WO2020124152A1

WO2020124152A1 - Assay for screening drugs

Info

Publication number: WO2020124152A1
Application number: PCT/AU2019/051405
Authority: WO
Inventors: John Wilson; Tom KOTSIMBOS; Dominic KEATING; David THORBURN; Ann FRAZIER
Original assignee: Monash University; Murdoch Childrens Research Institute
Priority date: 2018-12-19
Filing date: 2019-12-19
Publication date: 2020-06-25

Abstract

The present invention relates to assays, methods and kits for determining the efficacy of one or more pharmaceutical agents for improving mitochondrial function in an individual.

Description

Assay for screening drugs

Field of the invention

The present invention relates to assays and methods for determining the efficacy of one or more pharmaceutical agents for improving mitochondrial function in an individual.

Related application

This application claims priority from Australian provisional application AU 2018904831 , the contents of which are hereby incorporated by reference in their entirety.

Background of the invention

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in a nucleotide-gated, small conductance epithelial chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR) (Riordan et al (1989) 245(4922): 1066- 1073).

CFTR is a large, multi-domain glycoprotein consisting of two membrane- spanning domains, two nucleotide-binding domains that bind and hydrolyse ATP and a regulatory domain that gates the channel by phosphorylation. CFTR modulates chloride transport at the cell membrane, and CFTR expression is ubiquitous, being present in every nucleated cell.

While there are more than 1800 reported mutations in the CFTR gene, many of which do not currently have a reported phenotype, nearly 300 of these mutations are associated with CF. The most common mutation, AF508 (also referred to as p.F508del), results from a deletion (D) of three nucleotides which results in a loss of the amino acid phenylalanine (F) at the 508th amino acid residue of the protein. As a result, the protein does not fold normally and is more quickly degraded. Other mutations primarily alter channel gating (e.g., G551 D), conductance (e.g., R117H) or translation (e.g., G542X).

CF-causing mutations in the CFTR gene are those that prevent the channel from functioning properly, leading to a blockage of the movement of salt and water into and out of cells. As a result of this blockage, cells that line the passageways of the lungs, pancreas, and other organs produce abnormally thick, sticky mucus. This mucus obstructs the airways and glands, causing the characteristic signs and symptoms of CF. In addition, only thin mucus can be removed by cilia; thick mucus cannot, so it traps bacteria that give rise to chronic infections.

Complications of CF include thickened mucus in the lungs with frequent respiratory infections, and pancreatic insufficiency giving rise to malnutrition and diabetes. These conditions lead to chronic disability and reduced life expectancy.

Current treatments for CF focus on restoring CFTR function at the chloride channel. For example, pharmacological agents are aimed at reversing the downstream manifestations of CFTR mutations, such as salt/water imbalance, protein dysfunction and epithelial cellular abnormalities. Flowever, unexplained performance limitations also exist in many non-epithelial cells, including the contractile function of cardiac, skeletal and gastrointestinal musculature, immune cell function and bone metabolism.

The traditional approach to determining efficacy of a proposed CF-drug in a patient population is to conduct lengthy clinical trials. There is therefore a need for new approaches for identifying appropriate candidate drugs for treating CF.

In addition, because of the large number of different mutations in CFTR that can contribute to CF phenotypes, not all drugs will be useful for treating all CF-sufferers. Flowever, it can be costly and inconvenient for patients to be subjected to a range of different pharmacological agents prior to obtaining a clinical benefit. As such, there is also a need to develop a means for identifying“personalised” treatments for CF patients and new methods for predicting the efficacy of CF drugs in individual patients.

Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art. Summary of the invention

The present invention provides a method for evaluating the efficacy of an agent for treating Cystic Fibrosis (CF) in an individual, the method comprising

- providing a biological sample from an individual diagnosed with or suspected of having CF;

- contacting the biological sample with a candidate agent for treating CF in the individual, in conditions enabling the agent to modulate mitochondrial function in the sample;

- determining the mitochondrial function of the cells in the biological sample;

- comparing the mitochondrial function of the cells to the mitochondrial function of a control sample that has not been contacted with the agent; wherein an increase in mitochondrial function in the biological sample upon contact with an agent indicates that the agent is suitable for use in treating CF in the individual.

The present invention also provides a method for evaluating the efficacy of an agent for treating CF in an individual, the method comprising

- providing cells from an individual diagnosed with or suspected of having CF;

- contacting the cells with a candidate agent for treating CF in the individual in conditions enabling the agent to modulate mitochondrial function in the cells;

- determining the mitochondrial function of the cells;

- comparing the mitochondrial function of the cells to control cells that have not been contacted with the agent; wherein an increase in mitochondrial function in the cells upon contact with an agent indicates that the agent is suitable for use in treating CF in the individual.

The present invention also provides an in vitro method for evaluating the efficacy of an agent for treating CF in an individual, the method comprising

- contacting cells from an individual diagnosed with or suspected of having CF with a candidate agent for treating CF in the individual in conditions enabling the agent to modulate mitochondrial function in the cells; - determining the mitochondrial function of the cells;

- contacting a biological sample from an individual diagnosed with or suspected of having CF with an agent that is a candidate for treating CF in the individual, in conditions enabling the agent to modulate mitochondrial function in the sample;

- determining the mitochondrial function of the cells in the biological sample;

- comparing the mitochondrial function of the cells to a control sample that has not been contacted with the agent; wherein an increase in mitochondrial function in the biological sample upon contact with an agent indicates that the agent is suitable for use in treating CF in the individual.

The present invention also provides a method for evaluating the efficacy of an agent for improving CFTR function in an individual, the method comprising

- contacting a biological sample from an individual diagnosed or suspected of having impaired CFTR function, with an agent that is a candidate for improving CFTR function, in conditions enabling the agent to modulate mitochondrial function in the sample;

- determining the mitochondrial function of the cells in the biological sample;

- comparing the mitochondrial function of the cells to a control sample that has not been contacted with the agent; wherein an increase in mitochondrial function in the biological sample upon contact with an agent indicates that the agent is suitable for use in improving CFTR function in the individual. The individual may be diagnosed or suspected of having one or more mutations resulting in impaired CFTR function. The individual may or may not display overt symptoms of CF. The individual may be male or female, preferably male.

Still further, the present invention provides an in vitro method for evaluating the efficacy of an agent for treating CF in an individual, the method comprising

- contacting mitochondria isolated from an individual diagnosed with or suspected of having CF with an agent that is a candidate for treating CF in the individual, in conditions enabling the agent to modulate mitochondrial function;

- determining the mitochondrial function of the mitochondria;

- comparing the mitochondrial function of the mitochondria contacted with the agent, to a control sample of mitochondria that has not been contacted with the agent; wherein an increase in mitochondrial function upon contact with an agent indicates that the agent is suitable for use in treating CF in the individual.

The present invention also provides a method for evaluating the efficacy of an agent for improving exercise capacity in an individual, the method comprising

- contacting a biological sample or isolated cells from an individual diagnosed with or suspected of having CF with an agent that is a candidate for treating CF in the individual, in conditions enabling the agent to modulate mitochondrial function in the sample or cells;

- determining the mitochondrial function of the cells in the biological sample or in the isolated cells;

- comparing the mitochondrial function of the cells to a control sample that has not been contacted with the agent; wherein an increase in mitochondrial function in the cells upon contact with an agent indicates that the agent is suitable for use in improving the exercise capacity of the individual.

The present invention also provides a method for evaluating the efficacy of an agent for improving exercise capacity in an individual, the method comprising - contacting a biological sample or isolated cells from an individual diagnosed with or suspected of having one or more mutations in the gene encoding CFTR, with an agent that is a candidate for treating CF, in conditions enabling the agent to modulate mitochondrial function in the sample or cells;

- comparing the mitochondrial function of the cells to a control sample or control isolated cells that have not been contacted with the agent; wherein an increase in mitochondrial function in the cells upon contact with an agent indicates that the agent is suitable for use in improving the exercise capacity of the individual.

Further, the present invention provides an in vitro method for evaluating the efficacy of an agent for improving exercise capacity in an individual, the method comprising

- contacting mitochondria isolated from an individual diagnosed with or suspected of having one or more mutations in the gene encoding CFTR, with an agent that is a candidate for treating CF, in conditions enabling the agent to modulate mitochondrial function;

- determining the mitochondrial function of the mitochondria following contact with the agent;

- comparing the mitochondrial function of the mitochondria contacted with the agent, to a control sample of mitochondria that has not been contacted with the agent; wherein an increase in mitochondrial function upon contact with an agent indicates that the agent is suitable for use in improving the exercise capacity of the individual.

Further, the present invention provides an in vitro method for evaluating the efficacy of an agent to improve exercise capacity in an individual, the method comprising - contacting mitochondria isolated from an individual diagnosed with or suspected of having CF with an agent that is a candidate for treating CF in the individual, in conditions enabling the agent to modulate mitochondrial function;

In certain embodiments of the above methods, the individual has symptoms of CF. Alternatively, the individual may have no symptoms of CF.

