Targeted Transposons for Gene Therapy

用于基因治疗的靶向转座子

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：Discovery Genomics, Inc. 致力于开发睡美人 (SB) 转座子系统，作为将新基因序列整合到细胞和组织中以达到治疗目的的非病毒手段。在这里，我们建议开发一种方法，通过将靶向系链与转座子的质粒载体偶联，在全身注射后改善 SB 系统对特定细胞类型的靶向性。这种方法将允许“通用”载体可以携带任何基因并定向到任何细胞类型，而无需修改每个细胞靶标的转座酶或转座子。该项目将与 DGI 正在进行的 SBIR 项目相配合，在适当的动物模型系统中提供因子 VIII 和 IX 基因。因此，该项目具有高影响力的潜力，因为我们将开发的技术可用于使用“通用”非病毒载体系统治疗多种疾病，该系统的特性将适用于许多治疗性转基因。该项目的具体目标是： 1. 构建肝脏定向靶向系链（LTT）载体系统，以增加肝细胞对 SB 转座子的摄取。这一目标将通过构建 LTT 靶向系链来实现，该系链由与肝细胞特异性配体融合的 LexA DNA 结合结构域组成，并构建质粒 pKLAT2，该质粒包含 SB 转座子以及靶向系链所连接的多聚体 LexA 操纵子位点。可以绑定。图 2：评估 LTT 靶向系链在培养的肝细胞（HuH7 和 HepG2）中增强 pKLAT2 转座子载体的摄取和转座的效率。 HeLa 细胞将作为缺乏适当肝脏特异性受体的细胞的对照。图 3：评估 LTT 靶向系链增强 pKLAT2 转座子载体摄取和转座到小鼠肝细胞中的效率。