Targeted Transposons for Gene Therapy

用于基因治疗的靶向转座子

• 批准号: 7274374
• 负责人: PERRY B. HACKETT
• 金额: $ 17.4万
• 依托单位: DISCOVERY GENOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7274374
• 关键词: Adverse effects; Animal Model; Animals; Binding; Biodistribution; Biological; Biological Models; Cell Line; Cell membrane; Cells; Coupling; DNA; DNA Binding Domain; Development; Disadvantaged; Disease; Facility Construction Funding Category; Factor VIII; Gene Expression; Gene Transduction Agent; Genes; Genomics; Goals; Hela Cells; Hematopoietic; Hemophilia A; Hepatocyte; Hereditary Disease; Immune response; Infection; Injection of therapeutic agent; Ligands; Liver; Mammals; Methods; Modification; Molecular; Mus; Non-Viral Vector; Organ; Plasmids; Property; Proteins; Purpose; Site; Sleeping Beauty; Small Business Funding Mechanisms; Small Business Innovation Research Grant; System; Tail; Techniques; Testing; Therapeutic; Tissues; Transgenes; Transgenic Organisms; Transposase; Validation; Veins; Viral; Virus; base; cell type; cellular targeting; gene therapy; improved; non-viral gene therapy; receptor; therapeutic gene; therapeutic transgene; uptake; vector; viral DNA

DESCRIPTION (provided by applicant): Discovery Genomics, Inc. is focused on development of the Sleeping Beauty (SB) transposon system as a non-viral means of integrating new gene sequences in cells and tissues for therapeutic purposes. Here we propose to develop a method for improved targeting of the SB system to specific cell types following systemic injection by coupling a targeting tether to the plasmid carrier of the transposon. This approach will allow 'universal' vectors that could carry any gene and be directed to any cell type without modification of the transposase or transposon for each cellular target. This project will dovetail with DGI's ongoing SBIR project to deliver Factor VIII and IX genes in appropriate animal model systems. Thus, this project has high-impact potential because the techniques we will develop can be used for treatment of multiple diseases using a 'universal' non-viral vector system whose properties will be applicable to many therapeutic transgenes. The Specific Aims of the project are to: 1. Construct a Liver- directed Targeting Tether (LTT) vector system to increase uptake of SB transposons into liver cells. This aim will be accomplished by construction of a LTT targeting tether comprised of a LexA DNA-binding domain fused to a ligand specific for hepatocytes and construction of a plasmid, pKLAT2, that contains an SB transposon plus multimeric LexA operator sites to which the targeting tether can bind. 2: Evaluate the efficiency in cultured liver cells (HuH7 and HepG2) of a LTT targeting tether to enhance uptake and transposition of a pKLAT2 transposon vector. HeLa cells will serve as a control for cells that lack appropriate liver-specific receptors. 3: Evaluate the efficiency of a LTT targeting tether to enhance uptake and transposition of a pKLAT2 transposon vector into liver cells in mice.

描述(申请人提供)：发现基因组公司专注于开发睡美人(SB)转座子系统，作为一种将新基因序列整合到细胞和组织中用于治疗目的的非病毒手段。在这里，我们建议开发一种改进的方法，在全身注射后，通过将靶向系链连接到转座子的质粒载体来改进SB系统对特定细胞类型的靶向性。这一方法将允许携带任何基因的“通用”载体被定向到任何细胞类型，而不需要修改每个细胞靶标的转座酶或转座子。该项目将与华大基因正在进行的SBIR项目相吻合，在适当的动物模型系统中传递因子和IX基因。因此，这个项目具有很高的影响潜力，因为我们将开发的技术可以用于治疗多种疾病，使用一个“通用的”非病毒载体系统，其特性将适用于许多治疗性转基因。本项目的具体目标是：1.构建肝定向靶向系链(LTT)载体系统，以增加Sb转座子在肝细胞中的摄取。这一目的将通过构建LTT靶向系链，该锚链由融合到肝细胞特异性配体上的LexA DNA结合域组成，并构建包含SB转座子和靶向锚链可与之结合的多聚体LexA操纵子的质粒pKLAT2。2：在培养的肝细胞(Huh7和HepG2)中评价LTT靶向系链增强pKLAT2转座子载体摄取和转座的效率。HeLa细胞将作为缺乏适当肝脏特异性受体的细胞的对照。3：评估LTT靶向锚链增强pKLAT2转座子载体摄取和转座到小鼠肝细胞中的效率。