In any embodiment, the individual is male or female, preferably male.

In certain embodiments, the individual has a mutation in the gene encoding CFTR that does not affect chloride ion transport in the individual.

The present invention also provides an in vitro method for evaluating the efficacy of an agent to improve exercise capacity or exercise tolerance in an individual, the method comprising

- providing a biological sample, isolated cells or isolated mitochondria from an individual diagnosed with or suspected of having one or more mutations in the gene encoding CFTR;

- contacting the sample, cells or mitochondria with an agent that is a candidate for improving the function of CFTR, in conditions enabling the agent to modulate mitochondrial function;

- determining the mitochondrial function of the cells in the biological sample, or of the isolated cells or isolated mitochondria, following contact with the agent; - comparing the mitochondrial function of the cells or mitochondria to the function of control cells or mitochondria that have not been contacted with the agent; wherein an increase in the mitochondrial function upon contact with an agent, indicates that the agent is suitable for improving the exercise capacity or tolerance in the individual.

In certain embodiments, the individual has symptoms of cystic fibrosis. Alternatively, the individual may have no symptoms of CF.

The individual may have a mutation in CFTR that is known to be associated with CF, whether or not the individual exhibits symptoms of CF. Further, the individual may have a plurality of mutations in CFTR. The individual may be homozygous or heterozygous for any one or more CFTR mutations.

In certain embodiments, the agent is a pharmacological agent that is known or suspected to be useful for treating CF.

The present invention also provides a method for improving the treatment of CF in an individual, the method comprising

- providing a plurality of biological samples from an individual diagnosed with or suspected of having CF;

- contacting the samples with a plurality of agents that are candidates for treating the CF in the individual, in conditions enabling the agents to modulate mitochondrial function in the samples;

- determining the mitochondrial function of the cells in the biological samples;

- comparing the mitochondrial function of the cells in each of the biological samples; and

- selecting the agent that provides the greatest increase in mitochondrial function in the biological sample, for use in treating CF in the individual. The present invention also provides a method for determining the likelihood that an individual will benefit from a treatment for CF, the method comprising

- contacting the samples with a plurality of agents that are candidates for treating the CF in the individual, in conditions enabling the agent to modulate mitochondrial function;

- comparing the mitochondrial function of the cells in each of the biological samples; determining that the agent that provides the greatest increase in mitochondrial function in the biological sample has a greater likelihood of being useful for treating CF in the individual.

Preferably, each biological sample is contacted with a different agent.

It will be appreciated that any candidate agent that is known to or suspected of modulating CFTR function may be tested in accordance with the methods of the present invention.

The present invention also provides a method for improving the exercise capacity or exercise tolerance of an individual, the method comprising

- providing a biological sample from an individual diagnosed with or suspected of having one or more mutations in the gene encoding CFTR;

- contacting the biological sample with an agent that is a candidate for improving the function of CFTR, in conditions enabling the agent to modulate mitochondrial function in the sample;

- determining the mitochondrial function of the cells in the biological sample;

- comparing the mitochondrial function of the cells to a control sample that has not been contacted with the agent; determining to treat the individual with the agent if there is an increase in mitochondrial function upon contact of the biological sample with the agent, thereby improving the exercise capacity or tolerance of the individual.

- providing cells from an individual diagnosed with or suspected of having one or more mutations in the gene encoding CFTR;

- contacting the cells with an agent that is a candidate for improving the function of CFTR, in conditions enabling the agent to modulate mitochondrial function in the cells;

- determining the mitochondrial function of the cells following contact with the agent;

- comparing the mitochondrial function of the cells to a control sample that has not been contacted with the agent; determining to treat the individual with the agent if there is an increase in mitochondrial function upon contact of the cells with the agent, thereby improving the exercise capacity or tolerance of the individual.

In any method for improving the exercise capacity or exercise tolerance of an individual as described herein, the method further comprises the step of administering to the individual the agent determined to increase mitochondrial function.

In certain embodiments, the individual has a mutation in the gene encoding CFTR that does not affect chloride ion transport in the individual. In certain embodiments, the agent is a pharmacological agent that is known or suspected to be useful for treating CF.

The present invention provides a screening method for identifying an agent useful for treating CF in an individual, the method comprising:

- providing biological samples from a plurality of individuals having CF;

- contacting the biological samples with one or more agents that are candidates for treating CF, in conditions enabling the one or more agents to modulate mitochondrial function in the samples;

- determining the mitochondrial function of cells in the biological samples;

- comparing the mitochondrial function of the cells to a control sample that has not been contacted with the one or more agents; wherein an increase in mitochondrial function of the biological samples upon contact with an agent indicates that the agent is suitable for use in treating CF in an individual.

The present invention also provides a screening method for identifying an agent useful for improving the exercise capacity in an individual with CF, the method comprising:

- providing biological samples from a plurality of individuals having CF;

- determining the mitochondrial function of cells in the biological samples;

- comparing the mitochondrial function of the cells to a control sample that has not been contacted with the one or more agents; wherein an increase in mitochondrial function in the biological samples upon contact with an agent indicates that the agent is suitable for use in improving exercise capacity in an individual with CF.

The present invention provides a screening method for identifying an agent useful for treating CF in an individual, the method comprising: - providing cells having one or more mutations in the gene encoding CFTR;

- contacting the cell with one or more agents that are candidates for treating CF, in conditions enabling the one or more agents to modulate mitochondrial function in the cells;

- determining the mitochondrial function of cells;

- comparing the mitochondrial function of the cells to a control sample of the cells that has not been contacted with the one or more agents; wherein an increase in mitochondrial function of the cells upon contact with an agent indicates that the agent is suitable for use in treating CF in an individual.

The cells for use in the screening methods described herein may be cell lines in which one or more mutations have been introduced into the gene encoding CFTR. In certain embodiments, the cells may express a form of CFTR that has reduced or impaired function. The degree of CFTR functional impairment may be a 5% reduction in function, 10%, 20%, 40%, 50%, 75%, 80%, 90%, or 95% reduction in CFTR function. In certain embodiments, the cells may contain a mutation that results in a partial or complete loss-of-function (e.g., knock-out) of the CFTR protein.

In one example, the cells are CFBE14o-cells (airway cells homozygous for the AF508-CFTR allele). Alternatively, the cells may be 16FIBE14o-cells expressing one or more mutations in the gene encoding CFTR.

- providing cells from a plurality of individuals having CF;

- contacting the cells with one or more agents that are candidates for treating CF, in conditions enabling the one or more agents to modulate mitochondrial function in the cells;

- determining the mitochondrial function of cells;

- comparing the mitochondrial function of the cells to a control sample of the cells that has not been contacted with the one or more agents; wherein an increase in mitochondrial function in the cells upon contact with an agent indicates that the agent is suitable for use in improving exercise capacity in an individual with CF.

In any embodiment of the invention, the increase in mitochondrial function (e.g., oxygen consumption or glycolysis) that is indicative that the agent is suitable for any use described herein (including use in treating CF or for use in improving CFTR function, or for use in improving exercise capacity in an individual), is any statistically significant increase in mitochondrial function, as measured by any parameter described herein. Preferably the increase in mitochondrial function may be an increase of at least 5%, at least 10%, at least 20%, at least 50% or more in mitochondrial function, compared to controls.

In any embodiment, the increase in exercise capacity of an individual may be a statistically significant increase in exercise capacity, as measured by any parameter described herein. Preferably, the increase in exercise tolerance and exercise capacity is an increase of at least 5%, at least 10%, at least 20%, at least 50% or more, compared to controls.

In a preferred embodiment, the cells may be from a cell line that is engineered to express one or more mutations in the gene encoding CFTR. In one example, the cells are CFBE14o-cells (airway cells homozygous for the AF508-CFTR allele). Alternatively, the cells may be 16FIBE14o-cells expressing one or more mutations in the gene encoding CFTR.

In certain embodiments of the invention, the one or more mutations in the gene encoding CFTR are mutations that do not affect the ability of CFTR to transport chloride ions.

In a preferred embodiment of the invention, the cells are grown in tissue culture for the purposes of conducting the screening method.

In any embodiment,“modulating mitochondrial function” refers to the ability of an agent to increase or decrease mitochondrial function. The present invention is aimed at identifying agents which increase mitochondrial function. In any method of the invention described herein, mitochondrial function in a biological sample, in an organoid, in isolated cells or in isolated mitochondria is determined by measuring any one or more of: ADP-ATP exchange, oxygen consumption rate (OCR), change in membrane potential (Dy), Mitochondrial complex I- V amount and/or activity, reactive oxygen species production, membrane lipid peroxidation, glucose utilisation, or extracellular acidification rate (ECAR).

Preferably, the increase in mitochondrial function is an increase in Oxygen Consumption Rate (OCR), preferably wherein the OCR is determined according to the maximum respiratory capacity of the cell. As used herein, the terms “Oxygen Consumption Rate” and“Oxygen Consumption Index” may be used interchangeably. OCR is typically reported in units of pmol 02/minute.

