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Lay summary

EB 2012: Outcomes and Future

Every three years, DEBRA International brings together the leading EB research teams worldwide to survey progress made in developing therapies for EB and, in particular, to identify the opportunities and challenges that may arise over the next few years. This enables DEBRA International to understand expert opinion and to fine-tune its research strategy.

Following EB 2006 in Dublin, where gene therapy was centre stage, and EB 2009 in Vienna, where there was a strong showing from emerging cell therapies, EB 2012 was held in Marbella, bringing together around 120 delegates from Europe, North and South America, Australasia and Asia. In addition to scientists and clinicians, industry representatives and expert patients also played a leading role in the presentations and discussions.

These triennial conferences offer a great opportunity to see what advances have been made since the previous meeting, and EB 2012 was no exception. In his introductory speech, co-chair Prof Jouni Uitto identified some of the highlights since EB 2009, including the identification of new EB-associated genes, the increased understanding of ‘modifier’ genes which play a part in determining the severity of symptoms, and a number of new tools and technologies, such as TALENS and zinc finger nucleases, which create new opportunities for developing EB therapies. Most encouraging, of course, was the progress that is being made towards developing gene-correction and protein-replacement therapies and the emergence of early-stage clinical trials in cell-based therapies, including the use of fibroblasts, mesenchymal stem cells and bone marrow transplantation. From this baseline, the goal of the meeting was to derive a community consensus on the priorities for the next steps in EB research and therapy development.

The proceedings of the conference are currently being collated to be published in an international scientific journal; previous conferences have been reported in the prestigious Journal of Investigative Dermatology. This report is aimed at the general reader interested in progress in EB research.

Protein therapy

The first therapy area presented at EB 2012 was protein-replacement therapy with talks from Profs David Woodley and Mai Chen from the University of Southern California (USC) whose ground-breaking work in the laboratory identified the potential for this type of therapy in DEB. Prof Peter Marinkovich from Stanford University, whose team is also working on protein-replacement for RDEB in parallel with USC, presented research aimed at improving the quality of the collagen protein in preparation for scaled-up production for eventual clinical use. Dr Vitali Alexeev of Jefferson Medical College reported on very early work investigating the feasibility of protein-replacement therapy in JEB and, finally, Dr Mark De Souza of the biotech company Lotus Tissue Repair (recently acquired by pharma giant Shire) focused on the challenging pathway of getting a protein therapy for DEB to market.

Considerable grounds for optimism about the development of protein therapy as an effective treatment for EB came from these presentations, particularly for DEB. The physiology is understood: DEB is caused by the lack of a functioning level of the protein type VII collagen (C7), leading to a lack of anchoring fibrils to hold the skin layers together, and thus allowing the skin to shear. Theoretically, all that is needed is to get normal C7 molecules into the skin to strengthen it. In the laboratory, C7 has been injected into EB mouse and dog models of the human disease and both a homing of the injected protein to the sites of wounds and an increase in anchoring fibrils have been identified with no discernible side effects.

The aim now is to get protein therapy to a point where it can go to human clinical trial. As is well known, there is, rightly, strict regulation of when and how clinical trials can take place. It is felt that protein therapy may pose fewer obstacles to getting regulatory approval as no viruses or living cells are used and the protein appears to be stable and persistent. Both the USC and Stanford groups are now working on the protocols for clinical trial, are in discussion with the regulators and hope to be in trials within 12-24 months.

However, there is still work to be done. First, whilst injections into the skin of animal models have shown good effects, there are obvious limits, especially in the area of the skin treated by each set of injections. It is believed that infusing the protein into the blood circulation may be more effective, and work is continuing to develop this method of delivery. Second, it will be necessary to produce large amounts of C7 in an economic way if it is to become a viable treatment. This will require the protein to be very stable, and to remain fully functional, as it passes through the industrial processes needed to create a sufficient supply. Work is underway to identify and test methodologies to do this. Third, more work is needed to test possible toxicity and the development of antibodies to the injected protein which may reduce efficacy, and possibly safety, with the repeat injections needed over time.

On the positive side, many protein therapies are already licenced, such as insulin for diabetes, giving a track record that is encouraging. C7 would be the first structural protein (i.e. a protein which becomes part of the body tissue, unlike enzymes or hormones) to be delivered therapeutically but, in general, the manufacture and use of protein products is well understood and has gained regulatory approval in Europe and the USA.

