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 修复调节的几种（最多 9 种）蛋白质之一缺陷引起。患者表现出出生缺陷，在生命早期患有骨髓衰竭，并且容易患上包括白血病和实体瘤在内的癌症。 FA 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。我们提出将MaxCyte, Inc.的细胞装载技术与Discovery Genomics, Inc. (DGI)的DNA整合技术相结合，引入并表达FANC基因来治疗FA。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，以确定培养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 以及转座酶源高效加载到几种不同的造血细胞系（Jurkat、KB、K562）中，转座酶介导从新引入的质粒 DNA 转座到染色体 DNA。随着时间的推移，将通过流式细胞术检测基因表达，并进行分子分析（PCR、Southern印迹）以分析转座到染色体靶标的情况。在目标 2 中，DGI 将组装设计用于表达人类 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，测试这些细胞群对 DNA 损伤剂丝裂霉素 C 的敏感性降低以及通过分子遗传学分析进行转座。这些第一阶段的研究将为第二阶段针对造血干细胞（HSC）的联合细胞装载和 DNA 整合技术的进一步临床前开发奠定基础。病毒引入 HSC 的情况尚未有报道，并且需要我们的组合技术提供高效的细胞装载和整合能力。这里提出了针对范可尼贫血的初步开发细胞装载/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。