Gene therapy for cystic fibrosis

The report of the first patients with cystic fibrosis (CF) to receive cystic fibrosis transmembrane conductance regulator gene (CFTR) therapy appeared in 1993, and since then there have been more than 20 clinical trials of both viral and nonviral gene transfer agents. These have largely been single dose to either nose or lower airway and have been designed around molecular or bioelectrical outcome measures. Both transgene mRNA and partial correction of chloride secretion have been reported, although sodium hyperabsorption has not been improved. The U.K. Cystic Fibrosis Gene Therapy Consortium is focused on a clinical program to establish whether these proof-of-principle measures translate into clinical benefit. Here, we review the published literature, discuss the limitations to gene therapy in the CF airway, and consider issues influencing the design of clinical trial programs.     

Gene therapy for cystic fibrosis

Summary Recent studies have identified the underlying molecular defect in cystic fibrosis (CF). Reduced or absent cAMP-mediated chloride transport in epithelial-lined organs characterizes this disease. With the identification of the CF gene, gene therapy has become a potential novel form of treatment for this disease. This article reviews the rapid progress in CF research from the understanding of the bioelectric defect to the recently begun human gene therapy trials.     

Total plasma antioxidant capacity in cystic fibrosis

Several studies have demonstrated ongoing oxidative stress in cystic fibrosis (CF). With the complexity of the antioxidant network, measurement of individual antioxidants does not necessarily assess how they work in combination. One measure that has been proposed as a gauge of total plasma antioxidant capacity is the Trolox-equivalent antioxidant capacity (TEAC) of plasma. We decided to look at plasma TEAC levels in children with CF, and relate this measure to their nutritional status, lung function, and blood measurements of several known antioxidants. We hypothesized that values in general would be lower than healthy control values, especially during acute pulmonary exacerbations. Twenty-nine children were evaluated, five of whom were during an acute pulmonary exacerbation. Height and weight, expiratory spirometry, and lung volumes were assessed, as were serum concentrations of vitamins A and E, uric acid, albumin, and lymphocyte glutathione (GSH) concentrations. TEAC values for nonhospitalized patients (1.40 +/- 0. 20 mmol/L) were not different from laboratory control values (1.35 +/- 0.11 mmol/L), but greater than values for hospitalized patients (1.09 +/- 0.17 mmol/L). TEAC correlated with anthropometric values (height: r = 0.39, P < 0.03; weight: r = 0.50, P < 0.01; body mass index: r = 0.47, P < 0.01), and pulmonary function (forced expiratory volume in 1 sec: r = 0.43, P < 0.02; residual volume/total lung capacity: r = -0.42, P < 0.03), but not with age. Univariate correlation with blood measurements demonstrated a significant correlation of TEAC with uric acid (r = 0.49, P < 0.02), but not with albumin, vitamins A or E, or lymphocyte GSH. Multiple regression analysis demonstrated a correlation between TEAC and uric acid, albumin, and lymphocyte GSH in the non-hospitalized group (r(2) = 0.38, P < 0.03). We conclude that TEAC appears to represent a mixed antioxidant response, rather than response to a single antioxidant. While being responsive to oxidative stress, the mechanism of the response may differ between clinical situations, such that the clinical significance of changes in plasma TEAC remains to be defined.     

Exon-skipping antisense oligonucleotides for cystic fibrosis therapy

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), and the CFTR-W1282X nonsense mutation causes a severe form of CF. Although Trikafta and other CFTR-modulation therapies benefit most CF patients, targeted therapy for patients with the W1282X mutation is lacking. The CFTR-W1282X protein has residual activity but is expressed at a very low level due to nonsense-mediated messenger RNA (mRNA) decay (NMD). NMD-suppression therapy and read-through therapy are actively being researched for CFTR nonsense mutants. NMD suppression could increase the mutant CFTR mRNA, and read-through therapies may increase the levels of full-length CFTR protein. However, these approaches have limitations and potential side effects: because the NMD machinery also regulates the expression of many normal mRNAs, broad inhibition of the pathway is not desirable, and read-through drugs are inefficient partly because the mutant mRNA template is subject to NMD. To bypass these issues, we pursued an exon-skipping antisense oligonucleotide (ASO) strategy to achieve gene-specific NMD evasion. A cocktail of two splice-site–targeting ASOs induced the expression of CFTR mRNA without the premature-termination-codon–containing exon 23 (CFTR-Δex23), which is an in-frame exon. Treatment of human bronchial epithelial cells with this cocktail of ASOs that target the splice sites flanking exon 23 results in efficient skipping of exon 23 and an increase in CFTR-Δex23 protein. The splice-switching ASO cocktail increases the CFTR-mediated chloride current in human bronchial epithelial cells. Our results set the stage for developing an allele-specific therapy for CF caused by the W1282X mutation.