The involvement of a commercial partner, Lotus Tissue Repair (now acquired by Shire Human Genetic Therapies), also provides both a template for us to follow for the therapy development pipeline and an understanding of the costs involved. Lotus has raised approximately US$30 million to take their work forward: the cost of manufacturing the initial supplies of protein alone is estimated at around US$6 million, for example. It is clear that, in the case of all therapies, commercial partners will be needed to provide the funding and expertise needed to get them into the clinic.

Gene correction therapy

As is well known, the first proof of principle of a gene therapy in any form of EB was in Italy in 2006 when a single patient with non-Herlitz JEB had his thighs grafted with skin grown from his own skin cells which had had the JEB gene corrected in the laboratory. Although, some 7 years on, he remains healthy and the transplanted skin strong, regulatory bodies removed permission to use the method used to correct the cells because of cancer-risk concerns that had arisen with therapies for other conditions. Since this pilot clinical trial, a number of research groups have been working on developing safer methods to correct genes which, it is hoped, will be acceptable to the regulators for clinical trials.

Presentations were made at EB 2012 by Prof Alfred Lane from Stanford University, Prof Alain Hovnanian from INSERM/ the Necker Hospital and Dr Marcela del Rio from University Carlos III, Madrid. All spoke about their different approaches for gene therapy for RDEB and their current state of readiness.

The Stanford group presentation gave a good example of how long it can take to move therapies forward from both the technical and regulatory perspectives. Approval in principle of their planned approach was given by the NIH in March 2007 but it took until August 2009 before approval could be given for the next stages as the regulators required changes to the way the gene had been corrected and amendments to the proposed protocol. Human clinical trials require that materials used must be manufactured under highly controlled standards known as Good Manufacturing Practice (GMP), and it was not until 2010 that the vector (gene-delivery mechanism) could be produced under these conditions. The Food & Drug Administration (FDA) has now consented to an initial trial involving 5 adults and it is hoped that the first patient will be grafted in early 2013.

Similarly, Prof Hovnanian updated the meeting on GENEGRAFT, an EU-funded project in which DEBRA International is a partner, which aims to conduct a gene therapy trial for RDEB using skin equivalent grafts (i.e. skin engineered in the lab to resemble the natural multiple layers of normal skin) created using a modified vector for gene-delivery which automatically inactivates once it has done its job, and is therefore expected to be safer. This is a Phase I/II trial, and therefore aims to look at both safety, and give some early measure of efficacy too. Orphan Drug Designation was awarded in March 2009 – an orphan drug is one which addresses a rare disease which has severe consequences for the patient, and designation is important because it provides support and financial incentives to those who wish to further develop such drugs for rare conditions. Work since then has concentrated on laboratory testing to optimise the efficacy of the vector and the methodology to improve the production processes and transfer them to GMP standards. Concurrently, potential participants in the trial are being evaluated with the aim of identifying 3-6 suitable patients.

Similarly, Dr del Rio spoke about her work to develop bio-engineered human skin from epidermal stem cells and fibroblasts, and possibly mesenchymal stem cells, for grafting. She hoped to be able to undertake a clinical study in the near future in a patient with severe RDEB using the compassionate use regulations, which allow as-yet unapproved treatments still in development to be given to a limited number of named patients where no satisfactory alternative treatment exists.

As with protein therapy, there are grounds for optimism about all of the approaches to gene therapy but outstanding issues still remain, in particular whether the delivery mechanism can be shown to be sufficiently safe and effective. There will always be a balance of risk versus benefit, for any treatment but, until such issues can be resolved, regulators would be unlikely to approve systemic therapies (i.e. whole-body treatments delivered via the blood circulation) based on gene therapy using viral vectors for gene correction. For the foreseeable future, only a grafting approach for gene therapy is proposed; the therapy can only be used on limited, external areas of the body and so, if problems arise, could be more safely removed However, a grafting approach also limits the benefits for EB, and cannot be used to treat internal body linings which can also shear or blister.

At the time of the conference, no gene therapy product for any condition had gained marketing authorisation (licence to be sold and used therapeutically) in the USA or Europe. However, subsequently, the first authorisation has been granted by the European Medicines Agency for a gene therapy in another condition.