In any embodiment, the change or increase in OCR is preferably an increase of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%. Preferably the increase in OCR is at least about 10%.

In any embodiment, the change or increase in OCR is preferably an increase of at least 1.1 -fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5 fold, at least 1.6-fold, at least 1.7-fold, or at least 2-fold, at least a 5-fold, at least a 10-fold or greater.

Further, the increase in mitochondrial function may be an increase in glucose utilisation (glycolytic activity). In any embodiment, the change or increase in glycolytic activity may be an increase of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%. Preferably the increase in in glycolytic activity is at least about 10%.

In any embodiment, the change or increase in in glycolytic activity may be an increase of at least 1.1 -fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5 fold, at least 1.6-fold, at least 1.7-fold, or at least 2-fold, at least a 5-fold or at least a 10-fold or greater.

In any embodiment of the invention described above, the control sample, control cells or control mitochondria are preferably in the form of a biological sample, cells or mitochondria from the individual. (In other words, the individual serves as their own control).

Alternatively, the control sample, control cells or control mitochondria may be from one or more other individuals. Where the control sample, cells or mitochondria are from one or more other individuals, preferably those individuals have also been diagnosed with or are suspected of having CF. More preferably, the individuals have the same mutations in the CFTR gene or have mutations in the gene encoding CFTR which have substantially the same effect on CFTR function.

In any method of the invention, any biological sample comprising mitochondria may be used, including stored cells obtained from or organoids derived from CF patients and cultured or propagated ex vivo. The biological sample may also be a serum or plasma sample, a preparation of peripheral blood mononuclear cells (PBMCs), a muscle biopsy, an aspirate comprising airway cells, or a sample from any organ or tissue in the body comprising mitochondria.

In a preferred embodiment, the biological sample is enriched for mononuclear cells. In a preferred embodiment, the biological sample is a sample of PBMCs.

In a preferred embodiment of the invention, the biological sample or isolated cells have been treated to remove or reduce the numbers of neutrophils and red blood cells.

In any aspect of the present invention, the screening methods are performed in vitro or ex vivo.

The present invention also provides a kit for use in:

- evaluating the efficacy of an agent for treating CF in an individual,

- evaluating the efficacy of an agent for improving exercise capacity in an individual with one or more mutations in the gene encoding CFTR,

- selecting a treatment for CF in an individual,

- identifying an agent useful for treating CF or for identifying an agent useful for improving the exercise capacity in an individual with CF, the kit comprising:

- a means for determining the mitochondrial function in a population of cells or in isolated mitochondria; and

- one or more agents for modulating the function of CFTR in a population of cells.

Preferably, the kit also comprises reagents for isolating or extracting mitochondria, or cells comprising mitochondria from a biological sample obtained from an individual. In one embodiment, the kit comprises reagents for obtaining PBMCs from a peripheral blood sample of an individual.

Preferably the means for determining mitochondrial function include reagents for determining ADP-ATP exchange, oxygen consumption rate (OCR), change in membrane potential (Dy), Mitochondrial complex l-V amount and/or activity, reactive oxygen species production, membrane lipid peroxidation, glucose utilisation, or extracellular acidification rate (ECAR) in the cells or mitochondria.

Preferably, the kit also comprises written instructions for use of the kit in a method of the invention as described herein.

The present invention also provides for the use of agents in the manufacture of a kit as described herein.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Brief description of the drawings

Figure 1 A: Oxygen consumption rate as a measure of mitochondrial function in PBMCs from individuals having the F508del mutation in CFTR (n=7) and age- matched controls. Oxygen consumption was measured using the Seahorse XF24 Analyzer Agilent Technologies). Addition of oligomycin inhibits ATP synthase. FCCP uncouples feedback between OXPHOS and ATP synthase allowing for maximum proton pump function in an out of the mitochondria. Addition of antimycin A halts cellular respiration by inhibiting Complex III. Samples were run in triplicate and results were normalised to the protein content in each sample.

B: Principles of the OCR assay utilised in A: addition of the compounds oligomycin, FCCP, and antimycin A (or a mix of rotenone and antimycin A), enable measurement of ATP-linked respiration, maximal respiration, and non-mitochondrial respiration, respectively. Proton leak and spare respiratory capacity are then calculated using basal respiration and these parameters.

Figure 2 Glycolytic activity as a measure of mitochondrial function in PBMCs from CF patients (n=10) and age-matched controls (n=10). The y-axis shows glycolytic activity. The results suggest that mitochondria in PBMCs from control individuals have a better capacity to utilise glucose than PBMCs from CF patients.

Figure 3 Oxygen Consumption Rate (OCR) as a measure of mitochondrial function in PBMCs from CF patients and age-matched controls before and after treatment with Orkambi (lumacaftor and ivacaftor). Results show mitochondrial function is reduced in CF compared to control. Following treatment with Orkambi, oxygen consumption increases to better than in controls.

Detailed description of the embodiments

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

CFTR mutations

More than 1 ,700 different mutations have been identified in the CFTR gene. Of these, around 300 are thought to contribute to symptoms of CF. Generally, CFTR mutations are classified according to the following groups: Protein production mutations (Class 1 ), Protein processing mutations (Class 2), Gating mutations (Class 3), Conduction mutations (Class 4), Insufficient protein mutations (Class 5) and Decreased Stability of CFTR protein (Class 6).

Contemporary descriptions of the pathophysiology of CF centre on the chloride function of CFTR. This is reflected in the fundamental principle of CF treatments that target CFTR and which aim to correct the underlying defect in the cellular processing and chloride channel function. CFTR“correctors” are principally targeted at the F508del microprocessing (for example, acting as chaperones to ensure that the protein reaches the cell membrane), whereas“potentiators” are intended to restore cAMP-dependent chloride channel activity at the cell surface.

Without wishing to be bound by theory, the present inventors consider that in addition to its role as a chloride channel, CFTR functions as a driver of mitochondrial function. Although the specific molecular mechanism governing CFTR regulation of mitochondrial function remains to be elucidated, the inventors have found that mutations in the CFTR gene can have a profound effect on mitochondrial function; an effect that is independent of the effect on chloride ion efflux.

Previous approaches to treating individuals with CF have typically involved administration of a CF-treatment to the individual, and monitoring for a response. This type of approach can be financially burdensome and may also result in the individual being subjected to a number of different treatments, before an“optimal treatment” is identified. The methods of the present invention provide an advantage over past approaches, as they reduce the cost to the individual, both financially and physically. Candidate drugs for administration to the individual can be screened for their suitability for correcting CFTR dysfunction in the individual, using the methods of the present invention.

In addition, because of the large number of different mutations in CFTR that can contribute to CF phenotypes, not all drugs will be useful for treating all CF-sufferers. As mentioned, it can be costly and inconvenient for patients to be subjected to a range of different pharmacological agents prior to obtaining a clinical benefit. As such, the present invention provides a means for identifying “personalised” treatments for CF patients and a means for predicting the efficacy of CF drugs in individual patients.

Further, traditional approaches to identifying new candidate drugs for treating CF have involved conducting lengthy clinical trials. The present invention provides an advantage over these prior approaches, by providing an in vitro, ex vivo assay for determining CFTR-modulating drug efficacy through measurement of changes in mitochondrial function in cells obtained from individuals requiring treatment. Alternatively, the invention can be performed with cell lines expressing mutant forms of CFTR. Thus, measuring improvements in mitochondrial function as a proxy for determining improvements of CFTR function can provide a useful platform for easily and rapidly screening for suitable candidates for treating conditions caused by CFTR dysfunction, including CF. The present invention therefore provides a method for testing a panel of candidate drugs for treating symptoms of CF in an individual.

The inventors have undertaken studies that suggest that exercise tolerance and capacity in CF is limited by cell energetics. In fact, the inventors have shown that treatment with drugs designed to correct and potentiate CFTR chloride function, can also significantly increase mitochondrial function (for example, oxygen consumption) to levels that exceed those seen in controls.

Although there are a large number of CFTR mutations that do not manifest as CF, the inventors believe that many of these mutations may contribute to reduced mitochondrial function and cell energetics in individuals who do not exhibit symptoms of CF. The methods of the present invention are therefore useful for identifying an agent capable of improving cell energetics in an individual with one or more mutations in the CFTR gene. The methods of the present invention therefore also have utility in identifying treatments for improving mitochondrial function in a broad range of individuals who have reduced exercise capacity or tolerance (or have exercise intolerance) in the context of CFTR mutations, and not just individuals suffering from CF.

For example, individuals who carry one copy of a known CF-causing gene mutation, so-called“carriers” do not exhibit typical features of CF, but do have a risk of more rapid decline in lung function and have elevated sweat chloride secretion which is a feature of CF. It is known that CFTR function may be impaired by exogenous factors, such as cigarette smoking. Carriers who have CFTR functional impairment may potentially benefit from CFTR gene modulation, and may therefore also benefit from the methods of the present invention.

