Current research on cystic fibrosis gene therapy suggests that it will become an important treatment strategy INTRODUCTION:
Cystic fibrosis is an autosomal recessive disease, triggered by mutation in the gene CFTR i.e. cystic fibrosis transmembrane conductance regulator.
CFTR is an ABC gene i-e ATP-binding cassette (transporter) gene that encodes a protein. This CFTR protein is a chloride ion channel protein that controls the flow of chloride ions and water across the cells. This movement is important in generating sweat, digestive juices, as well as mucus secretion and its clearance.
This CFTR gene is located at q 31.2 locus of chromosome 7 long arm. The most common mutation in cystic fibrosis is removal of three nucleotides that results in loss of a nucleotide phenylalanine (F) at 508th position at the long arm of chromosome 7.
Depending upon the protein expression and function six classes of mutations have been identified in cystic fibrosis:
CLASS I: these are non-sense mutations that hinder the protein synthesis as they have premature stop codons.
CLASS II: these mutations are most commonly found in patients of cystic fibrosis that include the deletions of phenylalanine 508th del. As a result of this deletion, protein folding is reduced and as a result its transport to cell surface is impaired and it undergoes degradation within the cellular lysosomes.
CLASS III: in these mutations, proteins are normally present but are not expressed to a level that responds to intracellular signals.
CLASS IV: these are the mutations in which chloride channels are present but exchange is impaired due to pore abnormalities.
CLASS V: in these mutations, CFTR proteins are synthesized and are present on the cell surface but are not sufficient to conduct signals.
CLASS VI: in these mutations, proteins are synthesized normally but proteins have a shortened half-life.

Overall, these mutations, specifically del 508th phenylalanine, lead to abnormal CFTR proteins which are then responsible for the malfunctioning of chloride channels, i.e. decreased chloride secretion and increased sodium absorption, resulting in impaired mucociliary clearance.
PATTERN OF INHERITANCE:
Pattern of inheritance is an autosomal recessive disease. Cystic fibrosis develops when both alleles inherited from parents cannot produce normal functioning CFTR protein. If both the parents are unaffected carriers then chances of their child to develop cystic fibrosis are one in four (affected), while the chances of unaffected carrier children are two in four and of unaffected child are one in four. This shows autosomal pattern of inheritance.

SIGNS AND SYMPTOMS:
Patients with genetic defect of CFTR gene mainly present respiratory infections, starting from accumulation of thick and sticky mucus and impaired clearance leading to pneumonia, bronchiectasis causing pulmonary hypertension and eventually, heart failure.
These infections of the respiratory tract become chronic and irreversible sometimes, with more than 90% patients dying due to respiratory failure. These patients have poor growth and weight despite of normal food intake as digestion is also impaired due to lack of digestive enzymes and juices. Absorption of minerals and vitamins is impaired as well.
Cystic fibrosis, besides respiratory system, also affects pancreas which in turn cause diabetes. It also affects liver, kidney and intestines, causing impaired clotting, cirrhosis (nodular) of liver and malabsorption. Patients with cystic fibrosis may be infertile, especially males as they congenitally lack vas deferens and have sperm with low motility rate, whereas in females, infertility is due to thickened cervical mucus and malnutrition, leading to ovulatory failure and finally amenorrhea.
TREATMENT AND MANAGEMENT:
Management strategies that should be adopted to improve the life expectancy of patients of cystic fibrosis include the proactive treatments of respiratory tract infections and pulmonary rehabilitation mainly, as well as good and healthy diet and an active lifestyle. Quality of life of patients with cystic fibrosis should be maintained by limiting and treating secondary bacterial infections of respiratory tract.
Nowadays, the treatment and management plan for the patients of cystic fibrosis is multidisciplinary, as the disease involves multi organs like lungs, gastro intestinal tract, and reproductive organs. As most of the damage is to lungs, the main target therapy is for lungs, including antibiotics and chest physiotherapy that uses percussive methods. The aim behind these chest percussive therapies and mechanical techniques is to dislodge mucus plugs and clear the airway.
