Applying Laboratory Breakthroughs to Treat Pediatric Skin Diseases: Part 1


Amy Paller, MD, MS

At Maui Derm 2014, Dr Paller discussed how laboratory breakthroughs have enabled us to better treat skin disorders in the pediatric population. These discoveries come from the understanding of disease pathogenesis and our seeking of targeted interventions.

New discoveries and new technologies, e.g., gene sequencing, and analysis of RNA and protein expression, are increasing our understanding of skin disease and aiding in the development of new therapies.

Unraveling the Cause of Monogenetic Disorders

Discoveries from Sanger sequencing revolutionized prenatal diagnoses such as amniocentesis, chorionic villus sampling, and more recently for a limited set of disorders, maternal blood sampling. Preimplantation diagnosis is a technique that utilizes in vitro fertilization to pull out a single egg, extract the DNA, and distinguish whether or not the fertilized egg is normal or not and, thus, implant the eggs that will be normal.

Most dermatologists are familiar with Deep-Sequencing or “next-generation” sequencing technology, which is transforming our understanding of genetic diseases and how we can treat them.

Can next-generation sequencing help patients?

By simultaneously analyzing all candidate genes, we can detect low frequency mutations in known genes, mutations in newly associated genes, and low frequency SNPs in thousands of samples. We can also find genotype-phenotype correlations based on alterations in multiple genes. We can analyze epigenetic changes like methylation at base-pair level, histone modifications and protein-DNA interactions. This may provide us with a greater ability to predict responses to medications and personalized gene therapy.

Technologies in Trials Today

Gene therapy: Ex-vivo cell therapy

Researchers at Stanford are using this technology for recessive dystrophic epidermolysis bullosa (RDEB) in adults. One potential risk of ex vivo gene therapy is that the introduced protein may be viewed by the immune system as foreign, leading to a problem with the creation of autoantibodies. At Stanford, the researchers are only accepting patients (~60% of individuals with RDEB) who have the N-terminal domain of collagen VII—the immunogenic domain. This decreases the chance of the patient developing an autoimmune disorder. In these studies the concern about insertional oncogenesis (ie, that the viral insertion, which is random, could lead to interruption of an important tumor suppressor gene) is mitigated by the fact that any tumor that might developed would be highly visible and easily managed. On question is whether spraying the corrected keratinocytes onto a wound bed could be a substitute for grafting skin; this techniques has been used to introduce normal keratinocytes into graft sites for burn patients (Gerlach et al. Burns 2011;37:e19).

Stem Cell Therapy

Several years ago, the University of Minnesota began pioneering bone marrow transplants on children with RDEB, leading to evidence of incorporation of autologous stem cells, deposition of collagen VII at the dermal-epidermal junction, and some improved healing. Subsequent studies have used newer techniques to decrease the risk to patients and increase efficiency, including extending the therapy to junctional EB (JEB) patients. It is important to note that this procedure still carries a risk with variable success and slow improvement.

Revertant Mosaicism: “Natural” Gene Therapy

This term refers to a spontaneous correction of a loss-of-function mutation, leading to “carrier” phenotype. This has been seen extensively in patients with JEB because of mutations in collagen XVII, but has now been described in several other genetic disorders of the skin. This “natural gene therapy” may lead to expanding a small biopsy of patient cells to introduce as a graft or through use of iPS cells (see below) without requiring immunosuppression because the cells are otherwise the patient’s own cells.

Induced Pluripotent Stem Cells (iPS)

iPS cell technology allows one to take any cell (e.g., keratinocytes, fibroblasts) and de-differentiate it into a stem cell using well-known factors. You can then use other factors that will differentiate it into any type of cell that you want. As noted above, with regards to revertant mosaic areas, you can take those cells directly and turn them into stem cells and give someone back his own cells.

Many genetic diseases are dominant negative disorders, i.e., a genetic change in one allele is enough to disrupt function. Small interfering RNA (siRNA) is an approach to suppress the express of a target gene and has already been described in a human trial as a viable means to suppress gene expression and lead to clearance in pachyonychia congenita (Leachman et al. Mol Ther. 2010;18:442). siRNA specifically directed against the KRT6a mutation in a patient was injected into a tiny area of the plantar keratoderma using a left-right, double-blind analysis. Despite clear improvement, the injections were very painful, emphasizing the need for improved techniques for the delivery of genetic material through the epidermal barrier. Protrusion array devices utilize microneedles, often dissolvable, as a painless way to deliver genetic material or protein through the barrier. New creams that can be applied to the skin, including with the use of nanotechnology, are also under development as a means to deliver siRNA. TALENs and CRISPrs induce site-specific, double-stranded DNA breaks, then allow homologous-directed repair with the normal sequence. Although early in development, these techniques hold the promise of delivery of the normal sequence without the risk of random insertional oncogenesis.

Proteomic arrays (amino acid sequences encoded by mass spectrometry) and phosphoarrays (screens for activated proteins.) allow us to look at proteins directly and are great for drug discovery. ELISA assays have also become more sensitive. These technologies are allowing us to understand the specific pathways that are impacted in disease, leading to the possibility of targeted erapies for our patients with skin disease. With a better understanding of genes, we can develop newer therapies based on the pathways that they affect.