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Future treatments 

Future treatments
Future treatments

Uta Griesenbach

, Jane Davies

, and Eric Alton

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Subscriber: null; date: 16 July 2018

Key points

  • Increased understanding of CF pathophysiology is leading to new therapies

  • Several gene therapy trials have established proof-of-principle for gene transfer in the airways, but a gene therapy-based treatment has not yet been developed

  • Small molecule drugs directed at class-specific CFTR mutations are showing promise in early phase trials

  • Drugs aimed at restoring hydration of the airway surface have been shown to improve mucociliary clearance

  • Increasing numbers of antibiotics are being formulated for both nebulization and inhalation.

12.1 Introduction

CF is an attractive target for genetic and molecular therapies aimed at correcting the basic defect. It is a common single gene disorder with an abbreviated life expectancy, the site of major pathology (the lung) is easily accessible, and there is evidence to suggest that partial correction of the underlying defect would be able to achieve clinical benefit. We are increasingly defining the mechanisms by which defective CFTR results in clinical disease, and there are a number of points of intervention where novel therapies could effect a ‘cure’ (Figure 12.1). In reality, it has been much harder to circumvent the body's immune system and achieve effective gene transfer and expression. Increasingly, novel therapies are directed at alternative steps in the gene expression pathway.

Figure 12.1 Schematic presentation of CF pathophysiology and new treatment strategies that are currently being developed for the different aspects of disease aetiology.

Figure 12.1
Schematic presentation of CF pathophysiology and new treatment strategies that are currently being developed for the different aspects of disease aetiology.

12.2 Gene therapy

CF has been at the forefront of gene therapy research. Since cloning of the CFTR gene in 1989, 25 phase I/II clinical trials, involving approximately 420 CF patients, have been carried out using a variety of viral and non-viral gene transfer agents (GTAs). Although many of these trials established proof-of-principle for gene transfer in the airways, a gene therapy-based treatment has not yet been developed. Not surprisingly airway epithelial cells, which have evolved to keep foreign particles out of the lung, are difficult to transfect. The GTAs employed so far can be divided into those derived at least in part from viruses that typically infect the airway epithelium, and artificial liposome vectors, containing a copy of the CFTR gene. Viral vectors are typically highly effective at infecting and transfecting airway cells, but induce an immune response that limits repeat administration, and only some are capable of carrying the large CFTR gene. Artificial liposomal vectors on the other hand are designed to be administered repeatedly, but suffer from lower rates of transfection. Each of the two approaches has both merits and significant remaining challenges.

12.2.1 Clinical studies

Viral vectors

Despite encouraging results in nasal and pulmonary tissues of pre-clinical models, and despite being well-tolerated at low to intermediate doses in humans, adenovirus-mediated gene transfer in the absence of epithelial damage has been inefficient in CF patients. This is mainly due to the absence of the coxsackie-adenovirus receptor on the apical surface of the majority of human airway epithelial cells, and highlights the important differences in receptor distribution between animal models and man.

Adenoviruses were superseded by first generation adeno-associated viruses (AAV). Several clinical trials have been carried out in the nose, sinus and lungs of CF patients all demonstrating that one of these, AAV2, is safe even when repeatedly administered. However, a more recent phase II trial assessing efficacy of repeated administration (three doses one month apart) failed to significantly improve lung function and has been discontinued.

The use of viral vectors for chronic diseases such as CF is currently limited by effective cellular and humoral immune responses against the virus, which prevent re-administration of the vectors. Suitable strategies that prevent immune responses and are compatible with use in CF patients have so far not been identified.

Non-viral vectors

Nine clinical trials have evaluated non-viral gene transfer to nasal or lung epithelium. The majority of these studies have shown approximately 25% correction of the chloride transport defect in either the nasal or pulmonary epithelium (Figure 12.2). In contrast to viral GTAs, non-viral formulations are more likely to be able to be administered repeatedly, and proof-of-principle for this has been demonstrated in man. Despite these promising studies it is not known if non-viral gene transfer agents, which are less efficient than viral vectors, will be able to correct clinically relevant endpoints such as infection and inflammation in the CF lung. The UK CF Gene Therapy Consortium ( has identified the most efficient non-viral GTA currently available for airway transfection and will, over the next few years, assess whether repeated administration of this complex can alter clinically relevant endpoints in CF.

Figure 12.2 Partial correction of low chloride transport in lungs of CF patients after administration of a CFTR plasmid complexed with cationic lipid. “Pre” = chloride transport in CF lung before gene therapy, “Post” = chloride transport in CF lung after gene therapy, “Non-CF” = chloride transport in non-CF lung. Data shown are mean±SEM. Adapted from Alton E, et. al. (1999) Lancet 353: 947–54, with permission from Elsevier.

