Team:Heidelberg/project/cf
Introduction
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
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
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
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
Symptoms of Cystic Fibrosis
In the 1950s, most patients died in infancy or childhood
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
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
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
Transsplicing Ribozymes and Twinzymes
Amongst all small self-cleaving ribozymes, the hairpin ribozyme is very well characterized
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
Idea and Methodology
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 in the target mRNA 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 on its own results in a loss of cleavage and ligation activity
Assembly of Ribozymes
For the assembly of ribozymes with the repair oligonucleotide, we designed a ribozyme assembly vector. This vector is comprized of the Hammerhead – Twinzyme - HDV cassette and various numbers of Hammerhead - repair oligonucleotide - HDV cassettes. The self-cleaving activity of the Hammerhead and HDV ribozymes ensures that the mature Twinzyme is free of sequences potentially capable of compromizing its activity, such as the 5'- and 3'-UTRs and the polyA-tail.
The ribozyme assembly vector was designed by inserting a pCat promoter into the pSB1C3 plasmid along with a new cloning site containing restriction sites for insertion of the ribozyme and the repair oligonucleotide. By transferring 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
Sequencing of the assembled ribozyme construct proved extremely difficult, most likely due to extensive secondary structure formation. Neither one of several commercial sequencing services nor any other laboratory could obtain sequences of the plasmids. We therefore decided to screen a large number of candidates for functionality under the assumption that constructs possessing a correct sequence would be among the most efficient ones. We chose yeast as a eucaryotic model due to its rapid growth and easy handling.
Reporter Assay
We designed a fluorescent reporter to measure the Twinzyme's activity. To account for differences in protein expression due to variations in plasmid copy number, a ratiometric approach utilizing a fusion of two fluorescent proteins, mCherry and sfGFP, was chosen. The two FPs are linked by a fragment from the CFTR protein containing the ΔF508 mutation followed by a frameshift test region. The Twinzyme is designed to introduce a stop codon into the CFTR fragment, thus decreasing the GFP/mCherry fluorescence intensity ratio. In case the Twinzyme does not function as expected and a frameshift is introduced, the frameshift test region will translate into a hexahistidine stretch detectable by immunoblotting. 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 yellow 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
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
Ribozyme reaction:
According to the protocol provided to us by Darko Balke