In vivo model systems to develop filaggrin as a drug target in atopic disease

Lead Research Organisation: University of Dundee
Department Name: Unlisted

Abstract

We recently discovered the major gene that causes eczema and a host of other conditions associated with eczema, such as allergies and eczema-associated asthma. The filaggrin gene, which makes a protein essential for skin barrier formation, is completely deactivated by genetic mutations in about 10% of the population, leading to ?leaky? skin that allows foreign material such as bacteria, allergens and chemicals to enter the body. These foreign substances are seen by the immune system and this leads to inflammation of the skin (eczema) and as a secondary event, other systems such as the lungs (asthma). The filaggrin gene is an ideal target for two different types of drugs that can eithr rescue certain defective genes and switch them back on again, or boost the activity of a gene. We have shown that these drugs can indeed rescue or boost filaggrin production in cells and skin. This grant is designed to develop animal models that closely mimic the human filaggrin defects. This will allow us to dissect out the disease mechanisms and importantly, to test the new therapy systems aimed at this gene. This project has the potential to treat over 4 million people in the UK, or 10s of millions worldwide. If administered early, such drugs could prevent the onset of eczema and associated diseases, such as allergies and certain types of asthma and hay fever.

Technical Summary

We recently discovered that loss-of-function mutations in the filaggrin gene cause atopic dermatitis (eczema) and a host of other associated atopic conditions, such as allergies and eczema-associated asthma. Filaggrin is essential for skin barrier formation and is deactivated by genetic mutations in about 10% of the population, leading to permeable skin that allows antigens, allergens and chemicals to enter the body, leading to inflammation of the skin (eczema) and other organ systems (allergies and asthma). The filaggrin gene is the best target yet discovered for drugs such as gentamicin, negamycin and others that can rescue premature termination codon (nonsense) mutations by binding to the 16S ribosome subunit and allowing the ribosome to read-through the mutant stop codon. We have shown that this class of drug an indeed rescue filaggrin production in cultured cells and skin from filaggrin-null patients. A medicinal chemistry component, in house at nearby St Andrews University, is aimed at developing one new type of low toxicity, highly efficacious readthrough compound, negamycin, for testing in cell culture and animal models of filaggrin-null atopy. We have also shown that another class of small molecules, PPAR-gamma agonists, many of which there are known, are able to increase expression of the filaggrin gene in cultured cells. Since most individuals with filaggrin-related atopy are heterozygous carriers of null-mutations, these drugs might be used to boost expression of the normal allele to therapeutic effect. Mouse models of increasing complexity and similarity to the human disease will be developed for testing both read-through and filaggrin-boosting drugs in vivo using both reporter gene and humanized phenotypic readouts. This project has the potential to find new drugs aimed at treating about 4 million people in the UK, or several 10s of millions worldwide. If administered early, such drugs could prevent the onset of eczema and atopic diseases.

Publications

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publication icon
Bergboer JG (2010) Deletion of Late Cornified Envelope 3B and 3C genes is not associated with atopic dermatitis. in The Journal of investigative dermatology

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Brown SJ (2008) Filaggrin null mutations and childhood atopic eczema: a population-based case-control study. in The Journal of allergy and clinical immunology

 
Description Clinical impact of filaggrin research in dermatology
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
URL http://www.dermquest.com
 
Description Clinical reviews
Geographic Reach Multiple continents/international 
Policy Influence Type Citation in clinical reviews
URL http://europepmc.org/abstract/MED/19589816
 
Description European Dermatology Forum
Geographic Reach Europe 
Policy Influence Type Citation in other policy documents
 
Description Discovery Partnerships with Academia
Amount £260,000 (GBP)
Funding ID DPAc 440 
Organisation GlaxoSmithKline (GSK) 
Sector Private
Country Global
Start 07/2011 
End 03/2013
 
Description Industrial collaboration
Amount £200,000 (GBP)
Organisation Wave Life Sciences 
Sector Private
Country Unknown
Start 09/2015 
End 08/2017
 
Description Industrial collaboration
Amount £1,565,511 (GBP)
Organisation Pfizer Ltd 
Department Pfizer Rare Diseases Consortium
Sector Private
Country Unknown
Start 02/2015 
End 01/2017
 
