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In vivo Gene Correction of Keratin 14 Gene in EBS Mouse

EBS 小鼠角蛋白 14 基因的体内基因校正

基本信息

批准号：
7216336
负责人：
OLGA IGOUCHEVA
金额：
$ 7.4万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-04-01 至 2009-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7216336
关键词：
Affect Age Animal Model Animals Area Basement membrane Biopsy Bulla Categories Cells Characteristics Cutaneous DNA Sequence Detection Disease Dominant-Negative Mutation Epidermis Epidermolysis Bullosa Epidermolysis Bullosa Simplex Epithelial Gene Expression Regulation Gene Mutation Gene Targeting Genes Genomics Histology Human In Situ Inborn Genetic Diseases Keratin Knock-in Mouse Laboratories LacZ Genes Lead Localized Mammalian Cell Mediating Methods Modality Modeling Monitor Mucous Membrane Mus Mutate Mutation Neonatal Oligonucleotides Patients Phenotype Play Point Mutation Proteins Reporting Restriction fragment length polymorphism Role Side Site Skin Staining method Stains Stem cells Surface Technology Testing Therapeutic Tissues Transgenic Mice Transgenic Organisms Urinary tract Variant Viral animal mortality base clinical application design desire embryonic stem cell experience gastrointestinal gene correction gene repair gene therapy in vivo keratin 14, K14 keratin 5 keratinocyte mammalian genome mouse genome mouse model mutant prevent prototype research study respiratory skin disorder tissue culture transgene expression

项目摘要

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.

描述（由申请人提供）：寡核苷酸的基因修复会在哺乳动物细胞的基因组中产生靶向改变。我们在理解基于谷胱甘肽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。我们最近的发现包括：（1）小鼠皮肤中角质形成细胞的体内基因校正可能发生在比组织培养中更高的水平上;（2）各种形式的寡核苷酸，RNA-DNA寡核苷酸（RDO）和单链寡脱氧核苷酸（ODN），都能够进行基因改变。这些寡核苷酸的几个特征使它们在表皮中的体内应用具有吸引力。组织的可接近性使得可以局部施用治疗剂并监测治疗部位和对照部位。在我们的体内研究中，我们在小鼠皮肤中观察到令人惊讶的高水平和持久的基因改变。这些研究结果表明，阿托伐他汀为基础的基因修复可能能够表皮干细胞的基因校正和扩展，这些细胞可能会导致在表皮基因校正的明显高水平。基于这些结果，我们建议纠正在Dowling-Meara单纯性大疱性表皮病（EBS）患者中发现的鼠角蛋白14基因的显性突变。最近，Roop博士的实验室产生了一个优雅的转基因EBS模型，其中只在皮肤的焦点区域诱导显性负突变。在这里，我们将测试ODN是否可以纠正这些新生转基因小鼠的显性突变，以及是否可以通过原位应用ODN皮肤预防或减少水疱的诱导。一旦基底角质形成细胞得到纠正，这些细胞将具有超过突变细胞的增殖优势。如果成功的话，这一原理验证实验将导致寡核苷酸在EB患者的严重受影响区域进行治疗的潜在临床应用。