Difference between revisions of "Team:Heidelberg/project/cf"

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 <x-ref>WHO-2004</x-ref>, 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 <x-ref>WHO-2004</x-ref>.

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 <x-ref>WHO-2004</x-ref>.

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 <x-ref> Odolczyk-2014</x-ref>.

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 <x-ref>Amaral-2015</x-ref>. Other mutations can cause missplicing, loss of function or even completely absence of the protein <x-ref>Amaral-2015</x-ref>, <x-ref>WHO-2004</x-ref>.

Symptoms of Cystic Fibrosis

In the 1950s, most patients died in infancy or childhood <x-ref>Andersen-1958</x-ref>, in 2010 the suggested life expectancy of new-born CF-patients was 39 years <x-ref>MacKenzie-2010</x-ref>. 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 <x-ref>Andersen-1958</x-ref>. The most severe symptoms are the respiratory problems, which significantly reduce the life expectancy and gravely impairs the life quality of cystic fibrosis patients <x-ref>Andersen-1958</x-ref>, <x-ref>Cutting-2015</x-ref>. They are caused by thickening of mucus, which provides an ideal environment for bacterial infections <x-ref>Cutting-2015</x-ref>. 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 <x-ref>MacKenzie-2010</x-ref>. 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 <x-ref>Matthews1964</x-ref>. 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 <x-ref>Matthews1964</x-ref>. 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 <x-ref>Matthews1964</x-ref>. Dietaries were used to overcome the digestion problems and high chloride excretion <x-ref>Matthews1964</x-ref>, <x-ref>Haack2013</x-ref>. Antibiotics were often permanently used to fight lung infections, even though the bacteria showed resistances <x-ref>Matthews1964</x-ref>. Another option is the transplantation of organs, like the lungs <x-ref>Yankaskas1998</x-ref>. The classical way to diagnose cystic fibrosis is an electrical measurement of the chloride sweat <x-ref>Gibson1959</x-ref>. 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 <x-ref>Jones2010</x-ref> and pancreatic enzymes <x-ref>Haack2013</x-ref>, <x-ref>Somaraju2014</x-ref>. The most reknown example for a mucolytic used in CF treatment is Dornase alfa, a human recombinant DNAse (hrDNAse) <x-ref>Jones2010</x-ref>. 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 <x-ref>Somaraju2014</x-ref>. The medication of CF with bovine dornase was damped because of adverse effects like bronchospasms <x-ref>Raskin1968</x-ref>. 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 <x-ref>Somaraju2014</x-ref>. There are also novel approaches to treat CF such as medication to stabilize the ΔF508-CFTR-protein to prevent it from degradation <x-ref>Fuller2000</x-ref>.

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 <x-ref>Alton2015</x-ref> and viral gene therapy <x-ref>Drumm1990</x-ref>. 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 <x-ref>Alton2015</x-ref>. The non-viral gene therapy approaches show almost no adverse effects <x-ref>Alton2015</x-ref>. Viral-gene therapy also aims either to deliver the CFTR-gene into the cells <x-ref>Crystal1994</x-ref>. The two most used vectors are adeno- <x-ref>Crystal1994</x-ref> and adeno-associated virus vectors <x-ref>Aitken2001</x-ref>. 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 <x-ref>Crystal1994</x-ref>, <x-ref>Zuckeman1999</x-ref>. Also patients develop immunities against the viral particles after at least 3 applications <x-ref>Harvey1999</x-ref>.