Increased mitochondrial function may also result in a number of positive physiological effects, including increased metabolic rate, increased muscle mass (and decreased fat-mass), increased oxygen consumption during exercise (VO2 max), increased exercise capacity and tolerance. Therefore, the present invention has application in identifying particular methods of treatment of individuals having mitochondrial dysfunction characterised by alterations in CFTR activity or function.

As used herein“exercise capacity” refers to the maximum amount of physical exertion that a patient can sustain. An accurate assessment of exercise capacity requires that maximal exertion is sufficiently prolonged to have a stable (or steady state) effect on the circulation and that the pattern of patient response is consistent when exertion is repeated. The skilled person will be familiar with clinical measures for determining exercise capacity in an individual.

As used herein, “exercise tolerance” refers to the amount of time for which a person can sustain exercise.

Exercise capacity and tolerance can be measured in the clinic using cardiopulmonary exercise testing (CPET). The skilled person will be familiar with established protocols for performing CPET. Briefly, CPET includes infrared assessment of oxygen update while the patient undertakes exercise on an upright cycle ergometer using an incremental protocol. Workload is increased every minute to a set cadence and concluded if it cannot be maintained by the patient. Oxygen saturation is measured throughout the CPET. In addition, ventilator and cardiac data are recorded. Parameters that are measured during CPET include VO2 max, (the measurement of the maximum amount of oxygen that an individual can utilize during intense, or maximal exercise, measured as milliliters of oxygen used in one minute per kilogram of body weight (ml/kg/min)), total exercise energy (Watt), minute ventilation (VE), total exercise time, the point at which anaerobic metabolism becomes evident (anaerobic threshold, AT) and FEVi (forced expiration volume).

An increase in exercise capacity and tolerance is preferably any statistically significant increase in any one or more of the above listed parameters associated with exercise capacity and tolerance. Preferably, the increase is at least a 5%, 10%, 20%, 50% or greater increase in exercise capacity and/or tolerance compared to controls.

The inventors believe that drugs which target CFTR function may have utility in improving mitochondrial function. Importantly, this improvement in mitochondrial function can be observed in individuals even in the absence of any overt symptoms of chloride ion dysfunction (for example, as may occur in individuals with cystic fibrosis). As such, the present invention provides for a means for identifying drugs that can significantly improve the mitochondrial function (and therefore, downstream parameters such as exercise tolerability and capacity) in individuals who have one or more CFTR mutations.

As used herein, a“CFTR mutation” can include any mutation that alters the ability of CFTR to regulate mitochondrial function. These mutations may include mutations which: alter the amount of CFTR protein synthesised by the body’s cells, or which alter the amount of mature CFTR that can be used by the body’s cells (including for example, protein mis-folding mutations that lead to a premature degradation of the protein product or mutations that prevent proper processing and trafficking of the CFTR protein).

Examples of relevant CFTR mutations include missense, nonsense, deletions and splice mutations. Mutations may reduce synthesis of CFTR protein through a decrease in CFTR transcripts, a decrease in translation of CFTR transcripts, or by encoding a non-functional protein product. Mutations may also impact on the ability of the protein to be correctly processed by the cell (including transport to the mitochondria or cell surface). Other mutations may result in incorrect folding of the CFTR protein, which may result in premature degradation of the protein product or a reduction of the amount of CFTR protein at the cell surface, or relevant site of action. Still further CFTR mutations may impact on the 3-dimensional structure of the CFTR protein such that its activity is altered (including the ability to transport ions, regulate mitochondrial function)

Although it will be appreciated that the present invention is not limited with respect to the mutation of the gene encoding CFTR that may be present in a given cell, some specific examples of CFTR mutations include:

E56K, P67L, R74W, D110E, D110H, R117H, R117C, W1282X, G1349D, G178R, E193K, L206W, R347H, R352Q, A455E, F508del, G542X, S549R, S549N, G551 D, G551 S, D579G, S945L, S977F, F1052V, K1060T, A1067T, G1069R, R1070Q, R1070W, F1074L, W1282X, G1244E, S1251 N, S1255P, D1270N, N1303K, G1349D, D1152H, G1244E wherein‘X’ refers to any amino acid change. The skilled person will be familiar with the use of such nomenclature to describe mutations in the gene encoding CFTR.

A complete list of known CFTR mutations can also be obtained from the CFTR2.org database, which is continuously updated.

Further, the present invention is applicable where an individual has more than one mutation in the gene encoding CFTR, or where the individual is homozygous or heterozygous for any mutation in the gene encoding CFTR.

Measures of mitochondrial function

The present invention provides methods for determining whether one or more agents will be useful for a variety of purposes, by determining whether contacting cells or mitochondria with the agent, provides for a change in mitochondrial function.

It will be appreciated that the methods of the present invention contemplate the use of a range of different measures, for determining mitochondrial function (or dysfunction). For example, various assays can be used to determine oxygen consumption or glycolytic flux in live cells, which are used as indicators of mitochondrial function. The skilled person will be familiar with the range of assays that may be performed in order to ascertain mitochondrial function in a cell. One such assay includes the Extracellular Oxygen Consumption (OCR) Assay (which, for example, may utilise a fluorophore to determine oxygen consumption rates by cells as a means for determining mitochondrial activity). Intracellular oxygen assays, to determine intracellular oxygen levels can also be utilised as a means for comparing mitochondrial function between samples.

Preferably, the OCR measured is a measure of the maximum respiratory capacity of the cells, as described further herein.

Mitochondrial viability and activity levels can be determined by measuring mitochondrial membrane potential. For example, TMRE (tetramethylrhodamine ethyl ester) can be used to label active mitochondria. TMRE is a cell permeant, positively- charged red-orange dye that readily accumulates in active mitochondria due to their relative negative charge. Depolarised or inactive mitochondria have decreased membrane potential and fail to sequester TMRE. Other dyes that can be used to determine membrane potential are JC-1 and JC-10, which form red aggregates at high concentrations (unaggregated dye is green). Loss of membrane potential causes loss of dye and increased green fluorescence.

The activity of individual oxidative phosphorylation (OXPHOS) enzyme complexes (e.g., Mitochondrial complexes l-V) can be determined using immunocapture methods and toxicity assays. OXPHOS protein expression levels can also be determined using standard methods, including for example, quantitative western blot procedures.

Cells generate ATP via glycolysis independent of oxygen, producing lactic acid and protons. Thus, determining the level of glycolysis can be achieved through measurement of extracellular acidification rate (ECAR) using a pH sensor. ECAR is thus typically reported in units of milli pH (mpH/min), representing the change in pH over time, wherein mpH = 1/1000 pH units. Fatty Acid oxidation in cells can also be utilised for determining mitochondrial function.

Finally, ATP assays can be used to determine mitochondrial function, and are extremely sensitive. Flowever, such assays are not an ideal metric of mitochondrial function as cells strive to maintain a particular ATP budget and will adjust metabolism accordingly. Thus, alterations in ATP levels are usually only detectable during pathophysiological changes.

The skilled person will be familiar with, and have suitable access to commercial kits available for determining mitochondrial function in a given cell type. For example, various commercially produced kits are available for measuring OCR, ECAR, fatty acid oxidation, OXPFIOS activity and membrane potential. These kits are adaptable to be used with a variety of sources of mitochondria, including PBMCs and cultured cells.

Suppliers of such kits include Abeam (e.g.: the Abeam TMRE-Mitochondrial membrane potential assay kit ab113852, OXPFIOS enzyme complex activity assays, OXPFIOS protein expression assays), Agilent (e.g.: Seahorse XF Mito Stress Test®; Seahorse XF Extracellular flux Analyzer®), Enzo LifeSciences, (Mito-ID Extracellular O2 sensor kit) and Cayman Chemical. It will be appreciated that any commercially available kit can be used in accordance with the methods of the present invention.

The Agilent Seahorse XF cell Mito Stress® assay uses modulators of cellular respiration that target components of the electron transport chain to reveal key parameters of metabolic function. More specifically, the compounds, oligomycin, FCCP, and a mix of rotenone and antimycin A, are serially applied to cells to enable distinction between the different components that contribute to overall cellular respiration, including ATP-linked respiration, maximal respiration, and non-mitochondrial respiration, respectively. Proton leak and spare respiratory capacity are then calculated using basal respiration and these parameters. More specifically, the Oxygen Consumption Rate (OCR) is measured before and after the addition of inhibitors to derive several parameters of mitochondrial respiration. Initially, baseline cellular OCR is measured, from which basal respiration can be derived by subtracting non-mitochondrial respiration. Next oligomycin, a complex V inhibitor, is added and the resulting OCR is used to derive ATP-linked respiration (by subtracting the oligomycin rate from baseline cellular OCR) and proton leak respiration (by subtracting non-mitochondrial respiration from the oligomycin rate). Next carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP), a protonophore, is added to collapse the inner membrane gradient, allowing the ETC to function at its maximal rate, and maximal respiratory capacity is derived by subtracting non-mitochondrial respiration from the FCCP rate. Lastly, antimycin A and rotenone, inhibitors of complex III and I, are added to shut down ETC function, revealing the non-mitochondrial respiration. Mitochondrial reserve capacity is calculated by subtracting basal respiration from maximal respiratory capacity. A schematic depicting the contribution of each of the parameters to overall cellular respiration is provided in Figure 1 B of the instant application.