Figure 12.2
Partial correction of low chloride transport in lungs of CF patients after administration of a CFTR plasmid complexed with cationic lipid. “Pre” = chloride transport in CF lung before gene therapy, “Post” = chloride transport in CF lung after gene therapy, “Non-CF” = chloride transport in non-CF lung. Data shown are mean±SEM. Adapted from Alton E, et. al. (1999) Lancet 353: 947–54, with permission from Elsevier.

12.2.2 Pre-clinical developments

Viral vectors

Helper-dependent (‘gutted’) adenoviral vectors, which are depleted of all viral genes, have been developed, but do not currently prevent induction of immune responses. Research into the optimal AAV-vectors for airway transduction is being pursued actively. Various natural isoforms have been identified and assessed for airway transfection. In addition, molecular evolution techniques are currently utilized to generate novel AAV vectors. Strategies to overcome the AAV packaging problem, thereby allowing incorporation of stronger promoters in addition to the large CFTR gene, are being assessed. However, the feasibility of repeated AAV administration is still unresolved. Results have varied greatly, and may depend on the host, delivery route and AAV serotype tested.

Most recently, lentiviral vectors, which integrate into the genome of the target cell, have been developed for airway gene transfer. Gene expression in vivo persists in mouse airway epithelium for the lifetime of the animal. Importantly, lentiviruses can be repeatedly administered to the airways of mice, although it is currently unclear how the virus evades the immune system.

Non-viral vectors

Over the last few years there have not been many convincing advances in improving non-viral gene vectors for the airway gene transfer. However, improvements in the plasmids used for non-viral gene transfer are at least as important as improving the lipid or polymer vector. A good example is the generation of plasmids completely devoid of pro-inflammatory unmethylated ‘CpG’ sequences in the bacterial DNA. This has reduced inflammatory responses and prolonged gene expression in mouse models. The optimal combination of non-viral vector and plasmid DNA will ultimately determine efficiency and safety.

12.3 Mutation specific therapies

The classes of mutations and effect they have on protein production/ function have been discussed in Chapter 1 and are illustrated in Figure 1.2. Based on an increased understanding in this area, several novel approaches to therapy have been devised, some of which have reached the stage of clinical trials.

12.3.1 Class I mutations

These premature truncation (stop) mutations result in an absence of full-length CFTR protein. The initial observation that certain members of the aminoglycoside family could facilitate read-through of these mutations and subsequent translation of full-length protein, has led to synthetic drugs with similar properties being trialled. Such drugs do not affect the normal termination of translation in non-mutated genes as the normal DNA ‘stop’ sequences are flanked both upstream and downstream by specific enhancing sequences.

Of the newly discovered molecules, PTC124 has been shown partially to restore chloride secretion on nasal potential difference testing. Certain class I mutations may be more amenable to this type of correction than others and multi-centre phase III studies are currently underway to determine how these changes correlate with improvements in clinical status.

12.3.2 Class II mutations

The commonest cause worldwide of CF is a mutation in this class: Phe508del. Full length protein is produced but is mis-folded and therefore incorrectly processed: instead of trafficking to the apical cell surface it undergoes degradation. Early experiments confirmed that incubation at reduced temperature encouraged trafficking, after which Phe508del protein was able to function as a chloride channel, albeit at reduced levels of efficiency. Subsequent work has revealed increased rates of turnover at the apical cell membrane in addition to this trafficking problem.

The concept of certain molecules facilitating trafficking has led to the terms ‘molecular chaperoning’ and a search for co-called ‘corrector’ drugs. 4-phenylbutyrate has been demonstrated to increase CFTR function, although whether this is via inhibition of the natural degradation process, or an increase in CFTR expression is unclear. Early clinical trials of this agent provided proof-of-principle on the basis of changes in nasal potential difference, although clinical development has been limited by side effects.

Miglustat, an alpha glucosidase inhibitor, in clinical use for the inherited metabolic disorder Gaucher's disease, has been shown to restore chloride secretion in both cultured CF cells and CF mice. Results from a recent Phase II clinical trial are awaited.

High throughput screening has led several groups to identify potentially promising small molecule approaches; one such drug, VX809, is currently in clinical trial.