Transsplicing Ribozymes and Twinzymes

Amongst all small self-cleaving ribozymes, the hairpin ribozyme is very well characterized <x-ref>Fedor2000</x-ref><x-ref>Walter1998</x-ref><x-ref>Müller2012</x-ref>, with our knowledge about it being second only to the extensive characterization of the hammerhead ribozyme <x-ref>Hamman2012</x-ref><xref>Birikh1996</x-ref>. It is capable of both cleavage <x-ref>Gerlach1986</x-rev> and ligation<x-ref>Buzayan1986</x-ref>, the mechanisms and structural requirements of which are well-studied<x-ref>Fedor2000</x-ref><x-ref>Rupert2001</x-ref><x-ref>Walter1998</x-ref>. 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 <x-ref>Péret-Ruiz1999</x-ref><x-ref>Hampel1989</x-ref>, efficient ligases <x-ref>Paul2002</x-ref><x-ref>Ivanov2005<x-ref><x-ref>Kazakov2006</x-ref><x-ref>Vlassov</x-ref>, combinations of both<x-ref>Drude2007</x-ref><x-ref>Balke2014</x-ref><x-ref>Schmidt2000</x-ref> and ribozymes controlled by the addition of ligands<x-ref>Meli2003</x-ref><x-ref>Najafi-Shoushtari2006</x-ref>. 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 <x-ref>Komatsu1995</x-ref><x-ref>Komatsu1996</x-ref><x-ref>Komatsu1997a</x-ref>, and expanding its repertoire of cleavable sequences by the readjustment of tertiary interactions<x-ref>Drude2011</x-ref><x-ref>Balke2014</x-ref>. The hairpin ribozyme itself consists of two stems, each containing a bulge loop, termed A and B respectively<x-ref>Müller2011</x-ref>, where loop A contains the cleavage site, with an extended consensus sequence YNGUHN <x-ref>Balke2010</x-ref>, and loop B establishes tertiary interactions vital to ribozyme activity <x-ref>Kath-Schorr2012</x-ref><x-ref>Heldenbrand</x-ref><x-ref>Rupert2001</x-ref><x-ref>Pinard</x-ref>. From a map of those interactions, as given in <x-ref>Sumita2013</x-ref> 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-cleave<x-ref>Komatsu1997b</x-ref>, or to trans-cleave and ligate a target strand at multiple sites<x-ref>Schmidt2000</x-ref><x-ref>Balke2011</x-ref><x-ref>Balke2014</x-ref><x-ref>Drude2007</x-ref><x-ref>Welz2000</x-ref>. To this end, one can use both the standard version of the ribozyme, and the reverse-linked ribozyme<x-ref>Komatsu1995</x-ref><x-ref>Welz2000</x-ref><x-ref>Schmidt2000</x-ref><x-ref>Balke2011</x-ref><x-ref>Balke2014</x-ref><x-ref>Drude2007</x-ref>, 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 <x-ref>Sioud2003</x-ref>.

Idea

Over the last decades, numerous new techniques for the editing of DNA inside living cells have been developed. Tools like Cas9-based homologous recombination enable synthetic biologists to locate targets inside the genome and change complete sections of the DNA with greater than ever ease. Many chronical and hereditary diseases are caused by mutations in the DNA genome that are transcribed into mRNA and translated into altered proteins. As they are not able to fulfill their task correctly, the metabolism of the cell, and in the end the wellbeing of the patient is infringed by these changes. This challenge in mind, the new DNA editing tools gave hope to repair specific regions in the genome that are responsible for the malicious phenotypes. Despite vast improvements in the ability to target virtually any gene, the DNA editing gene therapy still has to overcome many challenges and and mediate certain risks. The strategy of changing the cells’ DNA requires the system to be highly reliable. A genetic instability caused by the unwanted insertion of the therapeutic gene can create permanent damage and may lead to an increased cancer risk or even turn out fatal. A promising way to circumvent this risks, however displaying the full potential of curing a disease by repair of the damaged sequences, is given by the idea of RNA gene therapy. In this strategy the required correction is done on mRNA level. As this type of therapy can be applied transiently, the side effects can be considered to be lower. (QUELLE) The straightforward strategy to change mRNAs relies on the use of mRNA editing ribozymes like trans-splicing ribozymes or highly engineered RNA cleaving and ligating systems (QUELLE).

Construction of Ribozymes

For our goal of treating cystic fibrosis, specifically repairing the deletion of phenylalanine 508, which is the prevalent cause for the disease, we engineered a series of Twinzymes to specifically excise and replace the affected RNA sequence. To this end, we selected cut sites, with a distance of 20bp at most, as distances above that cause the base-pairing between substrate and ribozyme to be too strong to permit efficient dissociation of the cleavage product. That constraint could potentially be relaxed by the introduction of mismatches between ribozyme and substrate. As no sites corresponding to the extended consensus sequence were found adjacent to the δF508 deletion site under said constraint, we used structure-based design to engineer ribozymes corresponding to target sites close to the consensus, differing by a substitution of the active site U by A. This mutation, if left untreated results in a loss of cleavage and ligation activity<x-ref>Péret-Ruiz1999</x-ref>. Taking into account the interaction map of the ribozyme<x-ref>Sumita2013</x-ref>, we choose nucleotides on the ribozyme strand, which most probably generate those interactions with the target strand. We do this for two target sites, conforming to the constraint of maximum distance of 20bp. A second stem with loop B is then added, which is either classical, or reverse-linked, depending on how much distance is left to the active site. If the distance is short, we choose the reverse-linked design. Next, we add a sequence complementary to the target in its edited state in between our ribozymes, linking them to form the Twinzyme. If we want to insert bases, there will be a bulge on the ribozyme strand, where the bases are to be inserted <x-ref>Balke2014</x-ref>. If we want to delete bases, there will be a bulge on the substrate strand, where bases are to be deleted<x-ref>Drude2007</x-ref>. Following these easy design principles, one can target any sequence compatible with the extended consensus. For sequences outside that consensus, interaction maps and crystal structures have to be consulted.

