Novel therapeutic approaches for the management of cystic fibrosis

Molecular Physiology Laboratory, Centre for Atherothrombosis & Metabolic Disease, Hull York Medical School, University of Hull Cottingham Road, Hull HU6 7RX, UK. Tel. +44.1482.465008. ku.ca.smyh@5jryh

Contributions: RJ, concepted the design of the paper and wrote most of the main text. AS, wrote significant portions of the paper, specifically within the emerging molecular therapies section; he also revised and edited the final draft. CH, provided additional information, revising, and helping draft the article. All contributors are satisfied with all aspects of the work.

Conflict of interest: RJ, AS and CH, have no competing interests, as we wrote this manuscript entirely by ourselves, and do not work for any corporation or government.

Availability of data and materials: Data was only gathered from previously published clinical trials, as referenced in the paper. This data was mainly used in the tables to highlight the efficiencies of treatment. No data was taken by the authors as this is a review article.

Ethics approval and consent to participate: As this was a review article it does not require consent due to the absence of participants.

This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (by-nc 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Abstract

Cystic fibrosis (CF) is a genetic condition characterised by the build-up of thick, sticky mucus that can damage many of the body’s organs. It is a life-long disease that results in a shortened life expectancy, often due to the progression of advanced lung disease. Treatment has previously targeted the downstream symptoms such as diminished mucus clearance and recurrent infection. More recently, significant advances have been made in treating the cause of the disease by targeting the faulty gene responsible. Hope for the development of potential therapies lies with ongoing research into new pharmacological agents and gene therapy. This review gives an overview of CF, and summarises the current evidence regarding the disease management and upcoming strategies aimed at treating or potentially curing this condition.

Introduction

Cystic fibrosis (CF) is a life-long condition characterised primarily by a build-up of mucus in the lungs and digestive tracts [1]. The body’s failure to remove this often leads to breathing difficulties and frequent lung infections [2]. Unfortunately, these manifestations become chronic as patients progress through life, with pulmonary disease and respiratory failure remaining the major cause of morbidity and mortality [3]. Currently, there is no cure for CF, however with good management, patients often live well into adulthood with the average predicted survival standing at 48 years for males and 43 years for females [4]. Yet, the demanding routine patients must go through to control their symptoms often has deleterious effects on their physical and mental wellbeing. Ongoing research is trying to explore how we can minimise these problems, focusing on how best to manage, treat and possibly cure CF..

The aim of this review is to critically discuss the current evidence on the therapeutic strategies for managing CF. Firstly, we provide an overview of CF in relation to its clinical presentation and pathophysiology and then look at the current options available for treatment. We will then explore future therapies, where emphasis may be given towards how we can target the underlying mechanisms of this genetic condition by focusing on pharmacological interventions that are currently undergoing investigation.

Prevalence of CF

Various countries have introduced registers which track the number of patients diagnosed with CF [5-7]. These help to measure, appraise and compare aspects of cystic fibrosis, its diagnosis, and its treatment regimen, thereby encouraging new procedures to deal with this disease. Most countries within Africa, South America and parts of Asia have a limited or non-existent system due to health authorities prioritising other important public health issues; poverty, malnutrition, civil war, and outbreaks of infectious diseases are given greater emphasis [8]. For these reasons, the most accurate data concerning the prevalence of CF is usually found in countries within Europe and North America.

Each year, the European Cystic Fibrosis Society Patient Registry collects data on CF patients and causative mutations from Europe and neighbouring countries [5]. In the 2017 report [9], the number of CF patients registered totalled 48,204, though the amount is certain to be higher due to coverage varying from different countries [10]. In Romania for example, there is an estimated 35% coverage in comparison to the UK which has a coverage of more than 95% [9]. Overall, the F508del mutation is the most frequent, with approximately 80% of patients detected as being F508del heterozygous and around 40% as being F508del homozygous. This is in comparison to the G542X allele mutation, the second most common, for which 2.69% of patients tested positive and N1303K with 2.19%, although it should be noted that the geographical distributions of these variants can differ.

