Research > Research Programs > Molecular Therapy of Genetic Dermatoses

Research program for molecular therapy of genetic dermatoses

Epidermolysis bullosa (EB) is a genetic dermatosis which is caused by mutations in the genes coding the structural proteins of the skin. If these structural proteins are affected qualitatively or quantitatively or are completely absent, the lightest mechanical loads lead to blistering on the skin and sometimes also on the mucous membranes.

Depending on the level of fissure formation, different sub-forms are distinguished. In the mildest form – EB simplex – it comes to a cytolysis of the basal keratinocytes caused by mutations in, for example, KRT5, KRT14 or PLEC. The junctional form – EB junctionalis – is characterised by the formation of a fissure in the area of lamina lucida and is caused mainly by mutations in COL17A1 or in the coding sequences for LAM-332. The dystrophic EB is characterised by the formation of a fissure in the area of lamina densa and is caused by mutations in COL7A1.

Photos (6): R. Hametner

Research

The causal therapy of Epidermolysis bullosa is possible by eliminating the cause and the consequences of the genetic changes in the skin and the mucous membranes. Despite the intensive research worldwide, it has not yet been possible to find a form of therapy of the disease. We use the newest scientific knowledge from the gene therapy area in order to come closer to this goal.

The short-term goal is to prepare our proprietary technology "Gene scissors" by experiments on cell cultures and laboratory animals for its use on people within the framework of an ex vivo gene therapy. Based on that, a safe and efficient therapy for EB patients must be developed in the long term in collaboration with other international research teams.

In addition to the gene therapy, it is worked in the immunology area on a tolerance induction, in order to prevent the rejection of the new proteins. Another focus is the subject of cancer and wound healing. In the dystrophic form of EB there is a risk of developing aggressive squamous cell carcinomas, which is particularly high after the 20th year of the life, so that the cancer therapy is an important subject.

In addition, we try to filter out by substance screening possible alternative therapeutic molecules which have already been approved as medicaments for other diseases, in order to achieve an improvement for the patients within a relatively short period of time.

Gene therapy

Since some of the structural proteins of the skin are very large and there are also dominant mutations to be corrected, we do not use any standard cDNA gene therapy but use SMaRT (Spliceosome mediated mRNA trans-splicing). In this way, the mutated parts of the effected genes can be exchanged in a targeted manner at RNA level. As a result, the size of the repair molecules can be significantly reduced. In addition, a possibility is offered for correcting the dominant mutations, and the corrected gene remains under endogenous control, because a correction at RNA level can take place only in cells in which the gene has expressed itself. In this way, the uncontrolled over-expression is prevented.

In contrast to Cis splicing, whereby the exons are joined together from a single gene, during trans splicing the exons are joined together from different premRNAs. This process takes place also in nature – for example in trypanosomes. In humans as well, the oestrogen receptor is coded by two different mRNAs which are then spliced together. We take advantage of this techniquewith which we are channelling in the gene one premRNA – a so-called RNA trans-splice moleule (RTM), which contains a part of the gene which we would like to exchange.

The RTM binds its mutated target-RNA with the help of a specific binding domain. Through the trans-splicing process, a hybrid-mRNA is formed, in which the mutated part of the endogeneous RNA is exchanged by the WT sequence of the RTM.

Trans-splicing technologies

3´trans-splicing
For correcting the mutations in the rear part of a given gene, 3´trans-splicing is used. To this purpose, a repair molecule is constructed which is bound in intron before the first exon which we want to replace.

5´trans-splicing
For correcting the mutations in the front part of a given gene, 5´trans-splicing is used. To this purpose, a repair molecule is constructed which is bound in intron after the last exon which we want to replace.

Internal exon replacement
For correcting individual exons in the middle, the technology of internal exon replacement is used. To this purpose, the repair molecule with bound domains is used for the introns next to the exon.

For the construction of the repair molecules (RTMs), a special fluorescent screening is used. Hereby a target molecule is constructed, which consists in one half of the GFP protein and the desired target intron of the gene that is to be corrected. This target, together with a bank of RTMs, which contains different binding domains and the other half of the GFP protein, is transfected in test cells .

In the case of a functional trans-splicing reaction, the two GFP halves are added together and positive cells can be detected. The functionally bound domains are then used for the endogenous correction in the patient's cells. As a final objective, the use on the patient is planned. This must take place during an ex-vivo gene therapy. For this, stem cells are taken from the patient, are corrected in vitro, then are expanded into transplants and are transplanted in the particularly affected areas of the patient.