Assembly of Ribozymes

For the assembly of ribozymes with the repair oligonucleotide, we designed a ribozyme assembly vector. This vector comprises the Hammerhead – Ribozyme - HDV cassette and besides that various numbers of Hammerhead - repair oligonucleotide - HDV cassettes.

To achieve this, we inserted a pCat promoter with a new cloning site in the pSB1C3 plasmid, with restriction sites for insertion of the ribozyme and the repair oligonucleotide. By recloning this cassette to yeast or mammalian backbones, expression in different chassis-systems is possible. In order to make insertion of multiple repair-oligonucleotide cassettes in a sequential fashion possible, we decided to use recognition sites of isocaudomeric restriction enzymes. See fig.

Ribozyme Assay Design

For screening a large number of candidates and getting the most effective ones we designed a screening assay based on a reporter construct. This is necessary as the very strong secondary structure of the ribozymal DNA renders sequencing of the constructs highly difficult. During the validation process we started for verification of the sequence, we tried many optimization conditions, yet unfortunately were unable to acquire a sequence. After the sequencing could not be performed by large sequencing companies, we tried different other labs and industrial groups, but despite the effort those we asked put into it, no sequence could be obtained. Consequently, we worked with multiple replicates per sample in each step, as we could not discard any sample without risk. This lead to a very large number of samples we needed to validate in yeast and hence was very time-consuming.

Reporter Assay

In order to visualize the ribozyme reaction, a fusion protein consisting of four parts has been designed as test-construct. The first part is a mCherry that emits red light when excited with UV-light. The second part fragment from the CFTR gene, which exhibits the ΔF508-deletion. Therefore the construct is called CFTR-test-construct. The third part is a frameshift test region. When no frameshift occurs, this region works as a short linker between the CFTR part and the following GFP part. In case a frameshift should be introduced in the CFTR region by the ribozyme acting upon the test-construct, the linker will be changed to a His-tag (this occurs for both frames the reading frame could be shifted to). By blotting a cell lysate, one can determine if frameshifts occurred. The fourth and last part is a GFP, which emits green light when excited with UV-light. The twin ribozymes can exchange a fragment from the CFTR-gene-region in the test construct with an insert and thereby add three bases. In our test assay we want to insert a stop codon in the position of the F508 with mRNA editing. Hence we predict the following possible outcomes of the reaction:

Controls:
Several controls were used. First a fluorescence positive control, that is transformed with the CFTR-test-construct, but not with ribozymes and inserts. The second control is a full negative control without the CFTR construct or the ribozymes.

Ribozymes that exchange the region properly:
The first (and desired) scenario is a fully and properly inserted stop codon at the ΔF508-site. This would result in no translation of the mRNA to GFP, but a continued expression of the mCherry. Therefore the mCherry red light signal stays the same, while the GFP green light signal damps. Also on a western blot a non-frameshifted, half-length region can be seen by detecting the mCherry with a proper antibody.

Ribozymes are unable to insert:
The second outcome that can be expected is that ribozymes are cutting, but not inserting the corresponding insert. Here the GFP signal decreases in response to the cutting, but the mCherry signal stays the same. We therefore get false positive results when the cells are only examined in FACS or under a fluorescence microscope. To check if the ribozymes have an insert, we performed …

Ribozymes are producing a frameshift:
The third scenario is that the ribozymes can cut and insert effectively, but insert 1-2 bases away from our predicted insertion site, producing a frameshift. This would result in a nonsense region in the GFP protein, but can be seen on a western blot with cell lysate by detecting the His-tag of the construct. We also would get false positive results, therefore every result that could be positive, i.e. the GFP emission is decreasing, but the RFP emission is stable, should be checked If It is exhibiting a cut site or a frameshift.

Ribozyme unable to cut:
Several of the ribozymes tested were not found to be functional at all or have a highly decreased activity. This would result in non-significant results when colonies with this ribozymes are compared with a control that is expressing the CFTR-construct only. This strong reduction or entire lack of functionality can be caused by non-optimized hairpins. We made a screening with 6 different constructs which were designed in non-double-excision and the double-excision (DE) versions, to find a candidate with a high activity. Double excision versions have a hepatitis delta virus ribozyme and a hammerhead ribozyme at one end each.

