Gene correction in hematopoitic stem cells using a retargetable meganuclease

描述（由申请人提供）：对分裂细胞（如造血干细胞）进行持续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，包括在不合适的位点上整合可能导致癌症，以及缺乏适当的基因调控元件。相比之下，通过同源重组的基因靶向可以将转基因放置在基因组中预定的、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，先前的研究表明，在目标位点引入双链断裂可以将哺乳动物细胞中的同源重组频率提高多达五个数量级。本研究旨在确定一种诱导基因靶向的最佳方法，这种方法是用一种可重靶向的内切酶特异性地切割小鼠基因组中的ROSA26位点。ROSA26是一个理想的转基因表达位点，因为它是无所不在的表达和非必需的。该内切酶来源于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，可以设计用于靶向大多数位点。首先，工程核酸内切酶在小鼠细胞系中诱导基因靶向的能力将被证明。绿色荧光蛋白将作为转基因模型。其次，将确定在造血干细胞中诱导基因校正的最佳方法，同时保持其重建髓鞘切除个体血液的能力。最后，将开发基于培养扩增和移植前流式细胞术分选的方法来增加造血细胞移植中正确靶向细胞的比例。目前旨在通过基因治疗某些遗传性血液疾病的方法存在缺陷，如可能致癌和疗效低。本提案旨在定义新的方法，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。

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.