Current projects

Gene therapy for EB dystrophicans
3' correction in collagen 7 (COL7A1)
Gen E-Rare Project 2013-2016: I 1175-B13
Dr. Eva Murauer, Patrizia Peking, MA

Objectives:
With the help of the SMaRT ("gene scissors") technology, mutations in the rear part (3´ of exon 65) COL7A1-Gen are corrected.

Status:
Functional 3´RTMs were already transduced virally in the patient's cells. In this way, the production of collagen-VII-protein in the treated cells could be restored. In addition, we have grown in the laboratory artificial skin with the repaired cells, in which the composition of the layers was again present. (Murauer et al.2010)

Overview:
In the next step, we will transplant the skin grown from the repaired skin cells on a mouse, in order to be able to observe for a long time the properties of the skin with respect to its strength, correct layering and functionality. To this purpose, we use a so-called SCID mouse in which no rejection can be expected due to the absence of an immune system.


5' correction in a COL7A1 gene
FWF individual project 2009-2012: P22039-B12
Dr. Elisabeth Mayr, Stefan Hainzl, MSc

Objective:
The objective of this project is the construction of optimal repair molecules for the front section of the gene, which can later be used as medicament.

Stand:
Both for human and for murine COL7A1 functional molecules existwhich lead both in a cell culture and also in artificial skin, to a correction of the patient's phenotypes. (Mayr et al. 2013; Mayr et al. in preparation)

Overview:
The available molecules are still optimised in order to reduce the side effects, and they must be tested in vivo on the mouse.


Gene therapy for EB junctionalis:
Correction in the COL17A1 gene
Mag. Alfred Klausegger

Objectives:
The objective is the correction of mutations in the rear section of COL17A1.

Stand:
In one cell line with the defined mutation 4003delTC in Col17A1-Gen, various molecules were tested on the basis of the SMaRT technique. It could be shown that in this cell line with the best correcting molecule a correction efficiency of about 50 per cent could be achieved.

Overview:
Now, a primary goal is to bring the proof of the corrected protein in cell line. This constitutes a prerequisite to be able to confirm the applicability of this technology within the framework of an ex-vivo gene therapy in the further steps in an animal experiment (mouse model).


Gene therapy for EB simplex:
Correction in KRT14 gene, Dowling-Meara type
Patricia Peking, MA, Thomas Lettner, PhD, Verena Wally, PhD

Objective:
The objective of this project is the construction of an optimal repair molecule for the front section of the keratin 14 gene which has mutated in the various forms of Epidermolysis bullosa simplex. The specific characteristic here is that keratin 14 in the EB-Type Dowling-Meara is dominantly inherited, which is a challenge for the conventional gene therapy.

Stand:
The repair molecules for keratin 14 were successfully produced and tested in patient cells. Subsequently, regulatory elements were exchanged as well as changes were made in order to achieve high specificity and efficiency.

Overview:
The next step is the testing of the repair molecules in mice. To this purpose, EB cells will be treated with the repair molecule, from which then human skin will be made. These pieces of skin are then transplanted on mice, in order to be able to examine their strength, safety and stability.


Development of gene therapy for EB patients: Optimisation of the SMaRT technology for the correction of the gene collagen 17 and 7
FWF individual project 2013-2016, P25304-B19
Dr. Ulrich Koller, Clemens Hüttner, MA

Objective:
Through the optimisation of the SMaRT technology, the gene areas of the genes collagen 17 and 7 must be corrected with higher efficiency. Thereby, in particular, the focus must be placed on the exchange of internal gene regions. Until now it was possible to correct either the front or the rear gene area of of various genes that have evolved in EB. As an example, the genes collagen 7, plectin or keratin 14 can be mentioned.

Stand:
The exchange of an internal gene section of the collagen 17 gene could be simulated on the basis of a repair model developed in the laboratory. The functionality of the repair model was confirmed by cell culture experiments. This permits the identification of highly functional repair molecules which can be considered for a correction of internal gene regions of the collagen 17 gene. (Koller et al. 2011)

Overview:
This repair model should be optimised further for the collagen 7 gene and used. The best repair components must be introduced in the cells of the patient by means of retroviruses and tested by using molecular biology techniques for their functionality.

