Developing targeted therapeutics for keratinizing skin disorders

The epidermis is the largest, most complex epithelial tissue in the human body and its mechanical integrity is vital in protecting the human body from harm. Keratinizing skin disorders are a large group of highly debilitating, difficult-to-manage hereditary skin conditions that present major treatment challenges in the clinic. Collectively these diseases affect ~1 in 2000 people, but because they are individually quite rare very little progress has been made towards developing effective treatments. The quality-of-life impacts for these patients are life-long and can be devastating, thus, the overall healthcare and societal burden is very great. This proposal aims to begin to tackle the major challenge of developing effective, long-term treatments for these conditions.

Most keratinizing disorders arise from single nucleotide mutations in one of several critically important structural molecules of the skin, which leads to the development of weakened, thick skin - epidermal fragility and hyperkeratosis. The great challenge in developing treatments for these disorders lies in selectively repairing or silencing the causative mutation. RNA interference (RNAi) is a remarkable natural cellular process that uses a unique class of molecules, called small interfering RNA (siRNA), to specifically and potently control gene activity. Harnessing the therapeutic potential of the RNAi pathway, several siRNAs that specifically target keratinizing skin disorder mutations have been identified. Unfortunately, the physical properties of siRNA molecules, they are very large and carry a negative charge, make epidermal delivery difficult. One of the major goals of this research proposal is to develop a patient-friendly way to delivery siRNA into the skin. We will use a unique in vivo reporter model, that produces a visually-trackable enzyme and facilitates real-time monitoring, to develop clinically-viable siRNA skin delivery methods. Once we have an efficient and effective mode of delivery, we will use it to test whether disease targeting siRNA treatments alleviate disease symptoms in an in vivo keratinizing skin disorder model displaying symptoms similar to the human disease. Our findings will provide the preclinical evidence required to progress siRNA therapeutics into clinical trials and, ultimately, patient prescribed treatments.

Because each condition is individually rare and multiple mutation-specific siRNAs will be required to treat each patient population, we feel that it is important to also explore the possibility to developing one common treatment for all of these disorders. New medicines aimed at a common disease feature, like hyperkeratosis, rather than the specific mutant gene, may allow treatment of several keratinizing disorders regardless of the genetic abnormality. Unfortunately, we do not understand why hyperkeratosis develops or how to stop it. Therefore, the other major goal of this research proposal is to define the molecules and identify the biological pathways that cause a single gene mutation to develop into the phenotypic end product of weak, thickened, blistering, painful skin. We will do this by examining unique in vivo keratinizing skin disorder models, that develop hyperkeratosis, using cutting edge molecular profiling techniques to assemble an in-depth inventory of the individual cellular components that are present in normal but not diseased states, and vise versa. This will enable us to identify the molecular mechanisms that trigger hyperkeratosis and begin to understand how hyperkeratosis is regulated. It is our hope that these findings will seed the development of a generic treatment for most, if not all, keratinizing skin disorders.

State-of-the-art methodologies will be used to study a group of highly debilitating inherited skin conditions called keratinizing skin disorders, for which there are no effective treatments. These diseases are caused by dominant-negative mutations in key epithelial structural molecule genes and are characterized by skin fragility and secondary hyperkeratosis. Collectively, the studies proposed here aim to develop cutaneous delivery systems for current and future therapeutics aimed at skin disorders and identify key molecular regulators of hyperkeratosis. Small interfering RNA (siRNA)-based therapeutics, which selectively inactivate the mutant allele, currently represent the most promising therapeutic intervention for these disorders, but delivering siRNA to the skin is challenging at best. Using a power combination of unique reporter and disease models, we aim to develop non-invasive ways to effectively deliver siRNA-based therapies into the skin. Combining our reporter model with innovative real-time imaging will allow us to rapidly test and fine-tune multiple siRNA formulation-based delivery approaches. The in vivo disease model will then be used to confidently assess, via phenotype resolution, whether these promising formulations effectively deliver potentially therapeutic siRNAs into the skin. This work will provide a strong preclinical data package to facilitate progression to clinical trials in the future. We will use powerful in vivo SILAC-based quantitative proteome profiling techniques to identify the active pathways, key molecular components and cellular processes altered under disease conditions in two, related in vivo hyperkeratosis models. Common key regulators of hyperkeratosis will be identified via molecular validation in an alternative in vivo disease model and a human keratinizing disorder tissue collection. These findings could spearhead the development of a generic treatment for most, if not all, keratinizing skin disorders.

This research will benefit:

1. Pharmaceutical companies:
The animal models described here are of benefit to companies seeking to test delivery of gene silencing therapies in the skin. To this end, two patents have already been filed relating to the work described here. Companies interested in using these biological tools will be encouraged to enter into licensing agreements, brokered by the University of Dundee's technology transfer agency, Research and Innovation Services (RIS). In particular, siRNA therapies, topical delivery strategies, reporter mouse models and new biological pathways uncovered may be of interest to industry. As new intellectual property arises from this project, this will be discussed with our RIS colleagues in a timely manner and further patents filed as required.

2. Other scientists:
As outlined above, other academic groups active in gene silencing therapy are likely to benefit from the output of this research.

3. The general public:
The College of Life Sciences actively engages with the local community through "Open Doors" days when the public can visit the research facilities and engage with the academic staff routinely to learn about the ongoing research activities and inspire potential students. DMM/DGEM actively participates in these College wide public engagements and my group regularly participates on behalf of the division.

4. Patient support groups:
DMM/DGEM is also actively involved in numerous charity events and patient meetings that provide support for individuals affected by keratinizing skin disorders. The division itself hosts support meetings for patients and families affected by pachyonychia congenita (PC) and epidermolysis bullosa simplex (EBS), which are jointly hosted by PC Project UK and DEBRA (Dystrophic Epidermolysis Bullosa Research Association), respectively, 2-3 times per year. At these meetings, we connect with patients to discuss ongoing research and the advances we are making towards new therapeutic strategies with the individuals that will actually benefit from our research efforts. These meetings are extremely informative and highly beneficial for researchers, clinicians, patients and their family members because the effects of living with such conditions are not often discussed. I will actively help with the organization and participate in these meeting. As our research advances rapidly towards new therapies for EPPK we are considering setting up an outreach program for the wider keratoderma community, given the numerous patients (4000-5000 people in the UK have EPPK, one of several keratodermas).

5. Patients:
In the longer term, if effective delivery of RNA or small molecule therapies arise from this project or follow-up projects downstream, these will be of enormous benefit to patients with devastating skin diseases that are presently incurable. Although these diseases are individually rare, the healthcare burden over the lifetime of these patients is considerable.