Transposon-mediated Gene Therapy for Fanconi Anemia

转座子介导的范可尼贫血基因治疗

• 批准号: 6787819
• 负责人: PERRY B. HACKETT
• 金额: $ 23.56万
• 依托单位: DISCOVERY GENOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6787819
• 关键词: biotechnology; cell line; clone cells; congenital aplastic anemia; electroporation; flow cytometry; gene delivery system; gene expression; gene therapy; hematopoietic stem cells; human genetic material tag; mitomycin C; polymerase chain reaction; reporter genes; southern blotting; technology /technique development; tissue /cell culture; transposon /insertion element

DESCRIPTION (provided by applicant): Fanconi anemia (FA) is an inherited recessive disorder caused by deficiency of one of several (up to 9) proteins involved in the regulation of DNA repair. Patients exhibit birth defects, suffer from bone marrow failure early in life, and are prone to develop cancers including leukemia and solid tumors. The only effective treatment for FA is allogeneic bone marrow transplant, but many patients lack a matched donor. We propose the treatment of FA by introduction and expression the FANC gene by combining the cell-loading technology of MaxCyte, Inc., with the DNA integrating technology of Discovery Genomics, Inc. (DGI). In Specific Aim 1, we will first test the combination of these two technologies by using reporter genes to ascertain long-term gene transfer and expression in cultured hematopoietic cells. DGI will assemble transposons designed for introduction and expression of red fluorescent protein (dsRed). MaxCyte will then use its electroporation technology for high efficiency loading of the transposon DNA into several different hematopoietic cell lines (Jurkat, KB, K562) along with a source of transposase which mediates transposition from the newly-introduced plasmid DNA into chromosomal DNA. Gene expression will be assayed over time by flow cytometry, and molecular analyses (PCR, Southern blot) will be conducted for analysis of transposition into chromosomal targets. In Aim 2, DGI will assemble transposons designed for expression of the human FANC-C and FANC-A genes. MaxCyte will then load these transposons into lymphoblastoid cell lines established from patients with Fanconi anemia type C or A, respectively, testing these cell populations for reduced sensitivity to the DNA damaging agent mitomycin C as well as for transposition by molecular genetic analysis. These Phase 1 studies will provide the basis for further preclinical development of the combined cell loading and DNA integrating technologies targeting hematopoietic stem cells (HSC) in Phase 2. These studies will be ground-breaking in that long-term expression of genes after non-viral introduction into HSC has yet to be reported, and will require the efficient cell loading and integrating capacities provided by our combined technologies. Initial development cell loading / DNA integration approach is proposed here for Fanconi anemia, subsequently providing the technical basis for treatment of other inherited (immunodeficiencies, hemoglobinopathies) or acquired (AIDS) diseases.

描述（由申请人提供）：范可尼贫血（FA）是一种遗传性隐性疾病，由参与DNA修复调控的几种（多达9种）蛋白质之一缺乏引起。患者表现出出生缺陷，在生命早期遭受骨髓衰竭，并且易于发展癌症，包括白血病和实体瘤。唯一有效的治疗方法是同种异体骨髓移植，但许多患者缺乏匹配的供体。我们提出了通过结合MaxCyte，Inc.的细胞加载技术引入和表达FANC基因来治疗FA，利用Discovery Genomics，Inc.的DNA整合技术，（DGI）。在具体目标1中，我们将首先通过使用报告基因来测试这两种技术的组合，以确定在培养的造血细胞中的长期基因转移和表达。DGI将组装设计用于引入和表达红色荧光蛋白（dsRed）的转座子。然后MaxCyte将使用其电穿孔技术将转座子DNA高效加载到几种不同的造血细胞系（Jurkat，KB，K562）中，沿着介导从新引入的质粒DNA转座到染色体DNA中的转座酶来源。将通过流式细胞术测定基因表达随时间的变化，并进行分子分析（PCR，Southern印迹）以分析转座至染色体靶标中的情况。在目标2中，DGI将组装设计用于表达人FANC-C和FANC-A基因的转座子。然后MaxCyte将这些转座子分别加载到从范可尼贫血C型或A型患者建立的淋巴母细胞系中，通过分子遗传分析测试这些细胞群对DNA损伤剂丝裂霉素C的敏感性降低以及转座。这些I期研究将为II期靶向造血干细胞（HSC）的细胞加载和DNA整合技术的进一步临床前开发提供基础。这些研究将是突破性的，因为在非病毒引入HSC后基因的长期表达尚未被报道，并且将需要我们的组合技术提供的有效细胞加载和整合能力。本文提出了用于范可尼贫血的初始开发细胞加载/ DNA整合方法，随后为治疗其他遗传性（免疫缺陷，血红蛋白病）或获得性（艾滋病）疾病提供了技术基础。