As used herein, Basal respiration refers to the oxygen consumption used to meet cellular ATP demand and resulting from mitochondrial proton leak. This parameter provides a measure of the energetic demand of the cell under baseline conditions.

As used herein, ATP production refers to the decrease in oxygen consumption rate upon injection of the ATP synthase inhibitor oligomycin and represents the portion of basal respiration that was being used to drive ATP production. This parameter provides a measure of the ATP produced by the mitochondria that contributes to meeting the energetic needs of the cell.

As used herein, H+ (Proton) leak refers to the remaining basal respiration that is not coupled to ATP production. Proton leak can be a sign of mitochondrial damage or can be used as a mechanism to regulate the mitochondrial ATP production.

As used herein, maximal respiration refers to the maximal oxygen consumption rate attained by adding the uncoupler FCCP. FCCP mimics a physiological“energy demand” by stimulating the respiratory chain to operate at maximum capacity, which causes rapid oxidation of substrates (sugars, fats, amino acids) to meet this metabolic challenge. This parameter provides a measure of the maximum rate of respiration that the cell can achieve. Preferably, the methods of the present invention involve identifying an increase in the maximal oxygen consumption rate using the method described herein. As used herein, spare respiratory capacity refers to the capability of the cell to respond to an energetic demand as well as how closely the cell is to respiring at its theoretical maximum. The cell's ability to respond to demand can be an indicator of cell fitness or flexibility. The spare respiratory capacity, defined as the difference between maximal respiration and basal respiration. Spare respiratory capacity is a measure of the ability of the cell to respond to increased energy demand.

As used herein, non-mitochondrial respiration refers to oxygen consumption that persists due to a subset of cellular enzymes that continue to consume oxygen after rotenone and antimycin A addition. This is important for obtaining an accurate measure of mitochondrial respiration.

Although the Seahorse kits provide a useful platform in which to determine the various parameters of mitochondrial OCR, the skilled person will be familiar with other methods for measuring each of the parameters of basal respiration, ATP-linked OCR, proton-leak OCR, maximal OCR, spare capacity OCR and non-mitochondrial respiration.

In addition the Seahorse XF Extracellular Flux Analyzer ® assay measures the two major energy-producing pathways of the cell simultaneously— mitochondrial respiration (oxygen consumption) and glycolysis (extracellular acidification)— in a sensitive microplate format.

The Seahorse XF® assays are label free and non-destructive, and therefore the cell plate can be subsequently reused in another assay or placed back into the incubator to make additional measurements at later time points. The skilled person will also be aware that it is possible to perform ATP or other viability assays on the same XF cell plate to generate additional information and/or normalize the XF data.

General methods for assaying mitochondrial function are described in Brand and Nicholls, (2011 ) Assessing mitochondrial dysfunction in cells, Biochem J, 435: 297-312. Other methods for assaying mitochondrial function are described in Lanza and Nair (2011 ) Mitochondrial metabolic function assessed in vivo and in vitro, Curr. Opin. Clin. Nutr. Metab. Care, 13: 511-517. The entire contents of these references are hereby incorporated by reference. Sources of mitochondria

It will be appreciated that the methods of the present invention have application using a wide range of sources of mitochondria, provided that the mitochondria are still functioning, and therefore capable of demonstrating a response to the drug candidates being tested. As such, the biological samples for use in the above-described methods, can be any fresh biological sample from an individual that include live cells having functioning mitochondria.

Examples of suitable biological samples acting as sources of active mitochondria include skeletal muscle biopsies, lung aspirates, or any cells derived from other patient sources (such as peripheral blood) or cell lines.

It will be appreciated that the methods of the present invention can also be performed on isolated mitochondria, obtained from the above-mentioned biological samples and cells.

The skilled person will be familiar with methods for obtaining suitable biological samples, and cells comprising mitochondria, for the purposes of determining mitochondrial function, and responses thereof to contact with candidate agents.

For example, methods for isolating mitochondria from small amounts of human skeletal muscle obtained by needle biopsy are described in Lanza and Nair (2009) Functional assessment of isolated mitochondria in vitro, Methods Enzymol., 457: 349- 372.

In a preferred embodiment, the source of mitochondria is a fresh peripheral blood sample, including a preparation of peripheral blood mononuclear cells (PBMCs) prepared therefrom. The skilled person will be familiar with methods for preparing PBMCs from blood samples, including using the Histopaque-1077 density gradient centrifugation method (McCoy, J.P., 1998. Handling, storage and preparation of human blood cells. Curr. Protocol. Cytom. 5:5.1.1 -5.1.13).

In further embodiments, the source of mitochondria may include one or more cell lines that have been engineered to express a gene encoding CFTR, wherein the gene comprises one or more mutations which affect CFTR function. Examples of suitable cell lines include any cell line in which CFTR can be expressed recombinantly. In some examples, the cell line may be 16HBE14o- airway cells, expressing either wild-type CFTR (for use as a control in experiments) or alternatively, expressing a gene encoding one or more mutant forms of CFTR. It will be appreciated that the gene encoding CFTR may comprise one or more mutations which affect CFTR function, such as those mutations previously described herein. CFBE41 o- cells are described in Ehrhardt et al. , Cell Tissue Res. 2006 Mar;323(3):405-15. Epub 2005 Oct 25, the contents of which are hereby incorporated by reference.

Other types of cells which may be studied in cell culture for the purpose of performing the methods of the present invention include: human bronchial epithelial cells, CFTR knockout cell lines, cell lines comprising one or more mutations in the gene encoding CFTR and others. The assays described in Dekkerset al. (2016) Science translational medicine. Jun 22;8(344):344 and Dekkers et al (2013) Nature medicine. Jul; 19(7):939-45, incorporated herein by reference, may be used to study human organoids, derived from tissues such as colonic epithelium from patients or carriers with CFTR mutations.

Agents and candidate agents

The present invention provides methods for screening of a large number of agents / candidate agents (the terms“agent” and“candidate agent”, and their plurals, are used interchangeably herein) for the purposes of determining whether the agents are suitable for use as therapeutics for improving mitochondrial function in relevant patient groups (such as individuals with CF, or individuals who do not have CF but have one or more mutations in the CFTR gene).

In addition, the invention provides methods for a “personalised medicine” approach, such that a panel of candidate agents can be screened to determine their suitability for increasing mitochondrial function in an individual in need thereof, prior to administration of the drug to the individual.

In one potential scenario, a patient may present at a clinic with symptoms of CF, wherein it is not yet clear which pharmacological intervention should be used to treat the patient. Adopting the methods of the present invention, a sample of peripheral blood may be obtained from the patient, and this sample is used to obtain peripheral blood mononuclear cells. The cells can be plated in a microplate and allowed to reach confluence. A control cell line (HEK293 cells) may also be included in the microplate.

Various candidate agents for treating CF (wherein the agents are known or thought to modulate CFTR activity) can then be tested for their suitability in treating the patient. Briefly, the cells are contacted with one of the agents (in triplicate). Control cells are not contacted with any agent but are contacted with vehicle. Following a suitable period of time, the mitochondrial function of the cells is determined using the Seahorse XF assay kit. Samples are run in triplicate and measured three times after the addition of the inhibitors provided in the Seahorse kit.

At the end of the assay, cell lysates are collected and the results normalised to the protein content of each well. The Oxygen Consumption Rate for each well can be determined using the manufacturer’s instructions for the Seahorse kit.

The agent that is identified to provide an increase in mitochondrial function in the PBMCs can then be selected for use in treating the patient.

In a further example, a patient may present at a clinic complaining of lethargy and an inability to sustain exercise. The patient is mildly overweight and a genetic test reveals that the patient has a mutation in the gene encoding CFTR. Flowever, the patient does not present with symptoms of CF.

A sample of peripheral blood can be obtained from the patient, and this sample used to obtain peripheral blood mononuclear cells. The cells can be plated in a microplate and allowed to adhere. A control cell line (FIEK293 cells) may also be included in the microplate.

Various candidate agents (thought to or known to modulate CFTR activity) can then be tested for their suitability in treating the patient. Briefly, the cells are contacted with one of the agents (in triplicate). Control cells are not contacted with any agent but are contacted with vehicle. Following a suitable period of time, the mitochondrial function of the cells is determined using the Seahorse XF assay kit. Samples are run in triplicate and measured three times after the addition of the inhibitors provided in the Seahorse kit. At the end of the assay, cell lysates are collected and the results normalised to the protein content of each well. The Oxygen Consumption Rate for each well is determined using the manufacturer’s instructions for the Seahorse kit.