Cancer and wound healing

In patients with recessive dystrophic Epidermolysis bullosa (RDEB), the risk for developing a squamous cell carcinoma is significantly higher. This can already happen at younger age and is a dangerous complication. Due to the high aggressiveness of the carcinoma, the traditional therapy possibilities such as chemotherapy or radiation therapy were less successful until now.


Cancer therapy for recessive EB dystrophicans
Dr. Christina Gruber, Dr. Ulrich Koller

Objectives:
We have developed a new alternative therapy based on the SMaRT technology, at which toxins were introduced in the tumor cells leading to the death of the cells.

Stand:
Marker genes, which function as ideal target structures for the SMaRT technology, were identified. Specially constructed RTMs can dock on these target structures and then introduce a provided toxin into the cells. Through the introduction of the toxin, the cells produce their own cytotoxin and thus kill themselves. These so-called marker genes do not exist in healthy cells, which results in a high specificity for the tumour being guaranteed. The functionality and specificity of these suicidal RTMs was already shown in a cell culture. (Gruber et al. 2011)

Overview:
We try with a specially developed screening method to improve the efficiency of the molecules and obtain higher tumour specificity. In addition, further in-vivo experiments on a mouse are planned.

Immunology

Induction of immunological tolerance

Dr. Iris Gratz (leave of absence), Dr. Victoria Reichl (on leave of absence since autumn 2013), Dr. Ana Sancho (on leave of absence since autumn 2013), Sophie Kitzmüller, MA

This project is important for persons who can not produce at all a certain protein in their skin due to null mutations of a single gene (e.g. missing Type 17 collagen in the case of junctional Epidermolysis bullosa, JEB). If gene therapy is performed on an affected person, as a result a new protein (Type 17 collagen) is produced in the skin of this patient. Thereby it can happen that the immune defence system of the affected person considers the new protein as a foreign body and as a danger. In this case, all cells that that successfully underwent gene-therapy are rejected and thus the gene therapy remains without any effect.

Objective:

The goal of this project is to prepare the immune system of the EB patient in such a way that after the successfully performed ex-vivo gene therapy the newly introduced, foreign protein is accepted and no defence reaction (rejection of the transplant) takes place.

Stand:

We have established a mouse model for the simulation of ex-vivo skin gene therapy in order to test the effectiveness of our approach for tolerance induction. The used transplant donors can produce the human Type 17 collagen in their skin. The recipient mice are treated correspondingly before the transplantation in order to prevent the rejection reaction to Type 17 collagen. On the basis of the pre-treatment, it is possible for us to increase the acceptance of the transplant also with respect to the new skin protein human collagen 17 from 0 per cent to 80 per cent.

Overview:

The results of this project serve as a base for the development of a specific pre-treatment of persons with Epidermolysis bullosa prior to performing ex-vivo skin gene therapy (targeted induction of immunological tolerance), in order to slow down very much or to prevent completely the action of the defence mechanisms of the body against the now functional protein.

Therapeutic target proteins

In order to provide relief for the patient as quickly as possible, we research in parallel with the causal therapy a therapy with small amounts of active substances ("small molecules"). There are often active substances which are already approved for other diseases, but they could help our patients as well. These medicaments are quickly available and can be used. To this purpose, we are searching for possible candidates and then test the medicaments on patient cells. These alternative therapeutic applications can in relatively short time become important means for achieving a relief in the expression of the EB.

EB simplex: Identification of alternative therapeutic target proteins
Dr. Verena Wally, Thomas Lettner, MA

Objective:
The changes in the keratin 14 gene have many effects on other genes of the affected cells. For example, inflammation paths are activated, whose blocking can be beneficial for the patients. Because of this, certain known substances are tested on EB cells in order to see whether in this way it would be possible to achieve a relief in the expression of the symptoms.

Stand:
Verboril is an approved medicament which is used for treatment of rheumatoid arthritis. Since the inflammation paths in the case of rheumatoid arthritis can also be found in the case of EBS-DM, this medicament was tested on patient cells. The active substance diacerein blocks an inflammation mediator that is responsible for some of the symptoms. By the treatment of the cells with verboril, but also with the pure active substance diacerein and certain antibodies, a change in the protein production sample in the direction to the healthy cells is achieved (Wally et al. 2012). This positive result was already implemented in a clinical study and the results have been published (Wally et al. 2013).

Overview:
The clinical study will be expanded and further substances are screened.