12.3.3 Class III mutations

These mutations result in full length protein which reaches its correct position on the apical cell surface but which fails to respond to activation by cAMP-mediated phosphorylation. These so-called ‘gating’ mutations therefore lead to a reduced chloride transport activity of the CFTR protein. Encouraging results have been reported for VX770, a member of the ‘potentiator’ class of drugs which increases chloride transport of the CFTR. In placebo controlled trials, VX770 has been shown to correct sweat chloride values into the non-CF range and to significantly improve chloride secretion, when assessed by nasal potential difference. Larger phase III clinical trials are currently being initiated to explore the clinical benefits associated with such electrophysiological changes.

12.4 Other agents

12.4.1 Alternative ion channels

Denufosol is a P2Y2 receptor agonist that increases chloride secretion through non-CFTR channels by increasing intracellular calcium concentrations. Early phase trials demonstrated improvements in nasal potential difference and mucociliary clearance and later small, but statistically significant, increases in lung function were reported. Multi-centre trials are underway.

12.4.2 Osmotic therapies

Mannitol is a non-absorbable osmotic sugar, which was shown in early phase studies to improve both mucociliary and cough clearance in patients with CF. It improved FEV1 over a short period of 2 weeks, and a phase III trial has recently been completed demonstrating improvements in FEV1 and frequency of pulmonary exacerbations over a 6 month period. Theoretical concerns of an increase in bacterial growth related to the nutritional functions of the compound appear to be unfounded.

12.5 New antimicrobial therapies

Although there are many antibacterial agents available for systemic use, there is a relative paucity of relevant drugs for topical use. Aztreonam has now been formulated for nebulization and in a series of well-conducted clinical trials has resulted in improvements in lung function compared to placebo; head-to-head trials are currently underway comparing aztreonam with nebulized tobramycin solution (TOBI®).

A liposomal formulation of the aminoglycoside amikacin has been developed for nebulisation, with promising initial results and trials of inhaled ciprofloxacin are also planned. Both colistin and tobramycin have been formulated as dry powders with an emphasis on rapid, easy delivery having the potential to improve quality of life.

Trial data confirming non-inferiority to licensed nebulized drugs is likely to be required before these drugs will be granted marketing authorization.

Trials of anti-pseudomonal vaccine approaches have, to date, been disappointing (see Chapter 3). An increased understanding of mechanisms of infection, such as quorum sensing and biofilms and the recognition, with molecular tools, that many more organisms exist within the CF lower airway than has been thought, may lead to new antimicrobial approaches in the future.

12.6 Assessing new treatments

The design of clinical trials in CF is becoming more complex and challenging. Whereas several decades ago, patients deteriorated rapidly and had a short life expectancy, predicted survival of today's children is around 40 years and many live relatively healthy lives, albeit at the cost of time spent on multiple treatments. This means that certain previously appropriate outcome measures (rate of decline in lung function and mortality) are now inappropriate for the vast majority of trials, and that more sophisticated surrogate measures have to be designed and applied.

Which of these surrogates are chosen, ranging from basic molecular assays to standard clinical measurements, will depend in part on the intervention being applied, on the severity of the group being studied, and the time period available over which change can be measured. Levels of CFTR transgene mRNA are limited in applicability to gene therapy trials, whereas antibody-based techniques to detect apically-localized CFTR would be useful in this context and also with small molecule drugs aimed directly at certain classes of CFTR mutation (e.g. premature stop mutations).

CFTR function is often assessed electrophysiologically using an in vivo technique, epithelial potential difference. Baseline measurements largely reflect sodium absorption and application of zero chloride solutions and isoprenaline (which stimulates cAMP) allows a highly sensitive assessment of chloride transport; this is the primary outcome measure in many of the trials outlined above. Alternatively, for systemically applied drugs, sweat electrolyte measurements provide a simple, non-invasive end-point assay.

Several strategies aimed at improving hydration of the airway surface have been assessed with mucociliary clearance scans, where clearance of inhaled labelled particles is followed over time. For larger, later phase studies, relevant outcome measures might include bacterial load, inflammatory markers (sputum, exhaled breath, blood, bronchoalveolar lavage), lung function testing (both conventional methods and more novel techniques such as the multiple breath washout), reduction in the frequency of exacerbations and CT scans. The choice of which of these measures to employ will depend crucially on the intervention being applied, the sensitivity of the assay to detect change and the phase of study.

12.7 Conclusions

Considerable progress in the development of new therapies based around the basic CF defect has been made in the two decades following cloning of the CF gene. Importantly, these therapies are aimed at multiple targets along the pathophysiological pathway, thereby spreading the risk of failure. Academics, charities and industry have combined in unique partnerships to address issues related to a clear unmet need but in the context of a relatively small patient population. There is considerable optimism, but this needs to be balanced by the recognition that no treatment aimed at the basic defect is yet in routine clinical use.


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