Ivafactor is a medicine recently approved for the treatment of cystic fibrosis. Aerosolized medications i.e. Dornase Alfa, hypertonic saline are used. Often, when the disease worsens mechanical breathing support (ventilator) is also required, and if mechanical breathing support fails then lung transplantation becomes necessary. Care must be taken as both of the lungs are necessary to be replaced. Replacing one lung might not be beneficial as it would easily get infected from the remaining lung.
DISCUSSION:
Another innovative approach and treatment strategy for cystic fibrosis is CFTR gene therapy. Gene therapy is a therapeutic technique that involves replacement, inactivation or knocking out of a mutant gene with the healthy and normal one to one fight against a disease.
GENE THERAPY:
It seems like an ideal treatment where basic pathology is rectified and genetic error is targeted. Initially, it was believed as an ideal and promising treatment but this method is a risky one and is still under study to be applicable regarding its safety and effectiveness.
Gene therapy concept regarding cystic fibrosis treatment is not a new concept; it is a method of manipulating cells at molecular level that involve the substitution of diseased/mutant gene with the healthy/working copy of gene into specific cells of the body. In this way the healthy genes would override the function of the mutant genome. This is the basic concept of the gene therapy.
Cystic fibrosis is a genetic disorder that affects multiple organs at the same time. The defect involved is of a single gene i-e CFTR gene (cystic fibrosis transmembrane conductance regulator). The main idea behind this gene therapy approach was replacement for this single mutant gene and introduction of a healthy gene in to the cells of the body, especially in lungs. Another vision was to lower the therapeutic burden of cystic fibrosis patients.
The transfer of normal CFTR gene into the affected cells usually results in the production of functional CFTR gene in the target cells without adverse effects. This was the basic idea behind the gene therapy, as different studies indicated that pulmonary manifestations due to cystic fibrosis can be overcome only by 5-10% expression of CFTR gene. So gene therapy was considered as a revolutionary approach for the treatment of cystic fibrosis.
Method of delivery of CFTR gene (either by using a viral vector or non-viral vector i.e. lipids) to the targeted cells i.e. respiratory tract is in situ delivery that involves direct administration of the genetic material in to the targeted tissue.
The new technique was tested for the gene transfer with the help of 1) Viral vectors CFTR gene therapy in animal models and clinical trails 2) Non-viral cationic liposomes CFTR gene therapy in animal models and clinical trials.
Viral vectors available are retrovirus, lentivirus adenovirus, adeno-associated virus.
Non-viral gene delivery is done by naked DNA, electroporation, magnetofection. In the case of cystic fibrosis, non-viral chemical delivery use cationic liposomes for CFTR gene transfer is undertaken and is still under process.
All the methods for gene delivery using vectors either viral or non-viral are not completely successful and have certain limitations. Depending on the demand of the expression of gene, vectors are chosen e.g. for short duration of expression of gene adenoviruses as vector are considered adequate but for longer expression as in the case of cystic fibrosis integrating vector with minimum immunological problems is suitable.
In CFTR gene therapy, ideal vector qualities to be considered were the lack of immunogenic response, the ability to integrate a specific location in a chromosome, and the ability to target the desired cells.
Gene transfer based therapeutic approach that use viral or non-viral vectors was considered feasible as the lining epithelium of respiratory tract was easily accessible by instillation or aerosol delivery.
Initially, the main obstacle in gene therapy was the cloning of the particular gene in cDNA but with dedicated efforts the goal was achieved. It was considered as the best remedy for the cystic fibrosis patients. But with the passage of time other factors like suitable vector, transgenic expression and vector biology and its interaction with the host were the hurdles and important challenges to be considered.
In primary clinical trials, virus vectors were used to deliver a normal functioning CFTR gene into lungs, but results were not successful. The main drawback with using viral vectors were the immunogenic responses in host cells.
In later trials, repeated gene therapy using a non-virus CTRF gene encased in a bubble of fat (lipids) were given to patients in a randomized controlled trial which was also a double blind trial. Gene treatment that was given to the patients was repeated nebulization of non-viral CFTR gene i.e. with plasmid DNA encoding the CFTR gene complexed cationic liposome.