The frequency of CF in North America is similar to Europe, affecting 1 in 2,500-3,500 people [11,12]. However, there are differences in its distribution across the North American continent. This is primarily linked to ethnic group; the Caucasian population has a higher prevalence than other ethnicities, including Hispanics, Native Americans and those of African or Caribbean ancestry [8]. Consequently, as with Europe, North America’s most prevalent mutation is the F508del mutation. The 2018 US Cystic Fibrosis Foundation Patients Registry report found that, in those genotyped, 84.7% of those in the registry had at least one F508del mutation, with 44.2% of those being homozygous for F508del [13]. There is a substantial drop in the next prevalent mutations which are G542X, G551D and R117H. These are found to be at a prevalence of 4.6%, 4.4% and 3.0% of the CF population, respectively. Due to its high frequency among Caucasian populations, it is the most common, potentially lethal inherited condition in this demographic [14].

Pathophysiology

Cystic fibrosis is an autosomal recessive disorder caused by a mutation on chromosome 7 at position q31.2 [15]. This location contains the cystic fibrosis transmembrane conductance regulator (CFTR) gene which is 189 kb in length with 27 exons and 26 introns. The protein itself is made of 1,480 amino acids and consists of five domains. There are two transmembrane domains that are each connected to a nucleotide binding domain (NBD) in the cytoplasm. The first NBD is connected to the second transmembrane domain by a regulatory ‘R’ domain. For the channel to open, serine residues on the ‘R’ domain must be phosphorylated by a cAMP-dependent protein kinase and ATP must bind to both NBDs and be subsequently hydrolysed [16]. In its open state, the channel is responsible for the transport of anions, specifically bicarbonate and chloride ions, across the cell membrane [17]. Physiologically, chloride and bicarbonate ions are secreted onto the epithelial surface by the CFTR channel resulting in the secretion of fluid through osmosis. Any impairment in CFTR function may therefore lead to a decrease in CFTR-mediated chloride and bicarbonate secretion. Water resorption follows, creating a thick, viscous mucus which accumulates on the epithelial surfaces of bodily organs such as the lungs, liver, intestines and pancreas [18]. The CFTR channel is also involved in the regulation of the epithelial sodium channel (ENaC), otherwise known as the amiloridesensitive sodium channel, found in the airways of the lungs [19].

While CF is a multi-organ disease, the dominant cause of morbidity and mortality is its effects on the lungs [20]. In the airways of the lungs, CFTR is highly expressed, where the buildup of mucus often causes breathing difficulties and increases the risk of infections. The airways are predominantly lined by pseudostratified columnar epithelium [21]. This epithelium is composed of numerous cell types including basal cells, club cells, ciliated cells, goblet cells, and pulmonary neuroendocrine cells (PNECs) amongst others. The pulmonary ionocytes are of particular importance to CF. These cells are derived from basal cells and are responsible for most of the CFTR expression in the airway epithelium. It is therefore thought that pulmonary ionocytes are heavily involved in fluid regulation and may play a critical role in the pathogenesis of CF [22].

Mucus build-up is primarily driven by dehydration of the airway surface liquid (ASL) that coats the epithelial cells of the airways. Hydration of the ASL is primarily regulated by CFTR-mediated anion secretion and ENaC-mediated sodium absorption. In CF, an increase in ENaC-mediated sodium absorption into the airway epithelial cells has been found alongside CFTR dysfunction, contributing to the dehydration of ASL [23]. This is thought to be due to the abolition of ENaC regulation by the CFTR channel, when CFTR is mutated and dysfunctional, as seen in CF [24]. The ASL is composed of a periciliary layer (PCL) that is covered with a mucus layer (ML) [25]. The ML is composed of mucins that form a gellike structure and are involved in trapping inhaled particles, while the PCL covers the cilia, preventing penetration by those inhaled particles. This allows the cilia to beat to remove inhaled particles through mucociliary clearance. Dehydration of the ASL leads to a decrease in PCL volume, resulting in the dehydrated ML compressing the cilia. This ultimately results in a decrease in the ability of the cilia to beat, thickened mucus, mucus stasis, and recurrent infections [26]. Over time this may progress into chronic lung disease and an overall decline in lung function.

CFTR is also strongly expressed in the sebaceous and eccrine sweat glands [15,27]. Defective CFTR results in reduced transport of sodium chloride in the absorptive duct and therefore results in saltier sweat. In the digestive tract, nutrition and vitamin deficiencies are common as mucus clogs the pancreas, preventing digestive enzymes from reaching the gut. Blockages of the small ducts in the liver and biliary system can lead to problems such as gallstones and liver disease, with the latter affecting roughly 30% of CF sufferers [28]. The reproductive systems are also not spared as the destruction of the vas deferens and epididymis are responsible for 95% of male CF patients being infertile [29]. In females, the majority are fertile but have thicker cervical mucus and may have ovulation issues due to poor nutrition.