Nonsense mutations account for about 20% of all known pathogenic genetic lesions within the coding region (1). Nonsense mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF) by impairing chloride conductance in the epithelial tissues of multiple organs including the lungs, the gastrointestinal tract, and the reproductive organs (2). The CFTR-W1282X mutation is the sixth most common CF-causing mutation and the second most common CF-causing nonsense mutation; it is found in 1.2% of CF patients worldwide and causes a severe form of CF if homozygous or combined with another CF-causing allele (3, 4). Nonsense mutations, including the CFTR-W1282X mutation, lead to reduction of functional proteins due to premature termination of translation and degradation of the messenger RNA (mRNA) by nonsense-mediated mRNA decay (NMD) (5).There have been significant strides in CF therapeutics development, with the most recent Food and Drug Administration (FDA)–approved treatment, Trikafta—a combination of CFTR correctors (elexacaftor and tezacaftor) and a potentiator (ivacaftor)—showing unprecedented improvements in the pulmonary exacerbations, survival, and quality of life for CF patients with at least one F508del mutation (6). Although almost 90% of CF patients may benefit from Trikafta (7), there are no approved targeted therapies for CF caused by nonsense mutations. Therefore, there is still a significant unmet need for CF treatments that can simultaneously address different classes of CFTR protein deficiencies, particularly the CFTR-W1282X mutation (8).One potential therapeutic modality for CFTR nonsense mutations is read-through compounds (RTCs), which increase the level of full-length protein by reducing the fidelity of the ribosome at the premature termination codon (PTC) (9). Ataluren is a nonaminoglycoside RTC with a favorable safety profile that was investigated in the context of several genetic diseases. Although it showed a therapeutic effect in Duchenne muscular dystrophy caused by nonsense mutations (9), it was not able to improve forced expiratory volume in clinical trials in CF patients with various CFTR nonsense mutations (10). Gentamicin is an aminoglycoside RTC that can increase full-length CFTR protein in vitro, whose clinical efficacy in CF is limited by NMD (11). Recently, a new class of RTC that induces read-through by depleting the translation release factor eRF1 showed efficacy in vitro (12). Despite active research, a clinically viable read-through approach for CF caused by nonsense mutation does not yet exist.Because the activity of CFTR-W1282X protein can be enhanced by CFTR modulators (4, 13, 14), combining NMD-inhibition strategies with CFTR modulators could potentially benefit patients. One such strategy involves antisense oligonucleotides (ASOs), which are short, synthetic, single-stranded DNA, RNA, or structural mimics of DNA/RNA that bind to complementary RNA to alter its functions (15). ASOs with uniformly modified nucleotides, such as 2′-O-methoxyethylribose-phosphorothioate (PS-MOE-ASOs), phosphorodiamidate morpholino oligomer (PMO), or 2′-O-methyl (2’-OMe), can sterically block the binding of proteins, nucleic acids, or other factors that are important for RNA metabolism and processing (15). On the other hand, chemically modified nucleotides in “gapmer” ASOs are spaced out by natural DNA nucleotides in order to induce RNase-H–mediated degradation of their target RNA (15). Antisense-mediated knockdown or small-molecule inhibition of SMG1 reduces NMD in human bronchial epithelial cells and improves expression and function of CFTR in cells harboring the W1282X mutation (16). However, general inhibition of NMD may not be the optimal strategy because the NMD machinery is essential for vertebrate development and regulates the expression of a subset of normal and physiologically functional mRNAs (17). Therefore, a targeted approach for restoring functional CFTR mRNA seems a more desirable therapeutic strategy.According to the “55-nucleotide rule,” mRNA with an exon–exon junction >55 nucleotides (nt) downstream of a stop codon is degraded by NMD, due to the binding of exon-junction complexes (EJCs) near each of the downstream exon–exon junctions (18). Attempts have been made to target the multiple exon–exon junctions downstream of the PTC on the CFTR-W1282X mRNA to inhibit NMD. Deleting the genic region downstream of the W1282X mutation using CRISPR-Cas9 genome editing prevents NMD of CFTR-W1282X mRNA and increases the truncated protein levels in human airway cells (19). PS-MOE ASOs designed to prevent EJC binding downstream of a PTC can attenuate NMD in a gene-specific manner (20). Recently, we showed that a cocktail of three ASOs designed to prevent EJC binding downstream of the W1282X mutation specifically increases the expression of CFTR-W1282X mRNA and CFTR protein function in human bronchial epithelial cells (21).An alternative strategy we describe here is to up-regulate a partially functional isoform of CFTR mRNA using ASOs that can modulate mRNA splicing. In recent years, ASOs drugs have been shown to be well suited for the treatment of various diseases (22). Recent FDA and European Medicines Agency regulatory approvals have demonstrated the clinical potential of ASOs for hereditary diseases. Several PMO ASOs approved to treat different causal mutations in Duchenne muscular dystrophy induce skipping of target exons in the mutant DMD pre-mRNA and promote the production of partially functional dystrophin protein (23). The FDA approval of nusinersen (Spinraza) in 2016 to treat spinal muscular atrophy and the rapid development of milasen, a personalized therapy for a single patient with neuronal ceroid lipofuscinosis 7 (CLN7, a form of Batten’s disease), further highlights the clinical potential of splice-switching ASOs (24, 25). Although there is no FDA-approved ASO therapy for CF at present, splice-switching ASOs designed to correct defective CFTR splicing have shown efficacy in patient-derived airway cells and nasal epithelial cells (26, 27).If CFTR exon 23 with the W1282X mutation is skipped using splicing-switching ASOs, the resulting CFTR-Δex23 mRNA should not be targeted by NMD, as exon 23 is in frame. The intrinsic activity of CFTR-Δex23 has not been reported, but it is expected to be less active than wild-type (WT) CFTR, based on the deletion’s location in the second nucleotide-binding domain (NBD2). On the other hand, this protein is expected to retain partial activity by analogy to the truncated CFTR-W1282X protein. CF-causing exon 23 splice-site mutations (4005 + 1G > A and +2T > C) have been identified (3); however, the intrinsic activity of CFTR-Δex23 cannot be inferred from the phenotypes of these mutations because whether they cause exon skipping, cryptic splice-site activation, and/or intron retention is unknown (28).As previously mentioned, CFTR modulators can increase the activity of a wide variety of mutant CFTR proteins (29), so CFTR-Δex23 protein’s chloride conductance may also be enhanced by CFTR modulators. Moreover, as low as 10% of normal CFTR function provides a significant therapeutic benefit for CF patients (30). Thus, increasing the expression of mutant CFTR protein with residual activity could be beneficial for CF patients. In this report, we show that CFTR-Δex23 protein retains partial activity. We also describe uniformly modified PS-MOE ASOs that target the splice sites flanking exon 23 and induce efficient exon 23 skipping, with the resulting CFTR-Δex23 protein increasing CFTR-mediated chloride conductance in human bronchial epithelial cells. This approach provides the basis for clinical development of antisense-directed exon skipping as a therapeutic strategy for CF caused by the W1282X mutation.     

