Gene Therapy for Autosomal Dominant Disorders of Keratin Alfred S. Lewin, Peter M. Glazer, Leonard M. Milstone  Journal of Investigative Dermatology Symposium Proceedings  Volume 10, Issue 1, Pages 47-61 (October 2005) DOI: 10.1111/j.1087-0024.2005.10207.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Columnar organization of the epidermis into discrete units with individual stem cells. Based onAlonso and Fuchs (2003). Journal of Investigative Dermatology Symposium Proceedings 2005 10, 47-61DOI: (10.1111/j.1087-0024.2005.10207.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Gene therapy for dominant diseases should either correct a mutant gene or suppress the synthesis of its product. Such therapies target either chromatin in the nucleus (DNA) or ribonucleoprotein in the cytoplasm (RNA). At the DNA stage, the ideal approach is gene correction, which may be mediated by oligonucleotides, DNA fragments or adeno-associated virus (AAV). Alternatively, genes may be knocked out using the same approaches or silenced using triplex-forming oligonucleotides or zinc-finger transcription factors. In the cytoplasm, antisense RNA (with RNase H) or ribozymes or siRNA can be used to digest transcripts and lead to their degradation. Alternatively, antisense DNA or triplex-forming oligonucleotides can interfere with the translation of the mRNA. Journal of Investigative Dermatology Symposium Proceedings 2005 10, 47-61DOI: (10.1111/j.1087-0024.2005.10207.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 The introduction of a premature stop codon in K6a using a triplex-forming oligonucleotide (TFO) and donor oligonucleotide. The TFO and donor oligos can be uncoupled or tethered together by a flexible linker. Journal of Investigative Dermatology Symposium Proceedings 2005 10, 47-61DOI: (10.1111/j.1087-0024.2005.10207.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Targeted gene repair using a zinc-finger endonuclease. Although this scheme shows the chromosomal sequence as the nuclease target, the donor sequence may also be cut. Journal of Investigative Dermatology Symposium Proceedings 2005 10, 47-61DOI: (10.1111/j.1087-0024.2005.10207.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 A hammerhead ribozyme specific for the F174S allele of K6a, which leads to pachyonychia congenita. The target sequence is shown in red, and the wild-type sequence is above it in black. Journal of Investigative Dermatology Symposium Proceedings 2005 10, 47-61DOI: (10.1111/j.1087-0024.2005.10207.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 An RNA replacement strategy using ribozymes. A ribozyme is designed to cleave both mutant and normal RNA, leading to the turnover of these transcripts by cellular nucleases. Silent mutations are introduced into a cDNA making it ribozyme-resistant (“hardened WT”). The genes for the ribozyme and the hardened wild-type are introduced together. This same strategy can apply to siRNA induced RNA interference. Journal of Investigative Dermatology Symposium Proceedings 2005 10, 47-61DOI: (10.1111/j.1087-0024.2005.10207.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Guidelines for design of siRNA. SeeMittal (2004). Journal of Investigative Dermatology Symposium Proceedings 2005 10, 47-61DOI: (10.1111/j.1087-0024.2005.10207.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions