SUMMARY OF RESEARCH BEING UNDERTAKEN

Epidermolysis Bullosa is a set of genetically inherited skin blistering conditions affecting 1 in 17,000 of the population. The various forms of EB Simplex are the most common with an estimated 2000 sufferers in the UK alone. The research group in Dundee led by Prof Lane are world leaders in EB Simplex research. Cadbury & Wellcome are funding other projects. In 1997 DebRA also received a grant from the UK National Lottery for a Gene Sequencer for use by this group. This is being used to identify the many different mutations in each individual case.

EB is the term given to a group of skin fragility disorders characterised by readily forming blisters in or underneath the epidermis and the loosening and shedding of the protective outer layer of the skin. Loss of this layer exposes the person to water loss and infection; repeated loss of the epidermis leads to progressive scarring and eventual loss of function due to mutilating tissue distortion produced by chronic scarring. The constant physiological trauma associated with fragile skin leads to general ill health and poor growth and a greatly reduced quality of life.

EB Simplex is the form of EB about which most is known. In EB Simplex the epidermal cell fragility is a consequence of mutations in keratin filament genes, which encode the reinforcing cytoskeleton - web like structure that prevents skin cells from collapsing and bursting under pressure.

Disorders like EB Simplex with a genetic origin are becoming increasingly important in western society, as the incidence and morbidity of other diseases is reduced by improving living standards and availability of antibiotics. A greater proportion of the general public is now becoming aware of EB so that although the incidence may not have increased, the perception of their impact certainly has.

Genetic disorders are almost by definition incurable as they arise from a systemic defect in the coding information of the body which is present in every cell: cells which regenerate to heal the damaged tissue all have the same fragility as the cells they are replacing. Therefore genetic disorders cannot be cured by direct topical medication. Gene therapy is therefore seen as the ultimate goal. However as yet there are no diseases for which gene therapy is routinely available.

Gene Therapy for Skin Disorders - EB

There are great advantages in using skin as a system in which to develop gene therapy, not only for EB related disorders. The epidermis is uniquely accessible: keratinocytes can be removed from the epidermis, grown in tissue culture and grafted back onto dermis where they can re-establish normal patterns of differentiation. They can be observed and removed again if any abnormal developments become evident. Keratinocytes are therefore unique as vector cells for gene delivery, even for non-skin disorders; their use for systemic delivery of novel gene products is already documented. Any advances in gene therapy strategies involving keratinocytes and skin cells may therefore be applicable to other disorders. This is important, as it will increase the market for findings arising from this research, thus increasing the chances of getting the pharmaceutical industry interested in the project. If the downstream stages of developing EB therapy are to be realised, serious financial input will be required that would be beyond the scope of basic research funding.

BACKGROUND TO RESEARCH

A New Approach - Transcriptional Supplementation

Traditional direct gene replacement strategies face many biological obstacles. The direct approach to gene therapy for dominant disorders such as EB Simplex would introduce a DNA construct into the affected cells which would integrate into the patient's genome at the mutant gene and substitute a normal sequence in the place of the mutant one. This "cut-and-paste" procedure must be done separately for each private mutation and has so far only been done on cells in tissue culture; expression of the introduced DNA is not very efficient or ceases after some time. There is also the risk of the site- directed sequence alterations accidentally interfering with other genes.

Another persistent problem with gene therapy for epidermal disorders arises from the rapid turnover time of normal keratinocytes. Any genetic construct introduced into basal cells would normally be shed from the epidermis in 1 - 3 weeks and the treatment would have to be repeated, unless the introduction of such reparative DNA constructs could be successfully targeted at a stem cell population from which long term regeneration of epidermis takes place. However whilst one can now enrich stem cells when isolating cells from epidermis, there are still no absolute markers for stem cells as a discrete population of cells. There is also evidence that stem cell behaviour is partly a product of the epidermal cell's environment and may therefore be a rather transient characteristic.

An alternative approach to gene therapy would be to reinforce or even bypass the faulty system without trying to repair it directly. The frequently episodic nature of skin blistering suggests that the fragility is often close to the threshold of functionality. Thus a patient's situation may be significantly improved by only a slight reinforcement of the skin cells. Most structural genes are encoded by multigene families with a degree of functional overlap between members. It may be possible to take advantage of this in gene therapy if one could induce or augment specific gene expression within a functionally related gene family. This approach is being used for models of Duchenne Muscular Dystrophy in which introduction of a modified closely related protein into cells with dystrophin mutations gives some improvement of the muscle phenotype.

SUMMARY OF RESEARCH BEING UNDERTAKEN

This research seeks to investigate a novel strategy for gene therapy for EB Simplex. The blisters caused by EB Simplex are localised and occur irregularly. This suggests that the epidermal cells are almost strong enough to withstand normal physical stress.

The aim is to test the idea that the clinical condition might be greatly improved by reinforcing the fragile cell instead of repairing it. It should be possible to switch on an extra gene of related function by introducing a small molecule into the skin cells. Such therapy would be easier to deliver and could provide a strategy which is not mutation specific (ie have to be tailored for each individual), and is therefore much cheaper than conventional gene therapy approaches.

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