Description MRC Developmental Pathway Funding Scheme (DPFS; Dundee portfolio)
Amount £100,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2009 
End 10/2011
 
Description Project Grant
Amount £81,000 (GBP)
Organisation British Skin Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2015 
End 12/2016
 
Description Wellcome Trust Strategic Award
Amount £5,869,875 (GBP)
Funding ID 098439/Z/12/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2012 
End 07/2017
 
Title Filaggrin mutant mouse 
Description An existing mouse strain with multiple mutations, known as matted-flaky tail (maft), was characterised as part of this project grant. The filaggrin gene was fully sequenced for the first time (the mouse genome data for filaggrin is incomplete) in this mouse and the 5303delA loss-of-function mutation identified. Genotyping assays for this mutation allowed removal of additional mutations in the maft mouse by backcrossing to obtain a "pure" filaggrin-deficient mutant mouse congenic on C57BL/6. This then facilitates properly controlled studies of homozygous, heterozygous filaggrin mutant mice versus wildtype littermates on a pure congenic mouse background. We are currently backcrossing the mutation onto other mouse strains, which may lead to variations in the phenotypes observed. These mice exhibit a skin barrier deficiency leading to abnormal transcutaneous antigen priming. These mice bear the immunological hallmarks of human atopic eczema - e.g elevated biomarkers such as Th2 cytokines. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2009 
Provided To Others? Yes  
Impact First description of filaggrin mutant mice and first experimental evidence that filaggrin deficiency leads to cutaneous antigen/allergen priming: Fallon PG, Sasaki T, Sandilands A, Campbell LE, Saunders SP, Mangan NE, Callanan JJ, Kawasaki H, Shiohama A, Kubo A, Sundberg J, Presland RB, Fleckman P, Shimizu N, Kudoh J, Irvine AD, Amagai M* and McLean WHI* (2009) A homozygous frameshift mutation in the murine filaggrin gene facilitates enhanced percutaneous allergen priming. Nat Genetics 41: 602-608 [Epub ahead of print 6th April 2009]. 
 
Title Filaggrin reporter gene mouse 
Description A transgenic mouse was generated whereby a firefly luciferase reporter gene is expressed under control of the human filaggrin promoter. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact This is the first reporter gene mouse for the filaggrin gene, allowing the in vivo assay of filaggrin expression levels. This has immediate applications in determining efficacy of small molecules aimed at up-regulation of filaggrin expression for therapy in ichthyosis vulgaris, xerosis (dry skin) and atopic eczema/allergy involving genetically determined filaggrin deficiency. In addition, these mice are a valuable tool for assay of the efficacy and cutaneous delivery of gene silencing agents such as siRNA or antisense RNA applied to skin disease. They are already finding use in two of our other MRC funded projects (MRC Milstein Award G0801742; and MRC Programme grant G0802780), adding value to these grants. 
 
Title Small molecules to read through nonsense mutations 
Description Following assay development and optimisation, a high-throughput screen of a small molecule library was performed, which led to identification of a number of hit series capable of reading through nonsense mutations in genes causing a wide range of genetic disease, including filaggrin deficiency. The current lead molecule following several rounds of medicinal chemistry is ~500 times more potent than the gold standard readthrough agent geneticin, but importantly is non-toxic and has good in vivo pharmacokinetic properties. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact No public impacts at this stage due to IP issues. 
 
Title Small molecules to upregulate filaggrin expression 
Description Following cell-based reporter assay development and optimisiation, a high throughput screen of a small molecule library was performed which identified hit series which upregulate human filaggrin expression. The lead series has been confirmed to upregulate filaggrin expressin at the mRNA and protein level in a variety of primary cell systems. These molecules have potential for development into drugs to treat atopic eczema and other filaggrin-related disorders, as well as representing a new tool to study filaggrin gene regulation. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact No public impacts yet due to IP issues. 
 
