GENE TARGETING APPROACH FOR BLOOD DISEASES

目前的大多数基因治疗都是基于“基因加成”策略，即将一个有功能的转基因盒子输送到细胞中，并从异构体或随机整合的分子中表达出来。一个潜在的更可取的策略是纠正它们正常染色体位置的突变。这种“基因矫正”方法的主要优点包括适当地调节基因表达和消除显性致病突变。基因矫正在与干细胞的体外培养和操作方法相结合时尤其有希望，因为即使是罕见的矫正细胞也有可能在移植和体内增殖后重建患病的器官系统。这项建议的目的是开发基因打靶方法，可以用来纠正细胞中能够重建造血系统的基因。一般来说，传统方法可以实现的基因损失率太低，无法考虑治疗应用，而且在造血干细胞的情况下，从未证明过基因靶向。在这项提议中，一种使用腺相关病毒(AAV)载体的新的基因打靶方法将被用于纠正干细胞群体中的基因。基因靶向分析将基于方便的报告基因的校正或特定位置插入而开发。AAV靶向载体将被包装在不同的衣壳血清型中，并测试它们纠正小鼠和人类造血细胞中基因的能力。通过将小鼠细胞移植到去髓小鼠中，或将人细胞移植到NOD/SCID小鼠中，并分析移植受者中正确报告基因的表达，将在转导的干细胞中证明基因靶向。还将使用来自肌肉和脑组织的替代来源的造血干细胞进行实验，这些来源的干细胞具有更好的体外培养性能，可能更容易受到AAV介导的基因靶向的影响。这些实验试图证明基因打靶可以在造血干细胞中实现，并建立最终可能适合于通过纠正治疗血液疾病的实验条件。

Most current gene therapy are based on a "gene addition" strategy, where a functional transgene cassette is delivered to cells and expressed from episomal or randomly integrated molecules. A potentially more desirable strategy would be to correct mutations at their normal chromosomal locations. Major advantages of this "gene correction" approach include properly regulated gene expression and the removal of dominant disease- causing mutations. Gene correction is especially promising when combined with methods for the ex vivo culture and manipulation of stem cells, since even rare corrected cells could have the potential to reconstitute a diseased organ system after transplantation and in vivo proliferation. The objective of this proposal is to develop gene targeting methods that can be used to correct genes in cells capable of reconstituting the hematopoietic system. In general, the gene corruption rates that can be achieved with conventional methods have been far too low to consider therapeutic applications, and in the case of hematopoietic stem cells, gene targeting has never been demonstrated. In this proposal, a novel gene targeting method employing adeno-associated virus (AAV) vectors will be used to correct genes in stem cell populations. Gene targeting assays will be developed based on the correction or site-specific insertion of convenient reporter genes. AAV targeting vectors will be packaged in different capsid serotypes and tested for their ability to correct genes in mouse and human hematopoietic cells. Gene targeting will be demonstrated in transduced stem cells by transplantation of murine cells in myeloablated mice, or human cells in NOD/SCID mice, and assaying for expression of the corrected reporter gene in transplant recipients. Experiments will also be performed with alternative sources of hematopoietic stem cells derived from muscle and brain tissue, which have improved ex vivo culture properties and may be more susceptible to AAV-mediated gene targeting. These experiments constitute an attempt to demonstrate that gene targeting can be achieved in hematopoietic stem cells, and establish experimental conditions that may ultimately be adapted towards the treatment of blood diseases by correction.