WO2019070759A1

WO2019070759A1 - Amino acid compositions and methods for treating cystic fibrosis

Info

Publication number: WO2019070759A1
Application number: PCT/US2018/054024
Authority: WO
Inventors: Sadasivan Vidyasagar; Reshu GUPTA; Astrid GROSCHE
Original assignee: University Of Florida Research Foundation Incorporated
Priority date: 2017-10-02
Filing date: 2018-10-02
Publication date: 2019-04-11

Abstract

Provided herein are amino acid compositions useful for increasing the translocation of the cystic fibrosis transmembrane conductance (CFTR) protein from the cytoplasm to the plasma membrane, particularly in epithelial cells. Methods for increasing the concentration of CFTR in the plasma membrane, increasing chloride ion transport, and increasing water transport are also provided. These compositions and methods are useful in treating cystic fibrosis in subjects bearing one or more mutations in the CFTR protein.

Description

AMINO ACID COMPOSITIONS AND METHODS FOR TREATING CYSTIC FIBROSIS

RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application, U.S. S.N. 62/566,873, filed October 2, 2017 which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Cystic fibrosis (CF) is the most common life-limiting genetic disorder that can affect the lungs, liver, pancreas, kidneys, and intestines. Cystic fibrosis is inherited in an autosomal recessive manner, where each parent carries a mutation in at least one allele of the gene encoding the cystic fibrosis transmembrane conductance (CFTR) protein, leading to one out of four progeny carrying two mutated copies (e.g., alleles) of the CFTR gene. Thus, more than 75% of patients are diagnosed with cystic fibrosis by age 2.¹ While the life expectancy of people living with cystic fibrosis has increased in recent years, the median age of survival is currently around 40 years. Current therapies often involve burdensome supportive treatments designed to manage the increased risk of lung infection and poor nutritional status. In addition, while small molecule therapies that treat the underlying genetic cause of CF have recently been approved, these therapies are minimally effective against the most common CFTR mutation, Phe508del, and are cost prohibitive at upwards of $300,000 annually per patient.²

[0003] While over 2,000 CFTR mutations have been identified in patients suffering from cystic fibrosis, the vast majority of cystic fibrosis diagnoses exhibit one or more of only a handful of muations.³ The most common mutation, Phe508del, is a deletion of 3 nucleotides, resulting in the loss of a single codon for the amino acid phenylalanine (three letter code: Phe, single letter code: F). This mutation results in defective CFTR protein processing (e.g., folding and trafficking to the plasma membrane), resulting in little to no membrane expression of CFTR, a chloride ion transporter protein involved in ion and water transport across the cell membrane. In addition, the small amount of Phe508del CFTR that successfully translocates to the plasma membrane is often functionally defective, as characterized by impaired chloride ion transport. In general, CFTR mutations lead to the dysregulation of the ion gradient across membranes, resulting in reduced osmotic pressure for water to flow out of the epithelial cells and manifestation of a thick mucus layer that covers the cells. The thick, nutrient rich mucus serves as an optimal environment for the trapping and growth of bacteria, such as Pseudomonas aeruginosa and Staphylococcus aureus, that lead to persistent infection that is often unresponsive to antibiotics. In addition, many CF patients also develop other lung diseases, such as bronchopulmonary aspergillosis and bronchiectasis, leading to increased morbidity and mortality.

[0004] While targeted therapies for cystic fibrosis caused by specific mutations in CFTR have been developed, many are only efficacious in patients with certain CFTR mutations, and adverse side effects have been reported.² One example is ivacaftor (VX-770, Kayldeco™), a small molecule potentiator of CFTR,⁴ as described in U.S. Patent Application Publication US 2014/0221424 and International Patent Application Number PCT/US2015/036691. However, ivacaftor is directed to the treatment of CF in patients with the missense mutation Gly551Asp (G551D) on at least one allele of the CFTR gene, which encompasses around 4 to 5% of patients suffering from CF.⁵ There is a clear, unmet need for therapies directed toward the treatment of cystic fibrosis, particularly in patients harboring the most prevalent CFTR mutation, Phe508del CFTR.

BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1A shows ileal sections depicting the formation of a normal cell junction complex observed by electron microscopy in non-irradiated epithelial cells.

[0006] Figure IB shows a schematic diagram with the various parts of a junction complex including a tight junction, intercellular space, gap junction, adherens junction, desmosome, and nucleus.

[0007] Figure 1C shows space formation between epithelial cells after 3 Gy irradiation as observed by electron microscopy.

[0008] Figure ID shows a schematic diagram depicting space formation between epithelial cells.

[0009] Figure IE shows ileal sections depicting the restored junction complex observed by electron microscopy in epithelial cells after 3 Gy irradiation and treatment with Enterade.

[0010] Figure 2 shows a graph of 16sr-RNA expression versus dosage. Exemplary amino acid composition Enterade (Isoleucine, Aspartic acid, Threonine, Lysine, Tyrosine, Serine, Valine, Glycine, Tryptophan) decreased bacterial translocation across intestinal mucosa.

[0011] Figure 3 shows a graph of fluorescence density of 4 and 10 kDa rhodamine- dextran. Exemplary amino acid composition Enterade decreased non-ionic particle translocation across intestinal mucosa. The exemplary amino acid-ORS or "AA-ORS" is a composition of threonine, valine, tryptophan, serine, and tyrosine, as free amino acids; and water; wherein the composition does not include free amino acid glutamine or a glutamine- containing dipeptide, or, if free amino acid glutamine and/ or a glutamine-containing dipeptide is present, the total concentration of the free amino acid glutamine and the glutamine-containing dipeptide is less than 50 mg/1; wherein the composition does not include glucose or, if glucose is present, the concentration of glucose is less than 1 g/1; and wherein the composition does not include free amino acid methionine or a methionine- containing peptide, optionally lysine, glycine, aspartic acid, and/or isoleucine, and optionally electrolytes, vitamins, minerals, and/or flavoring agents. AA-ORS decreased epithelial permeation of 4 & lOkD dextran.

[0012] Figure 4A shows cell survival expressed as crypt count following irradiation and treatment with AA-ORS versus saline.

[0013] Figure 4B shows the length of villus following irradiation and treatment with AA- ORS versus saline.

[0014] Figure 5A shows glucose-stimulated Na^" absorption at in 0 Gy and 5 Gy radiation using ²²Na upon treatment with AA-ORS versus saline.

[0015] Figure 5B shows a glucose-stimulated CI^" absorption at in 0 Gy and 5 Gy radiation using ³⁶C1 upon treatment with AA-ORS versus saline.

[0016] Figure 5C shows a magnified view of NHE3 expression (indicated by arrows) upon treatment with AA-ORS versus saline along the brush border membrane of villus epithelial cells (indicated by white arrows) using immunohistochemistry.

[0017] Figure 5D shows a Western blot for NHE3 protein upon treatment with AA-ORS at 0 and 5 Gy.

[0018] Figure 6 shows glucose-stimulated CI^" absorption at 0 Gy and 5 Gy radiation using ³⁶C1 upon treatment with AA-ORS versus water.

[0019] Figure 7 A shows a Western blot for Anol protein upon treatment with AA-ORS at 0, 0.5, 1, 3, 5, 7, and 9 Gy.

[0020] Figure 7B shows a magnified view after Anol visualization upon treatment with AA-ORS versus saline via immunohistochemistry.

[0021] Figure 8A shows the amiloride-insensitive current effect of basic buffer and various amino acid formulations (CF5AA, CF6AA, CF6AA-2, CF8AA, CF12AA, CF14AA).

[0022] Figure 8B shows the FSK peak current effect of basic buffer and various amino acid formulations (CF5AA, CF6AA, CF6AA-2, CF8AA, CF12AA, CF14AA).

[0023] Figure 8C shows the FSK- stimulated current effect of basic buffer and various amino acid formulations (CF5AA, CF6AA, CF6AA-2, CF8AA, CF12AA, CF14AA). [0024] Figure 8D shows the before CFTRinh 172 current effect of basic buffer and various amino acid formulations (CF5AA, CF6AA, CF6AA-2, CF8AA, CF12AA, CF14AA).

[0025] Figure 8E shows the CFTRinh 172-sensitive current effect of basic buffer and various amino acid formulations (CF5AA, CF6AA, CF6AA-2, CF8AA, CF12AA, CF14AA). Amount of current inhibited by specific exemplary CFTR inhibitors (amino acid formulations (CF5AA, CF6AA, CF6AA-2, CF8AA, CF12AA, CF14AA).

[0026] Figure 8F shows the CFTRinh 172-insensitive current effect of basic buffer and various amino acid formulations (CF5AA, CF6AA, CF6AA-2, CF8AA, CF12AA, CF14AA).

This signifies the chloride secretion by other chloride channels like anol.

[0027] Figure 9A shows a magnified view of epithelial cells after CFTR visualization via immunohistochemistry.

[0028] Figure 9B shows a magnified view of epithelial cells treated with Ringer solution after CFTR visualization via immunohistochemistry.

[0029] Figure 9C shows a magnified view of epithelial cells treated with CF5AA after CFTR visualization via immunohistochemistry showing how CFTR is trafficked onto the apical membrane of bronchial epithelial cells.

[0030] Figure 9D shows a magnified view of epithelial cells treated with CF6AA after CFTR visualization via immunohistochemistry showing how CFTR is trafficked onto the apical membrane of bronchial epithelial cells.

[0031] Figure 9E shows a magnified view of epithelial cells treated with CF6AA-2 after CFTR visualization via immunohistochemistry showing how CFTR is trafficked onto the apical membrane of bronchial epithelial cells.

[0032] Figure 9F shows a magnified view of epithelial cells treated with CF8AA after CFTR visualization via immunohistochemistry showing how CFTR is trafficked onto the apical membrane of bronchial epithelial cells.

[0033] Figure 9G shows a magnified view of epithelial cells treated with CF12AA after CFTR visualization via immunohistochemistry showing how CFTR is trafficked onto the apical membrane of bronchial epithelial cells.

[0034] Figure 9H shows a magnified view of epithelial cells treated with CF14AA after CFTR visualization via immunohistochemistry showing how CFTR is trafficked onto the apical membrane of bronchial epithelial cells.

[0035] Figure 10A shows fluorescence intensity at varying times for CRL-4011 cells treated with glucose. [0036] Figure 10B shows fluorescence intensity at varying times for CRL-4011 cells treated with glucose and NSP4.

[0037] Figure IOC shows fluorescence intensity at varying times for 4013 cells treated with glucose.

[0038] Figure 10D shows fluorescence intensity at varying times for 4013 cells treated with glucose and NSP4.

[0039] Figure 11 shows stimulation of [Ca²+]j via fluorescence intensity at varying times for CFBE cells via NSP4 and glucose. Background reading: 60.27993 F/Fo ratio shows how many fold change after NSP4 or glucose. F is peak value, Fo is basal value. Before calculating F and Fo, background reading needs to be subtracted from each. NSP4 F/F0: 1.09 Glucose F/Fo: 1.12.

SUMMARY OF THE INVENTION

[0040] The present disclosure recognizes the need to provide more cost-effective and efficacious treatment options for cystic fibrosis patients. The amino acid compositions described herein may be particularly useful in treating CF patients carrying at least one Phe508del mutation on an allele of the CFTR gene. When the CFTR gene is homozygous for the Phe508del mutation, little to no CFTR is trafficked to the plasma membrane, rendering treatment with a CFTR potentiator (e.g., ivacaftor), which increases the function of CFTR present in the plasma membrane, virtually ineffective. The present disclosure provides compositions shown to increase the translocation of both wild-type and Phe508del CFTR proteins from the cytoplasm to the plasma membrane. In particular, the compositions described herein are particularly effective at increasing the number of mutant CFTR proteins on the plasma membrane. In addition, methods for treating diseases in which CFTR dysfunction is present (e.g., cystic fibrosis) are also provided herein.

[0041] In another aspect, the present disclosure provides compositions comprising, consisting essentially of, or consisting of one or more free amino acids selected from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine for use in treating cystic fibrosis. In a further aspect, the present invention provides use of the compositions of amino acids to treat cystic fibrosis in a subject in need thereof.

[0042] In one aspect, the present disclosure provides compositions comprising, consisting essentially of, or consisting of, one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, or all six free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition comprises, consists essentially of, or consists of the free amino acids asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition further comprises cysteine. In certain embodiments, the composition further comprises histidine. In certain embodiments, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, or all eight free amino acids selected from asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine. In certain embodiments, the composition comprises, consists essentially of, or consists of the free amino acids asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine. In certain embodiments, the composition does not include cysteine. In certain embodiments, the composition does not include histidine. In certain embodiments, the composition does not include cysteine and does not include histidine. In certain embodiments, the composition further comprises, consists essentially of, or consists of one or more free amino acid selected from arginine, glycine, lysine, serine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or all twelve free amino acids selected from arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of the free amino acids arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition further comprises cysteine. In certain embodiments, the composition further comprises histidine. In certain embodiments, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, or all fourteen free amino acids selected from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition further comprises proline, phenylalanine, tryptophan, methionine, leucine, or alanine. In certain embodiments, the composition further comprises water. In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, or excipient.

[0043] In certain embodiments, the composition consists of the free amino acids asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition consists of the free amino acids asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine, and a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, or excipient. In certain embodiments, the composition consists of the free amino acids asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine, and an additional therapeutic agent.

[0044] In certain embodiments, the composition consists of the free amino acids asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine. In certain embodiments, the composition consists of the free amino acids asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine, and a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, or excipient. In certain embodiments, the composition consists of the free amino acids asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine, and an additional therapeutic agent.

[0045] In certain embodiments, the composition consists of the free amino acids arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition consists of the free amino acids arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine, and a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, or excipient. In certain embodiments, the composition consists of the free amino acids arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine, and an additional therapeutic agent.

[0046] In certain embodiments, the composition consists of the free amino acids arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition consists of the free amino acids arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine, and a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, or excipient. In certain embodiments, the composition consists of the free amino acids arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine, and an additional therapeutic agent.

[0047] Each of the free amino acids, if present in the composition, may be present in, for example, the following concentrations: threonine at about 0.4 to about 1.5, about 0.7 to about

1.3, or about 0.9 to about 1.1 grams/liter; valine at about 0.7 to about 1.7, about 0.9 to about 1.5, or about 1.1 to about 1.3 grams/liter; serine at about 0.6 to about 1.6, about 0.8 to about

1.4, about 1.0 to about 1.2 grams/liter; tyrosine at about 0.05 to about 0.4, or about 0.1 to about 0.3 grams/liter; and tryptophan at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter. In certain embodiments, the concentration is in grams of amino acid per liter of solution.

[0048] In one aspect, the present disclosure provides methods for increasing the number of cystic fibrosis transmembrane conductance regulator (CFTR) proteins present on the plasma membrane of a cell, the method comprising contacting the cell with an effective amount of a composition described herein.

[0049] In another aspect, the present disclosure provides methods for increasing the number of CFTR proteins on the plasma membrane of a cell, the method comprising contacting a cell with an effective amount of a composition described herein, wherein the effective amount increases the number of CFTR proteins present on the plasma membrane of the cell. In certain embodiments, the number of wild-type CFTR proteins on the plasma membrane of the cell increases. In certain embodiments, the number of mutant CFTR proteins on the plasma membrane of the cell increases. In certain embodiments, the number of Phe508del CFTR proteins on the plasma membrane of the cell increases. In certain embodiments, the number of both wild-type and mutant CFTR proteins on the plasma membrane of the cell increases. In certain embodiments, the number of both wild-type and Phe508del CFTR proteins on the plasma membrane of the cell increases.

[0050] In a further aspect, the present disclosure provides methods for increasing the number of CFTR proteins on the plasma membrane of a cell, the method comprising contacting a cell with an effective amount of a composition described herein, wherein the cell is an epithelial cell. In certain embodiments, the epithelial cell is a small intestine epithelial cell. In certain embodiments, the epithelial cell is a lung epithelial cell. In certain

embodiments, the lung epithelial cell is a bronchial epithelial cell. The bronchial epithelial cell may be obtained from a patient suffering from cystic fibrosis. In certain embodiments, the bronchial epithelial cell expresses mutant CFTR. In certain embodiments, the bronchial epithelial cell expresses Phe508del CFTR. In certain embodiments, the bronchial epithelial cell expresses wild-type CFTR. The cells may be in vitro, in vivo, or ex vivo.

[0051] In yet another aspect, the present disclosure provides methods for increasing chloride export from a cell, the method comprising contacting the cell with an effective amount of a composition as described herein.

