GENE TARGETING APPROACH FOR BLOOD DISEASES

大多数当前的基因疗法基于“基因添加”策略，其中功能性转基因盒被递送至细胞并从附加型或随机整合的分子表达。一个潜在的更可取的策略将是在正常的染色体位置纠正突变。这种“基因校正”方法的主要优点包括适当调节基因表达和去除显性致病突变。当与用于干细胞的离体培养和操作的方法结合时，基因校正是特别有希望的，因为即使是罕见的校正的细胞也可能具有在移植和体内增殖后重建患病器官系统的潜力。该提案的目的是开发基因靶向方法，其可用于校正能够重建造血系统的细胞中的基因。一般来说，用常规方法可以实现的基因破坏率太低，以至于不能考虑治疗应用，并且在造血干细胞的情况下，基因靶向从未得到证实。在这个建议中，一种新的基因靶向方法，采用腺相关病毒（AAV）载体将被用来纠正干细胞群体中的基因。基因靶向测定将基于方便的报告基因的校正或位点特异性插入来开发。将AAV靶向载体包装在不同的衣壳血清型中，并测试其在小鼠和人造血细胞中校正基因的能力。通过在骨髓清除小鼠中移植鼠细胞或在NOD/SCID小鼠中移植人细胞，并测定移植受体中校正的报告基因的表达，将在转导的干细胞中证明基因靶向。实验还将使用来自肌肉和脑组织的造血干细胞的替代来源进行，其具有改善的离体培养特性并且可能更容易受到AAV介导的基因靶向的影响。这些实验构成了证明基因靶向可以在造血干细胞中实现的尝试，并建立了最终可以通过校正而适于治疗血液病的实验条件。

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.