The status of gene therapy for cystic fibrosis

Cystic fibrosis (CF) has been a primary focus for gene therapy of lung diseases because the genetic cause is known and the airway epithelium is accessible for direct deoxyribonucleic acid (DNA) delivery. Soon after the mutated gene was identified in 1989, investigators demonstrated that transfer of a normal copy of the CF gene corrected ion transport abnormalities, thus validating the potential for use of gene therapy for this autosomal recessive disease. However, subsequent studies in a variety of in vitro and animal models, and more limited human studies, have revealed several obstacles to gene therapy for CF: (1) The incomplete understanding of CF lung disease pathogenesis, particularly the relative importance of ion transport and other cellular abnormalities (including glycoconjugate processing, pH regulation of intracellular organelles, and membrane trafficking), and of surface epithelial versus submucosal gland CF transmembrane regulator (CFTR) expression, generates uncertainty as to the necessary target cells for gene transfer and the optimum end point(s) for short-term human studies. (2) The airway epithelium has protective barriers against viral infection that impair gene transfer with several vectors, including recombinant viruses and DNA conjugates. Improvement in DNA transfer technology will be necessary for successful gene therapy. (3) Immune responses to recombinant viruses and inflammatory effects of bacterial DNA are only partially understood and appear to limit efficacy, particularly with repeated administration. Identification of these obstacles is prerequisite for progress, and recent studies with novel DNA delivery methods appear promising.     

Use of nonviral vectors for cystic fibrosis gene therapy

Over the last decade, three groups within the United Kingdom (Edinburgh, Oxford, and Imperial College, London) have undertaken key studies in the development of clinical gene therapy for cystic fibrosis. In 2001, catalyzed by the Cystic Fibrosis Trust, these groups came together to form the United Kingdom Cystic Fibrosis Gene Therapy Consortium. The Consortium has removed duplication and competition, developed core facilities playing to the respective strengths of the centers, and introduced the joint strategy described in this article. This is driven by a clinical trial program, with a product pipeline and the necessary development of novel preclinical and human assays. The program is milestone-related, has a structure that lies between the pharmaceutical industry and academia, and has as its endpoint negotiations with industry to undertake a phase III clinical trial of the identified product.     

Advances in cystic fibrosis gene therapy

PURPOSE OF REVIEW: The first cystic fibrosis gene therapy trials were carried out in 1993, and although proof-of-principle for gene transfer to the lungs was established, efficiency was generally low. The authors review the most recent advances in preclinical airway gene transfer and summarize the results from the latest clinical trials. RECENT FINDINGS: Recent clinical trials report encouraging results. Repeat administration of adeno-associated virus to the lung was safe. Nonviral nanoparticles used, for the first time, in the nose of cystic fibrosis patients were also safe and led to partial correction of the chloride transport defect in nasal epithelium. Important advances have been made in preclinical research, including the development of new viral and nonviral gene transfer agents and improved plasmid DNA. In addition, physical delivery methods, such a magnetofection and electroporation, are being assessed to improve nonviral gene transfer. SUMMARY: Considerable progress has been made in understanding and overcoming the problems associated with gene transfer to airway epithelial cells, the target cells for cystic fibrosis gene therapy. It has also been recognized that novel preclinical and clinical assays are crucial for the success of cystic fibrosis gene therapy, and considerable effort is currently being put into assay development and trial designs.     

Inhaled hypertonic saline as a therapy for cystic fibrosis

PURPOSE OF REVIEW: The beneficial effect of a short course of nebulized hypertonic saline on lung function for people with cystic fibrosis was first identified in 1996. At that time, competing hypotheses about the pathogenesis of cystic fibrosis lung disease predicted very different responses to long-term inhalation of hypertonic saline. RECENT FINDINGS: Recent benchtop research supports the hypothesis that the liquid layer lining the airways is depleted in cystic fibrosis. In addition to osmotically restoring this liquid layer, hypertonic saline improves the rheological properties of the mucus and stimulates cough. The net result is accelerated mucus clearance that is short-lived for single doses but sustained with regular inhalation. Long-term use improves lung function mildly but has marked benefits with respect to exacerbations, quality of life and absenteeism, without promoting infection or inflammation. SUMMARY: Hypertonic saline appears broadly applicable as an inexpensive therapy for most patients with cystic fibrosis.     

