Cystic Fibrosis and Causes of the Disease
Cystic fibrosis (CF) is a recessive monogenetic disorder, which is present in every ethnicity worldwide. Especially Europeans are affected with 1:2000 to 1:3000 people exhibiting cystic fibrosis WHO-2004, while Japanese people are least affected with an incidence rate of 1:100000 to 1:350000. In Germany the incidence rate for cystic fibrosis is 1:3300, in the USA it is 1:3500. Ireland has the highest incidence rate with 1:1800 people WHO-2004.
The disease can be a result of multiple different genetic mutations. At the moment 2001 mutations, which result in CF, are known. The most abundant mutation is the ΔF508, also called Phe508del or F508del mutation (http://www.genet.sickkids.on.ca/StatisticsPage.html, Statistics Page of the CFTR mutation database [accessed 29.08.2015]), which occurs in about 70% of western, central, northern and north-eastern Europe. In Germany, the prevalence of the mutation lies between 63.5% and 74.2% depending on the region. On the Faroe Islands 100% of cystic fibrosis patients exhibit the ΔF508 mutation WHO-2004.
The mutations leading to CF occur in one particular gene: The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The CFTR-gene encodes for a transmembrane anion channel, expressed in epithelial cells. The CFTR-protein is part of the ATP-binding cassette protein superfamily. Anions, especially chloride ions, are able to pass the membrane passively along the concentration gradient. ATP helps to stabilize the open channel to establish a higher anion flux through the channel. The CFTR consists of 5 domains: Two nucleotide binding domains (NBD1 and NBD2), two transmembrane domains (TMD1 and TMD2) and one regulatory domain (RD). The CFTR-channel is moreover regulated by protein kinase A (PKA) and ATP Odolczyk-2014.
Out of the 2001 mutation leading to CF the most common ΔF508-mutation leads to a misfolding of the CFTR-protein. This leads to a faster degradation, a reduction in function and consequently a damping in cell membrane integration rate Amaral-2015. Other mutations can cause missplicing, loss of function or even completely absence of the protein Amaral-2015, WHO-2004.
Symptoms of Cystic Fibrosis
In the 1950s, most patients died in infancy or childhood Andersen-1958, in 2010 the suggested life expectancy of new-born CF-patients was 39 years MacKenzie-2010. This is caused by the manifold symptoms patients suffer from: High chloride sweat, male infertility, digestion problems and obstipation, pancreatic insuffency, as well as liver problems, leading to liver cirrhosis Andersen-1958. The most severe symptoms are the respiratory problems, which significantly reduce the life expectancy and gravely impairs the life quality of cystic fibrosis patients Andersen-1958, Cutting-2015. They are caused by thickening of mucus, which provides an ideal environment for bacterial infections Cutting-2015. These infections will be fought by the human immune system, which leads to lysis of both, bacteria and immune cells and thereby to a further thickening of the mucus by released bacterial and cellular lysis products such as free DNA, parts of the cell walls and cell membranes MacKenzie-2010. Through the development of strategies and medication to fight the disorder, the child mortality of CF-patients could be strongly reduced.
Approaches in Mediaction and Therapy
The classical approach to medicate cystic fibrosis is a mixture of nebulization therapy, physiotherapy, physical exercise, dietaries and antibiotic therapy Matthews1964. In the nebulization therapy water is evaporated and dispensed in the breathing air of cystic fibrosis patients. This results in a higher fluidity of the lung mucus. With the nebulization therapy patients can cough up the thick mucus more easily, which results in a better respiratory function. Medication, like bronchodilatic drugs, mucolytics and antibiotics, was often given into the nebula Matthews1964. Another therapy is the physiotherapy and physical exercises in which helps with the removal of the mucus by massaging the airway or by loosen the mucus through higher lung function in physical exercise Matthews1964. Dietaries were used to overcome the digestion problems and high chloride excretion Matthews1964, Haack2013. Antibiotics were often permanently used to fight lung infections, even though the bacteria showed resistances Matthews1964. Another option is the transplantation of organs, like the lungs Yankaskas1998. The classical way to diagnose cystic fibrosis is an electrical measurement of the chloride sweat Gibson1959. An early diagnosis helps to stop the progression of CF through an earlier treatment. Without the sweat test the detection of the defects in infants or young children can be hard. This especially affects patients with less severe symptoms such as chronic cough, chronic diarrhoea and malnutrition.