Patients in group were given gene therapy i.e. 5ml of lipid mixture with plasmid DNA by nebulization while patients under control group were given simple saline solution by nebulizer. The gene therapy was given at monthly intervals over 1 year. Patients in both groups were also given antibiotics orally as well as intravenously during the trial.
The main standard test for both groups was FEV1 (forced expiratory volume in one second). But other outcomes were also considered like CT scan of lungs and quality of life of patients in both groups. According to the trial, the main analysis was per- protocol i.e. those who took the gene therapy were analyzed for the results to see the actual results and efficacy of the therapy.
RESULTS OF THE TRIAL:
Both the study groups were analyzed after 12 months. Results showed that the group with the gene therapy treatment had an increased FEV1 ratio as compared to the placebo group. It was also concluded that the gene therapy was able to stabilize the pulmonary symptoms. Overall, no gross and obvious adverse effects were noticed and mentioned by researchers. Gene therapy was accepted as modest and variable by the researchers.
It was said that at the end of the trail, increasing the dose of gene therapy could improve the lung functions more in addition with antibiotic courses. But still they did not consider it suitable for clinical practice, as there were certain drawbacks regarding its efficacy. It was said that further improvements in efficacy and CFTR gene combination with potentiators may be required before gene therapy is deemed suitable for clinical practice.
Scientists were not satisfied with the results of gene therapy with regards to its efficacy and it was not advised for clinical practice.
The clinical trials assessing the gene therapy treatment were small, and in order to clinically practice and fully assess the safety and efficacy, large clinical trials were needed.
As this therapy improved lung functions with repeated high dosage in the worst lung manifestations and played a role to lessen the degree of deterioration, it is a remarkable discovery for researchers regarding their scope to work more and improve the techniques of gene therapy.
Conversely, in cystic fibrosis, the defected gene has been recognized and clinical trials using DNA as an aerosol into the lung has been done. But what is still unclear is that whether the healthy gene by this method will reach the correct type of cell and will be able to express properly or not.
To conclude, while these clinical trials regarding gene therapy have revolutionary effects, there are still a few gaps that need to be filled for future clinical practices. Gaps regarding efficacy of gene therapy include gene manipulation with potentiators, more potent gene transfer vectors, and gene maximum expression at targeted cells.
It should be noted that this study using non-viral vector strategy of gene therapy provides a viable treatment. It can be considered as a breakthrough in the development of gene therapy treatment for cystic fibrosis, but for clinical practice further large trial compensation and gaps in study should be considered.
Researchers are planning and working on wave 2 product i.e. using lentivirus as a vector with modifications and safety measures to be used in clinical trials in future.

CONCLUSION:
Cystic fibrosis is a genetic autosomal recessive disorder, caused by mutation of CFTR gene i.e. cystic fibrosis trans-membrane conductance regulator gene that encodes a chloride ion channel protein. Mutant CFTR gene leads to decreased chloride secretion and impaired mucocilliary clearance Cystic fibrosis affects multiple organs but main manifestations are seen in lungs, but it also affects liver, pancreas, intestines and reproductive organs. Pattern of inheritance is autosomal recessive. Management strategies include the proactive treatment of airway infection, pulmonary rehabilitation throughout illness with either antibiotics, chest physiotherapy and in worst conditions mechanical breathing support. If mechanical breathing support fails then lung transplant is necessary. Another new approach for cystic fibrosis treatment is gene therapy i.e. CFTR gene. This process involved the replacement of diseased /mutant gene with the normal and healthy one. Initial trials using viral vectors were not successful but later on cationic liposomes (non-viral) vectors were used to transfer gene by nebulizer. This clinical trial was said to be modest and variable by the researchers as they concluded that gene therapy could be a breakthrough in treatment of cystic fibrosis. Results were satisfactory but not at the level that it could be used for clinical practice because there were gaps in the study. But researchers are working hard to revolutionize the world with gene therapy treatment of cystic fibrosis and there are possibilities that these gaps are filled and a breakthrough could be made in medical world regarding treatment of cystic fibrosis.
REFERENCES:
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