Cell therapies

It was pleasing to see that the hopes for cell therapies expressed at EB 2009 were not misplaced. In this session, Prof John McGrath of Guy's & St Thomas’ Trust and Prof Jakub Tolar of the University of Minnesota spoke about their existing or planned clinical trials whilst Dr Marius Wernig of Stanford University, Prof Dennis Roop of the University of Colorado and Dr Arabella Meixner from IMBA in Vienna spoke about the longer-term possibilities of the use of induced pluripotent stem cells (iPSC) as a potential cell therapy. Dr Marjon Pasmooj of the University of Groningen discussed revertant cells as an aid to therapy in some patients and Dr Paul Kemp of Intercytex looked at the commercial issues in developing cell therapy for EB, having worked with Prof McGrath on taking fibroblast therapy first through Phase I, and more recently through a Phase II/III trial, aiming to establish efficacy of the treatment.

Existing trials for cell therapy have shown some encouraging results so far. Prof McGrath reported on his work in which he injected fibroblasts derived from foreskins of unrelated donors into a small number of patients with RDEB and observed an improvement in healing in long-established wound sites. This has been followed by the Phase II/III observer-blinded clinical trial (i.e. the clinicians don't know whether a patient has received the treatment or a placebo 'dummy treatment' at the point where they assess skin improvement, and thus avoid unintentionally biasing results). At the time of the conference, the trial was about to be unblinded and the results analysed. He also spoke about his planned clinical trial to infuse mesenchymal stem cells derived from unrelated donors. Mesenchymal stem cells might be expected to have similar benefits to fibroblast therapy, as well as known anti-inflammatory and wound-healing enhancing effects.

Prof Tolar reported on his experience of using bone marrow transplantation (BMT) to treat 18 patients with RDEB and JEB. He acknowledged that BMT is a high risk:high yield strategy, i.e. as a systemic treatment, the potential benefits are significant but there is a high risk of mortality as the body's immune system has to be suppressed to prevent rejection of the unrelated cells. He was very clear that BMT is not a cure; where it is successful the patient still has symptoms but less severe than previously, showing an increase in C7 and anchoring fibrils with increased skin stability. On the other hand, the risk of death is not negligible – the overall survival rate is currently 73%. The team have made several adjustments to their protocols to try to reduce the risk and are continuing to do so.

The situation with regard to iPSC is somewhat different in that this is much further away from human application but there is a strong theoretical potential. Essentially, the aim is to take adult stem cells and re-programme them back to an earlier development stage, resembling embryonic stem cells, so that they can be directed to develop into cells of any body tissue, including the fibroblasts or keratinocytes of skin. The anticipated advantage of iPSC over stem cells from donors is that, because they are derived from the patient's own cells, they are less likely to be perceived as foreign by the patient's immune system and rejected – once genetically corrected, they would be suitable for introduction into the patient to produce whichever protein is deficient, and thus to treat EB systemically. Dr Wernig, a leader in developing iPSC technology for diverse conditions, is currently leading a US$11.7 million programme to develop iPSC for RDEB. Dr Meixner at IMBA has worked closely with Dr Wernig, introducing the iPSC technology for developing RDEB and JEB therapies to Austria. Prof Roop is leading the development of iPSC for EBS. All have had some success in the laboratory in creating iPSCs. The challenges now are to create a means of re-programming that is safe, stable and does not cause rejection and to develop efficient methods for differentiating genetically corrected iPSCs into specific tissues.

A problem for cell therapy using the patient's own cells, which would be the preferred course, is that they need to be genetically corrected to remove the EB defect in those cells. Dr Pasmooj, representing Prof Jonkman who was on sabbatical, discussed their work on revertant cells, i.e. cells from parts of the body of some patients with EB which have undergone a second mutation and thus appear to be normal having reversed the effects of the EB mutation. Previously, this was believed to be very uncommon but, over recent years, clinicians have identified larger numbers of patients exhibiting this phenomenon, particularly in non-Herlitz JEB, but also in RDEB. In those patients, use of the revertant cells would avoid the need for genetic correction. However, the current technical challenge is to produce enough revertant cells to be clinically useful from a small skin biopsy, by improving growth and selection of revertant cells, and creating better grafts from them.

Small molecule therapy

Dr John Common, from Professor Birgit Lane's lab (The A* STAR Institute of Medical Biology, Singapore) reviewed the mechanisms underpinning skin fragility in EBS, and the prospects for developing small-molecule drug therapies. These drugs would interrupt the damaging cascade of processes by which EBS cells respond inappropriately when stressed, thus becoming very fragile. Such pathway-intervention therapy would be independent of the specific mutation and could thus treat a wide variety of EBS patients. Prof Johann Bauer of the Paracelsus University in Salzburg reviewed existing examples of early-stage work where small-molecule drugs have been used, especially in EBS, to treat symptoms. These included the use of botulinum toxin (Botox) to treat sweat-worsened foot problems, tetracyclines to repress inflammatory cytokines and sulphoraphane to enhance synthesis of alternative keratins to replace those defective or missing in EBS.