Cooperation

Baum Christopher, Prof. Dr.
Medical University Hannover, Hannover, Germany

Breitenbach-Koller H, Prof. Dr.
University of Salzburg, Austria

Bruckner-Tuderman L, Prof. Dr.
Department of Dermatology, University Hospital Freiburg, Germany

De Luca Michele, Prof. Dr.
Centre for Regenerative Medicine "Stefano Ferrari", Modena, Italy

Hitzl Wolfgang, MMag. Dr.
Department of Biostatistics, Paracelsus Medical University, Salzburg, Austria

Hofbauer Peter, Dr.
Pharmacy of the University Hospital Salzburg, Austria

Krammer Barbara, Prof. Dr.
University of Salzburg, Austria

Lang Roland, Prof. Dr.
Dept. of Dermatology of the Paracelsus Medical University, Salzburg, Austria

Larcher Fernando, Dr.
CIEMAT / U714 CIBERER, Madrid, Spain

Mahnke Karsten, Prof. Dr.
University Medical Center Heidelberg, Germany

Mavilio Fulvio, Prof. Dr.
INSERM U634, Nice, France

Meneguzzi Guerrino, Prof. Dr.
INSERM U634, Nice, France

Mitchell Lloyd, Dr.
Retrotherapy LLC, Bethesda, Maryland, USA

Muss Wolfgang, Dr.
Dept. of Pathology, University Hospital of the Paracelsus Medical University, Salzburg, Austria

Redl Heinz, Prof. Dr.
LBI for Experimental and Clinical Traumatology, Austrian Cluster for Tissue Regeneration

South Andrew, Prof. Dr.
Division of Cancer Research, University of Dundee, UK

Thalhamer Josef, Dr.
University of Salzburg, Austria

Wang Wenxin, Dr.
NFB, NUIG Galway, Ireland

Weiß Lukas, Dr.
University Hospital of the Paracelsus Medical University, Austria

Wiche Gerhard, Prof. Dr.
University of Vienna, Austria

Wolkersdorfer Martin, Dr.
Pharmacy of the University Hospital Salzburg

Yancey Kim, Dr.
University of Texas Southwestern Medical Center, Dallas, Texas, USA

Academic distinctions and prizes

2012

ESDR Travel grant for the 42th Annual ESDR (European Society for Dermatological Research) Meeting in Venice, Italy, 2012; Wally V. 

2011

Paracelsus Science Award "Bronze" from the Paracelsus Medical University Salzburg, Ettinger M., Koller U. 2011

Paracelsus Science Award "Gold" from the Paracelsus Medical University Salzburg, Klausegger A.

Isidor Neumann poster-price from the Austrian Society for Dermatology and Venerology within the annual meeting 2011 in Linz; Gruber C.

UNILEVER Austrian Dermatology Award from the Austrian Society for Dermatology and Venerology - OEGDV: "A novel screening system improves genetic correction by internal exon replacement"; Koller U.

Josef Kyrle travel grant from the Austrian Society for Dermatology and Venerology – OEGDV; Murauer EM.

2010

Paracelsus Science Award "Bronze" from the Paracelsus Medical University Salzburg, Gruber C., Haim M., Koller U.

Paracelsus Science Award "Silver" from the Paracelsus Medical University Salzburg, Murauer EM., Wally V.

Paracelsus Science Award "Gold" and "Scientist of the year" from the Paracelsus Medical University Salzburg, Oender K.

UNILEVER Austrian Dermatology Award from the Austrian Society for Dermatology and Venerology - OEGDV: "K14 mRNA reprogramming for dominant epidermolysis bullosa simplex"; Wally V.

UNILEVER Austrian Dermatology Award from the Austrian Society for Dermatology and Venerology – OEGDV: "Functional repair of type VII collagen expression in vitro and in vivo.", Murauer EM.

ESDR Travel grant for the 40th Annual ESDR (European Society for Dermatological Research) Meeting in Helsinki, Finland, 2010, Murauer EM.

ESDR/SID Young Fellow Collegiality Award for the 70th Annual SID meeting in Atlanta, USA, Ettinger M.

2009

Isidor Neumann poster-price from the Austrian Society for Dermatology and Venerology within the annual meeting 2009 in Salzburg; Wally V.

Anton Luger Dissertation price from the Austrian Society for Dermatology and Venerology within the annual meeting 2009 in Vienna, "A suicide gene therapie approach to treat epidermolysis bullosa-associated skin cancer"; Gruber C.