The agent that is identified as providing an increase in mitochondrial function in the PBMCs would be selected for use in improving the exercise tolerance of the patient.

It will be appreciated that the present invention therefore contemplates the screening of a large number of candidate agents, either for application in the broader patient groups, or for individualised approaches to treatment.

It will be understood that the candidate agents may be small molecule drugs or may be protein (for example, an antibody or peptide) or nucleic-acid (for example, an inhibitory RNA or gene therapy) pharmacological agents.

The candidate agents are preferably modulators of CFTR, including CFTR potentiators, CFTR production corrector, CFTR activators, CFTR correctors or related agents. It will be understood that an agent that modulates CFTR function may do so by potentiating, correcting production of, activating, or correcting activity of CFTR function, and thereby improves CFTR function. Thus, an agent that improves CFTR function may be any agent that is a CFTR potentiator, CFTR production corrector, CFTR activator or CFTR corrector. Furthermore, an agent that modulates CFTR function will be understood to be an agent capable of modulating mitochondrial function, for example by increasing OCR and/or glycolytic capacity of mitochondria.

As used herein, a CFTR potentiator is an agent that restores cAMP-dependent chloride channel activity to mutant CFTRs at the cell surface.

As used herein, a CFTR corrector is a compound or treatment that promotes cell- surface expression of misfolded-CFTR. For example, CFTR correctors may act as “pharmacological chaperones” by interacting with mutants such as the F508del-CFTR, facilitating its folding and cellular processing. Alternatively, CFTR correctors may act as “proteostasis regulators” by modulating the cellular quality-control machinery to alter mutant-CFTR recognition and processing. As used herein, a CFTR activator is an agent that increases CFTR protein synthesis (by increasing CFTR gene expression).

In one embodiment, the candidate agent will be a drug that modulates or alters the activity, expression or function of the CFTR protein. Examples of candidate agents that are contemplated in the methods of the present invention include:

• ivacaftor (trade name Kalydeco®, Vertex Pharmaceuticals), also known as VX-770, CAS no: 873054-44-5, a CFTR potentiator that acts by improving the transport of chloride ions through the CFTR ion channel by binding to the channels directly, inducing a non-conventional mode of gating which in turn increases the probability that the channel is open (a CFTR “potentiator”);

• lumacaftor (VX-809, Vertex Pharmaceuticals), CAS no: 936727-05-8, acts as a chaperone during protein folding and increases the number of CFTR protein molecules trafficked to the cell surface (a CFTR“corrector”);

• tezacaftor (VX-661 , Vertex Pharmaceuticals), CAS no: 1152311 -62-0, a CFTR corrector, assists in trafficking of the CFTR protein to the correct position on the cell surface.

• VX-445 (Vertex Pharmaceuticals) a CFTR corrector;

• VX-659 (Vertex Pharmaceuticals)

• VX-152 (Vertex Pharmaceuticals)

• VX-440 (Vertex Pharmaceuticals)

• VX-371 (Vertex Pharmaceuticals)

• VX-561 (Vertex Pharmaceuticals)

• ABV-2222 (also known as GLPL2222, Galapagos NV or AbbVie), CAS no:

1918143-53-9, lUPAC: 4-((2R,4R)-4-(1 -(2,2-difluorobenzo[d][1 ,3]dioxol-5- yl)cyclopropane-1-carboxamido)-7-(difluoromethoxy)chroman-2-yl)benzoic acid, a CFTR“corrector”.

• GLPG-2737 (also known as ABV-2737), a CFTR corrector;

• GLPG-2851 (also known as ABV-2851 ), a CFTR corrector;

• GLPG-3067 (also known as ABV-3067), a CFTR potentiator;

• GLPG-2451 (also known as ABV-2737), a CFTR potentiator. It will be appreciated that the present invention also provides for screening to determine suitable combinations of drugs for use in treating a patient group, or individual. For example, the screening methods can be used to determine whether the combination of two, three, four or more drug candidates provide for optimal improvement in mitochondrial function.

In addition, the methods of the present invention allow for an easy, in vitro approach to determining synergism between various CFTR modulator agents. For example, determining the change in mitochondrial function in cells from an individual that are contacted with a combination of any two or more CFTR modulators, may assist in identifying synergistic combinations that address the particular CFTR mutations the individual has.

The skilled person will be familiar with methods for determining the appropriate concentration of relevant pharmacological agent for use in the methods of the present invention. Generally, the agents may be used in the range of between 0-20 mM, based on studies of plasma and sputum concentrations of known CF drugs following administration to CF patients. (See for example: Schneider et al. , (2016) Development of FIPLC and LC-MS/MS methods for the analysis of ivacaftor, its major metabolites and lumacaftor in plasma and sputum of cystic fibrosis patients treated with ORKAMBI or KALYDECO, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 1038:57-62, the contents of which are hereby incorporate by reference).

Once a suitable agent has been identified, it will also be within the purview of the skilled person to proceed to treat the individual requiring treatment, whether it be an individual with CF requiring treatment for CF, or an individual without CF but who has decreased exercise capacity, requiring an improvement in exercise capacity.

Accordingly, the present invention also provides a method of treating CF in an individual, the method comprising:

- providing an individual requiring treatment for CF;

- identifying a candidate agent for treating CF in the individual by: o contacting isolated cells obtained from the individual, with a candidate agent for treating CF in conditions enabling the agent to modulate mitochondrial function in the cells;

o determining the mitochondrial function of the cells;

o comparing the mitochondrial function of the cells to the mitochondrial function of a control sample of cells that has not been contacted with the agent;

- administering the agent to the individual when there is an increase in mitochondrial function in the cells upon contact with the agent, thereby treating the CF in the individual.

Further, the present invention also provides a method of treating CF in an individual, the method comprising:

- providing an individual requiring treatment for CF;

- identifying a candidate agent for treating CF in the individual by:

o contacting a biological sample obtained from the individual, with a candidate agent for treating CF in conditions enabling the agent to modulate mitochondrial function in the sample;

o determining the mitochondrial function of the cells in the biological sample;

o comparing the mitochondrial function of the cells to the mitochondrial function of a control sample that has not been contacted with the agent;

Further still, the present invention also provides a method of treating CF in an individual, the method comprising:

- providing an individual requiring treatment for CF;

- identifying a candidate agent for treating CF in the individual by: o contacting a cell having a mutation in the CFTR gene with a candidate agent for treating CF in conditions enabling the agent to modulate mitochondrial function in the cell;

o determining the mitochondrial function of the cell;

o comparing the mitochondrial function of the cell to the mitochondrial function of a control cell that has not been contacted with the agent;

The present invention also provides a use of an agent in the manufacture of a medicament for the treatment of CF, wherein the agent is identified by any method described herein.

The present invention also provides a use of an agent for treating CF in an individual in need thereof, wherein the agent is identified by any method described herein.

The present invention also provides a method for increasing the exercise tolerance of an individual, the method comprising:

- providing an individual having or suspected of having a mutation in the CFTR gene;

- identifying a candidate agent for increasing exercise tolerance in the individual by:

o contacting isolated cells obtained from the individual, with a candidate agent for modulating CFTR activity in conditions enabling the agent to modulate mitochondrial function in the cells;

o determining the mitochondrial function of the cells;

- administering the agent to the individual when there is an increase in mitochondrial function in the cells upon contact with the agent, thereby increasing the exercise tolerance in the individual.

Further, the present invention also provides a method of increasing the exercise tolerance of an individual, the method comprising:

o contacting a biological sample obtained from the individual, with a candidate agent for modulating CFTR activity in conditions enabling the agent to modulate mitochondrial function in the sample;

o determining the mitochondrial function of the cells in the biological sample;

Further still, the present invention provides a method of increasing the exercise tolerance of an individual, the method comprising:

o contacting a cell having a mutation in the CFTR gene with a candidate agent for increasing exercise tolerance in conditions enabling the agent to modulate mitochondrial function in the cell;

o determining the mitochondrial function of the cell;

o comparing the mitochondrial function of the cell to the mitochondrial function of a control cell that has not been contacted with the agent; - administering the agent to the individual when there is an increase in mitochondrial function in the cells upon contact with the agent, thereby increasing the exercise tolerance in the individual.

The present invention also provides a use of an agent in the manufacture of a medicament for increasing exercise tolerance in an individual, wherein the agent is identified by any method described herein.

The present invention also provides a use of an agent for increasing exercise tolerance in an individual in need thereof, wherein the agent is identified by any method described herein.

The agent / candidate agent may be any agent described herein.

