Development of antisense therapy for cystic fibrosis Manipulating CFTR expression with microRNA Target Site Blockers
Dr. Magali Taulan
Dr. Magali Taulan is an associate professor at the University of Montpellier in France. Her lab has identified antisense oligonucleotides that by simple addition to culture medium without transfection reagents de-repress CFTR expression and restore anion transport to cystic fibrosis primary nasal epithelial cell cultures. The oligonucleotides work by masking microRNA binding sites in the 3’UTR of the CFTR messenger.
What is the main focus of the research conducted in your lab?
Our research is focused on understanding how expression of the gene “cystic fibrosis transmembrane conductive regulator” (CFTR) is regulated. CFTR encodes an ABC transporter of chloride ions and mutations in this gene cause cystic fibrosis (CF). This rare autosomal recessive disease afflicts approximately 1 in 3000 people of Caucasian decent and there is currently no cure for this ultimately fatal disease that also has serious consequences for the quality of life of the patients. Some of the most frequent severe disease causing mutations actually result in partially active membrane transporters and several lines of evidence suggest that increased expression of mutant CFTR might be sufficient to restore almost normal levels of chloride transport. Thus a better understanding of how CFTR expression is regulated might identify new targets for treatment of CF.
You have recently published an article that describes regulatory features that govern foetal to adult changes in CFTR expression in lung tissue. Why were you interested in this?
The CFTR gene displays a tightly regulated tissue-specific and temporal expression. It is amazing that this gene that has dramatic consequences when mutated, is poorly expressed in mature lung. Interestingly, in fetal lung, the CFTR expression is more than four-fold higher than in adult tissue. We therefore wanted to identify trans-regulatory elements responsible for the attenuation of CFTR expression in adult tissue, since this insight might provide us with ideas of how to manipulate CFTR expression in CF patients.
The first half of your paper is dedicated to the identification of transcription factors responsible for downregulating CFTR expression in adult tissue. But then you focus on regulatory features of the 3’ UTR and in particular the involvement of miRNAs. What made you focus on microRNA regulation of CFTR expression?
For us it seemed more feasible to develop antisense oligonucleotides that interphere with microRNAs regulation of CFTR expression rather than inhibit transcription factors in the nucleus. Determining the importance of inhibitory motifs in the CFTR-3’UTR was therefore a primary step for us in developing new tools for the correction of some disease-causing mutations within CFTR.
Tell us how you went about identifying microRNAs involved in regulation of CFTR expression and what you found:
We cloned the CFTR 3’UTR downstream of a luciferase reporter and observed strong repression of Luciferase activity in multiple cell lines, demonstrating the presence of in cis
repressive elements. A paper had demonstrated the role of AU-rich elements on induced CFTR expression. In addition another paper demonstrated the role microRNAs on expression of the CFTR gene in colonic and pancreatic cell lines. Bioinformatically we identified several new AU-rich elements in addition to those previously described. Computational predictions also identified potential binding sites of 13 microRNAs and interestingly some of these overlapped with the AU-rich elements. We therefore characterized the potential of these miRNAs to interact with the CFTR 3’UTR using the luciferase reporter system and a combination of microRNA mimics and Exiqon LNA inhibitors. We found that exogenous miR-505, miR-943, miR-377, miR-145, miR-384 and miR-101 decreased luciferase activity in adult pulmonary cells, but interestingly not in primary fetal cells. In addition we found that miR-145 and miR-101 were strongly upregulated in adult lung tissue compared to fetal lung tissue making them ideal candidates for therapeutic intervention.
Rather than using microRNA inhibitors to derepress CFTR you chose to use Target Site Blockers that mask specific sites of interaction between CFTR mRNA and microRNAs. Why did you select this approach?
microRNAs regulate multiple genes, so microRNA inhibitors will have pleiotropic effects. Our objective is to derepress CFTR expression specifically. In theory a correctly designed antisense oligonucleotide masking say the miR-101 target site in the CFTR 3’UTR should be able to do this with surgical precision. We tested blockers of several microRNA binding sites and found that the miR-101 target site was particularly susceptible. With a target site blockers addressing this site we were able in adult lung cells to achieve 3-6 fold increase in luciferase activity and >2 fold increase in endogenous CFTR mRNA levels.
What have you done so far to demonstrate the therapeutic potential of stimulating CFTR expression with target site blockers and did you face any particular challenges in this regard and how did you overcome them?
We evaluated the effect of the target site blockers ex vivo because CFTR mutant mice do not develop the characteristic manifestations of human cystic fibrosis. To this aim we used human nasal cells obtained from epithelia of control individuals and CF patients homozygous for a severe mutation. As these cells cannot be transfected we took advantage of the high efficacy of LNA oligonucleotides that make it possible to achieve antisense activity by unassisted cellular uptake. We simply added medium containing either control oligonucleotide or target site blocker oligonucleotides, without any transfection reagent, directly to the upper compartment of Transwell-Clear supports in which well-differentiated primary cells were cultured. miR-101 target site blockers significantly increased CFTR mRNA and protein expression in cystic fibrosis epithelia compared to the control treated cells. Importantly, anion transport was also restored in cystic fibrosis cells to levels of non-cystic fibrosis cells.
Several recent publications suggest that microRNA inhibitors are promising therapeutic agents for treatment of a wide range of different diseases such as hepatitis C virus infection, artheroschlerosis, metabolic disorders, heart conditions and different types of fibrosis. What in your oppinion is the advantage of target site blockers over microRNA inhibitors as therapeutic agents?
Our objective is to specifically increase mutant CFTR expression in CF patients. If we used say a mir-101 inhibitor to achieve this we would also derepress a high number of other miR-101 targets increasing the risk of undesired side effects of the treatment. We hope that treatment with a target site blocker will stabilize CFTR transcripts and ultimately provide enough functional protein to improve the patients’ phenotype without disturbing other signalling cascades. So in cases where a therapeutic effect can be achieved by interupting microRNA interaction with a single mRNA then I think a target site blocker is the preferred option due to its highly target specific mode of action.
How do you feel about your results so far?
We checked by different ways our results and all of our findings demonstrated the positive action of these blockers on the CFTR restoration.
What do you find to be the main advantage of the LNA™ microRNA target site blockers from Exiqon?
In our hands, in addition to high affinity, LNA antisense oligonucleotides display excellent activity upon simple addition to the cell culture – without the need for transfection reagents – which is great advantage when working with primary cell cultures.
What would be your advice to colleagues about getting started with microRNA functional analysis?
I would suggest trying several protocols and to be very meticulous in control experiments.
In your opinion what is the most important factor for a successful microRNA functional analysis experiment?
Be sure you choose the right product and be meticulous in control experiments.
What are the next steps in the current project and how do you plan to perform them?
We plan to test different microRNA inhibitors, investigate other targets found on the 3’UTR of the CFTR gene and optimize already identified target site blockers.
What are the future perspectives for this research?
We will pursue the determination of inhibitory regulators involved in the control of the CFTR gene expression. Testing our target site blockers in a more adapted and preferably animal model is of key importance. The described work was recently published in European Respiratory Journal:
Read more in Viart et al. Transcription factors and miRNAs that regulate fetal to adult CFTR expression change are new targets for cystic fibrosis.