Cystic fibrosis (CF) is a genetic disorder occurring in about 1:2000 to 1:3000 people in Europe and about 1:100000 to 1:350000 in Japan
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 (for crystal structure see Fig. 2). 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 mutations 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 Medication 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
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 without viral-vectors, the side effects can be considered to be lower
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 comprised of the Hammerhead – Twinzyme - HDV cassette and various numbers of Hammerhead - repair oligonucleotide - HDV cassettes all transcribed into a single RNA. The Twinzyme and the repair oligonucleotides are released from the transcribed mRNA by the self-cleaving activity of the Hammerhead and HDV ribozymes.
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.
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.
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 (Fig. 4). 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 insert 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 either of two possible reading frames.
Both the reporter protein and the ribozymes were expressed from yeast centromeric plasmids under the control of the strong GPD promoter. A positive control expressing only the fluorescent reporter as well as a non-fluorescent negative control used for autofluorescence correction were included in the screening. Ribozyme activity was assayed by high-throughput flow cytometry.
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
According to the protocol provided to us by Darko Balke
Results from Yeast assay: Since Sanger sequencing of the cloned ribozymes proved impossible due to extensive secondary structure formation, several clones of each construct were used for all subsequent steps under the assumption that clones possessing the correct sequence would be among the most efficiently performing ones. As this led to an exponential increase in samples, we only assayed constructs expressing one copy of the repair oligonucleotide cassette. We screened the 12 CFTR ribozymes in both technical and biological replicates by high-throughput flow cytometry including a positive control expressing only the fluorescent reporter and a non-fluorescent negative control to correct for auto fluorescence. Only weak, if any, changes in GFP/mCherry fluorescence intensity could be detected (Fig. 1), indicating low cleavage and insertion efficiency. Given that we only assayed a kinetically suboptimal 1:1:1 ratio of reporter to ribozyme to repair oligonucleotide, and a repair efficiency of 5 - 10 % would suffice to completely alleviate clinical symptoms of cystic fibrosis, we selected the most promising candidates (CFTR 1C (4a), CFTR 2A (2c), CFTR 2C (1d), CFTR 2 DE T (2b), CFTR 2 DE T (3c), CFTR 2 DE C (1a 1 (a)), and CFTR 2 DE C (1c)) for a follow-up screen. While no significant change in GFP/mCherry ratio was observed, fluorescence intensities of both FPs dropped significantly in construct 2 DE C (1a 1(a)) (Figure 2), indicating dramatically reduced expression of the reporter construct. We therefore hypothesized that the ribozyme cleaves the target mRNA with high efficiency, but only a small minority of cleavage events is followed by an insertion reaction, rendering it difficult to observe. The cleaved, mRNA lacking the protective polyA-tail would be rapidly degraded, leading to the observed drop in expression. To determine the frequency of insertion events, we performed RNA-Sequencing of yeasts co-expressing the fluorescent reporter and construct 2DE C (1a 1(a)). While only 50 % of reporter construct mRNA was observed in ribozyme-expressing cells compared to the control (figure), indicating extremely high ribozyme activity, we found no evidence of insertion reactions.
Next Generation Sequencing: More exactly cells reporter as reference construct and cells with reporter and the ribozyme CFTR 2 DE C were screened with 75 Million reads of 70 bases each and data alligned to yeast BY4741s genome and the experssed reporter construct. The baseline important for comparing the data was generated by analysing the housekeeping genes oft the yeast strain with more than 1000 allignments. Th data shows that the candidate has a very high in-vivo cleavage activity, in two biological replicates at least 50% of mRNA has been cleaved without a ligation. In our target region there was in 803 reads no fragment found with the ATG insertion.
Click results: The in vitro assay was checked on a gel by scanning the fluorophore and the whole RNA by SYBR gold stain. As seen in the gel, the click reaction was not successful - so we could not see if there is a replacement by the ribozyme. With new click catalysts we have already shown a functional click of labeled RNA and habe to repeat the in vitro reaction.
In conclusion: We were able to show restored cleavage activity in vivo for non-consensus target sites involving a GA motif in the active site, instead of the usual GU. We have thus extended the hairpin ribozyme to sequences of the type YNGHHN, which were previously known to have only imparied funcionality. Furthermore, we were able to show the expression and activity of twin hairpin ribozymes in vivo, using a yeast promoter, as well as a hammerhead-HDV expression cassete. To date, the twin hairpin ribozyme was only known to cleave in vitro, which makes this not a trivial exercise, but a result in it's own right
Discussion and Outlook
Here we aimed to develop a novel, non-permanent gene therapy for cystic fibrosis by editing the CFTR mRNA using a twin hairpin ribozyme. We rationally engineered a new ribozyme targeting a fragment of the CFTR mRNA and, for the first time, showed cleavage of the mRNA to proceed at an extremely high rate in vivo. Even though no insertion events could be detected, our engineered ribozyme represents a first step towards that goal. Assuming that the rationally designed Twinzyme is able to catalyze fragment exchanges in principle, its failure to do so in vivo may result from a non-optimal ribozyme to repair oligonucleotide ratio. In theory, decreasing this ratio would result in a higher probability of a repair oligo binding to a ribozyme molecule currently engaging its substrate, thus enabling a trans-splicing reaction. This hypothesis could be easily tested in mammalian cells by transfection with different amounts of plasmids expressing either the ribozyme or the repair oligonucleotide. One must also consider that the Twin-ribozyme construct, synthesized in vitro and subsequently cloned into the expression vector, could not be sequenced. Since two active sites are required to perform the insertion reaction, and in vitro DNA synthesis is known to be highly error-prone, only one of the two active sites may be functional in the 2DEC construct, enabling cleavage of the target mRNA, but not insertion of the fragment. Given that a functional version of the Twin-ribozyme is now known, high-throughput screening of several hundred clones may be performed to identify a functional candidate. Alternatively, a yeast library could be generated from the DNA pool obtained from in vitro synthesis, and functional candidates could be isolated by fluorescence-aided cell sorting (FACS).