2008

Paracelsus Science Award "Bronze" from the Paracelsus Medical University Salzburg, Wally V.

Isidor Neumann poster-price from the Austrian Society for Dermatology and Venerology within the annual meeting 2008 in Vienna; Murauer EM.

Theodor-Koerner award from the Theodor Koerner Fondation Austria for the research project "Healing of butterfly children using an alternative gene therapy", Murauer EM

Paracelsus Science Award "Gold" and "Scientist of the year" from the Paracelsus Medical University Salzburg, Bauer JW

VWR International Prize from the jury of the Austrian Society for Biochemistry and Molecular Biology (ÖGBM), "5` Trans-splicing repair of the PLEC1 gene"; Wally V.

Fellowship for clinical and molecular research on Epidermolysis bullosa, Network Epidermolysis Bullosa, Gratz IK.

2007

International fellowship from the European Foundation for Research in Dermatology Fondation René Touraine; Murauer EM.

Team and contact

Univ.-Prof. Dr. Johann Bauer, MBA
Research Program in Molecular Therapy of Genodermatose
Interim head

Phone: +43 57255-57838
Tel.: +43 57255-24600

Mail: joh.bauer@salk.at
Publications
Dr. Elisabeth Mayr
Research Program in Molecular Therapy of Genodermatose
Research Coordinator

Phone: +43 (0) 57255-82413
Mail: mayr.lisi@gmx.at
Publications
Dr. Sigrid Arming
Research Program in Molecular Therapy of Genodermatose
Quality management

Phone: +43 (0) 57255-82413
Mail: s.arming@salk.at
Dr. Pamina Schlager
Research Program in Molecular Therapy of Genodermatose
Quality management (currently on maternity leave)

Mail: p.schlager@salk.at
Publications
Mag. Alfred Klausegger
Research Program in Molecular Therapy of Genodermatose

Phone: +43 (0) 57255-58294
Mail: a.klausegger@salk.at
Publications
Michael Ablinger, MSc
Research Program in Molecular Therapy of Genodermatose
Research Associate

Phone: +43 (0)57255-82407
Mail: m.ablinger@salk.at
Publications
Dr. Christina Gruber
Research Program in Molecular Therapy of Genodermatose
Research Associate

Phone: +43 (0) 57255-82412
Mail: c.gruber@salk.at
Publications
Stefan Hainzl, MSc
Research Program in Molecular Therapy of Genodermatose
Research Associate

Phone: +43 (0) 57255-80932
Mail: s.hainzl@salk.at
Publications
Dr. Thomas Kocher
Research Program in Molecular Therapy of Genodermatose
Research Associate

Phone: +43 (0) 57255-80932

Publications
Dr. rer. nat. Ulrich Koller
Research Program in Molecular Therapy of Genodermatose
Research Associate

Phone: +43 (0) 57255-80930
Mail: u.koller@pmu.ac.at
Publications
Dr. rer. nat. Mag. biol. Eva Murauer
Research Program in Molecular Therapy of Genodermatose
Research Associate

Phone: +43 (0) 57255-80930
Mail: e.murauer@salk.at
Publications
Josefina Pinon-Hofbauer, Ph.D.
Research Program in Molecular Therapy of Genodermatose
Research Associate

Phone: +43 (0) 57255-82412
Mail: j.d.pinon@salk.at
Publications
Dr. Victoria Reichl
Research Program in Molecular Therapy of Genodermatose
Research Associate

Phone: +43 (0) 57255-82414
Mail: v.reichl@salk.at
Publications
Dr. Birgit Tockner
Research Program in Molecular Therapy of Genodermatose
Research Associate

Phone: +43 (0) 57255-82412
Mail: b.tockner@salk.at
Publications
Bernadette Liemberger, MSc, BSc
Research Program in Molecular Therapy of Genodermatose
Research Associate

Phone: +43 (0) 57255-80933
Mail: b.liemberger@salk.at
Publications
Oliver March
Research Program in Molecular Therapy of Genodermatose
Ph.D. student

Phone: +43 (0) 57255-80931
Mail: o.march@salk.at
Dr. med. univ. Tobias Welponer Fries
Research Program in Molecular Therapy of Genodermatose
Ph.D. student

Phone: +43 (0) 57255-82412
Mail: tobias.welponer@alumni.pmu.ac.at
Publications