There are 2,102 known mutations of the CFTR gene [30], over 1,850 of which are known to be disease-causing [31]. These are separated into classes I-VI as shown in Table 1 . Patients with Class I-III mutations often have more severe clinical features due to either the absence of the CFTR protein at the cell surface, or the lack of efficient gating of the channel [32]. The most common mutation is the F508del mutation, which is a frameshift mutation caused by a deletion of phenylalanine at codon 508. As a class II mutation, it causes the misfolding of the CFTR protein, which is subsequently polyubiquitinated and destroyed by the cell proteasome [33]. Each mutation class confers a different defect in CFTR production and/or function, and therefore unique approaches are required when designing treatments that can target these alterations.

Diagnosis of CF

There are several tests that can be utilised for diagnosing CF. Babies are screened for CF using the heel prick test and, as a result, most children are diagnosed shortly after birth. The test itself involves taking blood from the baby’s heel at around 5 days of age, which is then sampled for several conditions including CF. The first stage of testing involves the measurement of Immunoreactive Trypsinogen (IRT) levels, which are raised in CF patients due to blockages of the pancreatic ducts [34]. If IRT is raised, mutation analysis of the CFTR gene is undertaken. To test positive, two copies of the faulty gene need to be identified, though further tests are required to confirm a diagnosis [35]. In this case, a sweat test will be ordered which measures the salt levels on the skin, as those with CF have higher chloride levels compared to the general population. The values of chloride and sodium in sweat are around 10-50 mmol/L in normal subjects, but in the patients with CF they are usually above 60 mmol/L and can be as high as 120 mmol/L [36].

Antenatal testing can be offered for potential parents who are aware they are both carriers. This may involve a chorionic biopsy or amniocentesis during pregnancy to identify the condition of the baby. Benefits of early diagnosis allow treatment to begin at birth, although these procedures can carry an increased risk of miscarriage.

New non-invasive techniques are available and can aid early diagnosis, although these are only accessible privately. The development of safer prenatal screening has been developed by the identification of circulating free foetal DNA in the maternal plasma in early gestation [37]. DNA reflecting both maternal and foetal material is collected from the maternal plasma, which is molecularly amplified by PCR and subsequently sequenced. The foetal DNA will be analysed to assess the inheritance of the CFTR mutation. Tests that can be offered include the paternal mutation exclusion when the parents carry different CF mutations, or where both parents are carriers of the same CF mutation. If the baby has inherited the paternal mutation, an invasive test may be required to see if the maternal mutation has also been inherited.

Cystic fibrosis can also be diagnosed later in life. This is because rarer mutations are not screened early on and may present with delayed symptoms that mimic other lung pathologies such as bronchitis, thus making an accurate diagnosis more challenging. Individuals may choose to get tested to determine if they carry a faulty CFTR gene, especially if they have a relative or history of CF in their family.

The nasal potential difference (NPD) test measures how well sodium and chloride flow across the mucous membrane lining the nasal cavity. NPD gives an in vivo measurement of CFTR function and sodium channel function in the respiratory epithelium [38]. This is helpful in distinguishing individuals with non-classic forms of CF with evidence of CFTR dysfunction, from individuals with normal CFTR function that are unlikely to have CF. This can therefore be used alongside sweat tests, especially in those with an intermediate sweat result and who may carry a rare or unknown mutation.

Clinical assessment of disease progression

Routine monitoring and assessments are crucial to assess any changes that can occur as patients progress through life. Comprehensive annual reviews are offered which include pulmonary, nutritional and psychological assessments [39]. Lung function is primarily singled-out as this has a significant impact on the patient’s quality of life, especially if their lung disease has significantly advanced. To assess this, pulmonary testing is carried out through spirometry. This measures forced expiratory volume in 1 second (FEV₁), forced expiratory flow (FEF)_25-75% and forced vital capacity (FVC), with oxygen saturation measured to further determine lung condition. Weight and height are used as key indicators in evaluating growth and nutritional status. These go alongside tests that look for CF-related disorders such as diabetes and liver disease [40]. Sputum samples are also taken for microbiological analysis or, if this is not possible, cough swabs or nasopharyngeal aspirates can be used. Any significant abnormalities found in any of these tests may call for clinicians to alter treatment.

Table 1.

CFTR mutation classes and required strategies for treating cystic fibrosis.