[0052] In a further aspect, the present disclosure provides a method for increasing chloride export from a cell, the method comprising contacting a cell with an amount of a composition described herein, wherein the cell is an epithelial cell. In certain embodiments, the epithelial cell is a small intestine epithelial cell. In certain embodiments, the epithelial cell is a lung epithelial cell. In certain embodiments, the lung epithelial cell is a bronchial epithelial cell. The bronchial epithelial cell may be obtained from a patient suffering from cystic fibrosis. In certain embodiments, the bronchial epithelial cell expresses mutant CFTR. In certain embodiments, the bronchial epithelial cell expresses Phe508del CFTR. In certain

embodiments, the bronchial epithelial cell expresses wild-type CFTR. The cells may be in vitro, in vivo, or ex vivo.

[0053] In another aspect, the present disclosure provides a method for increasing water export from a cell, the method comprising contacting the cell with an amount of a composition as described herein.

[0054] In a further aspect, the present disclosure provides a method for increasing water export from a cell, the method comprising contacting a cell with an amount of a composition described herein, wherein the cell is an epithelial cell. In certain embodiments, the epithelial cell is a small intestine epithelial cell. In certain embodiments, the epithelial cell is a lung epithelial cell. In certain embodiments, the lung epithelial cell is a bronchial epithelial cell. The bronchial epithelial cell may be obtained from a patient suffering from cystic fibrosis. In certain embodiments, the bronchial epithelial cell expresses mutant CFTR. In certain embodiments, the bronchial epithelial cell expresses Phe508del CFTR. In certain

[0055] In yet another aspect, the present disclosure provides a method for treating cystic fibrosis, the method comprising administering to a subject in need thereof a composition as described herein.

[0056] In a further aspect, the present disclosure provides a method for treating cystic fibrosis, the method comprising administering to a subject in need thereof a composition described herein, wherein the composition further comprises an additional therapeutic agent. In certain embodiments, the additional therapeutic agent is a small molecule drug, protein drug, or nucleic acid drug. In certain embodiments, the additional therapeutic agent is a small molecule drug. In certain embodiments, the additional therapeutic agent is a CFTR

potentiator, corrector, and/or read-through agent. In certain embodiments, the additional therapeutic agent is a CFTR potentiator. In certain embodiments, the CFTR potentiator is ivacaftor. In certain embodiments, the additional therapeutic agent is a CFTR corrector. In certain embodiments, the CFTR corrector is lumcaftor. The additional therapeutic agent may be included in any of the compositions described herein (e.g., the composition further comprises an additional therapeutic agent). The additional therapeutic agent may be administered concurrently with, prior to, or subsequently after, administration of any of the compositions described herein (e.g., combination therapy).

[0057] In a further aspect, the present disclosure provides a method for treating cystic fibrosis, the method comprising administering to a subject in need thereof a composition described herein, wherein the composition is formulated for administration by an enteral, pulmonary, inhalation, intranasal, or sublingual route. In certain embodiments, the composition is formulated for pulmonary administration to the subject. In certain

embodiments, the composition is formulated for enteral administration to the subject. In certain embodiments, the composition is formulated for oral administration to the subject.

[0058] In a further aspect, the present disclosure provides a method for treating cystic fibrosis, the method comprising administering to a subject in need thereof a composition described herein. In certain embodiments, the subject is suffering from cystic fibrosis in which the subject has a mutation in the CFTR gene. In certain embodiments, the subject is suffering from cystic fibrosis in which the subject is heterozygous for both wild-type CFTR and mutant CFTR. In certain embodiments, the subject is suffering from cystic fibrosis in which the subject is heterozygous for a first mutant CFTR and a second mutant CFTR (e.g., wherein the mutations are different). In certain embodiments, the subject is suffering from cystic fibrosis in which the subject is homozygous for mutant CFTR (e.g., both alleles have the same mutation). In certain embodiments, the subject is suffering from cystic fibrosis in which the subject is homozygous for wild-type CFTR (e.g., both alleles are not mutated). In certain embodiments, the mutant CFTR is a Gly542X, Gly551Asp, Arg553X, Argl l7His, 120del23, or Phe508del CFTR mutant, wherein X is any amino acid. In certain embodiments, the mutant CFTR is a Phe508del CFTR mutant. In certain embodiments, the CFTR protein present in the subject is least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of wild-type CFTR as provided by SEQ ID NO: 1. In certain embodiments, the CFTR protein present in the subject is least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of Phe508del CFTR as provided by SEQ ID NO: 2.

[0059] Information regarding diagnosis and treatments of various diseases, including cystic fibrosis and related pulmonary diseases, is found in Longo, D., et al. (eds.), Harrison's Principles of Internal Medicine, 18^th Ed.; McGraw-Hill Professional, 2011. Information regarding various therapeutic agents and human diseases, including pulmonary disease, is found in Brunton, L., et al. (eds.) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 12^th Ed., McGraw Hill, 2010 and/or Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill/ Appleton & Lange; 11^th edition (July 2009). All patents, patent applications, books, articles, documents, databases, websites, publications, references, etc., mentioned herein are incorporated by reference in their entirety. In case of a conflict between the specification and any of the incorporated references, the specification (including any amendments thereof) shall control. Applicants reserve the right to amend the specification based, e.g., on any of the incorporated material and/or to correct obvious errors. None of the content of the incorporated material shall limit the invention. Standard art-accepted meanings of terms are used herein unless indicated otherwise. Standard abbreviations for various terms are used herein.

[0060] The details of certain embodiments of the invention are set forth in the Detailed Description of Certain Embodiments, as described below. Other features, objects, and advantages of the invention will be apparent from the Definitions, Examples, Figures, and Claims.

DEFINITIONS

[0061] Descriptions and certain information relating to various terms used in the present disclosure are collected herein for convenience.

[0062] The term "agent" is used herein to refer to any substance, compound (e.g., molecule), supramolecular complex, material, or combination or mixture thereof. A compound may be any agent that can be represented by a chemical formula, chemical structure, or sequence. Example of agents, include, e.g., small molecules, polypeptides, nucleic acids (e.g., RNAi agents, antisense oligonucleotide, aptamers), lipids,

polysaccharides, etc. In general, agents may be obtained using any suitable method known in the art. The ordinary skilled artisan will select an appropriate method based, e.g., on the nature of the agent. An agent may be at least partly purified. In some embodiments an agent may be provided as part of a composition, which may contain, e.g., a counter-ion, aqueous or non-aqueous diluent or carrier, buffer, preservative, or other ingredient, in addition to the agent, in various embodiments. In some embodiments an agent may be provided as a salt, ester, hydrate, or solvate. In some embodiments an agent is cell-permeable, e.g., within the range of typical agents that are taken up by cells and acts intracellularly, e.g., within mammalian cells, to produce a biological effect. Certain compounds may exist in particular geometric or stereoisomeric forms. Such compounds, including cis- and irans-isomers, E- and Z-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (-)- and (+)- isomers, racemic mixtures thereof, and other mixtures thereof are encompassed by this disclosure in various embodiments unless otherwise indicated. Certain compounds may exist in a variety or protonation states, may have a variety of configurations, may exist as solvates [e.g., with water (i.e. hydrates) or common solvents] and/or may have different crystalline forms (e.g., polymorphs) or different tautomeric forms. Embodiments exhibiting such alternative protonation states, configurations, solvates, and forms are encompassed by the present disclosure where applicable. The term "agent" may also encompass a "therapeutic agent". The term "compound" and "agent" may be used interchangeably.

[0063] An "effective amount" or "effective dose" of an agent (or composition containing such agent) refers to the amount sufficient to achieve a desired biological and/or

pharmacological effect, e.g., when delivered to a cell or organism according to a selected administration form, route, and/or schedule. The phrases "effective amount" and

"therapeutically effective amount" are used interchangeably. As will be appreciated by those of ordinary skill in this art, the absolute amount of a particular agent or composition that is effective may vary depending on such factors as the desired biological or pharmacological endpoint, the agent to be delivered, the target tissue, etc. Those of ordinary skill in the art will further understand that an "effective amount" may be contacted with cells or administered to a subject in a single dose, or through use of multiple doses, in various embodiments. In certain embodiments, an effective amount is an amount that increases the trafficking of CFTR to the plasma membrane of a cell. In certain embodiments, an effective amount is an amount that increases the translocation of CFTR from the cytoplasm to the plasma membrane of a cell. In certain embodiments, an effective amount is an amount that increases the chloride ion export from a cell. In certain embodiments, an effective amount is an amount that increases water export from a cell. In certain embodiments, an effective amount is an amount that reduces the symptoms of and/or treats a lung disease. In certain embodiments, an effective amount is an amount that reduces the symptoms of and/or treats cystic fibrosis. [0064] The term "gene" refers to a locus (e.g., region) of DNA that is comprised of nucleotides. Generally, a gene contains multiple regions, including one or more upstream or downstream regulatory sequences (e.g., enhancer/silencer, promoter, 5' non-coding sequences, 3' non-coding sequences) that is normally required to initiate transcription, an open reading frame comprising one or more exons and one or more introns. An "exon" is any part of a gene that will encode part of the final mature RNA, which will be translated into a protein sequence. An "intron" is any part of a gene that is removed by RNA splicing during maturation of the final mature RNA. A "cryptic exon" is an exon that can introduce a premature translation stop codon into mature RNA or result in atypical splicing patterns. The term "gene" may refer to a nucleic acid fragment that expresses a protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. Accordingly, a chimeric gene or chimeric construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene not normally found in the host organism, but which is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.

[0065] The term "gene product" (also referred to herein as "gene expression product" or "expression product") encompasses products resulting from expression of a gene, such as RNA transcribed from a gene and polypeptides arising from translation of such RNA. It will be appreciated that certain gene products may undergo processing or modification, e.g., in a cell. For example, RNA transcripts may be spliced, polyadenylated, etc., prior to mRNA translation, and/or polypeptides may undergo co-translational or post-translational processing such as removal of secretion signal sequences, removal of organelle targeting sequences, or modifications such as phosphorylation, fatty acylation, etc. The term "gene product" encompasses such processed or modified forms. Genomic, mRNA, polypeptide sequences from a variety of species, including human, are known in the art and are available in publicly accessible databases such as those available at the National Center for Biotechnology Information (www.ncbi.nih.gov) or Universal Protein Resource (www.uniprot.org).

Databases include, e.g., GenBank, RefSeq, Gene, UniProtKB/SwissProt, UniProtKB/Trembl, and the like. In general, sequences, e.g., mRNA and polypeptide sequences, in the NCBI Reference Sequence database may be used as gene product sequences for a gene of interest. It will be appreciated that multiple alleles of a gene may exist among individuals of the same species. For example, differences in one or more nucleotides (e.g., up to about 1%, 2%, 3-5% of the nucleotides) of the nucleic acids encoding a particular protein may exist among individuals of a given species. Due to the degeneracy of the genetic code, such variations often do not alter the encoded amino acid sequence, although DNA polymorphisms that lead to changes in the sequence of the encoded proteins can exist. Examples of polymorphic variants can be found in, e.g., the Single Nucleotide Polymorphism Database (dbSNP), available at the NCBI website at w w w .ncbi . nlm.nih . go v/proj ects/S NP/ [Sherry, S. T., et al. (2001) dbSNP: The NCBI database of genetic variation. Nucl Acids Res, 29: 308-311 ; Kitts, A. and Sherry, S. (2009) The single nucleotide polymorphism database (dbSNP) of nucleotide sequence variation. In: The NCBI Handbook (Internet); McEntyre, J., Ostell, J., editors. Bethesda (MD): National Center for Biotechnology Information (US); 2002

(www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=handbook&part=ch5)]. Multiple isoforms of certain proteins may exist, e.g., as a result of alternative RNA splicing or editing. In general, where aspects of this disclosure pertain to a gene or gene product, embodiments pertaining to allelic variants or isoforms are encompassed, if applicable, unless indicated otherwise.

Certain embodiments may be directed to particular sequence(s), e.g., particular allele(s) or isoform(s).

[0066] The term "amino acid" encompasses all known amino acids comprising an amine (-NH2) functional group, a carboxyl (-COOH) functional group, and a side chain ("R") group specific to each amino acid. "Amino acids" encompasses the 21 amino acids encoded by the human genome (i.e., proteinogenic amino acids), amino acids encoded or produced by bacteria or single-celled organisms, and naturally derived amino acids. For the purposes of this disclosure, the conjugate acid form of amino acids with basic side chains (arginine, lysine, and histidine) or the conjugate base form of amino acids with acidic side chains (aspartic acid and glutamic acid) are essentially the same, unless otherwise noted. "Amino acids" also encompass derivatives thereof that retain substantially the same, or better, activity in terms of enhancing the effect of a composition of the present invention (e.g., increasing the number of CFTR proteins in the plasma membrane, increasing chloride ion export from a cell, treating cystic fibrosis). The derivatives may be, for example, enantiomers, and include both the D- and L- forms of the amino acids. The derivatives may be derivatives of "natural" or "non-natural" amino acids (e.g., β-amino acids, homo-amino acids, proline derivatives, pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring- substituted tyrosine derivatives, ring-substituted phenylalanine derivatives, linear core amino acids, and N-methyl amino acids), for example, selenocysteine, pyrrolysine, iodotyrosine, norleucine, or norvaline. Other amino acid derivatives include, but are not limited to, those that are synthesized by, for example, acylation, methylation, glycosylation, and/or halogenation of the amino acid. These include, for example, β-methyl amino acids, C-methyl amino acids, and N-methyl amino acids. The amino acids described herein may be present as free amino acids. The term "free amino acid" refers to an amino acid that is not part of a peptide or polypeptide (e.g., is not connected to another amino acid through a peptide bond). A free amino acid is free in solution, but may be associated with a salt or other component in solution.

[0067] The terms "protein," "peptide," and "polypeptide" are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. In some embodiments, a protein comprises a homodimer or a heterodimer. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. A protein may comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, e.g., a compound that can act as a nucleic acid cleavage agent. In some embodiments, a protein is in a complex with, or is in association with, a nucleic acid, e.g., RNA. In some embodiments, a protein comprises a ligand binding domain. In some embodiments, a protein comprises an active site (e.g., site of biological or enzymatic activity). In some embodiments, a protein comprises an allosteric site (e.g., site of a protein that can bind to a ligand that can be remote from an active site). Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual [4^th ed., Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, N.Y. (2012)], the entire contents of which are incorporated herein by reference.

[0068] "Identity" or "percent identity" is a measure of the extent to which the sequence of two or more nucleic acids or polypeptides is the same. The percent identity between a sequence of interest A and a second sequence B may be computed by aligning the sequences, allowing the introduction of gaps to maximize identity, determining the number of residues (nucleotides or amino acids) that are opposite an identical residue, dividing by the minimum of TGA and TGB (here TGA and TGB are the sum of the number of residues and internal gap positions in sequences A and B in the alignment), and multiplying by 100. When computing the number of identical residues needed to achieve a particular percent identity, fractions are to be rounded to the nearest whole number. Sequences can be aligned with the use of a variety of computer programs known in the art. For example, computer programs such as BLAST2, BLASTN, BLASTP, Gapped BLAST, etc., may be used to generate alignments and/or to obtain a percent identity. The algorithm of Karlin and Altschul (Karlin and

Altschul, Proc Natl Acad Sci USA, 87: 22264-2268, 1990) modified as in Karlin and

Altschul, Proc Natl Acad Sci USA, 90: 5873-5877,1993 is incorporated into the NBLAST and XBLAST programs of Altschul et al. [Altschul, et al. (1990) JMol Biol, 215: 403-410]. In some embodiments, to obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. [Altschul, et al. (1997) Nucleic Acids Res, 25: 3389- 3402]. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs may be used. See the Web site having URL www.ncbi.nlm.nih.gov and/or McGinnis, S. and Madden, TL, W20-W25 Nucleic Acids Research, 2004, Vol. 32, Web server issue. Other suitable programs include CLUSTALW [Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nuc Acid Res, 22: 4673-4680], CLUSTAL Omega [Sievers, F., Wilm, A., Dineen, D., et al. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Sys Biol, 7:

doi: 10.1038/msb.2011.75], and GAP (GCG Version 9.1; which implements the Needleman & Wunsch, 1970 algorithm [Needleman, S. B. and Wunsch, C D. (1970) J. Mol Biol, 48: 443- 453]). Percent identity may be evaluated over a window of evaluation. In some embodiments a window of evaluation may have a length of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, e.g., 100%, of the length of the shortest of the sequences being compared. In some embodiments a window of evaluation is at least 100; 200; 300; 400; 500; 600; 700; 800; 900; 1,000; 1,200; 1,500; 2,000; 2,500; 3,000; 3,500; 4,000; 4,500; or 5,000 amino acids. In some embodiments no more than 20%, 10%, 5%, or 1% of positions in either sequence or in both sequences over a window of evaluation are occupied by a gap. In some embodiments no more than 20%, 10%, 5%, or 1% of positions in either sequence or in both sequences are occupied by a gap.

