Abnormal Hemoglobin Synthesis -- Mechanism and Detection

血红蛋白合成异常——机制与检测

• 批准号: 6542371
• 负责人: YUET Wai KAN
• 金额: $ 39.48万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6542371
• 关键词: adeno associated virus group; clinical research; diagnosis design /evaluation; diagnostic tests; disease /disorder model; erythrocytes; flow cytometry; gene expression; gene mutation; gene targeting; genetic disorder diagnosis; genetic promoter element; genetically modified animals; hemoglobin; hemoprotein biosynthesis; human fetus tissue; human genetic material tag; laboratory mouse; postmortem; prenatal diagnosis; sickle cell anemia; thalassemia; transfection /expression vector

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.

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延续。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中的主题现在将由以前已成为独立调查员的学员进行，因此本提案将继续只追求第一和第三个目标。hi Aim 1，我们已经完成了斑点杂交诊断在美国人群中常见的血红蛋白病和地中海贫血突变的研究。我们建议发展和完善利用母体血液分离的胎儿细胞进行产前诊断的方法。我们的初步研究表明，这是一种很有前途的方法，但要使这种测试切实可行，还需要做更多的工作。我们将使用噬菌体展示技术分离胎儿有核红细胞和人胚胎血红蛋白特异性单链抗体。我们将研究使用图像分析和激光捕获显微解剖，以促进胎儿有核红细胞的检索。这些事态发展将大大提高这一程序的有效性。本提案的第二个目的是探索子宫内基因传递的可能性-地中海贫血是子宫内基因治疗的良好模型，因为纯合子-地中海贫血的病理变化通常出现在出生前。在我们的实验室里，我们通过敲除小鼠内源性的α -珠蛋白基因，制作了α -地中海贫血的小鼠模型。通过杂交不同的菌株，我们得到了有3、2、1或没有α -珠蛋白基因的老鼠，它们模仿了人类α -地中海贫血的临床表现。我们将使用AAV载体和含有控制人a-珠蛋白基因的β -g1obin LCR的慢病毒载体在妊娠第15周注射到胎鼠体内。我们将对这些小鼠进行跟踪观察。α -珠蛋白基因与拯救疾病表型的关系。这种方法不仅对地中海贫血有用，而且对其他遗传性疾病也有用，比如在子宫内就开始发病的OTC缺乏症。