Rationale for hypertonic saline therapy for cystic fibrosis lung disease

Cystic fibrosis (CF) is caused by alterations in the CF transmembrane conductance regulator ( CFTCR) gene. More than 1400 mutations in the CFTCR gene have been described, but the most common mutation (noted in 70% of CF chromosomes) is DeltaF508. Alterations in the CFTCR gene result in deranged sodium and chloride ion transport channels. This leads to failure of airway epithelia to hydrate their surfaces normally, particularly in response to infectious or toxic insults. Additional effects include mucus adhesion to airway surface, chronic inflammation, and infections. The concept that airway surface dehydration can cause CF-like lung disease is supported by in vitro data and in vivo animal models. Rehydrating airway surfaces may reduce or prevent lung injury and damage. Short- and longer term studies have shown that inhalation of hypertonic saline is well tolerated and improves lung function, reduces exacerbations, and improves quality of life in CF patients. This review discusses the importance of airway epithelial sodium and chloride channels in the pathogenesis of CF, and strategies (particularly the use of inhaled hypertonic saline) to reverse or minimize lung inflammation and injury in this disease.     

Current status of gene therapy for cystic fibrosis pulmonary disease

Cystic fibrosis (CF) is a common lethal genetic disorder that affects all ethnic populations; however, it is most prevalent in Caucasians. Intensive basic research over the last 20 years has resulted in a wealth of information regarding the CF gene, its protein product and the mutational basis of disease. This increased understanding has lead to the development of gene therapy for the treatment of CF pulmonary disease. Delivery of the CF gene to the airway requires direct in vivo transfer using vectors encoding for normal CF transmembrane regulator (CFTR) protein. Several vectors are currently available for CF gene transfer and include both viral (adenoviruses, adeno-associated viruses) and non-viral (liposomal) systems. Initial clinical trials with each of these vectors have demonstrated that gene transfer to the CF airway is possible. The efficiency of transfer and duration of expression, however, have been limited. The effects of gene transfer on correction of the basic ion transport defects have also been highly variable and inconsistent, irrespective of the vector. Currently, the risk of severe immunological reactions is the primary factor limiting the clinical advancement of gene therapy. Both the adenoviral and liposomal vectors are associated with significant acute inflammatory reactions. The adenoviruses and adeno-associated viruses also elicit humoral immune responses that significantly reduce the efficiency of transgene expression and increase the risk of readministration. Several strategies are under investigation to improve the efficiency of gene transfer to the CF airway. These include overcoming local barriers in the lung, circumventing the immune response and improving vector internalization and/or uptake. Application of gene transfer in the child and possibly the fetus are also potential future clinical applications of gene therapy. However, despite considerable research with gene therapy, there is little evidence to suggest that a well tolerated and effective gene transfer method is imminent and aggressive use of conventional pharmacological therapies currently offer the greatest promise in the treatment of patients with CF.     

Lentiviral vectors and cystic fibrosis gene therapy

Cystic fibrosis (CF) is a chronic autosomic recessive syndrome, caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, a chloride channel expressed on the apical side of the airway epithelial cells. The lack of CFTR activity brings a dysregulated exchange of ions and water through the airway epithelium, one of the main aspects of CF lung disease pathophysiology. Lentiviral (LV) vectors, of the Retroviridae family, show interesting properties for CF gene therapy, since they integrate into the host genome and allow long-lasting gene expression. Proof-of-principle that LV vectors can transduce the airway epithelium and correct the basic electrophysiological defect in CF mice has been given. Initial data also demonstrate that LV vectors can be repeatedly administered to the lung and do not give rise to a gross inflammatory process, although they can elicit a T cell-mediated response to the transgene. Future studies will clarify the efficacy and safety profile of LV vectors in new complex animal models with CF, such as ferrets and pigs.     