[0069] A "variant" of a particular polypeptide or polynucleotide has one or more additions, substitutions, and/or deletions with respect to the polypeptide or polynucleotide, which may be referred to as the "original polypeptide" or "original polynucleotide," respectively. An addition may be an insertion or may be at either terminus. A variant may be shorter or longer than the original polypeptide or polynucleotide. The term "variant" encompasses

"fragments". A "fragment" is a continuous portion of a polypeptide or polynucleotide that is shorter than the original polypeptide. In some embodiments a variant comprises or consists of a fragment. In some embodiments a fragment or variant is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more as long as the original polypeptide or polynucleotide. A fragment may be an N-terminal, C-terminal, or internal fragment. In some embodiments a variant polypeptide comprises or consists of at least one domain of an original polypeptide. In some embodiments a variant polypeptide or

polynucleotide comprises or consists of a polypeptide or polynucleotide that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical in sequence to the original polypeptide or polynucleotide. In some embodiments a variant polypeptide or polynucleotide comprises or consists of a polypeptide or polynucleotide that is over at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the original polypeptide or

polynucleotide. In some embodiments the sequence of a variant polypeptide comprises or consists of a sequence that has N amino acid differences with respect to an original sequence, wherein N is any integer up to 1%, 2%, 5%, or 10% of the number of amino acids in the original polypeptide, where an "amino acid difference" refers to a substitution, insertion, or deletion of an amino acid. In some embodiments a substitution is a conservative substitution. Conservative substitutions may be made, e.g., on the basis of similarity in side chain size, polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues involved. In some embodiments, conservative substitutions may be made according to Table A, wherein amino acids in the same block in the second column and in the same line in the third column may be substituted for one another other in a conservative substitution. Certain conservative substitutions are substituting an amino acid in one row of the third column corresponding to a block in the second column with an amino acid from another row of the third column within the same block in the second column.

Table A

[0070] In some embodiments, proline (P), cysteine (C), or both are each considered to be in an individual group. Within a particular group, certain substitutions may be of particular interest in certain embodiments, e.g., replacements of leucine by isoleucine (or vice versa), serine by threonine (or vice versa), or alanine by glycine (or vice versa).

[0071] In some embodiments a variant is a biologically active variant, i.e., the variant at least in part retains at least one activity of the original polypeptide or polynucleotide. In some embodiments a variant at least in part retains more than one or substantially all known biologically significant activities of the original polypeptide or polynucleotide. An activity may be, e.g., a catalytic activity, binding activity, ability to perform or participate in a biological structure or process, etc. In some embodiments an activity of a variant may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, of the activity of the original polypeptide or polynucleotide, up to approximately 100%, approximately 125%, or approximately 150% of the activity of the original polypeptide or polynucleotide, in various embodiments. In some embodiments a variant, e.g., a biologically active variant, comprises or consists of a polypeptide at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to an original polypeptide or over at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or 100% of the original polypeptide. In some embodiments an alteration, e.g., a substitution or deletion, e.g., in a functional variant, does not alter or delete an amino acid or nucleotide that is known or predicted to be important for an activity, e.g., a known or predicted catalytic residue or residue involved in binding a substrate or cofactor. Variants may be tested in one or more suitable assays to assess activity.

[0072] The term "small molecule" as used herein, is an organic molecule that is less than about 2 kilodaltons (kDa) in mass. In some embodiments, the small molecule is less than about 1.5 kDa, or less than about 1 kDa. In some embodiments, the small molecule is less than about 800 daltons (Da), 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, or 100 Da. Often, a small molecule has a mass of at least 50 Da. In some embodiments, a small molecule is non- polymeric. In some embodiments, a small molecule is not an amino acid. In some embodiments, a small molecule is not a nucleotide. In some embodiments, a small molecule is not a saccharide. In some embodiments, a small molecule contains multiple carbon-carbon bonds and can comprise one or more heteroatoms and/ or one or more functional groups important for structural interaction with proteins (e.g., hydrogen bonding), e.g., an amine, carbonyl, hydroxyl, or carboxyl group, and in some embodiments at least two functional groups. Small molecules often comprise one or more cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures, optionally substituted with one or more of the above functional groups. In certain embodiments, the small molecule is a therapeutically active agent such as a drug (e.g., a molecule approved by the U.S. Food and Drug

Administration as provided in the Code of Federal Regulations (C.F.R.)). The small molecule may also be complexed with one or more metal atoms and/or metal ions. In this instance, the small molecule is also referred to as a "small organometallic molecule." Preferred small molecules are biologically active in that they produce a biological effect in animals, preferably mammals, more preferably humans. Small molecules include, but are not limited to, radionuclides and imaging agents. In certain embodiments, the small molecule is a drug. Preferably, though not necessarily, the drug is one that has already been deemed safe and effective for use in humans or animals by the appropriate governmental agency or regulatory body. For example, drugs approved for human use are listed by the FDA under 21 C.F.R. §§ 330.5, 331 through 361, and 440 through 460, incorporated herein by reference. All listed drugs are considered acceptable for use in accordance with the present invention.

[0073] The terms "composition" and "formulation" are used interchangeably.

[0074] The term "administer," "administering," or "administration" refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound described herein, or a composition thereof, in or on a subject.

[0075] A "subject" may be any vertebrate organism in various embodiments. A subject may be individual to whom an agent is administered, e.g., for experimental, diagnostic, and/or therapeutic purposes or from whom a sample is obtained or on whom a procedure is performed. In some embodiments a subject is a mammal, e.g., a human, non-human primate, or rodent (e.g., mouse, rat, rabbit). In certain embodiments, the subject is a human. The human may be of either sex and may be at any stage of development. In certain embodiments, a subject has been diagnosed with cystic fibrosis. In certain embodiments, a subject has been diagnosed with cystic fibrosis caused by a CFTR mutation. In certain embodiments, a subject has been diagnosed with cystic fibrosis caused by a Phe508del CFTR mutation.

[0076] "Treat," "treatment", "treating" and similar terms as used herein in the context of treating a subject refer to providing medical and/or surgical management of a subject.

Treatment may include, but is not limited to, administering an age or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically undertaken in an effort to alter the course of a disease (which term is used to indicate any disease, disorder, syndrome or undesirable condition warranting or potentially warranting therapy) in a manner beneficial to the subject. The effect of treatment may include reversing, alleviating, reducing severity of, delaying the onset of, curing, inhibiting the progression of, and/or reducing the likelihood of occurrence or recurrence of the disease or one or more symptoms or manifestations of the disease. A therapeutic agent may be administered to a subject who has a disease or is at increased risk of developing a disease relative to a member of the general population. In some embodiments a therapeutic agent may be administered to a subject who has had a disease but no longer shows evidence of the disease. The agent may be administered, e.g., to reduce the likelihood of recurrence of evident disease. A therapeutic agent may be

administered prophylactically, i.e., before development of any symptom or manifestation of a disease. "Prophylactic treatment" refers to providing medical and/or surgical management to a subject who has not developed a disease or does not show evidence of a disease in order, e.g., to reduce the likelihood that the disease will occur or to reduce the severity of the disease should it occur. The subject may have been identified as being at risk of developing the disease (e.g., at increased risk relative to the general population or as having a risk factor that increases the likelihood of developing the disease).

[0077] The terms "condition," "disease," and "disorder" are used interchangeably.

[0078] An "effective amount" of a compound described herein refers to an amount sufficient to elicit the desired biological response. In certain embodiments, an effective amount is an amount sufficient to increase chloride ion transport. In certain embodiments, an effective amount is an amount sufficient to modulate (e.g., increase) the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein (e.g., wild-type CFTR or mutant CFTR). In certain embodiments, an effective amount is an amount sufficient to modulate (e.g., increase) the function of Phe508del CFTR. In certain embodiments, an effective amount is an amount sufficient to increase the translocation of CFTR from the cytoplasm to the plasma membrane. An effective amount of a compound described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. In certain embodiments, an effective amount is a therapeutically effective amount. In certain embodiments, an effective amount is a prophylactic treatment. In certain embodiments, an effective amount is the amount of a compound described herein in a single dose. In certain embodiments, an effective amount is the combined amounts of a compound described herein in multiple doses.

[0079] A "therapeutically effective amount" of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and/or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, a therapeutically effective amount is an amount sufficient for increasing chloride ion transport (e.g., increasing chloride export from an epithelial cell). In certain embodiments, a therapeutically effective amount is an amount sufficient for increasing water transport (e.g., increasing water export from an epithelial cell). In certain embodiments, a therapeutically effective amount is an amount sufficient for modulating (e.g., increasing) the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein (e.g., wild-type CFTR or mutant CFTR). In certain embodiments, a therapeutically effective amount is an amount sufficient to modulate (e.g., increase) the function of Phe508del CFTR. In certain embodiments, a therapeutically effective amount is an amount sufficient to increase the translocation of CFTR from the cytoplasm to the plasma membrane. In certain embodiments, a therapeutically effective amount is an amount sufficient for treating a lung disease. In certain embodiments, a therapeutically effective amount is an amount sufficient for treating cystic fibrosis.

[0080] As used herein, the term "salt" refers to any and all salts, and encompasses pharmaceutically acceptable salts. [0081] The term "carrier" may refer to any diluent, adjuvant, excipient, or vehicle with which a composition of the present disclosure is administered. Examples of suitable pharmaceutical carriers are described in Remington 's Essentials of Pharmaceuticals, 21^st ed., Ed. Felton, 2012, which is herein incorporated by reference.

[0082] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.

[0083] Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition. The exact amount of a composition comprising amino acids required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound, mode of administration, and the like. An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, any two doses of the multiple doses include different or substantially the same amounts of a compound described herein. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, tissue, or cell. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, tissue, or cell. In certain embodiments, a dose (e.g. , a single dose, or any dose of multiple doses) described herein includes independently between 0.1 μg and 1 μg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a composition comprising amino acids described herein. In certain

embodiments, a dose described herein includes independently between 1 mg and 3 mg, inclusive, of a composition comprising amino acids described herein. In certain

embodiments, a dose described herein includes independently between 3 mg and 10 mg, inclusive, of a composition comprising amino acids described herein. In certain

embodiments, a dose described herein includes independently between 10 mg and 30 mg, inclusive, of a composition comprising amino acids described herein. In certain

embodiments, a dose described herein includes independently between 30 mg and 100 mg, inclusive, of a composition comprising amino acids described herein.

[0084] Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

[0085] The composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents or therapeutic agents, which may be useful as, e.g. , combination therapies. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include small organic molecules such as drug compounds (e.g. , compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful for treating and/or preventing a disease (e.g., proliferative disease, lung disease, gastrointestinal disease, hematological disease,

neurological disease, painful condition, psychiatric disorder, or metabolic disorder). In certain embodiments, the additional therapeutic agent is an agent useful for treating a lung disease. In certain embodiments, the additional therapeutic agent is an agent useful for treating cystic fibrosis. In certain embodiments, an agent useful for treating cystic fibrosis may be ivacaftor (Kalydeco®), lumacaftor (Orkambi®), ataluren, or tezacaftor. In certain embodiments, the additional therapeutic agent is ivacaftor. In certain embodiments, the additional therapeutic agent is lumacaftor. In certain embodiments, the additional therapeutic agent is an agent useful for treating cystic fibrosis, managing the symptoms associated with cystic fibrosis, or for treating diseases or infections which occur concurrently with cystic fibrosis (e.g., bacterial infection, viral infection, bronchiolitis, asthma, etc.). Additional therapeutics useful for the purposes of this disclose include, but are not limited to, dornase alfa systemic (Pulmozyme), azithromycin (e.g., Zithromax, Zmax), aztreonam (Cayston, Azactam), tobramycin (e.g., TOBI®, Nebcin, Bethkis, Kitabis Pak, TOBI Podhaler), Amikin, pancrelipase (Creon, Zenpep, Pancreaze, Viokase, Pertzye, Ultresa, Pangestyme EC, Panocaps), gentamicin (Garamycin), and pancreatin. Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered together with each other and/or with the compound or composition described herein in a single dose or administered separately in different doses. The particular combination to employ in a regimen will take into account compatibility of the compound described herein with the additional pharmaceutical agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually. In certain embodiments, the compounds described herein or pharmaceutical compositions can be administered in combination with an anti-cancer therapy including, but not limited to, surgery, radiation therapy, transplantation (e.g., stem cell transplantation, bone marrow transplantation), immunotherapy, and chemotherapy.

[0086] The term "lung disease" or "pulmonary disease" refers to a disease of the lung. Examples of lung diseases include, but are not limited to, bronchiectasis, bronchitis, bronchopulmonary dysplasia, interstitial lung disease, occupational lung disease, emphysema, cystic fibrosis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), asthma (e.g., intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma), chronic bronchitis, chronic obstructive pulmonary disease (COPD), emphysema, interstitial lung disease, sarcoidosis, asbestosis, aspergilloma, aspergillosis, pneumonia (e.g., lobar pneumonia, multilobar pneumonia, bronchial pneumonia, interstitial pneumonia), pulmonary fibrosis, pulmonary tuberculosis, rheumatoid lung disease, pulmonary embolism, and lung cancer (e.g., non-small-cell lung carcinoma (e.g., adenocarcinoma, squamous-cell lung carcinoma, large-cell lung carcinoma), small-cell lung carcinoma).

[0087] Also encompassed by the disclosure are kits (e.g., pharmaceutical packs). The kits provided may comprise a pharmaceutical composition or compound described herein and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a

pharmaceutical composition or compound described herein. In some embodiments, the pharmaceutical composition or compound described herein provided in the first container and the second container are combined to form one unit dosage form. In certain embodiments, a kit described herein further includes instructions for using the kit.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Compositions comprising amino acids that increase translocation of CFTR to the plasma membrane

[0088] The compositions provided herein increase the translocation of both wild-type and mutant CFTR proteins from the cytoplasm to the plasma membrane. In particular, the compositions described herein are particularly effective at increasing the number of

Phe508del CFTR proteins on the plasma membrane, leading to more chloride ion and water secretion from epithelial cells to combat the formation of thick mucus, a hallmark of cystic fibrosis.

[0089] In one aspect, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine; and optionally, pharmaceutically acceptable carriers, buffers, electrolytes, adjuvants, excipients, and/or an additional therapeutic agent. In certain embodiments, the composition comprises, consists essentially of, or consists of the free amino acids asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition consists essentially of, or consists of only the specified free amino acids and no other free amino acids, or a negligible amount of other free amino acids. The compositions include, in certain embodiments, derivatives of the amino acids that are derivatives of "natural" or "non-natural" amino acids. The compositions include, in certain embodiments, salts and/or prodrugs of the amino acids. In certain embodiments, the composition further comprises the free amino acid histidine. In certain embodiments, the composition further comprises the free amino acid cysteine. In certain embodiments, the composition further comprises the free amino acids histidine and cysteine. In certain embodiments, the composition comprises, consists essentially of, or consists of the free amino acids asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine. In certain embodiments, the composition further comprises, consists essentially of, or consists of one or more of the free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, and alanine.

[0090] In yet another aspect, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, or all six free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine; and optionally, pharmaceutically acceptable carriers, buffers, electrolytes, adjuvants, excipients, and/or an additional therapeutic agent. In certain embodiments, the composition further comprises the free amino acids histidine or cysteine. In certain embodiments, the composition further comprises the free amino acid histidine. In certain embodiments, the composition further comprises the free amino acid cysteine. In certain embodiments, the composition further comprises the free amino acids histidine and cysteine. In certain embodiments, the composition further comprises, consists essentially of, or consists of one or more of the free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, or alanine.

[0091] In another aspect, the composition comprises, consists essentially of, or consists of only one free amino acid selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition comprises, consists essentially of, or consists of asparagine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of aspartic acid as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of isoleucine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of threonine as a free amino acid. [0092] In another embodiment, the composition comprises, consists essentially of, or consists of any two free amino acids selected from asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine. In certain embodiments, the composition comprises, consists essentially of, or consists of: asparagine and aspartic acid; asparagine and cysteine; asparagine and glutamic acid; asparagine and glutamine; asparagine and histidine; asparagine and isoleucine; asparagine and threonine; aspartic acid and cysteine; aspartic acid and glutamic acid; aspartic acid and glutamine; aspartic acid and histidine;

aspartic acid and isoleucine; aspartic acid and threonine; glutamic acid and cysteine; glutamic acid and glutamine; glutamic acid and histidine; glutamic acid and isoleucine; glutamic acid and threonine; glutamine and cysteine; glutamine and histidine; glutamine and isoleucine; glutamine and threonine; isoleucine and cysteine; isoleucine and histidine; isoleucine and threonine; threonine and cysteine; or threonine and histidine. The combinations disclosed in this paragraph are hereby disclosed in further combination with a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, and/or eighteenth free amino acid selected from alanine, arginine, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine.