Description Mouse skin barrier immunology 
Organisation Trinity College Dublin
Country Ireland 
Sector Academic/University 
PI Contribution We developed the flaky tail mouse mutant as a model of filaggrin-related eczema by devising methods to sequence the mouse filaggrin gene for the first time and identifying the 5303delA loss-of-function mutation, equivalent to many simialr mutations found in human patients with ichthyosis vulgaris and/or eczema. This allowed back-crossing of the mutation onto different mouse strains, permitting properly controlled experiments of skin barrier using these mice.
Collaborator Contribution This year, we published the first mouse model of eczema based on a mutation in the filaggrin gene (Fallon et al., Nature Genetics, 41: 602-608, 2009). We were the first to do this despite fierce international competition from the two groups in the USA, another in Germany and another in Japan. My partners at Trinity College Dublin, Prof Alan Irvine and in particular, Prof Padraic Fallon, have been instrumental in the immunological characterisation of filaggrin-deficient mice generated in the course of this MRC grant. Prof Fallon performed a number of key experiments without which we would not have been able to get this important work into print ahead of the rest of the world.
Impact This study led to the following high-impact paper: Fallon PG, Sasaki T, Sandilands A, Campbell LE, Saunders SP, Mangan NE, Callanan JJ, Kawasaki H, Shiohama A, Kubo A, Sundberg J, Presland RB, Fleckman P, Shimizu N, Kudoh J, Irvine AD, Amagai M and McLean WHI (2008) A homozygous frameshift mutation in the murine filaggrin gene facilitates enhanced percutaneous allergen priming. Nat Genetics 41: 602-608 [Epub ahead of print 6th April 2009].
Start Year 2006
 
Description Roslin 
Organisation University of Edinburgh
Department The Roslin Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Developing systems for ex vivo culture of skin.
Collaborator Contribution Provision of discarded pig skin following ova harvesting.
Impact This collaboration is helping us optimise an ex vivo skin culture system. No final outcome yet.
Start Year 2015
 
Description WaVe 
Organisation WaVe Biosciences
Country United States 
Sector Private 
PI Contribution We established an informal collaboration with WaVe Biosciences in 2014 to test their proprietary "stereopure" antisense oligonucleotides using in vitro and in vivo model systems we have available.
Collaborator Contribution None as yet but we anticipate obtaining "stereopure" oligonucleotides for testing in 2015.
Impact None as yet.
Start Year 2014
 
Description DEBRA Annual Conference, Weybridge, UK 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach National
Primary Audience Patients, carers and/or patient groups
Results and Impact More than 100 patients with the inherited skin blistering disorder epidermolysis bullosa simplex (EBS) attended 1-day meetings in Weybridge, South of England, and Staffordshire, South of England, to learn about the molecular basis of their disease and hear about progress being made in therapy development.

Owing to the success of this meeting and the preceding one in Scotland, a follow-up meeting was organised to spread this information to patients in the north of England in November 2012. These EBS patient meetings will now be an annual event.
Year(s) Of Engagement Activity 2012,2015
 
Description DEBRA UK research leadership visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Patients, carers and/or patient groups
Results and Impact Visits from DEBRA UK Research Managers and lead fundraisers to laboratories, with updates on current research and future planning.
Year(s) Of Engagement Activity 2015,2016
 
Description Open Doors Day, College of Life Sciences, Dundee 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Attended College of Life Sciences Open Doors Day where scientists explain their research to a few hundred members of the general public.
Year(s) Of Engagement Activity 2011
 
Description Press release about Pfizer Collaboration 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Press release on establishing new research partnership with Pfizer to develop read-through drugs to treat cystic fibrosis and a range of other genetic diseases:
http://www.dundee.ac.uk/research/main/news/20150206091048/978.html
National and international press coverage; local radio interviews
Year(s) Of Engagement Activity 2015
URL http://www.dundee.ac.uk/research/main/news/20150206091048/978.html
 
Description Press release about discovery of 10 eczema genes 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Press release on identification of 10 new eczema genes; local and national press coverage; local radio interviews; international web coverage
www.dundee.ac.uk/research/main/news/20151020115352/1006.html
Year(s) Of Engagement Activity 2015
URL http://www.dundee.ac.uk/research/main/news/20151020115352/1006.html
 
Description Press release about election to The Royal Society 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Press release about election to The Royal Society:
http://www.dundee.ac.uk/news/2014/professor-irwin-mclean-elected-to-royal-society.php
Several press articles and local radio coverage
Year(s) Of Engagement Activity 2014
URL http://www.dundee.ac.uk/news/2014/professor-irwin-mclean-elected-to-royal-society.php
 