The used drugs to fight cystic fibrosis belong mostly to the following classes: Bronchodilatorica, mucolytics, antibiotics and enzymes. Prominent examples of CF medications are dornase alfa Jones2010 and pancreatic enzymes Haack2013, Somaraju2014. The most reknown example for a mucolytic used in CF treatment is Dornase alfa, a human recombinant DNAse (hrDNAse) Jones2010. Especially long DNA strands strongly increase the viscosity of the mucus. In the 1950s the loosening of mucus by a bovine DNAse was proved the first time Somaraju2014. The medication of CF with bovine dornase was damped because of adverse effects like bronchospasms Raskin1968. In 1990 Dornase alfa was produced for the first time. It displayed significantly lower side effects than the bovine dornase, because of the decreased immune response due to its nature as human protein Somaraju2014. There are also novel approaches to treat CF such as medication to stabilize the ΔF508-CFTR-protein to prevent it from degradation Fuller2000.
Other approaches to treat cystic fibrosis want to cure cystic fibrosis, by targeting its cause, with gene therapy. Gene therapy approaches to medicate CF can be divided in two categories: Non-viral gene therapy Alton2015 and viral gene therapy Drumm1990. Non-viral gene therapy for cystic fibrosis works via the application of the CFTR-gene. One trial for non-viral gene therapy, working via the application of a CFTR-gene liposome complex, is currently in the clinical study phase 2b. Here the patient gets a statistically relevant, but only small effect. The gene-liposome complex has been applied every 28 days for 1 year Alton2015. The non-viral gene therapy approaches show almost no adverse effects Alton2015. Viral-gene therapy also aims either to deliver the CFTR-gene into the cells Crystal1994. The two most used vectors are adeno- Crystal1994 and adeno-associated virus vectors Aitken2001. Viral vectors lead often to inflammatory responses or common cold symptoms. This is a reaction of immune system on the administration of the viral particles Crystal1994, Zuckeman1999. Also patients develop immunities against the viral particles after at least 3 applications Harvey1999.
Transsplicing Ribozymes and Twinzymes
Amongst all small self-cleaving ribozymes, the hairpin ribozyme is very well characterized Fedor2000Walter1998Müller2012, with our knowledge about it being second only to the extensive characterization of the hammerhead ribozyme Hamman2012Birikh1996. It is capable of both cleavage Gerlach1986 and ligationBuzayan1986, the mechanisms and structural requirements of which are well-studiedFedor2000Rupert2001Walter1998. This knowledge of both structure and activity of the hairpin allows us to modify and adapt it to the needs of specific goals. As of this time, numerous adaptions of the hairpin including, but not limited to trans-cleavage enabled ribozymes Péret-Ruiz1999Hampel1989, efficient ligases Paul2002Ivanov2005Kazakov2006Vlassov, combinations of bothDrude2007Balke2014Schmidt2000 and ribozymes controlled by the addition of ligandsMeli2003Najafi-Shoushtari2006. Also, the hairpin ribozyme itself has been redesigned numerous times, based on structural data, yielding different or even more efficient versions, by stabilizing its active conformation Komatsu1995Komatsu1996Komatsu1997a, and expanding its repertoire of cleavable sequences by the readjustment of tertiary interactionsDrude2011Balke2014. The hairpin ribozyme itself consists of two stems, each containing a bulge loop, termed A and B respectivelyMüller2011, where loop A contains the cleavage site, with an extended consensus sequence YNGUHN Balke2010, and loop B establishes tertiary interactions vital to ribozyme activity Kath-Schorr2012HeldenbrandRupert2001Pinard. From a map of those interactions, as given in Sumita2013 and a target for cleavage and ligation, one could in principle use structure-based design to engineer a corresponding trans-acting ribozyme to catalyse that reaction. Furthermore, the hairpin ribozyme has been shown to be capable of working as multiple copies in tandem, either to both self-cleave and trans-cleaveKomatsu1997b, or to trans-cleave and ligate a target strand at multiple sitesSchmidt2000Balke2011Balke2014Drude2007Welz2000. To this end, one can use both the standard version of the ribozyme, and the reverse-linked ribozymeKomatsu1995Welz2000Schmidt2000Balke2011Balke2014Drude2007, which allows for additional flexibility when designing these so-called 'Twin Ribozymes' or 'Twinzyme'.
Using Twinzymes as Therapeutical Approach
Twin ribozymes are a new application for the treatment of cystic fibrosis. Through mRNA-editing, we could establish a ribozymal gene therapy and revolutionize the treatment of CF. Compared to the viral and non-viral therapy our ribozymal gene-therapy/mRNA-editing has several benefits. Like the non-viral therapy most problems with the immune systems can be evaded by application through liposomes or nanoparticles, like it was done for siRNA Sioud2003.