[0093] In another embodiment, the composition comprises, consists essentially of, or consists of any three free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition comprises, consists essentially of, or consists of: asparagine, isoleucine, and cysteine; asparagine, isoleucine, and glutamine; asparagine, isoleucine, and histidine; asparagine, isoleucine, and aspartic acid; asparagine, isoleucine, and glutamic acid; asparagine, isoleucine, and threonine; isoleucine, cysteine, and glutamine; isoleucine, cysteine, and histidine; isoleucine, cysteine, and aspartic acid; isoleucine, cysteine, and glutamic acid; isoleucine, cysteine, and threonine; asparagine, cysteine, and glutamine; asparagine, cysteine, and histidine; asparagine, cysteine, and aspartic acid; asparagine, cysteine, and glutamic acid; or asparagine, cysteine, and threonine. For the sake of brevity, all of the combinations of any three free amino acids are not being parsed out.

[0094] In another embodiment, the composition comprises, consists essentially of, or consists of any four free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition comprises, consists essentially of, or consists of: asparagine, isoleucine, cysteine, and glutamine;

asparagine, isoleucine, cysteine, and histidine; asparagine, isoleucine, cysteine, and aspartic acid; asparagine, isoleucine, cysteine, and glutamic acid; asparagine, isoleucine, cysteine, and threonine; asparagine, cysteine, glutamine, and histidine; asparagine, cysteine, glutamine, and aspartic acid; asparagine, cysteine, glutamine, and glutamic acid; asparagine, cysteine, glutamine, and threonine; asparagine, isoleucine, glutamine, and histidine; asparagine, isoleucine, glutamine, and aspartic acid; asparagine, isoleucine, glutamine, and glutamic acid; asparagine, isoleucine, glutamine, and threonine; isoleucine, cysteine, glutamine, and histidine; isoleucine, cysteine, glutamine, and aspartic acid; isoleucine, cysteine, glutamine, and glutamic acid; or isoleucine, cysteine, glutamine, and threonine. For the sake of brevity, all of the combinations of any four free amino acids are not being parsed out.

[0095] In another embodiment, the composition comprises, consists essentially of, or consists of any five free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition comprises, consists essentially of, or consists of: asparagine, isoleucine, cysteine, glutamine, and histidine; asparagine, isoleucine, cysteine, glutamine, and aspartic acid; asparagine, isoleucine, cysteine, glutamine, and glutamic acid; asparagine, isoleucine, cysteine, glutamine, and threonine; asparagine, cysteine, glutamine, histidine, and aspartic acid;

asparagine, cysteine, glutamine, histidine, and glutamic acid; asparagine, cysteine, glutamine, histidine, and threonine; asparagine, isoleucine, glutamine, histidine, and aspartic acid;

asparagine, isoleucine, glutamine, histidine, and glutamic acid; asparagine, isoleucine, glutamine, histidine, and threonine; asparagine, isoleucine, cysteine, histidine, and aspartic acid; asparagine, isoleucine, cysteine, histidine, and glutamic acid; asparagine, isoleucine, cysteine, histidine, and threonine; asparagine, isoleucine, cysteine, glutamine, and aspartic acid; asparagine, isoleucine, cysteine, glutamine, and glutamic acid; or asparagine, isoleucine, cysteine, glutamine, and threonine. For the sake of brevity, all of the combinations of any five free amino acids are not being parsed out.

[0096] In another embodiment, the composition comprises, consists essentially of, or consists of any six free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition comprises, consists essentially of, or consists of: asparagine, isoleucine, cysteine, glutamine, histidine, and aspartic acid; asparagine, isoleucine, cysteine, glutamine, histidine, and glutamic acid; asparagine, isoleucine, cysteine, glutamine, histidine, and threonine; asparagine, cysteine, glutamine, histidine, aspartic acid, and glutamic acid; asparagine, cysteine, glutamine, histidine, aspartic acid, and threonine; asparagine, isoleucine, glutamine, histidine, aspartic acid and threonine; asparagine, isoleucine, glutamine, aspartic acid, glutamic acid, and threonine; or asparagine, isoleucine, cysteine, histidine, glutamic acid, and threonine. For the sake of brevity, all of the combinations of any six free amino acids are not being parsed out.

[0097] In another embodiment, the composition comprises, consists essentially of, or consists of any seven free amino acids selected from asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine. In certain embodiments, the composition comprises, consists essentially of, or consists of: asparagine, isoleucine, cysteine, glutamine, histidine, aspartic acid, and glutamic acid; asparagine, isoleucine, cysteine, glutamine, histidine, aspartic acid, and threonine; isoleucine, cysteine, glutamine, histidine, aspartic acid, glutamic acid, and threonine; asparagine, cysteine, glutamine, histidine, aspartic acid, glutamic acid, and threonine; or isoleucine, cysteine, glutamine, histidine, aspartic acid, glutamic acid, and threonine. For the sake of brevity, all of the combinations of any seven free amino acids are not being parsed out.

[0098] In another embodiment, the composition comprises, consists essentially of, or consists of any eight free amino acids selected from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of: asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine; asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, and isoleucine; arginine, asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, lysine, and threonine; or arginine, asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, serine, and threonine. For the sake of brevity, all of the combinations of any eight free amino acids are not being parsed out.

[0099] In another embodiment, the composition comprises, consists essentially of, or consists of any nine free amino acids selected from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of: asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, and threonine; asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, lysine, and threonine; or arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine. For the sake of brevity, all of the combinations of any nine free amino acids are not being parsed out.

[00100] In another embodiment, the composition comprises, consists essentially of, or consists of any ten free amino acids selected from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of: arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, and threonine; asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, serine, and threonine; or asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, and threonine. For the sake of brevity, all of the combinations of any ten free amino acids are not being parsed out.

[00101] In another embodiment, the composition comprises, consists essentially of, or consists of any eleven free amino acids selected from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of: asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine; asparagine, aspartic acid, cysteine, glutamic acid, glutamine, isoleucine, lysine, serine, threonine, tyrosine, and valine; asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, serine, threonine, and tyrosine; arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, serine, threonine, tyrosine, and valine; arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, threonine, tyrosine, and valine; or arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, and valine. For the sake of brevity, all of the combinations of any eleven free amino acids are not being parsed out.

[00102] In another embodiment, the composition comprises, consists essentially of, or consists of any twelve free amino acids selected from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of: arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine; asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine; or arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, histidine, isoleucine, serine, threonine, tyrosine, and valine. For the sake of brevity, all of the combinations of any twelve free amino acids are not being parsed out.

[00103] In another embodiment, the composition comprises, consists essentially of, or consists of any thirteen free amino acids selected from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of: arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine; or arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. For the sake of brevity, all of the combinations of any thirteen free amino acids are not being parsed out.

[00104] In another embodiment, the composition comprises, consists essentially of, or consists of any fourteen free amino acids selected from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of: arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. For the sake of brevity, all of the combinations of any fourteen free amino acids are not being parsed out.

[00105] In one aspect, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine; and optionally, pharmaceutically acceptable carriers, buffers, electrolytes, adjuvants, excipients, and an additional therapeutic agent. In certain embodiments, the composition comprises, consists essentially of, or consists of asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine. In certain embodiments, the composition consists essentially of, or consists of only the specified free amino acids and no other free amino acids, or a negligible amount of other free amino acids. The compositions include, in certain embodiments, derivatives of the amino acids that are derivatives of "natural" or "non-natural" amino acids. The compositions include, in certain embodiments, salts and/or prodrugs of the amino acids. In certain embodiments, the composition further comprises one or more free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, and alanine.

[00106] In another aspect, the composition comprises, consists essentially of, or consists of only one free amino acid selected from asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine. In certain embodiments, the composition comprises, consists essentially of, or consists of asparagine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of aspartic acid as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of cysteine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of histidine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of isoleucine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of threonine as a free amino acid. In certain embodiments, the composition does not include cysteine as a free amino acid. In certain embodiments, the composition does not include histidine as a free amino acid.

[00107] In one aspect, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine; and optionally, pharmaceutically acceptable carriers, buffers, electrolytes, adjuvants, excipients, and/or an additional therapeutic agent. In certain embodiments, the composition further comprises, consists essentially of, or consists of arginine, glycine, lysine, serine, tyrosine, or valine. In certain embodiments, the composition further comprises arginine. In certain embodiments, the composition further comprises glycine. In certain embodiments, the composition further comprises lysine. In certain embodiments, the composition further comprises serine. In certain embodiments, the composition further comprises tyrosine. In certain embodiments, the composition further comprises valine. In certain embodiments, the composition further comprises one or more free amino acids selected from arginine, glycine, lysine, serine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition consists essentially of, or consists of only the specified free amino acids and no other free amino acids, or a negligible amount of other free amino acids. The compositions include, in certain embodiments, derivatives of the amino acids that are derivatives of "natural" or "non-natural" amino acids. The compositions include, in certain embodiments, salts and/or prodrugs of the amino acids.

[00108] In certain embodiments, the composition further comprises histidine or cysteine. In certain embodiments, the composition further comprises histidine. In certain embodiments, the composition further comprises histidine and cysteine. In certain embodiments, the composition comprises, consists essentially of, or consists of arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition consists essentially of, or consists of only the specified free amino acids and no other free amino acids, or a negligible amount of other free amino acids. The compositions include, in certain

embodiments, derivatives of the amino acids that are derivatives of "natural" or "non-natural" amino acids. The compositions include, in certain embodiments, salts and/or prodrugs of the amino acids. In certain embodiments, the composition further comprises one or more free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, and alanine.

[00109] In another aspect, the composition comprises, consists essentially of, or consists of only one free amino acid selected from arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of arginine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of asparagine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of aspartic acid as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glycine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of isoleucine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of lysine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of serine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of threonine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of tyrosine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of valine as a free amino acid.

[00110] In another aspect, the composition comprises, consists essentially of, or consists of only one free amino acid selected from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of arginine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of asparagine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of aspartic acid as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of cysteine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glycine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of histidine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of isoleucine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of lysine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of serine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of threonine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of tyrosine as a free amino acid. In certain embodiments, the composition comprises, consists essentially of, or consists of valine as a free amino acid.

[00111] In yet another aspect, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, or all six free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine; and optionally, pharmaceutically acceptable carriers, buffers, electrolytes, adjuvants, excipients, and/or an additional therapeutic agent. In certain embodiments, the composition further comprises the free amino acids histidine or cysteine. In certain embodiments, the composition further comprises the free amino acid histidine. In certain embodiments, the composition further comprises the free amino acid cysteine. In certain embodiments, the composition further comprises the free amino acids histidine and cysteine. In certain embodiments, the composition further comprises one or more of the free amino acids selected from arginine, glycine, lysine, serine, tyrosine, and valine. In certain embodiments, the composition further comprises arginine as a free amino acid. In certain embodiments, the composition further comprises glycine as a free amino acid. In certain embodiments, the composition further comprises lysine as a free amino acid. In certain embodiments, the composition further comprises serine as a free amino acid. In certain embodiments, the composition further comprises tyrosine as a free amino acid. In certain embodiments, the composition further comprises valine as a free amino acid. In certain embodiments, the composition further comprises the free amino acids arginine, glycine, lysine, serine, tyrosine, and valine. In certain embodiments, the composition does not include cysteine. In certain embodiments, the composition does not include histidine. [00112] In yet another embodiment, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or all twelve amino acids selected from arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine; and optionally, pharmaceutically acceptable carriers, buffers, electrolytes, adjuvants, excipients, and/or an additional therapeutic agent. In certain embodiments, the composition further comprises one or more free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, and alanine.

[00113] In yet another embodiment, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, or all fourteen free amino acids selected from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine; and optionally, pharmaceutically acceptable carriers, buffers, electrolytes, adjuvants, excipients, and/or an additional therapeutic agent. In certain embodiments, the composition further comprises one or more of the free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, and alanine.

[00114] In yet another embodiment, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, or all five free amino acids selected from aspartic acid, serine, threonine, tyrosine, and valine; and optionally, pharmaceutically acceptable carriers, buffers, electrolytes, adjuvants, excipients, and/or an additional therapeutic agent. In certain embodiments, the composition consists essentially of, or consists of only aspartic acid, serine, threonine, tyrosine, and valine, and no other free amino acids, or a negligible amount of other free amino acids. In certain embodiments, the composition further comprises one or more of the free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, and alanine.

[00115] In yet another embodiment, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, or all five free amino acids selected from alanine, aspartic acid, isoleucine, glutamic acid, and glutamine; and optionally, pharmaceutically acceptable carriers, buffers, electrolytes, adjuvants, excipients, and/or an additional therapeutic agent. In certain embodiments, the composition consists essentially of, or consists of only alanine, aspartic acid, isoleucine, glutamic acid, and glutamine, and no other free amino acids, or a negligible amount of other free amino acids. In certain embodiments, the composition further comprises one or more of the free amino acids selected from proline, phenylalanine, tryptophan, methionine, and leucine.

[00116] In yet another embodiment, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, or all five free amino acids selected from alanine, aspartic acid, glutamic acid, lysine, and proline; and optionally,

pharmaceutically acceptable carriers, buffers, electrolytes, adjuvants, excipients, and/or an additional therapeutic agent. In certain embodiments, the composition consists essentially of, or consists of only alanine, aspartic acid, glutamic acid, lysine, and proline, and no other free amino acids, or a negligible amount of other free amino acids. In certain embodiments, the composition further comprises one or more of the free amino acids selected from

phenylalanine, tryptophan, methionine, and leucine.

[00117] In yet another embodiment, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, or all six free amino acids selected from arginine, aspartic acid, glutamine, isoleucine, proline, and tyrosine; and optionally, pharmaceutically acceptable carriers, buffers, electrolytes, adjuvants, excipients, and/or an additional therapeutic agent. In certain embodiments, the composition consists essentially of, or consists of only arginine, aspartic acid, glutamine, isoleucine, proline, and tyrosine, and no other free amino acids, or a negligible amount of other free amino acids. In certain embodiments, the composition further comprises one or more of the free amino acids selected from phenylalanine, tryptophan, methionine, leucine, and alanine.

[00118] In yet another embodiment, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all nine free amino acids selected from aspartic acid, glycine, isoleucine, lysine, serine, threonine, tryptophan, tyrosine, and valine; and optionally, pharmaceutically acceptable carriers, buffers, electrolytes, adjuvants, excipients, and/or an additional therapeutic agent. In certain embodiments, the composition consists essentially of, or consists of only aspartic acid, glycine, isoleucine, lysine, serine, threonine, tryptophan, tyrosine, and valine, and no other free amino acids, or a negligible amount of other free amino acids. In certain embodiments, the composition further comprises one or more of the free amino acids selected from proline, phenylalanine, methionine, leucine, and alanine.

[00119] In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine, and further comprises one or more free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, and/or alanine. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, valine, proline, phenylalanine, tryptophan, methionine, leucine, and alanine. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more free amino acids selected from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, valine, proline, phenylalanine, tryptophan, methionine, leucine, and alanine. In certain embodiments the composition comprises, consists essentially of, or consists of one or more free amino acids selected from, arginine, cysteine, glycine, isoleucine, lysine, serine, threonine, tyrosine, valine, proline and leucine, and no other free amino acids. In certain embodiments the composition comprises, consists essentially of, or consists of glycine, lysine, proline, cysteine, threonine, leucine, isoleucine, arginine, tyrosine, serine, and valine, and no other free amino acids.

[00120] Non-limiting examples of compositions comprising amino acids that increase translocation of CFTR to the plasma membrane are provided in Table B below.

[00121] Table B: Compositions Comprising Amino Acids

[00122] Each of the free amino acids, if present in the composition, may be present in, for example, the following concentrations: proline at about 0.4 to about 1.5, about 0.7 to about 1.3, about 0.9 to about 1.1 grams/liter, or about 1.5 to about 1.7 grams/liter; glutamic acid at about 0.7 to about 1.7, about 0.9 to about 1.5, about 1.1 to about 1.3 grams/liter, or about 1.5 to about 1.7 grams/liter; glutamine at about 0.6 to about 1.6, about 0.8 to about 1.4, about 1.0 to about 1.2 grams/liter, or about 1.5 to about 1.7 grams/liter; leucine at about 0.05 to about 0.4, about 0.1 to about 0.3 grams/liter, or about 1.5 to about 1.7 grams/liter; alanine at about 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter; aspartic acid at about 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter; phenylalanine at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter; histidine at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter; threonine at about 0.4 to about 1.5, about 0.7 to about 1.3, about 0.9 to about 1.1 grams/liter, or about 1.5 to about 1.7 grams/liter; isoleucine at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter; asparagine at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter; tryptophan at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter.

