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2004 PC Project Grant Award Leonard Milstone, M.D. Yale University Project Title: Inactivation of Keratin 6a by Sequence-Specific Gene Targeting 
Biography Leonard Milstone received his B.S. from Yale College and his M.D. from Yale Medical School in Connecticut. He performed his Internship at the University of Portland Medical School Hospital where he began his Dermatology Residency. He worked with Dr. Joram Piatigorsky at the NICHHD Bethesda Laboratory of Molecular Genetics as a Research Associate before completing his residency at Yale-New Haven Hospital. He also spent a sabbatical year working with Dr. Elaine Fuchs at the University of Chicago in Illinois.
He is currently a Professor in the Department of Dermatology at Yale University as well as an Attending Physician of Yale-New Haven Hospital in Connecticut. He was previously Attending Physician and Chief of Dermatology Service at Veterans Affairs Medical Center.
He has been an Assistant editor and is currently Associate Editor for the Journal of Investigative Dermatology. Additionally he is Assistant Editor for the Journal of Dermatologic Science and a NIAMS as hoc grant reviewer.
His research interests focus on the biology of normal and abnormal keratinocytes. He studies disorders of keratinization in patients who have disorders of keratinization and, in the laboratory, in animal models and cultured epidermal keratinocytes. Active projects in the lab involve 1) molecular targeting of genes to correct specific gene defects; 2) dermatoremediation of systemic disease, for example by harnessing epidermal desquamation to treat iron overload.
Abstract Pachyonychia congenita is a rare skin disease that can be quite debilitating in some individuals. It is usually caused by mutations in one of three keratin genes KRT6A, KRT16 or KRT17. Pachyonychia congenita is unusual in that experiments in animals suggest that elimination of the mutant keratin protein might be curative, since other related proteins can assume the function of the eliminated protein. Therefore, the long-term goal of this project is a treatment strategy that targets the KRT6A gene so that it is never translated into protein. Furthermore, if the induced change in the gene is permanent, then repeated use of even a low efficiency method might achieve a beneficial result.
It is known that short pieces of DNA can be used to direct small changes in the DNA of cells. Unfortunately, this is usually a very rare event in mammalian cells. A method has been described recently, which has the potential to increase the frequency of such changes to levels that might be clinically significant. This method is called triplex-mediated DNA modification. The key reagent is called a triplex-forming oligonucleotide (TFO), a short piece of DNA that forms triple-stranded DNA.
The specific aims of this project are to use short pieces of DNA to introduce permanent changes in the keratin 6A gene and then to show that such altered genes can no longer make a mutant protein. The hypothesis we will test is that TFOs significantly increase the frequency of desired gene alteration. |
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