The agent may be administered by any appropriate route, depending on the formulation of the agent. Suitable routes of administration include oral, intravenous, intramuscular, topical, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion, as well as in vivo electroporation. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.The exact amount of the agent to be administered required will vary from subject to subject, depending on upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination i.e. other drugs being used to treat the patient), and the severity of the particular disorder undergoing therapy. Thus, it may not be possible to specify an exact therapeutically effective amount. However, an appropriate therapeutically effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. In some embodiments, a therapeutically effective amount of any compound described herein for a human subject lies in the range of about 250 nmoles/kg body weight/dose to 0.005 nmoles/kg body weight/dose. Preferably, the range is about 250 nmoles/kg body weight/dose to 0.05 nmoles/kg body weight/dose. In some embodiments, the body weight/dose range is about 250 nmoles/kg, to 0.1 nmoles/kg, about 50 nmoles/kg to 0.1 nmoles/kg, about 5 nmoles/kg to 0.1 nmol/kg, about 2.5 nmoles/kg to 0.25 nmoles/kg, or about 0.5 nmoles/kg to 0.1 nmoles/kg body weight/dose. In some embodiments, the amount is at, or about, 250 nmoles, 50 nmoles, 5 nmoles, 2.5 nmoles, 0.5 nmoles, 0.25 nmoles, 0.1 nmoles or 0.05nmoles/kg body weight/dose of the compound. Dosage regimes are adjusted to suit the exigencies of the situation and may be adjusted to produce the optimum therapeutic dose.

The skilled person will be able to assess whether or not the method of treatment has been successful by observing signs or symptoms of CF, or exercise tolerance, as are known in the art and as described herein.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Examples

Example 1 : Mitochondrial dysfunction in cystic fibrosis

Control and homozygous AF508 CF patients were recruited to donate one blood sample. PBMCs were isolated using Flistopaque-1077 density gradient centrifugation. This process also removed neutrophils and red blood cells from the sample. Cell counting confirmed that less than 0.05 x 10⁴ neutrophils per pi remained in cell suspension.)

Oxygen consumption and glycolysis were measured in real-time using the Seahorse XF24 Analyzer (Agilent Technologies) by the addition of various inhibitors of the oxidative phosphorylation pathway in the PBMCs in each cell population. This provided measures of cellular activity including basal cell respiration, maximal oxygen consumption, non-mitochondrial cellular respiration and extracellular acidification rate. Briefly, Oxygen Consumption Rate (OCR) was measured before and after the addition of inhibitors to derive several parameters of mitochondrial respiration. Initially, baseline cellular OCR was measured, from which basal respiration was derived by subtracting non-mitochondrial respiration. Next oligomycin, a complex V inhibitor, was added and the resulting OCR was used to derive ATP-linked respiration (by subtracting the oligomycin rate from baseline cellular OCR) and proton leak respiration (by subtracting non-mitochondrial respiration from the oligomycin rate). Next carbonyl cyanide-p-trifluoromethoxy-phenyl-hydrazon (FCCP), a protonophore, was added to collapse the inner membrane gradient, allowing the ETC to function at its maximal rate, and maximal respiratory capacity was derived by subtracting non-mitochondrial respiration from the FCCP rate. Lastly, antimycin A and rotenone, inhibitors of complex III and I, were added to shut down ETC function, revealing the non-mitochondrial respiration. Mitochondrial reserve capacity was calculated by subtracting basal respiration from maximal respiratory capacity.

Samples were run in triplicate and measured three times after the addition of inhibitors. A control cell line (HEK293) was used on each plate and at the end of the assay, cell lysate was collected and results normalised to the protein content of each well.

Statistical analyses was by a Student’s t-test.

Results (Figure 1A) showed control subjects (n=7) have higher stimulated maximal oxygen consumption compared with CF subjects (n=7) by 24% (p < 0.1 ).

Figure 1 B shows the principle of the Seahorse XF analysis system.

Purified PBMCs in CF showed sub-optimal stimulated oxygen consumption compared with controls.

Figure 2 shows the results for glycolytic capacity in controls (n=10) versus CF patients (n=10). When oxidative phosphorylation was blocked by the addition of oligomycin, glycolytic capacity (i.e. , the capacity to produce lactate) of CF patients was lower than for controls. The results suggest a better capacity of controls to utilise glucose, than CF subjects. Example 2: Oxygen consumption in PBMCs after treatment with Orkambi®

Similarly to the process described in Example 1 , peripheral blood samples were obtained from a control individual, and an individual with CF (AF508 mutation), before and after treatment with Orkambi ® (lumacaftor and ivacaftor). As for example 1 , oxygen consumption was measured in real-time using the

Seahorse XF24 Analyzer (Agilent Technologies) by the addition of various inhibitors of the oxidative phosphorylation pathway in the PBMCs in each cell population.

The results shown in Figure 3 clearly show higher stimulated maximal oxygen consumption in the control group, compared with the CF patient prior to treatment. Flowever, following 6 months’ of treatment with Orkambi (ivacaftor / lumacaftor) at the standard dose of 2 tablets twice daily, the stimulated maximal oxygen consumption in PBMCs derived from the CF patient was greater than for control or pre-treatment values.

The results indicate that administration of CFTR corrector and potentiator can significantly improve mitochondrial function in PBMCs of CF patients.

Claims

1. A method for evaluating the efficacy of an agent for treating Cystic Fibrosis (CF) in an individual, the method comprising:

- determining the mitochondrial function of the cells in the biological sample;

2. A method for evaluating the efficacy of an agent for treating CF in an individual, the method comprising:

- providing cells from an individual diagnosed with or suspected of having CF;

- determining the mitochondrial function of the cells;

3. An in vitro method for evaluating the efficacy of an agent for treating CF in an individual, the method comprising:

- contacting cells or mitochondria isolated from an individual diagnosed with or suspected of having CF with a candidate agent for treating CF in the individual in conditions enabling the agent to modulate mitochondrial function; - determining the mitochondrial function of the cells or of the isolated mitochondria;

- comparing the mitochondrial function of the cells or isolated mitochondria to control cells or mitochondria that have not been contacted with the agent; wherein an increase in mitochondrial function upon contact with an agent indicates that the agent is suitable for use in treating CF in the individual.

4. A method for evaluating the efficacy of an agent for improving exercise capacity in an individual, the method comprising:

- contacting a biological sample, isolated cells or isolated mitochondria from an individual with an agent that is a candidate for treating CF or for modulating CFTR function, in conditions enabling the agent to modulate mitochondrial function in the sample or cells;

- determining the mitochondrial function of the cells in the biological sample or in the isolated cells or isolated mitochondria;

- comparing the mitochondrial function to a control sample that has not been contacted with the agent; wherein an increase in mitochondrial function upon contact with an agent indicates that the agent is suitable for use in improving the exercise capacity of the individual.

5. The method according to claim 4, wherein the individual is diagnosed with or is suspected of having CF.

6. The method according to claim 4 or 5, wherein the individual has symptoms of CF.

7. The method according to any one of claims 1 to 6, wherein the individual is diagnosed with or is suspected of having one or more mutations in the gene encoding CFTR.

8. The method according to claim 7, wherein the individual has a mutation in the gene encoding CFTR that does not affect chloride ion transport in the individual.

9. The method according to any one of the preceding claims, wherein the individual has a plurality of mutations in the gene encoding CFTR.

10. The method according to any one of the preceding claims, wherein the individual is homozygous for any one or more mutations in the gene encoding CFTR.

11. A method for improving the treatment of CF in an individual, the method comprising:

- contacting the samples with a plurality of agents that are candidates for treating CF in the individual, in conditions enabling the agents to modulate mitochondrial function in the samples;

- determining the mitochondrial function of the cells in each of the biological samples;

- selecting the agent that provides the greatest increase in mitochondrial function in the biological sample, for use in treating CF in the individual,

- administering the selected agent to the individual, thereby improving the treatment for CF in the individual.

12. A method for determining the likelihood that an individual will benefit from a treatment for CF, the method comprising:

- contacting the samples with a plurality of agents that are candidates for treating CF in the individual, in conditions enabling the agent to modulate mitochondrial function;

- comparing the mitochondrial function of the cells in each of the biological samples; wherein the agent that provides the greatest increase in mitochondrial function in the biological sample has a greater likelihood of being useful for treating CF in the individual.

13. A method for improving the exercise capacity or exercise tolerance of an individual, the method comprising:

- providing a biological sample from an individual in whom exercise capacity or exercise tolerance is to be improved;

- determining the mitochondrial function of the cells in the biological sample;

- comparing the mitochondrial function of the cells to a control sample that has not been contacted with the agent; wherein an increase in mitochondrial function upon contact of the biological sample with the agent indicates that the agent is suitable for use in improving the exercise capacity or tolerance of the individual.

14. The method according to claim 13, wherein the individual has been diagnosed with or is suspected of having one or more mutations in the gene encoding CFTR.

