Gene targeting is designed to produce predefined alterations in the genome of eukaryotic cells. Advantage of gene targeting is that repaired gene can be maintained, expressed, and regulated as the normal endogenous gene and that it can rescue a dominant mutation. Viral gene replacement presents a problem of constitutive and unregulated expression of gene product even if such gene delivery is efficient to target cells. There are also safety concerns including immune reaction and generation of replication competent virus. An experimental strategy has been developed for a site-specific correction of single base mutations of target sequence by using an RNA-DNA oligonucleotide (RDO) (Yoon et al., 1996). Several characteristics of RDO make it attractive for in vivo application in epidermis. The accessibility of the tissue makes it possible to administer therapeutics locally, to monitor both the treated site and a control. We demonstrated the tyrosinase gene correction in melanocytes by RDO was inheritable in vitro (Alexeev and Yoon, 1998) and in vivo (Alexeev et al., 2000). During the course of in vivo study, we observed a surprisingly high level of genotypic correction from skin biopsy. We also found an increasing level of gene correction when skin biopsy was taken from older animals, suggesting that RDO is capable of epidermal stem cell gene conversion and that expansion of such cells may result in an apparent high level of gene conversion in epidermis. In this case, the high epidermal turnover may make the in vivo application RDO much more attractive than in vitro. Most murine models for genodermatoses with known genetic defects do not resemble the mutations found in human genetic diseases since they contain a large alteration of the mouse genome. Moreover, in some cases, they do not survive long enough to be useful because the entire body function is affected.
In Aim 1, we plan to generate a dominant mutation found in several genes involved in cutaneous genetic diseases at localized area of murine skin by in vivo application of RDO.
In Aim II, we plan to mutate the COL7A1 gene using the grafted human skin to SCID mouse by localized application of RDO.
In Aim III, we plan to develop a new strategy of the targeted gene conversion using a single-stranded deoxyoligonucleotide, which is capable of correcting a single point mutation in mammalian cells, similar extent as RDO (Igoucheva et al., 2000). With this new finding, a relatively short oligodeoxynucleotide can be envisioned to make a sequence-specific change in the target sequence in mammalian cells. Emerging technologies of non-viral gene delivery methods, topical application, particle bombardment, and in vivo electroporation will be incorporated throughout the project. Therapeutic nucleic acid repair strategy will be useful not only generating an animal model for testing therapy modalities but also a potential clinical application of oligonucleotides to severely affected areas of genodermatoses patients for treatment. Furthermore, a capability to establish cells containing a specific alteration will provide valuable cellular models in skin biology.
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