Abnormal Hemoglobin Synthesis -- Mechanism and Detection

描述（由申请人提供）：本申请是我们对镰状细胞性贫血和地中海贫血中正常和异常血红蛋白合成研究的继续。上一个赠款期的项目包括三个目标。1)镰状细胞性贫血和地中海贫血的DNA诊断。2)珠蛋白基因表达的控制。3)珠蛋白基因转移。由于目标2中的主题现在将由已成为独立调查员的以前的受训人员继续进行，因此本提案将继续只追求第一和第三个目标。目的1：我们已经完成了斑点杂交诊断血红蛋白病和地中海贫血突变的研究，这些突变在美国人群中很常见。我们建议开发和完善的产前诊断方法，从胎儿细胞分离使用母体血液。我们的初步研究表明，这是一种很有前途的方法，但还需要做更多的工作，使这种测试实用。我们将利用噬菌体展示技术来分离针对胎儿有核红细胞和人胚胎血红蛋白的单链抗体。我们将探讨使用图像分析和激光捕获显微切割，以促进胎儿有核红细胞的检索。这些发展将大大提高这一程序的效用。该建议的第二个目的是探索宫内基因递送的可能性，因为纯合子α-地中海贫血的病理变化通常在出生前出现，所以α-地中海贫血是宫内基因治疗的良好模型。在我们的实验室中，我们通过敲除内源性小鼠α-珠蛋白基因来制作α-地中海贫血小鼠模型。通过杂交不同的品系，我们得到了具有3，2，1或没有α-珠蛋白基因的小鼠，它们模仿了人类α-地中海贫血的临床表现。我们将使用含有控制人α-珠蛋白基因的β-珠蛋白LCR的AAV载体和慢病毒载体注射到妊娠第15周的胎鼠中。我们将跟踪观察这些小鼠的表达。α-珠蛋白基因和拯救疾病表型。这种方法不仅适用于α-地中海贫血，而且适用于其他遗传性疾病，如OTC缺乏症，这种疾病始于子宫内。

DESCRIPTION (provided by applicant): This application is a continuation on our studies of normal and abnormal hemoglobin synthesis in sickle cell anemia and thalassemia. The project in the previous grant period consisted of three aims. 1) DNA diagnosis of sickle cell anemia and thalassemia. 2) Control of globin gene expression. 3) Globin gene transfer. Because the topics in Aim 2 will now be carried on by previous trainees who have become independent investigators, this proposal will continue to pursue only the first and third aims. hi Aim 1 we have completed our studies on dot blot hybridization for the diagnosis of the mutations in hemoglobinopathies and thalassemia that are commonly found in the American population. We propose to develop and refine methods of prenatal diagnosis from fetal cells isolated using maternal blood. Our preliminary studies show that this is a promising approach, but much more work is needed to make this test practical. We will use phage display to isolate single chain antibodies specific for fetal nucleated red cells and human embryonic hemoglobin. We will investigate the use of image analysis and laser capture microdissections to facilitate the retrieval of fetal nucleated red cells. These developments will greatly enhance the usefulness of this procedure. The second aim of this proposal is to explore the possibility of in utero gene delivery alpha-thalassemia is a good model for in utero gene therapy because pathological changes in homozygous alpha-thalassemia usually appear before birth. In our laboratory, we have made mouse models of alpha-thalassemia by knocking out the endogenous mouse alpha-globin genes. By crossing various strains, we have mice that have 3,2,1 or no alpha-globin gene and they mimic the clinical manifestation of human alpha-thalassemia. We will use AAV vectors and lentiviral vectors containing the beta-g1obin LCR controlling the human a-globin gene to inject into fetal mice at the 15th week of gestation. We will follow these mice to observe the expression of the. alpha-globin gene and the rescue the disease phenotype. Such an approach could be useful not only for alpha-thalassemia but also for other genetic disorders such as OTC deficiency where the disease begins in utero.