQ ID NO: 3.
47. The method of claim 46, wherein the polypeptide has at least 90% sequence identity to SEQ ID NO:3.
48. The method of claim 47, wherein the polypeptide has at least 95% sequence identity to SEQ ID NO:3.
49. The method of claim 48, wherein the polypeptide has at least 99% sequence identity to SEQ ID NO:3.
50. The method of any one of claims 1-49, wherein the polypeptide has at least 85% identity to amino acids 34-981 of SEQ ID NO: 1.
51. The method of claim 50, wherein the polypeptide has at least 90% sequence identity to amino acids 34-981 of SEQ ID NO: 1.
52. The method of claim 51, wherein the polypeptide has at least 95% sequence identity to amino acids 34-981 of SEQ ID NO: 1.
53. The method of any one of claims 1-52, wherein the KL1 domain has at least one, and up to 5%, altered, inserted or deleted amino acids compared with the amino acid sequence of SEQ ID NO: 3.
54. The method of any one of claims 1-53, wherein the polypeptide further includes a domain that is at least 95% identical in sequence to SEQ ID NO: 5.
55. The method of any one of claims 1-54, with the proviso that the polypeptide is not a fusion protein that also contains a fibroblast growth factor (FGF).
56. The method of any one of claims 1-55, wherein the polypeptide has the sequence of SEQ
ID NO: 6.
57. The method of any one of claims 1-56, wherein the dose is a polypeptide administered subcutaneously (s.c.).
58. The method of claim 57, wherein the polypeptide is glycosylated.
59. The method of claim 58, wherein the polypeptide is both N-glycosylated and O- glycosylated.
60. The method of any one of claims 57-59, wherein the dose is at least 5 μg/kg s.c. and no more than 50 μg/kg s.c.
61. The method of claim 60, wherein the dose is at least 7.5 μg/kg s.c.
62. The method of claim 61, wherein the dose is at least 10 μg/kg s.c.
63. The method of claim 62, wherein the dose is at least 15 μg/kg s.c.
64. The method of claim 63, wherein the dose is at least 20 μg/kg s.c.
65. The method of claim 64, wherein the dose is at least 25 μg/kg s.c.
66. The method of any one of claims 60-65, wherein the dose is no more than 40 μg/kg s.c.
67. The method of claim 66, wherein the dose is no more than 30 μg/kg s.c.
68. The method of claim 67, wherein the dose is no more than 25 μg/kg s.c.
69. The method of claim 68, wherein the dose is no more than 20 μg/kg s.c.
70. The method of claim 59, wherein the dose is at least 350 μg and no more than 3500 μg.
71. The method of claim 70, wherein the dose is at least 500 μg.
72. The method of claim 71, wherein the dose is at least 750 μg.
73. The method of claim 72, wherein the dose is at least 1000 μg.
74. The method of claim 73, wherein the dose is at least 1500 μg.
75. The method of claim 74, wherein the dose is at least 2000 μg.
76. The method of claim 75, wherein the dose is at least 2500 μg.
77. The method of any one of claims 70-76, wherein the dose is no more than 3000 μg.
78. The method of claim 77, wherein the dose is no more than 2500 μg.
79. The method of claim 78, wherein the dose is no more than 2000 μg.
80. The method of claim 79, wherein the dose is no more than 1500 μg.
81. The method of claim 80, wherein the dose is no more than 1000 μg.
82. The method of any one of claims 1-56, wherein the dose is a polynucleotide.
83. The method of claim 82, wherein the polynucleotide is administered subcutaneously.
84. The method of claim 82 or claim 83, wherein the polynucleotide is an mRNA.
85. The method of claim 84, wherein the mRNA comprises: a) at least one 5 ’cap structure; b) a 5’-UTR; c) an mRNA open reading frame encoding the polypeptide; d) a 3’UTR; and e) a poly- A region of at least 100 nucleotides in length.
86. The method of claim 84 or claim 85, wherein the dose is administered in a pharmaceutical formulation that further comprises a pharmaceutically acceptable carrier for the polynucleotide.
87. The method of claim 86, wherein the formulation comprises a lipid nanoparticle, polymer, liposome, dendrimer, exosomes, or a lipid.
88. The method of claim 82 or claim 83, wherein the polynucleotide is an expression vector.
89. The method of claim 88, wherein the expression vector is a viral expression vector.
90. The method of claim 89, wherein the viral expression vector is a retroviral vector, a lentiviral vector, an AAV vector, or an adenoviral vector.
91. The method of claim 90, wherein the viral expression vector is an AAV vector.
92. The method of claim 90, wherein the viral expression vector is a retroviral vector.
93. The method of claim 90, wherein the viral expression vector is a lentiviral vector.
94. The method of any one of claim 89-93, wherein the viral expression vector is engineered to preferentially transduce at least one target tissue following parenteral administration.
95. The method of claim 94, wherein the at least one target tissue is liver, muscle, or central nervous system (CNS) cells.
96. The method of claim 95, wherein the at least one target tissue is CNS cells.
97. The method of claim 96, wherein the viral expression vector crosses the blood brain barrier following parenteral administration.
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