Gene correction in hematopoitic stem cells using a retargetable meganuclease

使用可重定位大范围核酸酶对造血干细胞进行基因校正

基本信息

批准号：
7744710
负责人：
Michael J Volles
金额：
$ 5.77万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-01-01 至 2010-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7744710
关键词：
Aminoglycosides Biological Assay Blood Blood Cells Bone Marrow Bone Marrow Purging Bone Marrow Transplantation Cancer Etiology Cell Fraction Cell Transplants Cells Chromosomes Cleaved cell Data Disadvantaged Disease Drug Formulations Electroporation Embryo Engineering Enzymes Event Fibroblasts Flow Cytometry Frequencies Gene Targeting Genes Genome Green Fluorescent Proteins Hematological Disease Hematopoietic Hematopoietic stem cells Homing Image Individual Inherited Life Location Luciferases Mammalian Cell Measures Methods Modeling Monitor Mouse Cell Line Mus Mutation Plasmids Population Procedures Proteins Protocols documentation Publishing Regulation Regulatory Element Relative (related person)Retroviridae Sampling Side Site Sorting - Cell Movement Specificity Stem cells Subfamily lentivirinae Time Toxic effect Transfection Transgenes Transgenic Organisms Transplant Recipients Transplantation Variant Wild Type Mouse antibiotic G 418 base cell growth congenic design endonuclease established cell line gene correction gene therapy homologous recombination in vivo lentiviral integration lipofection mammalian genome mouse genome mutant reconstitution research study stem therapeutic transgene transgene expression vector

项目摘要

DESCRIPTION (provided by applicant): Persistent gene therapy in dividing cells such as hematopoietic stem cells generally requires that the therapeutic transgene be integrated into a chromosome, for example, using a retrovirus. There are several drawbacks to this approach, including the possibility that integration at an inopportune locus can cause cancer, and a lack of appropriate gene regulatory elements. In contrast, gene targeting by homologous recombination allows one to place a transgene into a predetermined, safe, location in the genome. The difficulty in making use of gene targeting for gene therapy is that it occurs only rarely in most cells. However, it has previously been shown that introducing a double strand break at the site to be targeted can increase the homologous recombination frequency by up to five orders of magnitude in mammalian cells. This proposal seeks to define optimal methods for inducing gene targeting with a retargetable endonuclease that specifically cuts at the ROSA26 locus in the mouse genome. ROSA26 is an ideal site for transgene expression because it is ubiquitously expressing and non-essential. The endonuclease is derived from the ICrel enzyme and has sufficient specificity to be able to target particular sites in a mammalian genome, and is sufficiently modifiable that it can be designed to target most loci. First, the ability of the engineered endonuclease to induce gene targeting in mouse cell-lines will be demonstrated. Green fluorescent protein will be used as a model transgene. Second, optimal methods to induce gene correction in hematopoietic stem cells while maintaining their ability to reconstitute the blood of a myelpablated individual will be determined. Finally, methods for increasing the fraction of correctly targeted cells in a hematopoietic cell transplant, based on expansion in culture and selection with flow cytometry sorting prior to transplant, will be developed. Current methods aimed at genetically curing certain inherited blood diseases have drawbacks, such as the possibility of causing cancer and low efficacy. This proposal seeks to define new methods to more precisely correct the inherited mutations that cause these disorders, thereby eliminating some of the dangers and disadvantages of current gene therapy protocols.

描述（由申请人提供）：分裂细胞（例如造血干细胞）中的持久基因治疗通常需要将治疗性转基因整合到染色体中，例如使用逆转录病毒。这种方法有几个缺点，包括在不适当的位点进行整合可能导致癌症，以及缺乏适当的基因调控元件。相比之下，通过同源重组进行基因打靶允许将转基因置于基因组中预定的、安全的位置。利用基因靶向进行基因治疗的困难在于它在大多数细胞中很少发生。然而，之前的研究表明，在哺乳动物细胞中，在目标位点引入双链断裂可以将同源重组频率提高多达五个数量级。该提案旨在定义使用可重定位核酸内切酶诱导基因靶向的最佳方法，该酶专门切割小鼠基因组中的 ROSA26 位点。 ROSA26 是转基因表达的理想位点，因为它普遍表达且非必需。该核酸内切酶源自 ICrel 酶，具有足够的特异性，能够靶向哺乳动物基因组中的特定位点，并且具有足够的可修饰性，可以设计为靶向大多数基因座。首先，将证明工程化核酸内切酶在小鼠细胞系中诱导基因靶向的能力。绿色荧光蛋白将用作模型转基因。其次，将确定在造血干细胞中诱导基因校正的最佳方法，同时保持其重建骨髓切除个体血液的能力。最后，将开发基于培养物扩增和移植前流式细胞术分选的选择来增加造血细胞移植中正确靶向细胞比例的方法。目前旨在通过基因治疗某些遗传性血液疾病的方法存在一些缺点，例如可能致癌且疗效较低。该提案旨在定义新方法，以更精确地纠正导致这些疾病的遗传突变，从而消除当前基因治疗方案的一些危险和缺点。