Selectable Gene Editing for Muscular Dystrophy

肌营养不良症的选择性基因编辑

• 批准号: 7345644
• 负责人: Eric B. Kmiec
• 金额: $ 10.01万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7345644
• 关键词: Achievement; Adverse event; Affect; Alleles; Allogenic; Animal Model; Antibiotics; Antibodies; Antigens; Applications Grants; Autologous; Back; Biological Models; CD34 gene; Cell Differentiation process; Cell Line; Cell Transplants; Cell membrane; Cells; Chromosomes, Human, Pair 14; Clinical; Cloning; Competence; Complement; Complementary DNA; Complex; Condition; Coupled; Couples; DNA; DNA Sequence; Data; Defect; Detection; Development; Disease; Dystrophin; Engineering; Female; Fluorescence; Frequencies; Gene Delivery; Gene Expression Regulation; Gene Transfer; Generations; Genes; Genetic Enhancer Element; Goals; Grant; Human; Immune response; Immune system; Individual; Inherited; Interphase Cell; Investigation; Knowledge; Label; Laboratories; Learning; Link; MM form creatine kinase; Maintenance; Mammalian Cell; Measurement; Measures; Methodology; Methods; Mitotic; Modeling; Modification; Molecular Genetics; Mus; Muscle; Muscle Cells; Muscle Fibers; Muscular Dystrophies; Mutate; Mutation; Myoblasts; Natural Selections; Nonsense Mutation; Normal Cell; Numbers; Oligonucleotides; Oncogenic; Parents; Patients; Pattern; Peptides; Phenotype; Plasmids; Population; Positioning Attribute; Process; Proteins; Protocols documentation; RNA Splicing; Reader; Recombinants; Relative (related person); Reporter; Reporter Genes; Reporting; Resistance development; Site; Site-Directed Mutagenesis; Skeletal Muscle; Solid; Staging; Staining method; Stains; Stem cells; Structure; Surface; System; Techniques; Technology; Testing; Therapeutic; Time; Tissues; Transfection; Transplantation; Universities; Variant; Viral; Viral Vector; Work; Yeasts; base; chromophore; clinically significant; design; experience; expression vector; falls; gene repair; gene therapy; in vivo; indium arsenide; interest; male; mutant; novel; novel strategies; precursor cell; promoter; repaired; research study; response; restoration; satellite cell; success; uptake; vector

DESCRIPTION (provided by applicant): Inherited diseases, occurring in solid tissues, are often caused by simple recessive mutations derived from two carrier parents with a normal phenotype. In X-linked diseases, hemizygous males with a mutant allele will be affected while the female carrier is not. Gene therapy for recessive diseases has typically centered on a DNA-based gene system to deliver the normal allele in a mammalian expression vector or via an allogenic cell transplant. Numerous problems have been identified with these gene complementing systems arising from the lack of proper gene regulation, poor distribution of the transfecting vector, antigenic response from host immune system and the oncogenic potential of viral integration. These adverse events coupled with our long-standing interest in using the efficient endogenous DMA repair systems to correct mutations in mammalian cells led us to develop a new approach to gene therapy. This alternative strategy employs a synthetic oligonucleotide to direct the exchange of the mutant base in the affected gene or allele. The process, termed, gene repair or gene editing, has proven to be efficacious in a number of model systems and disease targets, including muscular dystrophy. The work in DMD, however, has not achieved levels of correction that enable therapeutic benefit. Thus, a primary challenge for this technique is to increase the frequency of repair or to isolate cells that have been corrected and expand them in culture. In this proposal, we aim to carry out preliminary investigations into the feasibility of meeting both challenges using primary muscle cells obtained from mdxScv and normal mice. We will examine the most efficient way of transfecting both a reporter plasmid and an oligonucleotide and achieving high levels of correction in either dividing or differentiating cells. We aim to develop a dual correction protocol in which a mutant reporter gene eYFP or DsRFP and the dystrophin mutation are simultaneously repaired. We shall select cells displaying functional gene editing activity by the emergence of fluorescence and assess the coordinate repair of the dystrophin mutation at the phenotypic and genotypic levels. Our long term goal is to develop a protocol in which primary muscle cells can be isolated and propagated ex vivo, then eventually transplanted back into the patient.

描述（由申请人提供）： 遗传性疾病发生在固体组织中，通常是由两个具有正常表型的载体父母得出的简单隐性突变引起的。在X连锁的疾病中，在女性携带者没有的情况下，将影响具有突变等位基因的半合子男性。隐性疾病的基因治疗通常以基于DNA的基因系统为中心，以在哺乳动物表达载体或通过同种异体细胞移植中递送正常等位基因。由于缺乏适当的基因调节，转染载体的分布差，宿主免疫系统的抗原反应以及病毒整合的致癌潜力而引起的这些基因补充系统已经发现了许多问题。这些不良事件加上我们对使用有效的内源性DMA修复系统纠正哺乳动物细胞突变的长期兴趣，这使我们开发了一种新的基因治疗方法。这种替代策略采用合成寡核苷酸来指导受影响基因或等位基因中突变基碱的交换。该过程称为基因修复或基因编辑，已被证明在许多模型系统和疾病靶标（包括肌肉营养不良）中有效。但是，DMD中的工作尚未达到可带来治疗益处的校正水平。因此，该技术的主要挑战是增加修复的频率或隔离已纠正并在培养中扩展的细胞。在此提案中，我们旨在对使用MDXSCV和正常小鼠获得的原代肌肉细胞应对这两种挑战的可行性进行初步研究。我们将研究转染报告基质粒和寡核苷酸的最有效方法，并在分裂或分化细胞中实现高水平的校正。我们旨在制定双重校正方案，其中突变报告基因EYFP或DSRFP和肌营养不良蛋白突变得到同时修复。我们将通过荧光的出现来选择显示功能基因编辑活性的细胞，并评估表型和基因型水平上肌营养不良蛋白突变的坐标修复。我们的长期目标是开发一种方案，在该方案中可以将原代肌肉细胞分离和传播离体，然后最终将其移回患者。