[00123] In certain embodiments, the amino acids of the composition are free amino acids. In certain embodiments, the amino acids of the composition are L-amino acids. In certain embodiments, the amino acids of the composition are D-amino acids. In certain

embodiments, the amino acids of the composition are a combination of D- and L-amino acids.

[00124] In certain embodiments, the amino acids of the compositions described herein may be prodrugs of the free amino acids. The term "prodrugs" refers to compounds that have cleavable groups and become by solvolysis or under physiological conditions the compounds described herein, which are pharmaceutically active in vivo.

[00125] In certain embodiments, the amino acids of the compositions described herein may be salts of amino acids (i.e., amino acid salts). Amino acids may be in salt form with cations (e.g., salts of amino acids with negatively charged side chains in solution (e.g., glutamate and aspartate)), anions (salts of amino acids with positively charged side chains in solution (e.g., lysine, arginine, histidine)), and inorganic compounds. Exemplary amino acid salts are listed in Fleck M and Petrosyan AM, Salts of Amino Acids, 1^st Ed; Springer International

Publishing, 2014, which is herein incorporated by reference. [00126] In certain embodiments, the composition further comprises water.

[00127] In certain embodiments, the composition further comprises a buffer. Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium

glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

[00128] In certain embodiments, phosphate ions, such as H₂P0₄ ^", HP0₄ ^2", and P0₄ ³ , are used to buffer the composition of the subject invention. In certain embodiments, the therapeutic composition uses HCO3^" or CO3^2" as a buffer. In other embodiments, the therapeutic composition does not use HCO3^" or CO3^2" as a buffer.

[00129] In certain embodiments, the composition comprises one or more electrolytes selected from, for example, Na⁺; K⁺; HCO3 ; CO3^2"; Ca²⁺; Mg²⁺; Fe²⁺; CI^"; phosphate ions, such as H₂P0₄ ^", HP0₄ ^2", and P0₄ ³ ; zinc; iodine; copper; iron; selenium; chromium; and molybdenum. In an alternative embodiment, the composition does not contain HCO3^" or CO3^2". In another alternative embodiment, the composition comprises HCO3^" and CO3^2" at a total concentration of less than 5 mg/1, or concentrations lower than 5 mg/1. In certain embodiments, the composition does not contain electrolytes. In certain embodiments, the composition does not contain carbohydrates (e.g., di-, oligo-, or polysaccharides). In certain alternative embodiments the composition does not comprise one or more, or any, of Na⁺; K⁺; HCO3 ; CO3^2"; Ca²⁺; Mg²⁺; Fe₂; CI^"; phosphate ions, such as H₂P0₄ ^", HP0₄ ^2", and P0₄ ^3"; zinc; iodine; copper; iron; selenium; chromium; and molybdenum.

[00130] In certain embodiments, the composition further comprises sugars, vitamins, electrolytes, minerals, proteins, or lipids. In certain embodiments, the composition further comprises sugars. In certain embodiments, the composition further comprises vitamins. In certain embodiments, the composition further comprises electrolytes. In certain embodiments, the composition further comprises minerals. In certain embodiments, the composition further comprises proteins. In certain embodiments, the composition further comprises lipids. [00131] In certain embodiments, the composition does not contain one or more of the ingredients selected from oligo-, polysaccharides, and carbohydrates; oligo-, or polypeptides or proteins; lipids; small-, medium-, and/or long-chain fatty acids; and/or food containing one or more above-mentioned nutrients.

[00132] The composition may have a pH ranging from about 2.5 to about 8.5. In certain embodiments, the pH of the composition ranges from about 2.5 to about 6.5, about 3.0 to about 6.0, about 3.5 to about 5.5, about 3.9 to about 5.0, or about 4.2 to about 4.6. In other embodiments, the pH of the composition ranges from about 6.5 to about 8.5, about 7.0 to about 8.0, or about 7.2 to about 7.8. In certain embodiments, the composition has a pH from, for example, about 2.5 to about 8.5. In certain embodiments, the composition has a pH from about 2.5 to about 6.5, about 2.5 to about 6.0, about 3.0 to about 6.0, about 3.5 to about 6.0, about 3.9 to about 6.0, about 4.2 to about 6.0, about 3.5 to about 5.5, about 3.9 to about 5.0, or about 4.2 to about 4.6. In other embodiments, the pH is about 6.5 to about 8.5, about 7.0 to about 8.5, about 7.0 to about 8.0, about 7.2 to about 8.0, or about 7.2 to about 7.8. In certain embodiments, the pH is about 7.3 to about 7.5. In certain embodiments, the pH is about 7.3 to about 7.4. In certain embodiments, the pH is about 7.4 to about 7.5. In certain embodiments, the pH is about 7.4.

[00133] In certain embodiments, the total osmolarity of the composition is from about 100 mosm to 280 mosm, or any value therebetween. In certain embodiments, the total osmolarity is from about 150 msom to 260 mosm. In another embodiment, the composition has a total osmolarity that is any value lower than 280 mosm.

[00134] In certain embodiments, the composition is sterile.

[00135] Compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include bringing compounds of the compositions described herein (i.e., the free amino acid(s)) into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit.

[00136] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition described herein will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. The composition may comprise between 0.1% and 100% (w/w) active ingredient.

[00137] A compound or composition, as described herein, can be administered in combination with one or more additional pharmaceutical agents (e.g. , therapeutically and/or prophylactically active agents). The compounds or compositions can be administered in combination with additional pharmaceutical agents that improve their activity (e.g. , activity (e.g. , potency and/or efficacy) in treating a disease in a subject in need thereof, in preventing a disease in a subject in need thereof, in reducing the risk to develop a disease in a subject in need thereof, and/or in inhibiting the activity of a protein kinase in a subject or cell), improve bioavailability, improve safety, reduce drug resistance, reduce and/or modify metabolism, inhibit excretion, and/or modify distribution in a subject or cell. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, and/or it may achieve different effects. In certain embodiments, a pharmaceutical composition described herein including a compound described herein and an additional pharmaceutical agent shows a synergistic effect that is absent in a pharmaceutical composition including one of the compound and the additional pharmaceutical agent, but not both.

[00138] The compositions described herein can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents or therapeutic agents, which are different from the compound or composition and may be useful as, e.g. , combination therapies. Pharmaceutical agents include therapeutically active agents.

Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include small organic molecules such as drug compounds (e.g. , compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful for treating and/or preventing a disease (e.g. , proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, lung disease, or metabolic disorder). In certain embodiments, the additional pharmaceutical agent is useful for treating a lung disease. In certain embodiments, the additional pharmaceutical agent is useful for treating cystic fibrosis. Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered together with each other and/or with the compound or composition described herein in a single dose or administered separately in different doses. The particular combination to employ in a regimen will take into account compatibility of the compound described herein with the additional pharmaceutical agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

[00139] In certain embodiments, the compositions are administered concurrently with, prior to, or subsequent to one or more additional therapeutic agents, wherein the additional therapeutic agent is a CFTR potentiator, corrector, or read-through agent. A "CFTR potentiator" is a compound that increases the function of a CFTR on the plasma membrane of a cell. These compounds may be useful for treating cystic fibrosis mutations that result in CFTR proteins that are correctly trafficked to the plasma membrane, but do not function properly (e.g., channel gating is defective, ATP binding is reduced or abrogated, and/or chloride transport is reduced). In certain embodiments, the CFTR potentiator is ivacaftor (VX-770, Kalydeco^®). A "CFTR corrector" is a compound that improves the intracellular processing and translocation of mutant CFTR, allowing more protein to reach the plasma membrane. In certain embodiments, the CFTR corrector is lumacaftor (VX-809, Orkambi^®). In certain embodiments, the CFTR corrector is tezacaftor (VX-661). A "CFTR read-through agent" is a compound that promotes transcription in the presence of a premature termination codon (PTC) mutation resulting from a point mutation in the CFTR gene sequence.

Normally, the PTC would result in a truncated CFTR protein which is not properly processed and/or is malfunctioning (e.g., channel gating is defective, ATP binding is reduced or abrogated, and/or chloride transport is reduced). In certain embodiments, the CFTR read- through agent is ataluren (Translarna™). In certain embodiments, a composition comprising amino acids as described herein is administered in combination with ivacaftor. In certain embodiments, a composition comprising amino acids as described herein is administered in combination with lumacaftor. In certain embodiments, a composition comprising amino acids as described herein is administered in combination with ataluren. In certain embodiments, a composition comprising amino acids as described herein is administered in combination with ivacaftor and lumacaftor. In certain embodiments, a composition comprising amino acids as described herein is administered in combination with ivacaftor and ataluren. In certain embodiments, a composition comprising amino acids as described herein is administered in combination with lumacaftor and ataluren. In certain embodiments, the compositions are administered alone, i.e., are not administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents or therapeutic agents. In certain embodiments, the compositions are administered in the absence of a CFTR potentiator, corrector, or read- through agent.

[00140] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include a buffering agent.

[00141] In certain embodiments, a composition comprising amino acids described herein may be provided in powdered form and reconstituted for administration to a subject. A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, or from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder and/or using a self-propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[00142] Liquid dosage forms for oral and parenteral administration include

pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g. , cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the conjugates described herein are mixed with solubilizing agents such as Cremophor^®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

[00143] Pharmaceutical compositions described herein formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations can be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range from about 0.1 to about 200 nanometers.

[00144] Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition described herein. Another

formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered by rapid inhalation through the nasal passage from a container of the powder held close to the nares.

[00145] Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) to as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

Methods for increasing translocation of CFTR to the plasma membrane

[00146] The present disclosure provides compositions for use for increasing the translocation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein from the cytoplasm to the plasma membrane. In general, without wishing to be bound by any particular theory, mutations in the CFTR protein (e.g., Phe508del) may disrupt correct processing, folding, and/or trafficking to the plasma membrane, resulting in a lower number of CFTR proteins on the plasma membrane and impaired chloride ion transport function in cells expressing the mutated CFTR protein. Impaired chloride ion transport results in an imbalanced osmotic profile, such that water is not being drawn out of the cells expressing the mutated CFTR protein via osmosis due to the abnormally high intracellular concentration of chloride ions. This results in formation of a thick mucus layer covering the cells, a hallmark of cystic fibrosis.

[00147] Thus, in one aspect, the present disclosure provides a method for increasing the number of CFTR proteins on the plasma membrane of a cell, the method comprising contacting the cell with an effective amount of a composition comprising amino acids of the present disclosure. In certain embodiments, the cell is an epithelial cell. In certain

embodiments, the epithelial cell is a small intestine epithelial cell or lung epithelial cell. In certain embodiments, the epithelial cell is a lung epithelial cell. In certain embodiments, the lung epithelial cell is a bronchial epithelial cell. The bronchial epithelial cell may be a normal human bronchial epithelial cell (NHBE) or a diseased human bronchial epithelial cell (DHBE). A diseased human bronchial epithelial cell may be obtained from a human donor diagnosed with a lung disease (e.g., asthma, COPD, cystic fibrosis). In certain embodiments, the bronchial epithelial cells are expressing wild-type CFTR. In certain embodiments, the bronchial epithelial cells are expressing mutant CFTR. In certain embodiments, the bronchial epithelial cells are expressing both wild-type and mutant CFTR (i.e., one allele in the CFTR gene encodes wild-type CFTR and the second allele contains a mutation (e.g., Phe508del CFTR)). In certain embodiments, the bronchial epithelial cells are expressing the Phe508del mutant CFTR. The cells may be present in vitro, in vivo, or ex vivo. [00148] In certain embodiments, the number of wild-type CFTR proteins on the plasma membrane increases. In certain embodiments, the number of mutant CFTR proteins on the plasma membrane increases. In certain embodiments, the number of Gly542X mutant CFTR proteins on the plasma membrane increases, where X is any amino acid. In certain

embodiments, the number of Gly551Asp mutant CFTR proteins on the plasma membrane increases. In certain embodiments, the number of Arg553X mutant CFTR proteins on the plasma membrane increases, where X is any amino acid. In certain embodiments, the number of Argl 17His mutant CFTR proteins on the plasma membrane increases. In certain embodiments, the number of 120del23 mutant CFTR proteins on the plasma membrane increases. In certain embodiments, the number of Phe508del mutant CFTR proteins on the plasma membrane increases. In certain embodiments, the number of CFTR proteins that are at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of wild-type CFTR as provided by SEQ ID NO: 1 on the plasma membrane increases. In certain embodiments, the number of CFTR proteins that are at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of Phe508del CFTR as provided by SEQ ID NO: 2 on the plasma membrane increases.

[00149] In certain embodiments, the present disclosure provides a method for increasing the number of CFTR proteins on the plasma membrane of a cell, the method comprising contacting the cell with an effective amount of a composition described herein. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition comprises two or more, three or more, four or more, five or more, or all six free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition comprises, consists essentially of, or consists of asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition further comprises one of more of the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, and alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, and/or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids valine and/or alanine.

[00150] By "negligible amount" it is meant that the amino acid present has no effect on the CFTR protein. Or, in certain embodiments, even if the amino acid is present in the composition, it is not present in an amount that would affect the translocation of CFTR to the plasma membrane, chloride ion transport, or the therapeutic effect of treating a subject in need thereof. In certain embodiments, a negligible amount is an amount wherein the total concentration of the amino acid is less than 100 mg/1, 50 mg/1, 10 mg/1, 5 mg/1, 1 mg/1, 0.5 mg/1, 0.1 mg/1, or 0.01 mg/1. In certain embodiments, a negligible amount is an amount wherein the total concentration of the amino acid is less than 100 mg/1. In certain

embodiments, a negligible amount is an amount wherein the total concentration of the amino acid is less than 50 mg/1. In certain embodiments, a negligible amount is an amount wherein the total concentration of the amino acid is less than 10 mg/1. In certain embodiments, a negligible amount is an amount wherein the total concentration of the amino acid is less than 5 mg/1. In certain embodiments, a negligible amount is an amount wherein the total concentration of the amino acid is less than 1 mg/1. In certain embodiments, a negligible amount is an amount wherein the total concentration of the amino acid is less than 0.5 mg/1. In certain embodiments, a negligible amount is an amount wherein the total concentration of the amino acid is less than 0.1 mg/1. In certain embodiments, a negligible amount is an amount wherein the total concentration of the amino acid is less than 0.01 mg/1.

[00151] In certain embodiments, the present disclosure provides a method for increasing the number of CFTR proteins on the plasma membrane of a cell, the method comprising contacting the cell with an effective amount of a composition described herein. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition further comprises the free amino acids histidine or cysteine. In certain embodiments, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, or all eight free amino acids selected from asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine. In certain

embodiments, the composition comprises, consists essentially of, or consists of asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine. In certain embodiments, the composition further comprises one or more free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, and alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, and/or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids valine and/or alanine. [00152] In certain embodiments, the present disclosure provides a method for increasing the number of CFTR proteins on the plasma membrane of a cell, the method comprising contacting the cell with an effective amount of a composition described herein. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition further comprises the free amino acids arginine, glycine, lysine, serine, tyrosine, or valine. In certain embodiments, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or all twelve free amino acids selected from arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition further comprises one or more free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, and alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids valine and/or alanine.

[00153] In certain embodiments, the present disclosure provides a method for increasing the number of CFTR proteins on the plasma membrane of a cell, the method comprising contacting the cell with an effective amount of a composition described herein. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition further comprises the free amino acids histidine or cysteine. In certain embodiments, the composition further comprises one or more free amino acids selected from arginine, glycine, lysine, serine, tyrosine, and valine. In certain embodiments, the composition further comprises one or more of the free amino acids selescted from arginine, cysteine, glycine, histidine, lysine, serine, tyrosine, and valine. In certain embodiments, the composition further comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, or all fourteen free amino acids selected from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition further comprises one or more free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, and/or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids valine and/or alanine.

[00154] In certain embodiments, compositions of the present disclosure comprise one or more free amino acids that are essential (e.g., necessary) for increasing the translocation of CFTR to the plasma membrane. In certain embodiments, compositions of the present disclosure may include one or more free amino acids that are not essential for increasing the translocation of CFTR to the plasma membrane (i.e., do not directly stabilize CFTR), but may instead provide alternative beneficial properties to the composition (e.g., maintain a specific pH or osmolarity).