These effects can also occur in parallel, so frameshifts can also occur while the majority of the ribozyme is working or some ribozymes will exchange the fragments properly, while most will just cut the fragment out of the CFTR-test-construct. There are several model organisms the reporter assay was carried out.

Yeast

We chose yeast as an easy eukaryotic model organism for the first ribozyme assays. Thereby we are able to access the ribozyme activity in an eukaryotic environment, while we are able to screen multiple candidates due to the relatively fast growth rate and an easy transformation procedure. The yeasts were co-transformed with a p415-plasmid {Mumberg 1995 #175} containing the CFTR-test-construct and a p413 plasmid {Mumberg 1995 #175} with the ribozymes and inserts. Both, the CFTR-construct and the ribozyme and inserts were overexpressed with a GPD promoter {Mumberg 1995 #175}. The ribozymes and inserts were transcribed to the same mRNA strand and the insert is post-translationally cut out by a Hepatitis Delta Virus (HDV) ribozyme and a hammerhead (HH) ribozyme, which are located at one end each. The ribozymes had an excised version, called a double excision (DE) ribozyme and a non-excised version. DE ribozymes were also cut out by HDV and HH. The selection of the proper colonies was performed on SD-His/Leu medium and plates. As positive control, yeast that was only transformed with the p415 with the CFTR-test-construct was used, in this case no insertion of the stop codon is possible. The negative control were yeast that were transformed with an empty p413 and an empty p415 backbone. Therefore, they only show the auto-fluorescence. The measurement of the yeast fluorescence was carried out in two different ways. The first way is to use FACS to screen yeast cultures with different ribozymes for their ribozymal functionality. The second way is to measure the fluorescence under a fluorescence microscope.

Cell Culture

To establish a functional test in human cells the model cell cultures HEK (Human Embryonic Kidney Cells) and HELA (Henrietta Lacks cells) were used. The reporter assay in human cell culture mirrors the conditions in patients much more than performing it in yeast cells. The cell culture assay was made by transfection of two different constructs. First the pIRES vector with the CFTR-construct and second the pIRES vector with BFP and our ribozymes. The ribozymes that seemed promising in the yeast reporter assay were cloned in the pIRES vector. Here also DE and non-DE ribozymes were used. The positive control is only transfected with the CFTR-test-construct and pIRES + BFP without ribozymes. Negative controls are transfected cells with the pIRES +BFP without ribozymes and non-transfected cells. The cell fluorescence and thereby the functionality of the ribozymes is measured via microscopy.

In vitro Assay

As the functionality of the twin ribozyme has only been demonstrated in in vitro setups until now, an important approach to take is to test the ribozyme reaction in a gel based assay. To visualize the reaction, an azide-modified repair-oligonucleotide should be inserted in the substrate RNA (the region of the CFTR around the d508 mutation) by our ribozyme-candidates. Thereby, we want to prove that the ribozyme is not only able to excise, but also to insert the repair-oligonucleotide. This approach should be seen as orthogonal, yet complementary insofar as by either the in vitro or the in vivo approach alone, there are possible outcomes that are difficult to distinguish. By combining and comparing the results obtained by both strategies, we not only want to be able to explain what the ribozymes are able to perform and if they are suitable as possible RNA editing agents, but furthermore that the results obtained are reproducible in different systems.

Construction of internal-labeled repair-oligonucleotide-sequences:
We wanted to build a modified repair oligonucleotide, based on our repair-oligonucleotide sequences in the ribozyme vectors. To achieve this, the 27bp-long repair-oligonucleotide-part of the type 2 – ribozymes are amplified in two separate parts – with one missing G-nucleotide in the middle of the sequence. Using overhangs at the PCR primers, a T7 promoter was added to 5´ of each part to facilitate the following in-vitro transcription to RNA fragments.
After purification of in-vitro transcription, a single azide-modified G-nucleotide was added to the 3’ of part 1 by using a poly-A-poymerase. The two parts were then ligated using splinted ligation with a DNA-splint covering the site of ligation. (Conditions as described in <x-ref>Winz2012</xref>). Due to low efficiency of the performed labeling and ligation, we decided to order the repair oligonucleotides with an internal G-azide modification for using them in the ribozyme reaction.

Ribozyme reaction:
According to the protocol provided to us by Darko Balke <x-ref>Balke2012</xref>, the ribozyme reaction was performed in a 40mM TRIS buffer with 10mM MgCl2. Six different ribozymes, the belonging repair oligonucleotides and the substrate RNA were incubated in equimolar concentration at 37° for eight hours. Afterwards a FAM-alkine fluorphore was added by copper click to the G-azide. By gel analysis, we could quantify how much of the repair oligonucleotide was inserted and compare these results with in vivo work.