Owing to the illness of Prof McLean from the University of Dundee, he was unable to attend and give an update of siRNA therapy development for EBS. In Dundee, there is a strong Drug Discovery Unit where existing libraries of pharmaceutical compounds can be rapidly screened to try to identify any that may have a beneficial effect on EB wound healing and other symptoms. Research is underway to look at either systemic application, if this is appropriate to the mode of action of the drug, or topically, which would have advantages in being non-invasive, and easy for a patient to apply daily, or weekly in the form of creams, for example. Developing effective methods of topical application for EB, and other conditions, does remain a clinical challenge.

It was noted that there is interest from pharmaceutical and biotechnology companies in small-molecule therapy for EB and that DEBRA is funding some work on this as well. It is hoped that small-scale trials may be possible within a couple of years.

Cancer

Squamous cell carcinomas (SCC) constitute the major cause of early death in people with RDEB, so understanding why these cancers occur and why they are so much more severe in their consequences in people with EB is at the heart of DEBRA-supported research. While this seemed to be an intractable problem at EB 2006, some glimmers of understanding were appearing at EB 2009, three years ago in Vienna. Since then, some common messages are starting to emerge, notably that it is the disrupted 'microenvironment' of EB skin, and RDEB skin in particular, which promotes cancer initiation and spread. Dr Andrew South of the University of Dundee and Prof Edel O'Toole of the Royal London Hospital presented their work on the drivers causing these cancers to proliferate and spread. A number of mutations have been identified as being implicated in this and signalling mechanisms between cells are becoming better understood. The inflammation associated with chronic skin wounds trying to heal is an aspect many researchers have focused on. Several researchers' presentations pointed to a molecule, TGFbeta, known to have a role in wound healing but also cancer development, which is raised in inflamed EB skin, and orchestrates a series of changes in cells through a cascade of molecules passing messages 'cell signalling'. Dr Venu Mittapalli (Univ Freiburg, Germany) showed that TGFbeta stiffens the skin matrix, leading to invading tumours: targeting TGFbeta signalling may have a therapeutic potential and reduce the risk of RDEB-associated cancers. Dr Cedric Gaggioli (Univ Nice, France) outlined a role for other cell signalling events linking scarring which is prevalent in RDEB both to the activation of fibroblasts into becoming more invasive, and structural changes in the skin.

This work is at the level of basic research but the hope is that, once the mechanisms are understood, therapies can be developed to interfere with the signalling process, preventing SCC spread.

Translational issues

It is a fundamental element of DEBRA International's research strategy to recognise that patient groups can only take the process of therapy development so far and that, in most cases, for a therapy to be readily available in the clinic, it will require partnership with industry which has the financial resources and expertise to bring products to market. A number of pharmaceutical and biotechnology companies were represented at EB 2012 and several speakers came from this background.

These contributions reinforced the messages given to DEBRA by industry in the past; that EB has many of the attributes that companies are looking for when making investment decisions in rare diseases but that the research needs to develop further, and fill gaps in knowledge. In addition, characteristics of the condition over a lifetime need to be documented through structured clinical studies to provide validated measures on which clinical trials can be based, and to evaluate potential therapies robustly. DEBRA has initiated a study to gain better understanding of the natural history of different types of EB and, at EB 2012, brought together several research groups developing robust measures of severity and quality of life, to develop a consensus framework on such issues.

Conclusion

The outcome of the meeting in many ways reinforces the expert opinions that DEBRA has received previously. There is the potential for patient benefit in all of the therapeutic approaches discussed at the conference and we should be optimistic that all or some will come to fruition. In virtually all cases there is the need for both further basic and applied research and DEBRA will continue to be required to be a significant funder until work reaches the point where external funders see a commercial opportunity that warrants investment.

DEBRA's present estimation is that a war chest of several million dollars/ euros will be needed in 18-24 months to make significant investment in those forms of therapy that have made the furthest strides towards clinical applicability to help take them to the point where they can attract sufficient external funding.

We hope that, by the time of EB 2015, significant advances will have been made in what is available in the clinic.

John Dart & Clare Robinson
January 2013

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