Long-term tobramycin aerosol therapy in cystic fibrosis

The long-term efficacy and safety of aminoglycoside aerosol therapy for Pseudomonas aeruginosa colonization/infection in cystic fibrosis has not been fully investigated. In the present study, 14 patients with cystic fibrosis, ages 8-19 years (mean: 13.3 years), received tobramycin aerosol therapy for a mean duration of 20 months. Eighty milligrams of a tobramycin solution were inhaled twice daily after physiotherapy via a jet nebulizer. After 1 year, weight for height increased significantly by 2.9% of the predicted normal, and the Kraemer clinical score increased by 2.1 points (P less than 0.05). The frequency of hospital admissions decreased from 2.0 to 1.3 per patient, respectively, during the years before and after the study onset. The antibody response to P. aeruginosa elastase, exotoxin A, and alkaline phosphatase showed a reduction in serum titers against one or more enzymes in eight patients. The best long-term results after 12-38 months of treatment were obtained in moderately ill children. No evidence of ototoxicity or renal damage was observed. Although intermittent bacterial resistance occurred in five patients after 10-21 months of tobramycin inhalation, this was not associated with clinical deterioration. The study demonstrates the safety and clinical efficacy of long-term tobramycin aerosol therapy. Double-blind studies with larger patient cohorts are required to determine the value of aminoglycoside inhalation as an adjunct to the established therapeutic regimens.     

Rational parameters for antibiotic therapy in patients with cystic fibrosis

Summary Data from the literature and the authors' experiences were used to review aspects of antibiotic therapy of patients with cystic fibrosis; attention was paid toin vitro antimicrobial susceptibility tests and assessment of therapy directed against mucoidPseudomonas aeruginosa. The heterogeneity ofP. aeruginosa within single sputa with respect to antibiotic susceptibility is stressed. Quantitative viable counts of bacteria based on an analysis of homogenised sputum is recommended. The mode ofin vivo growth of mucoidP. aeruginosa is discussed to explain the survival of hypersusceptibleP. aeruginosa in vivo, and the clinical benefit observed in the absence of a significant reduction of the pathogen. The value of ceftazidime in the treatment of exacerbations due toHaemophilus influenzae is emphasised. The social benefits from oral administration of ciprofloxacin also emphasises that the patient's quality of life must also be considered.

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Parameter für eine rationelle Antibiotikatherapie bei Patienten mit zystischer Fibrose

Zusammenfassung In einer Übersicht werden Daten aus der Literatur und eigene Erfahrungen in der Antibiotikatherapie bei Patienten mit zystischer Fibrose dargestellt. DieIn vitro-Testung und klinische Beurteilung der Therapie von Infektionen durch mukoide Stämme vonPseudomonas aeruginosa finden besondere Beachtung.P. aeruginosa-Isolate aus einer einzigen Sputumprobe weisen im Hinblick auf ihre Antibiotikaempfindlichkeit eine bemerkenswerte Heterogenität auf. Keimzahlenbestimmungen sollten an homogenisierten Sputumproben vorgenommen werden. DasIn vivo-Wachstumsverhalten mukoiderP. aeruginosa-Stämme gibt Aufschluß über die Beobachtung, daß hochempfindliche Stämme vonP. aeruginosa in vivo überleben. Auf das Phänomen der klinischen Besserung ohne signifikante Erregerreduktion wird eingegangen. Hervorgehoben wird der Wert von Ceftazidim in der Therapie akuter Exazerbationen durchHaemophilus influenzae. Der soziale Gewinn einer oralen Therapie mit Ciprofloxacin weist auf die Bedeutung der Lebensqualität für den Patienten hin.