In vivo Gene Correction of Keratin 14 Gene in EBS Mouse

描述(由申请人提供)： 由寡核苷酸修复的基因在哺乳动物细胞的基因组中产生有针对性的改变。我们在理解基于寡核苷酸的基因修复机制和将该策略应用于皮肤基因修复方面取得了重大进展。我们的最新发现包括：(1)小鼠皮肤角质形成细胞的体内基因校正可能发生在比组织培养更高的水平上；(2)各种形式的寡核苷酸，如RNA-DNA寡核苷酸(RDO)和单链寡核苷酸(ODN)都能引起基因改变。这些寡核苷酸的一些特性使它们在体内应用于表皮很有吸引力。组织的可获得性使局部给药和监测治疗部位和对照部位成为可能。在我们的活体研究中，我们在小鼠皮肤中观察到了令人惊讶的高水平和持久的基因变化。这些发现表明，基于寡核苷酸的基因修复可能能够纠正表皮干细胞的基因，而这种细胞的扩增可能会导致表皮中明显高水平的基因纠正。基于这些结果，我们建议纠正Dowling-Meara单纯性大疱性表皮松解症(EBS)患者中发现的小鼠角蛋白14基因的显性突变。最近，鲁普博士的实验室制作了一个优雅的转基因EBS模型，在该模型中，只在皮肤的局部区域诱导了显性负突变。在这里，我们将测试ODN是否可以纠正这些新生转基因小鼠中的显性突变，以及是否可以通过将ODN原位应用于皮肤来预防或减少水泡的诱导。一旦基底角质形成细胞被矫正，这类细胞将比突变细胞具有增殖优势。如果成功，这项原则验证实验将导致寡核苷酸潜在的临床应用于EB患者严重感染区域的治疗。

DESCRIPTION (provided by applicant): Gene repair by oligonucleotides produces targeted alterations in the genome of mammalian cells. We have made significant progress toward understanding the mechanism of oligonucleotide-based gene repair and applying the strategy to gene repair of skin. Our recent findings include; (1) in vivo gene correction of keratinocytes in murine skin may occur at a higher level than that in tissue culture and (2) various forms of oligonucleotides, RNA-DNA oligonucleotide (RDO) and single-stranded oligodeoxynucleotides (ODN), are both capable of gene alteration. Several characteristics of these oligonucleotides make them attractive for in vivo application in epidermis. Accessibility of the tissue makes it possible to administer therapeutics locally and to monitor both the treated and control sites. During our in vivo study, we observed a surprisingly high level and long-lasting gene alteration in murine skin. These findings suggest that oligonucleotide-based gene repair may be capable of epidermal stem cell gene correction and expansion of such cells may result in an apparently high level of gene correction in epidermis. Based on these results, we propose to correct the dominant mutation in murine keratin 14 gene found in Dowling-Meara Epidermolysis Bullosa Simplex (EBS) patients. Recently, Dr. Roop's laboratory generated an elegant transgenic EBS model where a dominant negative mutation was induced only in a focal area of skin. Here, we will test whether ODN can correct a dominant mutation in these neonatal transgenic mice and whether induction of blistering can be either prevented or diminished by in situ application of ODN to skin. Once basal keratinocytes are corrected, such cells will have proliferative advantage over mutated cells. If successful, this proof-of-principle experiment will lead to a potential clinical application of oligonucleotides to severely affected areas of EB patients for treatment.