[00155] In one aspect, the method of increasing the number of CFTR proteins on the plasma membrane of a cell involves increasing the translocation of CFTR from the cytoplasm of the cell to the cell membrane, the method comprising contacting the cell with an effective amount of a composition comprising amino acids of the present disclosure. In one embodiment, the method leads to an increase in the number of CFTR proteins on the plasma membrane of the treated cell. An increase in the number of CFTR proteins on the plasma membrane can be determined by comparing the cells contacted with a composition comprising amino acids described herein to untreated cells (e.g., control cells). For example, a western blot may be used to compare the presence and amount of CFTR in membrane vesicles isolated from epithelial cells contacted with a composition comprising amino acids as described herein versus the amount of CFTR in membrane vesicles isolated from untreated epithelial cells (e.g., control cells). Conducting a western blot analysis as described herein is within the ability of a person of ordinary skill in the art. Additional related techniques that may be used to determine the expression level of the CFTR protein in a sample include dot blot analysis, immunohistochemistry, immunocytochemistry, and enzyme-linked

immunosorbent assay (ELISA), among others. [00156] The CFTR protein is an ABC transporter protein that functions as an ATP-gated ion channel. When activated, CFTR allows chloride ions (CI^"), and other negatively charged ions such as thiocyanate ([SCN]^"), to flow down their electrochemical gradient (e.g., passive diffusion or passive transport). Mutations in CFTR, such as, for example, Gly551X, where X represents any amino acid (e.g., Gly551Asp), result in a CFTR protein characterized by defective ion channel gating function. The most common CFTR mutation, Phe508del, results in a CFTR protein that lacks the codon for phenylalanine 508 and cannot properly fold or traffick to the plasma membrane. Thus, in one aspect, the present disclosure provides a method for increasing the number of CFTR proteins on the plasma membrane of a cell and increasing the transport of chloride ions across the cell membrane (e.g., export of chloride ions from the cell), the method comprising contacting a cell with a composition comprising amino acids as described herein.

[00157] Thus, in one aspect, the present disclosure provides a method for increasing chloride ion export from a cell, the method comprising contacting the cell with an effective amount of a composition described herein. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition comprises two or more, three or more, four or more, five or more, or all six free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition comprises, consists essentially of, or consists of asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition further comprises one of more of the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, and alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, and/or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids valine and/or alanine.

[00158] In another aspect, the present disclosure provides a method for increasing chloride ion export from a cell, the method comprising contacting the cell with an effective amount of a composition described herein. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition further comprises the free amino acids histidine or cysteine. In certain embodiments, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, or all eight free amino acids selected from asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine. In certain embodiments, the composition comprises, consists essentially of, or consists of asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine. In certain embodiments, the composition further comprises one or more free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, and alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, and/or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids valine and/or alanine.

[00159] In yet another aspect, the present disclosure provides a method for increasing chloride ion export from a cell, the method comprising contacting the cell with an effective amount of a composition described herein. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition further comprises the free amino acids arginine, glycine, lysine, serine, tyrosine, or valine. In certain embodiments, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or all twelve free amino acids selected from arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition further comprises one or more free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, and alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids valine and/or alanine.

[00160] In yet another aspect, the present disclosure provides a method for increasing chloride ion export from a cell, the method comprising contacting the cell with an effective amount of a composition described herein. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition further comprises the free amino acids histidine or cysteine. In certain embodiments, the composition further comprises one or more free amino acids selected from arginine, glycine, lysine, serine, tyrosine, and valine. In certain embodiments, the composition further comprises one or more of the free amino acids selected from arginine, cysteine, glycine, histidine, lysine, serine, tyrosine, and valine. In certain

embodiments, the composition further comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, or all fourteen free amino acids selected from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition further comprises one or more free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, and/or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids valine and/or alanine.

[00161] In certain embodiments, the export of chloride from a cell is further enhanced by contacting the cell with a CFTR potentiator, corrector, or read-through agent. In certain embodiments, the export of chloride from a cell is increased by contacting the cell with a combination therapy comprising a composition described herein and a CFTR potentiator. In certain embodiments, the export of chloride from a cell is increased by contacting the cell with a combination therapy comprising a composition described herein and a CFTR corrector. In certain embodiments, the export of chloride from a cell is increased by contacting the cell with a combination therapy comprising a composition described herein and a CFTR read-through agent. In certain embodiments, the export of chloride from a cell is increased by contacting the cell with a combination therapy comprising a composition described herein and ivacaftor.

[00162] The flow of ions, such as chloride ions, across a membrane is one biological mechanism used to regulate the flow of water across a semi-permeable membrane (e.g., a cell membrane). Osmosis describes a process by which solvent (e.g., water) molecules flow from an area of low solute concentration to an area of higher solute concentration to balance the concentration on each side of a semi-permeable membrane (e.g., passive diffusion of water). When the chloride ion concentration in a cell increases due to malfunction of the CFTR, water molecules do not flow out of the cell and into the surrounding mucus membrane, resulting in the formation of thick mucus. Thus, in a further aspect, the present disclosure provides methods and compositions for increasing the flow of water out of a cell (e.g., osmosis), the method comprising contacting a cell with a composition comprising amino acids as described herein.

[00163] Thus, in one aspect, the present disclosure provides a method for increasing the export of water from a cell, the method comprising contacting a cell with a composition described herein. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition comprises two or more, three or more, four or more, five or more, or all six free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition comprises, consists essentially of, or consists of asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition further comprises one of more of the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, and alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, and/or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids valine and/or alanine.

[00164] In another aspect, the present disclosure provides a method for increasing the export of water from a cell, the method comprising contacting a cell with a composition described herein. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition further comprises the free amino acids histidine or cysteine. In certain embodiments, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, or all eight free amino acids selected from asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine. In certain embodiments, the composition comprises, consists essentially of, or consists of asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine. In certain embodiments, the composition further comprises one or more free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, and alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, and/or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids valine and/or alanine.

[00165] In yet another aspect, the present disclosure provides a method for increasing the export of water from a cell, the method comprising contacting a cell with a composition described herein. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition further comprises the free amino acids arginine, glycine, lysine, serine, tyrosine, or valine. In certain embodiments, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or all twelve free amino acids selected from arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition further comprises one or more free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, and alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids valine and/or alanine.

[00166] In yet another aspect, the present disclosure provides a method for increasing the export of water from a cell, the method comprising contacting a cell with a composition described herein. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition further comprises the free amino acids histidine or cysteine. In certain embodiments, the composition further comprises one or more free amino acids selected from arginine, glycine, lysine, serine, tyrosine, and valine. In certain embodiments, the composition further comprises one or more of the free amino acids selected from arginine, cysteine, glycine, histidine, lysine, serine, tyrosine, and valine. In certain embodiments, the composition further comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, or all fourteen free amino acids selected from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition further comprises one or more free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, and/or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids valine and/or alanine.

[00167] In certain embodiments, the export of water from a cell is further enhanced by contacting the cell with a CFTR potentiator, corrector, or read-through agent. In certain embodiments, the export of water from a cell is increased by contacting the cell with a combination therapy comprising a composition described herein and a CFTR potentiator. In certain embodiments, the export of water from a cell is increased by contacting the cell with a combination therapy comprising a composition described herein and a CFTR corrector. In certain embodiments, the export of water from a cell is increased by contacting the cell with a combination therapy comprising a composition described herein and a CFTR read-through agent. In certain embodiments, the export of water from a cell is increased by contacting the cell with a combination therapy comprising a composition described herein and ivacaftor.

Methods for treatment of cystic fibrosis

[00168] As described above, the flow of chloride ions through the cystic fibrosis transmembrane conductance regulator (CFTR) protein is crucial for maintenance of fluid levels in the cell and surrounding mucosal membrane, particularly in the lungs and small intestines. Mutations in the CFTR protein (e.g., Phe508del) that disrupt CFTR processing, folding, and trafficking to the plasma membrane result in a lower number of functioning CFTR proteins on the plasma membrane. The biological result is the build-up of a thick mucus layer covering the epithelial cell layer, promoting bacterial growth and preventing epithelial cells from obtaining nutrients from the surrounding fluid. Ultimately, respiratory and lung diseases, such as cystic fibrosis, may develop in patients carrying one or more CFTR gene mutations.

[00169] The subject can be, for example, a human suffering from a lung disease. In certain embodiments, the subject is suffering from cystic fibrosis. In certain embodiments, the underlying genetic cause of the cystic fibrosis may be the Phe508del mutation in one or more alleles of the CFTR gene. The human may also be suffering from additional complications that often occur concurrently with cystic fibrosis, such as bacterial infections, viral infections, asthma, and chronic respiratory failure, among others. Thus, the compositions comprising amino acids disclosed herein may also be useful in managing the symptoms and other complications in a subject with cystic fibrosis.

[00170] In certain embodiments, the methods described herein lead to increased survival of a patient suffering from a lung disease (e.g., cystic fibrosis). The methods and compositions described herein may also be useful for improving the therapeutic outcome of patients with cystic fibrosis.

[00171] Thus, in one aspect, the present disclosure provides compositions for use for treatment of cystic fibrosis, wherein the compositions are administered to a subject in need thereof (e.g., a subject with cystic fibrosis). In certain embodiments, a subject is suffering from cystic fibrosis in which wild-type CFTR of SEQ ID NO: 1 is present. In certain embodiments, the subject is suffering from cystic fibrosis in which a CFTR protein that is at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of wild-type CFTR as provided by SEQ ID NO: 1 is present. In certain embodiments, the subject is suffering from cystic fibrosis in which mutant CFTR is present. In certain embodiments, the subject is suffering from cystic fibrosis in which the mutant CFTR is a Gly542X, Gly551Asp, Arg553X, Argl 17His, 120del23, or Phe508del mutant, wherein X represents any amino acid. In certain embodiments, the subject is suffering from cystic fibrosis in which both wild-type CFTR and mutant CFTR are present. In certain embodiments, the subject is suffering from cystic fibrosis in which a CFTR protein that is at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of Phe508del CFTR as provided by SEQ ID NO: 2 is present. In certain embodiments, the subject is suffering from cystic fibrosis in which the mutant CFTR is Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the subject is suffering from cystic fibrosis in which both wild-type CFTR of SEQ ID NO: 1 and Phe508del CFTR of SEQ ID NO: 2 are present. The compositions described herein may be administered with one or more additional therapeutic agents, e.g., combination therapy, to further increase the therapeutic benefit of the compositions described herein.

[00172] In a further aspect, the present disclosure provides a method for treating cystic fibrosis, the method comprising administering to a subject in need thereof a composition described herein. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition comprises two or more, three or more, four or more, five or more, or all six free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition comprises, consists essentially of, or consists of asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition further comprises one of more of the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, and alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, and/or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids valine and/or alanine. In certain embodiments, the composition further comprises a CFTR potentiator, corrector, or read-through agent. In certain embodiments, the composition further comprises a CFTR potentiator. In certain embodiments, the composition further comprises a CFTR corrector. In certain embodiments, the composition further comprises a CFTR read-through agent. In certain embodiments, the composition further comprises ivacaftor. In certain embodiments, the composition does not further comprise a CFTR potentiator, corrector, or read-through agent.

[00173] In yet another aspect, the present disclosure provides a method for treating cystic fibrosis, the method comprising administering to a subject in need thereof a composition described herein. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition further comprises the free amino acids histidine or cysteine. In certain embodiments, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, or all eight free amino acids selected from asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine. In certain embodiments, the composition comprises, consists essentially of, or consists of asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine. In certain embodiments, the composition further comprises one or more free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, and alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, and/or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids valine and/or alanine. In certain embodiments, the composition further comprises a CFTR potentiator, corrector, or read- through agent. In certain embodiments, the composition further comprises a CFTR potentiator. In certain embodiments, the composition further comprises a CFTR corrector. In certain embodiments, the composition further comprises a CFTR read-through agent. In certain embodiments, the composition further comprises ivacaftor. In certain embodiments, the composition does not further comprise a CFTR potentiator, corrector, or read-through agent.

[00174] In yet another aspect, the present disclosure provides a method for treating cystic fibrosis, the method comprising administering to a subject in need thereof a composition described herein. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition further comprises the free amino acids arginine, glycine, lysine, serine, tyrosine, or valine. In certain embodiments, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or all twelve free amino acids selected from arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition further comprises one or more free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, and alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids valine and/or alanine. In certain embodiments, the composition further comprises a CFTR potentiator, corrector, or read-through agent. In certain embodiments, the composition further comprises a CFTR potentiator. In certain embodiments, the composition further comprises a CFTR corrector. In certain embodiments, the composition further comprises a CFTR read-through agent. In certain embodiments, the composition further comprises ivacaftor. In certain embodiments, the composition does not further comprise a CFTR potentiator, corrector, or read-through agent.

[00175] In yet another aspect, the present disclosure provides a method for treating cystic fibrosis, the method comprising administering to a subject in need thereof a composition described herein. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine. In certain embodiments, the composition further comprises the free amino acids histidine or cysteine. In certain embodiments, the composition further comprises one or more free amino acids selected from arginine, glycine, lysine, serine, tyrosine, and valine. In certain embodiments, the composition further comprises one or more of the free amino acids selected from arginine, cysteine, glycine, histidine, lysine, serine, tyrosine, and valine. In certain embodiments, the composition further comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, or all fourteen free amino acids selected from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition comprises, consists essentially of, or consists of arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine. In certain embodiments, the composition further comprises one or more free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids proline, phenylalanine, tryptophan, methionine, leucine, and/or alanine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acids valine and/or alanine. In certain embodiments, the composition further comprises a CFTR potentiator, corrector, or read-through agent. In certain embodiments, the composition further comprises a CFTR potentiator. In certain embodiments, the composition further comprises a CFTR corrector. In certain embodiments, the composition further comprises a CFTR read-through agent. In certain embodiments, the composition further comprises ivacaftor. In certain embodiments, the composition does not further comprise a CFTR potentiator, corrector, or read-through agent. In certain

embodiments, the amino acids in the composition enhance the function of the additional agent. In certain embodiments, the amino acids in the composition enhance the function of the additional agent. In certain embodiments, the amino acids in the composition enhance the function of the additional CFTR potentiator agent. In certain embodiments, the amino acids in the composition enhance the function of the additional CFTR corrector agent.

CFTR Gene and Gene Products and CFTR Mutants

[00176] As discussed above, the compositions comprising amino acids as described herein effectively increase the translocation of CFTR to the plasma membrane. In particular, the compositions comprising amino acids as described herein can effectively increase the translocation of mutant (e.g., Phe508del) CFTR to the plasma membrane. The compositions result in an increase in the number of CFTR proteins on the plasma membrane, providing a method for increasing chloride ion transport across the epithelial cell membrane to maintain membrane hyperpolarization and transmembrane water transport. These compositions are useful for the treatment of lung diseases, such as cystic fibrosis, or diseases which result from dysregulation of fluid transport in other epithelial cells, such as those in the pancreas and small intestine.

[00177] Sequences of the CFTR gene products of interest herein often comprise or consist of sequences encoded by human CFTR genes, although sequences of non-human mammalian homologs may be used in certain embodiments. In general, the sequence of a CFTR protein or CFTR RNA often comprises or consists of a sequence of a human CFTR. In certain embodiments, the sequence of a gene product of a CFTR gene comprises or consists of a naturally occurring sequence. It will be appreciated that a genetic locus may have more than one sequence or allele in a population of individuals. In some embodiments a naturally occurring sequence is a standard sequence. Unless otherwise indicated, a sequence listed in the Reference Sequence (RefSeq) Database as a reference sequence for a protein that is referred to herein by a particular name, abbreviation, or symbol, is considered to be a "standard sequence." If a sequence has been updated subsequent to the time of the present disclosure a version current at the time of the present disclosure or an updated version thereof may be used in certain embodiments. It will be appreciated that a genetic locus may have more than one sequence or allele in a population of individuals. In some embodiments a naturally occurring sequence differs from a standard sequence at one or more amino acid positions. A naturally occurring polynucleotide or polypeptide whose sequence differs from a standard sequence and that performs the normal function(s) of the polynucleotide or polypeptide may be referred to as having a "normal sequence". [00178] The CFTR gene is approximately 189 kb in length and is comprised of 27 exons and 26 introns. In certain embodiments, the CFTR protein is the full-length, wild-type CFTR. The CFTR may be a mammalian (e.g., human) CFTR. In certain embodiments, the sequence of a CFTR or variant thereof used in the compositions and methods described herein comprises the sequence of a naturally occurring CFTR protein or a biologically active variant thereof. A biologically active variant of an androgen receptor protein may contain one or more additions, substitutions, and/or deletions relative to the sequence of a naturally occurring CFTR protein. In some embodiments, the sequence of a CFTR protein comprises a standard CFTR sequence. The full-length CFTR protein is 1480 amino acids in length and has five domains: two transmembrane domains, one intracellular nucleotide binding domain (NBD) connected to each transmembrane domain, and one intracellular regulatory "R" domain. Full-length, wild-type CFTR has the following standard amino acid sequence (GenBank and NCBI Reference Sequence Accession Number: NG_016465.4):

MQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWD RELASKKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLLGRIIASYDPDNKEE RSIAIYLGIGLCLLFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISI GQLVS LLS NNLNKFDEGLALAHF VWIAPLQ V ALLMGLrWELLQ AS AFC GLGFLIVLA LFQAGLGRMMMKYRDQRAGKISERLVITSEMffiNIQSVKAYCWEEAMEKMIENLRQ TELKLTRKA A Y VRYFNS S AFFFS GFFV VFLS VLP Y ALIKGIILRKIFTTIS FCIVLRM A V TRQFPWAVQTWYDSLGAINKIQDFLQKQEYKTLEYNLTTTEVVMENVTAFWEEGFG ELFEKAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINFKIERGQLLAVAGSTGAG KTSLLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRYRSVIKA CQLEEDIS KFAEKDNIVLGEGGITLS GGQRARIS LARA VYKD ADLYLLDSPFG YLD VL TEKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPD FSSKLMGCDSFDQFSAERRNSILTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKR KNS ILNPINS IRKFS IVQKTPLQMNGIEEDS DEPLERRLS LVPDS EQGE AILPRIS VIS TGP TLQ ARRRQS VLNLMTHS VNQGQNIHRKTT AS TRKVS LAPQ ANLTELDIYS RRLS QET GLEISEEINEEDLKECFFDDMESIPAVTTWNTYLRYITVHKSLIFVLIWCLVIFLAEVAA S LV VLWLLGNTPLQDKGNS THS RNNS Y A VIITS TS S Y Y VFYIY VG V ADTLL AMGFFR GLPLVHTLITVSKILHHKMLHSVLQAPMSTLNTLKAGGILNRFSKDIAILDDLLPLTIF DFIQLLLIVIGAIAVVAVLQPYIFVATVPVIVAFIMLRAYFLQTSQQLKQLESEGRSPIF THLVTSLKGLWTLRAFGRQPYFETLFHKALNLHTANWFLYLSTLRWFQMRIEMIFVI FFIAVTFISILTTGEGEGRVGIILTLAMNIMSTLQWAVNSSIDVDSLMRSVSRVFKFID MPTEGKPTKSTKPYKNGQLSKVMIIENSHVKKDDIWPSGGQMTVKDLTAKYTEGGN AILENIS FS IS PGQRVGLLGRTGS GKS TLLS AFLRLLNTEGEIQIDG VS WD S ITLQQWRK AFGVIPQKVFIFSGTFRKNLDPYEQWSDQEIWKVADEVGLRSVIEQFPGKLDFVLVD GGCVLSHGHKQLMCLARSVLSKAKILLLDEPSAHLDPVTYQIIRRTLKQAFADCTVIL CEHRIE AMLEC QQFL VIEENKVRQ YDS IQKLLNERS LFRQ AIS PS DR VKLFPHRNS S KC KS KPQIA ALKEETEEE VQDTRL (SEQ ID NO: 1)

[00179] In certain embodiments, the present disclosure provides compositions comprising amino acids useful for increasing the number of CFTR proteins on the plasma membrane. In certain embodiments, the CFTR protein is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more, e.g. 100%, identical in sequence to wild-type CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR protein is at least 70% identical in sequence to wild-type CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR protein is at least 80% identical in sequence to wild-type CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR protein is at least 90% identical in sequence to wild-type CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR protein is at least 95% identical in sequence to wild-type CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR protein is at least 96% identical in sequence to wild-type CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR protein is at least 97% identical in sequence to wild-type CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR protein is at least 98% identical in sequence to wild-type CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR protein is at least 99% identical in sequence to wild-type CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR protein is at least 99.5% identical in sequence to wild-type CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR protein is at least 100% identical in sequence to wild-type CFTR of SEQ ID NO: 1.

[00180] In certain embodiments, the CFTR protein is a variant or fragment of the full- length CFTR of SEQ ID NO: 1. The term "variant" also encompasses splice variants of CFTR that result from alternative splicing of the CFTR gene. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of the full-length CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 50% of the full-length CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 60% of the full- length CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 70% of the full-length CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 80% of the full-length CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 90% of the full-length CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 95% of the full-length CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 96% of the full-length CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 97% of the full-length CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 98% of the full-length CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 99% of the full-length CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 99.5% of the full-length CFTR of SEQ ID NO: 1. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 100% of the full-length CFTR of SEQ ID NO: 1. These variants may be biologically active variants of wild-type CFTR, such that the ion channel gating function and/or the chloride transport function of the CFTR is retained in the variant. These variants may be biologically inactive variants of wild-type CFTR, such that the ion channel gating function and/or the chloride transport function of the CFTR is abrogated (e.g., not functional) in the variant.

[00181] In certain embodiments, a CFTR protein is a mutant CFTR protein, e.g., the sequence of the protein comprises the sequence of a naturally occurring mutant form of CFTR. The mutant CFTR may be a mammalian (e.g., human) CFTR mutant. The mutant CFTR may result from a nonsense, frameshift, or mRNA splicing mutation. Over 2,000 mutations in the CFTR gene have been discovered, many of which are clinically relevant and/or lead to a disease (e.g., cystic fibrosis) phenotype. See Bobadilla JL et al., 2002, Human Mutation, 19; pp. 575-606, which is herein incorporated by reference, for additional CFTR gene mutations for which administration of a composition of the present disclosure may prove useful. In certain embodiments, the mutant CFTR is a Gly542X, Gly551Asp, Arg553X, Argl l7His, 120del23, or Phe508del mutant, wherein X represents any amino acid. In certain embodiments, the mutant CFTR is a Gly542X mutant, wherein X represents any amino acid. In certain embodiments, the mutant CFTR is a Gly551Asp mutant. In certain embodiments, the mutant CFTR is a Arg553X mutant, wherein X represents any amino acid. In certain embodiments, the mutant CFTR is a Argl 17His mutant. In certain embodiments, the mutant CFTR is a 120del23 mutant. In certain embodiments, the mutant CFTR is a Phe508del mutant. In certain embodiments a human subject harbors a CFTR mutation in at least one allele of the gene encoding the CFTR protein (e.g., one allele encodes wild-type CFTR and one allele encodes Phe508del CFTR). In certain embodiments, a human subject harbors a CFTR mutation in at least two of the alleles of the gene encoding the CFTR protein. In certain embodiments, a human subject harbors the same mutation in at least two of the alleles of the gene encoding the CFTR protein (e.g., both alleles encode a Phe508del mutant, i.e., homozygous). In certain embodiments, a human subject harbors different mutations in at least two of the alleles of the gene encoding the CFTR protein (e.g., one allele encodes a Phe508del mutant and one allele encodes a Gly551Asp mutant, i.e., heterozygous). In certain embodiments a cell harbors a CFTR mutation in at least one allele of the gene encoding the CFTR protein (e.g., one allele encodes wild-type CFTR and one allele encodes Phe508del CFTR). In certain embodiments, a cell harbors a CFTR mutation in at least two of the alleles of the gene encoding the CFTR protein. In certain embodiments, a cell harbors the same mutation in at least two of the alleles of the gene encoding the CFTR protein (e.g., both alleles encode a Phe508del mutant, i.e., homozygous). In certain embodiments, a cell harbors different mutations in at least two of the alleles of the gene encoding the CFTR protein (e.g., one allele encodes a Phe508del mutant and one allele encodes a Gly551Asp mutant, i.e., heterozygous). In certain embodiments, the cell is an epithelial cell. In certain embodiments, the epithelial cell is a lung epithelial cell. In certain embodiments, the lung epithelial cell is a bronchial epithelial cell. In certain embodiments, the bronchial epithelial cell is derived from a patient with cystic fibrosis.

[00182] The Phe508del mutant is the most common mutant among patients with cystic fibrosis. The Phe508del mutant results from a deletion mutation in which the codon for the phenylalanine at amino acid position 508 is deleted, resulting in a CFTR protein that lacks residue 508 (e.g., is 1479 amino acids in length). Phe508del CFTR has the following standard amino acid sequence (GenBank and NCBI Reference Sequence Accession Number:

(NM_000492.3(CFTR):c.l521_1523delCTT):

MQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWD

RELASKKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLLGRIIASYDPDNKEE

RSIAIYLGIGLCLLFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISI

GQLVS LLS NNLNKFDEGLALAHF VWIAPLQ V ALLMGLrWELLQ AS AFC GLGFLIVLA

LFQAGLGRMMMKYRDQRAGKISERLVITSEMIENIQSVKAYCWEEAMEKMIENLRQ

TELKLTRKA A Y VRYFNS S AFFFS GFFV VFLS VLP Y ALIKGIILRKIFTTIS FCIVLRM A V

TRQFPWAVQTWYDSLGAINKIQDFLQKQEYKTLEYNLTTTEVVMENVTAFWEEGFG ELFEKAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINFKIERGQLLAVAGSTGAG

KTSLLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIGVSYDEYRYRSVIKAC

QLEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVLT

EKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPDFS

SKLMGCDSFDQFSAERRNSILTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKRK

NS ILNPINS IRKFS IVQKTPLQMNGIEEDS DEPLERRLS LVPDS EQGE AILPRIS VIS TGPT

LQARRRQSVLNLMTHSVNQGQNIHRKTTASTRKVSLAPQANLTELDIYSRRLSQETG

LEISEEINEEDLKECFFDDMESIPAVTTWNTYLRYITVHKSLIFVLIWCLVIFLAEVAAS

LV VLWLLGNTPLQDKGNS THS RNNS Y A VIITS TS S Y Y VF YIY VG V ADTLLAMGFFRG

LPLVHTLITVSKILHHKMLHSVLQAPMSTLNTLKAGGILNRFSKDIAILDDLLPLTIFDF

IQLLLIVIGAIAVVAVLQPYIFVATVPVIVAFIMLRAYFLQTSQQLKQLESEGRSPIFTH

LVTS LKGLWTLRAFGRQP YFETLFHKALNLHT ANWFLYLS TLRWFQMRIEMIF VIFFI

AVTFISILTTGEGEGRVGIILTLAMNIMSTLQWAVNSSIDVDSLMRSVSRVFKFIDMPT

EGKPTKSTKPYKNGQLSKVMIIENSHVKKDDIWPSGGQMTVKDLTAKYTEGGNAIL

ENISFSISPGQRVGLLGRTGSGKSTLLSAFLRLLNTEGEIQIDGVSWDSITLQQWRKAF

GVIPQKVFIFSGTFRKNLDPYEQWSDQEIWKVADEVGLRSVIEQFPGKLDFVLVDGG

CVLSHGHKQLMCLARSVLSKAKILLLDEPSAHLDPVTYQIIRRTLKQAFADCTVILCE

HRIEAMLECQQFLVIEENKVRQYDSIQKLLNERSLFRQAISPSDRVKLFPHRNSSKCKS

KPQIAALKEETEEEVQDTRL (SEQ ID NO: 2)

[00183] In certain embodiments, a CFTR protein is a mutant CFTR protein, e.g., the sequence of the protein comprises the sequence of a naturally occurring mutant form of CFTR. In certain embodiments, the mutant CFTR protein is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more, e.g. 100%, identical in sequence to Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the mutant CFTR protein is at least 70% identical in sequence to Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the mutant CFTR protein is at least 80% identical in sequence to Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the mutant CFTR protein is at least 90% identical in sequence to Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the mutant CFTR protein is at least 95% identical in sequence to Phe508del CFTR of SEQ ID NO: 2. In certain

embodiments, the mutant CFTR protein is at least 96% identical in sequence to Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the mutant CFTR protein is at least 97% identical in sequence to Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the mutant CFTR protein is at least 98% identical in sequence to Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the mutant CFTR protein is at least 99% identical in sequence to Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the mutant CFTR protein is at least 99.5% identical in sequence to Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the mutant CFTR protein is at least 100% identical in sequence to Phe508del CFTR of SEQ ID NO: 2.

[00184] In certain embodiments, the CFTR protein cannot be properly processed by traditional cellular machinery in the endoplasmic reticulum (ER). In certain embodiments, the CFTR protein cannot fold properly. In certain embodiments, the CFTR protein cannot be properly trafficked to the plasma membrane (e.g., remains in the cytoplasm or in the endoplasmic reticulum). In certain embodiments, the CFTR protein displays impaired ion channel gating functionality, i.e., cannot open properly for chloride ions to be transported out of the cell.

[00185] In certain embodiments, the CFTR protein is a variant or fragment of Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of the Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 50% of the

Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 60% of the Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 70% of the Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 80% of the Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 90% of the Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 95% of the Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 96% of the Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 97% of the Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 98% of the Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 99% of the Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 99.5% of the Phe508del CFTR of SEQ ID NO: 2. In certain embodiments, the CFTR variant comprises or consists of a polypeptide that is over at least 100% of the Phe508del CFTR of SEQ ID NO: 2. These variants may be biologically active variants of Phe508del CFTR, such that the ion channel gating function and/or the chloride transport function of the CFTR is retained in the variant. These variants may be biologically inactive variants of Phe508del CFTR, such that the ion channel gating function and/or the chloride transport function of the CFTR is abrogated (e.g., not functional) in the variant.

[00186] In some embodiments a mammalian nucleic acid sequence, e.g., a human nucleic acid sequence, e.g., a human DNA sequence encoding a CFTR protein (e.g., wild-type CFTR, Phe508del CFTR) may be codon optimized for increased expression in a cell. In certain embodiments, a sequence encoding a CFTR protein may be codon optimized for increased expression in an epithelial cell. In certain embodiments, the epithelial cell is a small intestine epithelial cell. In certain embodiments, the epithelial cell is a lung epithelial cell. In certain embodiments, the lung epithelial cell is a bronchial epithelial cell. The bronchial epithelial cell may be derived from a subject with cystic fibrosis.

[00187] In some embodiments, a CFTR protein provided in a purified form. In some embodiments, a CFTR protein is provided in the form of a cell lysate. In some embodiments, a CFTR protein is provided in the form of a tissue homogenate.

EXAMPLES

[00188] In order that the invention described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the methods, compositions, and systems provided herein and are not to be construed in any way as limiting their scope.

General Experimental

[00189] Ussing chamber equipment for cell culture: EM-CSYS-8 Ussing Chamber System, P2300 Chambers, P2302 Sliders, VCC MC8 Multichannel Voltage/ current Clamp, P2020 Electrodes, and DM MC6 Single Channel Electrode Input Module and Dummy Membranes (Physiologic Instruments, San Diego, CA; Figurel-2).

[00190] Electrodes: Silver/silver chloride (Ag/AgCl) electrodes placed in 4% agar-Ringer buffer-containing electrode tips

[00191] Ringer solution: 115 mM NaCl, 25 mM NaHC03-, 2.4 mM K2HP04, 0.4 mM KH2P04, 1.2 mM MgC12, 1.2 mM CaC12, and 20 mM HEPES. NaOH to adjust pH to 7.4. Osmolarity 290-300 mOsm. Example 1. Effects of selected amino acids on CFTR translocation from the cytoplasm to the plasma membrane of a cell

[00192] To study the effect of amino acids on CFTR translocation from the cytoplasm to the plasma membrane, studies were first done using isolated small intestinal loops that were filled with ringer solution containing each of the free amino acids or ringer solution containing a combination of free amino acids. Ringer solution containing each of the individual amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) were used in the isolated intestinal loops and incubated for a period of 45 minutes. The tissues were then isolated, the volume of fluid in the intestinal loops was measured and the tissues were homogenized for protein

characterization using western blot analysis. Western blot analysis showed that certain amino acids increased the protein levels of CFTR in the brush border membrane vesicles isolated from small intestinal epithelial cells. When these amino acids were used in combination in isolated intestinal loops, there was a significant increase in CFTR protein on brush border membrane vesicles isolated from the small intestinal villus epithelial cells.

Example 2. Effect of amino acids on paracellular permeability and inflammation

[00193] Mice were radiated at various radiation dose and then treated with the amino acids for a period of 6 days. Mice were sacrificed at the end of 6 days and tissues collected for analysis. Electron microscopy of ileal sections showed enterade® tightened the mucosal barrier and decreased paracellular permeability (Figures 1A to IE). Following radiation exposure, the mice were treated with the amino acids formulation and plasma samples collected at the end of 6 days for 16sr RNA analysis. AA-ORS (Enterade) decreased bacterial translocation across intestinal mucosa as shown by Figure 2. Finally, Enterade decreased non- ionic particle translocation across intestinal muscose (Figure 3).