15. A screening method for identifying an agent useful for treating CF in an individual, the method comprising:

- providing biological samples from a plurality of individuals having CF;

- determining the mitochondrial function of cells in each of the biological samples;

16. A screening method for identifying an agent useful for treating CF in an individual, the method comprising:

- providing cells that are engineered to express one or more mutations in the gene encoding CFTR;

- contacting the cells with one or more agents that are candidates for treating CF, in conditions enabling the one or more agents to modulate mitochondrial function in the samples;

- determining the mitochondrial function of the cells;

- comparing the mitochondrial function of the cells to a control sample that has not been contacted with the one or more agents; wherein an increase in mitochondrial function of the cells upon contact of the cell with an agent indicates that the agent is suitable for use in treating CF in an individual.

17. The method according to claim 16, wherein the cells are from a cell line that includes one or more mutations which reduce or impair CFTR function by at least 5%, at least 10%, at least 20%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, or at least 95%.

18. A screening method for identifying an agent useful for improving the exercise capacity in an individual with CF, the method comprising:

- providing biological samples from a plurality of individuals having CF;

- determining the mitochondrial function of cells in the biological samples;

- comparing the mitochondrial function of the cells to a control sample that has not been contacted with the one or more agents; wherein an increase in mitochondrial function in the biological samples upon contact of the biological samples with an agent indicates that the agent is suitable for use in improving exercise capacity in an individual with CF.

19. A screening method for identifying an agent useful for improving the exercise capacity in an individual with CF, the method comprising:

- providing cells from a plurality of individuals having CF;

- determining the mitochondrial function of cells;

- comparing the mitochondrial function of the cells to a control sample of the cells that has not been contacted with the one or more agents; wherein an increase in mitochondrial function in the cells upon contact of the cells with an agent indicates that the agent is suitable for use in improving exercise capacity in an individual with CF.

20. The method according to any one of the preceding claims, wherein the control sample, control cells or control mitochondria are in the form of a biological sample, cells or mitochondria from the individual.

21. The method according to any one of claims 1 to 19, wherein the control sample, control cells or control mitochondria are from one or more other individuals.

22. The method according to claim 21 wherein the individuals have been diagnosed with or are suspected of having CF.

23. The method according to claim 22, wherein the individuals have the same mutations in the CFTR gene or have mutations in the gene encoding CFTR which have substantially the same effect on CFTR function.

24. The method according to any one of the preceding claims wherein the biological sample is a serum or plasma sample, a preparation of peripheral blood mononuclear cells (PBMCs), a muscle biopsy, an aspirate comprising airway cells, or a sample or organoid derived from any organ or tissue in the body comprising mitochondria.

25. The method according to claim 24, wherein the biological sample is enriched for mononuclear cells.

26. The method according to claim 25, wherein the biological sample is a sample of PBMCs.

27. The method according to any one of the preceding claims wherein the biological sample or isolated cells have been treated to remove or reduce the numbers of neutrophils.

28. A screening method for identifying an agent useful for improving the exercise capacity in an individual with CF, the method comprising:

- providing cells that are engineered to express one or more mutations in the gene encoding CFTR.

- contacting the cells with one or more agents that are candidates for treating CF or for modulating CFTR activity, in conditions enabling the one or more agents to modulate mitochondrial function in the cells;

- determining the mitochondrial function of cells;

29. The method according to claim 28, wherein the one or more mutations in the gene encoding CFTR is a mutation that does not affect the ability of CFTR to transport chloride ions.

30. The method according to any one of the preceding claims wherein mitochondrial function is determined by measuring one or more of: ADP-ATP exchange, oxygen consumption rate (OCR), change in membrane potential (Dy), Mitochondrial complex l-V activity, reactive oxygen species production, membrane lipid peroxidation, glucose utilisation, or extracellular acidification rate (ECAR).

31. The method according to any one of the preceding claims, wherein an increase in mitochondrial function is an increase in OCR of at least 5%, at least 10%, at least 25%, at least 50%, at least 75% or at least 100% compared to controls.

32. The method according to any one of claims 1 to 30, wherein an increase in mitochondrial function is at least a 1.1 -fold, at least a 1.5-fold, at least a 2-fold, at least a 3-fold, at least a 5-fold, or at least a 10-fold increase in OCR compared to controls.

33. The method according to any one of claims 1 to 32, wherein an increase in OCR is an increase in the maximal respiratory capacity of the cells, as herein defined.

34. The method according to any one of claims 1 to 30, wherein an increase in mitochondrial function is an increase in glucose utilisation of at least 5%, at least 10%, at least 25%, at least 50%, at least 75% or at least 100% compared to controls.

35. The method according to any one of claims 1 to 30, wherein an increase in mitochondrial function is at least a 2-fold increase in glucose utilisation, at least a 3-fold, 5 -old, or 10-fold increase in glucose utilisation compared to controls.

36. A kit for use in:

- evaluating the efficacy of an agent for treating CF in an individual,

- selecting a treatment for CF in an individual,

- a means for determining the mitochondrial function in a population of cells or in isolated mitochondria; and - one or more agents for modulating the function of CFTR in a population of cells.

37. The kit according to claim 36, wherein the kit also comprises reagents for isolating or extracting mitochondria, or cells comprising mitochondria from a biological sample obtained from an individual. In one embodiment, the kit comprises reagents for obtaining PBMCs from a peripheral blood sample of an individual.

38. The kit according to claim 36 or 37, wherein the means for determining mitochondrial function include reagents for determining ADP-ATP exchange, oxygen consumption rate (OCR), change in membrane potential (Dy), Mitochondrial complex I- V activity, reactive oxygen species production, membrane lipid peroxidation, glucose utilisation, or extracellular acidification rate (ECAR) in the cells or mitochondria.

39. The kit according to any one of claims 36 to 38, wherein the kit also comprises written instructions for use of the kit in a method of any one of claims 1 to 35.

40. A method of treating CF in an individual, the method comprising:

- providing an individual requiring treatment for CF;

- identifying a candidate agent for treating CF in the individual by: o contacting isolated cells obtained from the individual, with a candidate agent for treating CF in conditions enabling the agent to modulate mitochondrial function in the cells; o determining the mitochondrial function of the cells; o comparing the mitochondrial function of the cells to the mitochondrial function of a control sample of cells that has not been contacted with the agent;

41. A method of treating CF in an individual, the method comprising: providing an individual requiring treatment for CF;

- identifying a candidate agent for treating CF in the individual by: o contacting a biological sample obtained from the individual, with a candidate agent for treating CF in conditions enabling the agent to modulate mitochondrial function in the sample; o determining the mitochondrial function of the cells in the biological sample; o comparing the mitochondrial function of the cells to the mitochondrial function of a control sample that has not been contacted with the agent;

42. A method of treating CF in an individual, the method comprising:

- providing an individual requiring treatment for CF;

- identifying a candidate agent for treating CF in the individual by: o contacting a cell having a mutation in the CFTR gene with a candidate agent for treating CF in conditions enabling the agent to modulate mitochondrial function in the cell; o determining the mitochondrial function of the cell; o comparing the mitochondrial function of the cell to the mitochondrial function of a control cell that has not been contacted with the agent;

43. Use of an agent in the manufacture of a medicament for the treatment of CF, wherein the agent is identified by any method of claims 1 to 35.

44. Use of an agent for treating CF in an individual in need thereof, wherein the agent is identified by any method of claims 1 to 35.

45. A method for increasing the exercise tolerance of an individual, the method comprising:

- identifying a candidate agent for increasing exercise tolerance in the individual by: o contacting isolated cells obtained from the individual, with a candidate agent for modulating CFTR activity in conditions enabling the agent to modulate mitochondrial function in the cells; o determining the mitochondrial function of the cells; o comparing the mitochondrial function of the cells to the mitochondrial function of a control sample of cells that has not been contacted with the agent;

46. A method of increasing the exercise tolerance of an individual, the method comprising:

- identifying a candidate agent for increasing exercise tolerance in the individual by: o contacting a biological sample obtained from the individual, with a candidate agent for modulating CFTR activity in conditions enabling the agent to modulate mitochondrial function in the sample; o determining the mitochondrial function of the cells in the biological sample; o comparing the mitochondrial function of the cells to the mitochondrial function of a control sample that has not been contacted with the agent;

47. A method of increasing the exercise tolerance of an individual, the method comprising:

- identifying a candidate agent for increasing exercise tolerance in the individual by: o contacting a cell having a mutation in the CFTR gene with a candidate agent for increasing exercise tolerance in conditions enabling the agent to modulate mitochondrial function in the cell; o determining the mitochondrial function of the cell; o comparing the mitochondrial function of the cell to the mitochondrial function of a control cell that has not been contacted with the agent;

48. Use of an agent in the manufacture of a medicament for increasing exercise tolerance in an individual, wherein the agent is identified by any method of claims 1 to 35.

49. Use of an agent for increasing exercise tolerance in an individual in need thereof, wherein the agent is identified by any method of claims 1 to 35.

50. An agent for use in treating CF in an individual or for increasing the exercise tolerance of an individual, wherein the agent is identified by any method of claims 1 to 35.