June 2017 Newsletter

A brief history of the cystic fibrosis therapy…

Oscar Moran – Istituto di Biofisica, CNR, Genova, Italy

Cystic fibrosis (CF) is the most common se­vere recessive hereditary disease in Cauca­sians. In Europe, CF affects 1 in 2000–3000 newborns. CF has a complex and greatly variable clinical expression, involving airways, pancreas, male genital system, intestine, liver, bone, and kidney. Some of the disease symp­toms associated with its mortality were rec­ognised in the Middle Ages: medieval folklore predicted death for a child who tasted “salty” when kissed. Such infants were thought to be “hexed”. Indeed, in 1606, Juan Alonso y de los Ruices de Fontecha, professor of medi­cine at Alcalá de Henares in Spain, wrote that it was known that the fingers taste salty after rubbing the forehead of the bewitched child.

Dorothy Andersen of the Babies’ Hospital New York was the first doctor to give the dis­ease its earliest definitive description in 1938, introducing the term cystic fibrosis. In 1948, Sydney Farber of the Children’s Hospital Bos­ton introduced the term mucoviscidosis, that is often preferred outside the English speaking world. The gene responsible for CF was identi­fied in 1989, opening a new era of understand­ing the disease and improve the treatment. In this way, CF view has changed from been considered lethal in childhood, to the nowa­days when patients attained median survivals of 50 years of age. This net improvement has occurred thanks to the early diagnosis through neonatal screening, and an aggressive symp­tomatic therapy. But still we have no cure and no effective control of its inexorable destruc­tion of the lungs and pancreas.

The gene CFTR (cystic fibrosis transmem­brane conductance regulator) codes for a protein that expresses in the apical membrane of the epithelial cells. Its principal function is to control the flux of anions (chloride and bi­carbonate) across the membrane, defining the salt, water contents and the acidity of the fluid that lies in the surface of the epithelium (pericil­iary fluid), as the bronchial lumen or pancreatic ducts. Failure of anion transport will reduce the periciliary fluid and will produce thick mu­cus, blocking conducts and facilitating chron­ic infections in the airways. More than 2000 mutations has been identified, and the defects were classified in six classes according to their mechanism. CFTR defects can include incom­plete synthesis of the protein, protein process­ing flaws, ion transport defects.

The cure of CF must involve the correction of the CFTR defect. Several strategies have been undertaken in the last 30 years. First attempts consisted on finding cellular anion transport mechanism that could be over-activated to replace the dysfunctional CFTR. However, this approach quickly proved to be impractical because of difficulties to find a good cellular target with the appropriate pharmacology. A genetic therapy was also explored, affording the difficulties of introducing a huge DNA frag­ment into the target cells, beside the addition­al concerns regarding the use of viral vectors in human patients.

…And, Why anionophores?

The first real advance on the CFTR ther­apy was the search of a pharmacological therapy. At the beginning of the century the American CF Foundation invested a lot of money in high throughput screening (HTS) programs for searching CFTR cor­rectors and potentiators. Potentiators are those drugs capable to stimulate a dam­aged CFTR to transport anions. Correctors, also named “chemical chaperonines”, are substances able to induce a CFTR with pro­cessing defects to assemble and become functional in the cell membrane. Indeed, the most common CFTR mutation, F508del, re­sults in impairing the correct protein folding and docking to the plasma membrane. The HTS tested hundreds of thousands of com­pounds in cellular preparations to find those with the CFTR potentiator or corrector char­acteristics. The final result was a Ivacaftor, a potentiator that can be successfully used on patients having one of ten specific mu­tations. Unfortunately, patients carrying one of these 10 mutations represent less than 6% of the CF patients. Differently, correctors have demonstrated to be more difficult to find. Identification of Lumacaftor, a promising molecule that should correct the missfolding of F508del, a mutation carried by more than 60% of the patients, produced excellent results in preclinical studies. However, clini­cal trials demonstrated that treatment with Lumacaftor does not produce any improve­ment in patients. Another drug, Ataluren, has demonstrated to be efficient for CF patients with mutations that introduce a stop in the CFTR gene, resulting a truncated- non func­tional- protein. However, mutations that can be successfully treated with Ataluren are relatively frequent in Ashkenazi Jewish (9%), but represent less than 4% of the European patients.