Description Press release regarding Buchanan Medal 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Press release on Buchannan Medal of the Royal Society, local and national press coverage
http://www.dundee.ac.uk/news/2015/professor-irwin-mclean-awarded-buchanan-medal-by-royal-society.php
Year(s) Of Engagement Activity 2015
URL http://www.dundee.ac.uk/news/2015/professor-irwin-mclean-awarded-buchanan-medal-by-royal-society.php
 
Description Press release, Wellcome Trust Strategic Award to Dundee Dermatology and Genetic Medicine 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Media interviews about the Wellcome Trust's major funding boost for dermatology research at the University of Dundee
e.g. http://www.bbc.co.uk/news/uk-scotland-tayside-central-18883942
Year(s) Of Engagement Activity 2012
 
Description Press release, drug discovery aimed at filaggrin 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Media interviews regarding funding for drug discovery in relation to the filaggrin gene
e.g. http://news.bbc.co.uk/1/hi/scotland/tayside_and_central/7014517.stm
Year(s) Of Engagement Activity 2007
 
Description Press release, filaggrin is a major gene in peanut allergy 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Media interviews regarding the discovery of the major gene defect underlying peanut allergy
e.g. http://www.bbc.co.uk/news/uk-scotland-tayside-central-12698727
Year(s) Of Engagement Activity 2011
 
Description Press release, filaggrin mutant mouse 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Media interviews regarding the discovery of the filaggrin mutant mouse model of eczema
e.g. http://news.bbc.co.uk/1/hi/scotland/tayside_and_central/7985307.stm
Year(s) Of Engagement Activity 2009
 
Description Progress Educational Trust, London 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Health professionals
Results and Impact Prof McLean presented the filaggrin-eczema-ichthyosis story and participated in a panel discussion. This included presenting the mouse model generated in this grant, as well as preliminary data showing that filaggrin therapy is feasible, another output of this grant. The audience consisted of healthcare professionals, educators, social scientists, lay people, policymakers and media.

As a consequence of this meeting, I have been invited to participate in a TV production aimed at secondary school education (led by Nowgen - a Centre for Genetics in Healthcare, a not-for-profit organisation which is affiliated to the Central Manchester NHS Trust and The University of Manchester. Nowgen is funded by the Wellcome Trust to run a 3 year Schools Genomics Programme, which aims to influence the way that genetics is taught in schools.) In addition, I have been asked for and have provided slides from my presentation to number of genetics and other healthcare educators for inclusion in a variety of courses. I had a lengthy conversation with a science journalist from the Sunday Times about the role of skin barrier in eczema and allergy.
Year(s) Of Engagement Activity 2009
 
Description Public Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact 65 people attended the public lecture.

Invited to do a follow-up lecture in 2014
Year(s) Of Engagement Activity 2013
URL http://www.cafesciencedundee.co.uk/?p=1235
 
Description Public Lecture (British Association for the Advancement of Science) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact "Are you thick skinned enough? Dermatology Discoveries in Dundee". Audience of about 60 people, all from the Tayside region general public. 60 min lecture plus 30 min discussion.
Year(s) Of Engagement Activity 2017
 
Description Public Lecture, Comber Rotary Club, County Down, Northern Ireland. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Public talk hosted by a Rotary Club. "Studying inherited skin disease through thick and thin". Audience of about 30-40 Rotarians and interested public. 30 min lecture plus 15 min discussion.
Year(s) Of Engagement Activity 2017
 
Description Public Talk, South West Scotland 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact By direct invitation, a 45 min presentation was given to about 100 members of the public involved in fund-raising for skin disease charities, particularly relating to the filaggrin gene in ichthyosis, common dry skin and eczema/allergy, as well as outlining our efforts to develop therapies based on this gene. Lengthy discussion followed - about 15 min formal and 2 hours informal.

Small financial donation to the lab was received from this group.
Year(s) Of Engagement Activity 2010
 
Description Undergraduate Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact Undergraduate Open Day, School of Life Sciences
Year(s) Of Engagement Activity 2015