RETROVIRAL TRANSFER OF ANKYRIN FOR SPHEROCYTOSIS

目前以造血干细胞为靶点的基因治疗面临的挑战包括实现高转导效率、转导细胞的长期植入和转基因的长期表达。为了实现这些目标，我们计划通过含有锚蛋白基因的逆转录病毒载体研究干细胞转导，作为基因治疗和纠正遗传性溶血性贫血的模型。我们将使用nb/nb小鼠作为溶血性贫血模型，研究干细胞周期状态和低剂量（100 cGy）宿主照射（最小骨髓消融）对转导细胞移植的影响。遗传性溶血性贫血，包括地中海贫血和镰状细胞性贫血，影响着全世界的大量人群，并导致显著的发病率和生存率降低。在小鼠中有几种自然发生的遗传性溶血性贫血，类似于人类的遗传性球形红细胞增多症。其中一个这样的突变，nb/nb小鼠，表现出明显的锚蛋白缺乏，锚蛋白是一种210 kDa的蛋白质，可以将红细胞膜骨架锚定在脂质双分子层上。应用逆转录病毒载体将正常锚蛋白cDNA转移到nh/nb小鼠骨髓祖细胞中，以纠正红细胞缺陷，改善贫血。鼠/人杂交cDNA由人类锚蛋白基因启动子、3带和spectrin结合的大部分小鼠结构域的编码序列，以及人类调控结构域的可选拼接（2.2带）版本组成。将追求下列目标：1)比较pG1-Ank与含有锚蛋白cDNA的pG1-Ank/rev在LTRs正反方向的表达；2)检测从正常骨髓祖细胞和nb/nb转导的骨髓祖细胞中衍生的分化红细胞；3)通过改变细胞因子孵育时间将转导的骨髓祖细胞移植到最低程度清髓的正常受体中，以优化移植。4)转导nb/nb小鼠骨髓细胞，将这些细胞移植到微量清髓的nh/nb受体，以改善溶血性贫血。所开发的方法，包括插入编码大蛋白的cDNA，使用非清髓性程序进行遗传疾病的移植，在细胞因子中培养干细胞但保留移植的能力，以及实现组织特异性基因表达的能力，与遗传性人类疾病的基因治疗方法的发展直接相关。

The current challenges in gene therapy with the hematopoietic stem cell as target include achievement of high transduction efficiency, long-term engraftment of transduced cells, and long-term expression of the transgene. Toward development o methods to achieve these aims, we plan to study stem cell transduction by a retroviral vector containing the ankyrin gene as a model for gene therapy and correction of hereditary hemolytic anemias. We-will use the nb/nb mouse as our hemolytic anemia model and study the impact of stem cell cycle status and low dose (100 cGy) host irradiation (minimal myeloablation) on engraftment of transduced cells. Hereditary hemolytic anemias, including thalassemia and sickle cell anemia, affect large populations worldwide, and result in significant morbidity and reduced survival. There are several naturally occurring inherited hemolytic anemias in mice which are analogous to the human disorder, hereditary spherocytosis. One such mutant, the nb/nb mouse, exhibits marked deficiency in ankyrin, a 210 kDa protein that anchors the red cell membrane skeleton to the lipid bilayer. This application proposes to transfer by retroviral vector the cDNA for normal ankyrin to marrow progenitor cells from nh/nb mice to correct the erythrocyte defect and improve the anemia. The murine/human hybrid cDNA consists of the human ankyrin gene promoter, most of the coding sequence of the murine domains for band 3 and spectrin binding, and the alternatively spliced (band 2.2) version of the human regulatory domain. The following objectives will be pursued: 1) to compare expression obtained with pG1-Ank to pG1-Ank/rev that contain ankyrin cDNA in the forward or reverse orientations between the LTRs, 2) to examine differentiating erythroid cells derived from both normal and nb/nb transduced marrow progenitors, 3) to engraft the transduced marrow progenitors in minimally myeloablated normal recipients with modification of the cytokine incubation time to optimize engraftment, and 4) to transduce bone marrow cells from the nb/nb mouse and engraft these cells in minimally myeloablated nh/nb recipients to improve the hemolytic anemia. The methods developed, including insertion of the cDNA encoding a large protein, the use of non-myeloablative procedures for transplant of genetic diseases, the ability to incubate stem cells in cytokines yet preserve engraftment, and the achievement of tissue-specific gene expression have direct relevance to the development of gene therapy approaches to inherited human disorders.