The strategy to develop a pharmacological CF treatment targeted to defined CFTR mu­tations, even when it succeeds, covers a lim­ited population of CF patients. An exception would be to find a good drug to cure patients with the F508del mutation, but it has been unsuccessful so far. Thus, our proposal to use synthetic anion transporters, named ani­onophores, to replace non-functional CFTR, is opening new possibilities for the CF thera­py. The idea is very simple: to apply topically the anionophores in the bronchial epithelium, that is among the most hit tissue, should re­cover the anion transport, and consequently the correct periciliary fluid layer and mucus properties. One, very important, advantage of this approach is that the treatment would be independent on the mutation that pro­duces the disease, and consequently would cover nearly the entire patient population.

We started from some compounds known to transport anions across the membranes, as the derivatives of natural products as pro­digiosin or tambjamine. These substances have been proposed as immuno-suppres­sive, antibiotic, antimalarial and anti-tumoral drugs. However, chemical modifications of these compounds, and as well as other compounds belonging to different chemical families, have resulted on new molecules that maintain, or even improve, the anion transport capacity, but reduce their toxicity. We have already identified a set of com­pounds with appropriate characteristics, that represent the leading substances for drug candidates. It remains, indeed, a long way to propose a real “drugable” molecule to be introduced in a clinical test pipeline, but we are confident that our strategy, with the advantage of the multidisciplinary collabora­tion in our project consortium, will help us to reach our objectives.

WP 5-Preclinical Studies Directed differentiation and genetic modification of novel iPS cell lines from CF patients

As mentioned, Cystic Fibrosis is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regu­lator (CFTR) gene. CFTR encodes a cyclic adenosine monophosphate (c-AMP)-dependent, phosphorylation-regulated chloride channel required for transport of chlo­ride and other ions through cell membranes. The disease is characterized by abnormal fluid and electrolyte mobility across the epithelia of numerous tissues. The first mani­festations occur in early childhood, generally affecting the respiratory tract, and later extending to other organs. Al­though several animal models exist, human genetic varia­bility cannot be reproduced in them. Therefore, the genera­tion of patient-specific iPS cells would be an important tool that can be used in disease modeling and drug discovery.

We reprogrammed human skin fibroblasts and keratino­cytes from two CF patients carrying the p.F508del muta­tion and one healthy donor. Reprogramming was achieved by retroviral transduction with four (c-MYC, KLF4, OCT4 and SOX2), or three (KLF4, OCT4 and SOX2) of the clas­sic Yamanaka’s factors. The resulting iPS cells were karyo­typed and tested by immunocytochemistry for stemness. Differentiation was induced by embryoid body formation and by treatment with a defined combination of growth factors.

A total of 30 independent CF and 18 wild type hiPSC lines were obtained from the three donors’ fibroblasts and ke­ratinocytes. The lines with better morphology and prolifera­tion rates were further characterized. All of them showed strong expression of stemness markers (NANOG, OCT4, SSEA4 and TRA-1-60) and, following embryoid body for­mation, were able to differentiate into ectoderm (NESTIN, TUJ1), mesoderm (GATA4, α-ACTININ) and endoderm (α-FETOPROTEIN (AFP), SOX17). In addition, directed dif­ferentiation into foregut and hindgut endoderm, the pro­genitors of lung, liver, and intestine endoderm, was con­firmed by the presence of the FOXA2, SOX2 and CDX2 cell markers.

Our hiPSC lines can be used for disease modeling and drug testing, as well as for the development of new cell-based regenerative therapies. In addition, CF hiPSC directed dif­ferentiation into fore- and hindgut endoderm demonstrates the dispensability of the CFTR gene during in vitro early endoderm development.

Next project meeting and Workshop in Copenhagen

TAT-CF will hold its next periodic meeting in the July 2017. The meeting of the Steering Committee will include the review of the project progress, milestones and planning of actions for the next six months together with the second TAT-CF Workshop. During these workshops there will be scientific presentations by TAT-CF partners to enhance the collaboration among partners, reinforce the sense of unity of the consortium and serve as learning experience for TAT-CF recruited researchers, at an operational level. Additionally, the project will benefit from the Common Exploitation Booster service implementation, a training service set up by the European Commission which will enhance the Exploitation strategy which is being developed by the Consortium. The meeting will be hosted by Bioneer at the Faculty of Pharmacy.

TAT :CF presented at IRDiRC 3rd Conference held in Paris

TAT:CF participated in the last IRDiRC Conference held in Paris in January 2017. We presented two posters, “TAT-CF: Novel therapeutic approaches for the treatment of cystic fibrosis based on small molecule transmembrane anion transporters through open innovation” and “Facilitated transmembrane transport, a viable therapeutic approach for channelopathies?”. IRDiRC conference joints all the major relevant stakeholders in rare diseases worldwide, meaning a great opportunity to let them know about TAT:CF aims and strategies.