Example 3. Effect of amino acids on cell proliferation

[00194] Mice were radiated at various radiation dose and then treated with the amino acids for a period of 6 days. Mice were sacrificed at the end of 6 days and tissues collected for analysis. Amino acids increased crypt count & villous length following irradiation (Figures 4A to 4B)

Example 4: Effect of amino acids on electrolyte absorption [00195] Mice were radiated at various radiation dose and then treated with the amino acids for a period of 6 days. Mice were sacrificed at the end of 6 days and tissues collected for analysis. Paraffin embedded tissues at 5-μιη thickness were used. Cell nuclei were stained with DAPI (blue). A minimum of five well-oriented villi were used. Immunoblots were repeated four time. Values are means + SEM from n = 4, * indicates statistically significant difference (p<0.05) from saline treated animals.

[00196] Experiments were carried out to study the effect of glucose-stimulated Na⁺ absorption (Figures 5A, 5C, and 5D). The experiments showed that amino acids increased electrolyte absorption and its transporters. Experiments were carried out to study the effect of glucose-stimulated CI^" absorption (Figures 5B and 6) and showed amino acids decreased net chloride absorption. Finally, the effect of amino acids on Anol chloride secreation was studied (Figures 7A to 7B) and shows that amino acids decreased chloride secretion. Anol protein expression increased on the brush border membrane with increasing radiation dose and correlated with increased chloride secretion associated with radiation. Treatment using the amino acid formulation decreased the anol expression in the brush border membrane thus suggesting increased translocation into the cytoplasm. Anol expression was high in the cytoplasm with amino acid treatment.

Example 5: Comparing normal and cystic fibrous human bronchial epithelial cells via Ussing chamber

[00197] Experiments were done on primary bronchial epithelial cells grown in culture. The cells were optained from CF and non-CF patients. Cells were grown to 80% confluence in culture dishes and the they were transferred to snapwell permeable inserts. Cells were grown to mature and differentiated in permeable cell culture supports for -30 days in air-media interface. Thereafter the cells were studied in Ussing chamber for measuring the

transepithelial current and resistance.

[00198] P2300 chambers were mounted in the EM-CSYS-8 Ussing chamber system. P2302 sliders were inserted with snapwell filter dummies, and Ag/AgCl electrodes were placed into the chambers. Ag/AgCl electrodes were connected with the VCC MC8 multichannel voltage/current clamp for measuring transepithelial voltage, and passing current. The Ussing chamber system was pre-heated to 37°C with a connected circulating water bath, and 5mL Ringer solution was added to both reservoirs of each chamber. 95% 0₂ and 5% CO supply were connected to the chambers, and allowed for adequate oxygenation and mixed the solution within the reservoirs continuously. The electrodes were calibrated to 0 mV at clamped voltage mode. The dummies were replaced by snap well filters with cell cultures, sliders were mounted in Ussing Chambers, and finally, 5mL of pre-warmed ringer solution was added to each chamber reservoir. Cell cultures were maintained in Ringer solution at 37 °C, and continuously oxygenized with 95% 0₂ and 5% CO2. The cells were equilibrated for 10-15 min. TEER and Isc were recorded after 30min, 60min, and 90min.

[00199] Current recorded in after the cell culture inserts were mounted in Ussing chambers. The current was measured in the presence of each of the amino acids and various formulations (Figures 8A-8F). Amiloride was added to block the sodium channel (ENaC). Amiloride-insensitive current is the chloride current at the basal state that is devoid of any sodium channel activity. Some amino acids increased the basal chloride current suggesting increased chloride channel trafficking to the membrane (Figure 8A). The cells having basal chloride recordings were stimulated by increasing the intracellular cAMP using forskolin. Some amino acids showed increased Forskolin-stimulated current while others showed decreased forskolin-stimulated current suggesting increased CFTR translocation and function when showing increased current (Figures 8B and 8C). The current in relation to CFTR for various amino acid formulations and basic buffer was studied (Figure 8D to 8F). Figure 8E shows the amount of current inhibited by specific CFTR inhibitor. Figure 8F shows that CFTR inhibitor-insensitive current represents chloride secretion by other chloride channels like anol.

Example 6: Effect of amino acid compositions on CFTR

Figures 8A to 8H show how CFTR are trafficked onto the apical membrane of bronchial epithelial cells.

[00200] Example 7: Effect of glucose and NSP4 on CRL-4011 and 4013 cells

[00201] Fluorescence was used to study the effect of glucose and NSP4 on various cells, including CRL-4011, 4013, and CFBE (Figures 10A to 11). It was found that both glucose & NSP4 increased intracellular Ca²⁺ for all three cell types.

EQUIVALENTS AND SCOPE

[00202] In the claims articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[00203] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms "comprising" and "containing" are intended to be open and permits the inclusion of additional elements or steps.

[00204] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[00205] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims. Where the claims or description relate to a product (e.g., a composition of matter), it should be understood that methods of making or using the product according to any of the methods disclosed herein, and methods of using the product for any one or more of the purposes disclosed herein, are encompassed by the present disclosure, where applicable, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where the claims or description relate to a method, it should be understood that product(s), e.g., compositions of matter, device(s), or system(s), useful for performing one or more steps of the method are encompassed by the present disclosure, where applicable, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[00206] Where ranges are given herein, embodiments are provided in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also understood that where a series of numerical values is stated herein, embodiments that relate analogously to any intervening value or range defined by any two values in the series are provided, and that the lowest value may be taken as a minimum and the greatest value may be taken as a maximum. Where a phrase such as "at least", "up to", "no more than", or similar phrases, precedes a series of numbers herein, it is to be understood that the phrase applies to each number in the list in various embodiments (it being understood that, depending on the context, 100% of a value, e.g., a value expressed as a percentage, may be an upper limit), unless the context clearly dictates otherwise. For example, "at least 1, 2, or 3" should be understood to mean "at least 1, at least 2, or at least 3" in various embodiments. It will also be understood that any and all reasonable lower limits and upper limits are expressly

contemplated where applicable. A reasonable lower or upper limit may be selected or determined by one of ordinary skill in the art based, e.g., on factors such as convenience, cost, time, effort, availability (e.g., of samples, agents, or reagents), statistical considerations, etc. In some embodiments an upper or lower limit differs by a factor of 2, 3, 5, or 10, from a particular value. Numerical values, as used herein, include values expressed as percentages. For each embodiment in which a numerical value is prefaced by "about" or "approximately", embodiments in which the exact value is recited are provided. For each embodiment in which a numerical value is not prefaced by "about" or "approximately", embodiments in which the value is prefaced by "about" or "approximately" are provided. "Approximately" or "about" generally includes numbers that fall within a range of 1% or in some embodiments within a range of 5% of a number or in some embodiments within a range of 10% of a number in either direction (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would impermissibly exceed 100% of a possible value). It should be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but the invention includes embodiments in which the order is so limited. In some embodiments a method may be performed by an individual or entity. In some embodiments steps of a method may be performed by two or more individuals or entities such that a method is collectively performed. In some embodiments a method may be performed at least in part by requesting or authorizing another individual or entity to perform one, more than one, or all steps of a method. In some embodiments a method comprises requesting two or more entities or individuals to each perform at least one step of a method. In some embodiments performance of two or more steps is coordinated so that a method is collectively performed. Individuals or entities performing different step(s) may or may not interact.

[00207] Section headings used herein are not to be construed as limiting in any way. It is expressly contemplated that subject matter presented under any section heading may be applicable to any aspect or embodiment described herein.

[00208] Embodiments or aspects herein may be directed to any agent, composition, article, kit, and/or method described herein. It is contemplated that any one or more embodiments or aspects can be freely combined with any one or more other embodiments or aspects whenever appropriate. For example, any combination of two or more agents, compositions, articles, kits, and/or methods that are not mutually inconsistent, is provided. It will be understood that any description or exemplification of a term anywhere herein may be applied wherever such term appears herein (e.g., in any aspect or embodiment in which such term is relevant) unless indicated or clearly evident otherwise.

REFERENCES

1. O'Sullivan BP and Freedman SD (2009) Cystic Fibrosis. Lancet, 373; pp. 1891-904.

2. Brodlie M, Haq IJ, Roberts K, Elborn JS (2015) Targeted therapies to improve CFTR function in cystic fibrosis. Genome Medicine, 7; doi: 10.1186/sl3073-015-0223-6. 3. Corvol H, Thompson KE, Tabary O, et al. (2015) Translating the genetics of cystic fibrosis to personalized medicine. Transl Res; doi: 10.1016/j.trsl.2015.04.008.

4. McPhail GL and Clancy JP (2013) Ivacaftor: the first therapy acting on the primary cause of cystic fibrosis. Drugs Today, 49; pp. 253-260.

5. Ramsey BW, Davies J, McElvaney NG, et al. (2011) A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. N Engl J Med, 365; pp. 1633-1672.

Claims

CLAIMS What is claimed is:

1. A method for treating cystic fibrosis, the method comprising administering to a subject in need thereof an effective amount of a composition comprising, consisting essentially of, or consisting of one or more amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and/or threonine.

2. The method of claim 1, wherein the composition comprises, consists essentially of, or consists of two or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine.

3. The method of claim 1, wherein the composition comprises, consists essentially of, or consists of three or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine.

4. The method of claim 1, wherein the composition comprises, consists essentially of, or consists of four or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine.

5. The method of claim 1, wherein the composition comprises, consists essentially of, or consists of five or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine.

6. A method for treating cystic fibrosis, the method comprising administering to a subject in need thereof an effective amount of a composition comprising, consisting essentially of, or consisting of asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine.

7. The method of any one of claims 1-6, wherein the composition further comprises the free amino acids histidine and/or cysteine.

8. A method for treating cystic fibrosis, the method comprising administering to a subject in need thereof an effective amount of a composition comprising, consisting essentially of, or consisting of asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine.

9. The method of any one of claims 1-8, wherein the composition further comprises one or more free amino acids selected from arginine, glycine, lysine, serine, tyrosine, and/or valine.

10. A method for treating cystic fibrosis, the method comprising administering to a subject in need thereof an effective amount of a composition comprising, consisting essentially of, or consisting of arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine.

11. The method of any one of claims 1-10, wherein the composition further comprises the free amino acids cysteine and/or histidine.

12. A method for treating cystic fibrosis, the method comprising administering to a subject in need thereof an effective amount of a composition comprising, consisting essentially of, or consisting of arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine.

13. The method of any one of claims 1-12, wherein the composition further comprises one or more free amino acids selected from proline, phenylalanine, tryptophan, methionine, leucine, and/or alanine.

14. The method of any one of claims 1-13, wherein the amino acids are L- amino acids.

15. The method of any one of claims 1-14, wherein the composition further comprises water.

16. The method of any one of claims 1-15, wherein the composition further comprises a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, or excipient.

17. The method of any one of claims 1-16, wherein the composition is sterile.

18. The method of any one of claims 1-17, wherein the composition is formulated for administration by an enteral, pulmonary, inhalation, intranasal, or sublingual route.

19. The method of any one of claims 1-18, wherein the subject is suffering from cystic fibrosis in which wild-type CFTR is present.

20. The method of any one of claims 1-19, wherein the subject has a mutation in the CFTR gene.

21. The method of any one of claims 1-19, wherein the subject is suffering from cystic fibrosis in which both wild-type and mutant CFTR are present.

22. The method of any one of claims 1-21, wherein the subject is suffering from cystic fibrosis in which a CFTR protein that is at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of wild-type CFTR as provided by SEQ ID NO: 1 is present.

23. The method of any one of claims 19-22, wherein the CFTR is a Gly542X,

Gly551Asp, Arg553X, Argl l7His, 120del23, or Phe508del CFTR mutant, wherein X is any amino acid.

24. The method of claim 23, wherein the CFTR is a Phe508del CFTR mutant.

25. A method for increasing the number of cystic fibrosis transmembrane conductance regulator (CFTR) proteins present on the plasma membrane of a cell, the method comprising contacting the cell with an effective amount of a composition comprising, consisting essentially of, or consisting of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine.

26. The method of claim 25, wherein the composition comprises, consists essentially of, or consists of asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine.

27. The method of claim 25, wherein the composition comprises, consists essentially of, or consists of asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine.

28. The method of claim 25, wherein the composition comprises, consists essentially of, or consists of arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine.

29. The method of claim 25, wherein the composition comprises, consists essentially of, or consists of arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine.

30. The method of any one of claims 25-29, wherein the effective amount increases the number of CFTR proteins present on the plasma membrane of the cell.

31. The method of any one of claims 25-30, wherein the number of wild-type CFTR proteins on the plasma membrane increases.

32. The method of any one of claims 25-30, where in the number of mutant CFTR proteins on the plasma membrane increases.

33. The method of any one of claims 25-32, wherein number of CFTR proteins that are at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of wild-type CFTR as provided by SEQ ID NO: 1 on the plasma membrane increases.

34. The method of any one of claims 32-33, wherein one or more of the CFTR proteins are a Gly542X, Gly551Asp, Arg553X, Argl l7His, 120del23, or Phe508del mutant, wherein X is any amino acid.

35. The method of claim 34, wherein one or more of the CFTR proteins are a Phe508del CFTR mutant.

36. The method of any one of claims 25-35, wherein chloride ion export from the cell is increased.

37. The method of any one of claims 25-36, wherein water export from the cell is increased.

38. The method of any one of claims 25-37, wherein the cell is an epithelial cell.

39. The method of claim 38, wherein the epithelial cell is a lung epithelial cell.

40. The method of claim 39, wherein the lung epithelial cell is a bronchial epithelial cell.

41. The method of claim 40, wherein the bronchial epithelial cell was obtained from a subject suffering from cystic fibrosis.

42. The method of any one of claims 1-41 further comprising administering an additional therapeutic agent.

43. The method of claim 42, wherein the additional therapeutic agent is a small molecule drug, protein drug, or nucleic acid drug.

44. The method of claim 42 or 43, wherein the additional therapeutic agent is a CFTR potentiator, corrector, or read-through agent.

45. The method of claim 44, wherein the additional therapeutic agent is a CFTR potentiator.

46. The method of claim 45, wherein the CFTR potentiator is ivacaftor.

47. The method of any one of claims 1-46, wherein the composition comprises, consists essentially of, or consists of one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine, and no other free amino acids.

48. The method of any one of claims 1-46, wherein the composition comprises, consists essentially of, or consists of one or more free amino acids selected from, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, and threonine, and no other free amino acids.

49. The method of any one of claims 1-46, wherein the composition comprises, consists essentially of, or consists of one or more free amino acids selected from, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, lysine, serine, threonine, tyrosine, and valine, and no other free amino acids.

50. The method of any one of claims 1-46, wherein the composition comprises, consists essentially of, or consists of one or more free amino acids selected from, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, tyrosine, and valine, and no other free amino acids.

51. The method of any one of claims 1-46, wherein the composition comprises, consists essentially of, or consists of one or more free amino acids selected from, aspartic acid, threonine, tyrosine, serine, and valine, and no other free amino acids.

52. The method of any one of claims 1-46, wherein the composition comprises, consists essentially of, or consists of one or more free amino acids selected from, isoleucine, glutamine, glutamic acid, alanine, and aspartic acid, and no other free amino acids.

53. The method of any one of claims 1-46, wherein the composition comprises, consists essentially of, or consists of one or more free amino acids selected from, lysine, proline, glutamic acid, alanine, and aspartic acid, and no other free amino acids.

54. The method of any one of claims 1-46, wherein the composition comprises, consists essentially of, or consists of one or more free amino acids selected from, arginine, aspartic acid, proline, tyosine, glutamine, and isoleucine, and no other free amino acids.

55. The method of any one of claims 1-46, wherein the composition comprises, consists essentially of, or consists of one or more free amino acids selected from, isoleucine, aspartic acid, threonine, lysine, tyrosine, serine, valine, glycine, and tryptophan, and no other free amino acids.

56. The method of any one of claims 1-46, wherein the composition comprises, consists essentially of, or consists of one or more free amino acids selected from, arginine, cysteine, glycine, isoleucine, lysine, serine, threonine, tyrosine, valine, proline and leucine, and no other free amino acids.

57. Use of a composition to treat cystic fibrosis in a subject in need thereof, wherein the composition comprises one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine.

58. A kit comprising:

a composition comprising, consisting essentially of, or consisting of, one or more free amino acids selected from asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, and threonine; and

instructions for administering to a subject or contacting a biological sample with the composition.