ROLE OF ENDOTHELIN-1 INSICKLEACUTE CHEST SYNDROME

镰状细胞性贫血（SCA）的特征是反复的血管闭塞和溶血，主要是由于 缺氧时形成镰刀形红细胞。对这种疾病的治疗主要是对症治疗。骨粒_ 移植是一种治疗方式，仅限于少数具有匹配供体的人，并且具有潜在的副作用。增加 通过长期施用羟基脲在RBC中表达"抗镰状化" 7-珠蛋白降低了 镰刀事件。在造血干细胞（HSC）中使用β-珠蛋白基因的基因治疗可以提高造血干细胞（HSC）的存活率。 RBC永久性地来源于基因修饰的HSC。血红蛋白病的基因治疗 载体存在载体不稳定、低滴度和可变表达的问题。的到来 更好的载体，改进的基因转移技术，更好地理解干细胞和载体生物学，基因 对于像SCID和血友病B这样的疾病，治疗已经从实验室走向了临床。最近开发的， 慢病毒载体将非分裂的HSC包裹，并稳定地输出高水平表达所需的大基因组片段。 调节"珠蛋白"基因表达。自失活（SIN）慢病毒载体甚至更有利： 当整合到细胞中时，末端重复序列被删除，完全灭活病毒转录，这是一个理想的特征， 高度谱系限制基因的表达，并且另外提高了生物安全性。我们最近在推1：- GFP的显着谱系特异性和长期表达以及小鼠红细胞生成的治疗性校正 用SIN-慢病毒载体治疗原发性和继发性小鼠卟啉病。我们想扩展这些结果，并检查 SIN-慢病毒载体携带人γ-珠蛋白基因和红系调节元件基因的特性 转移到HSC中，导致RBC中β-珠蛋白的高水平、稳定和持续表达。研究目的 1）开发携带人γ-珠蛋白基因和红系调节元件的SIN-慢病毒载体， 它们在MEL细胞中稳定传递和高水平表达。2)确定效力、谱系特异性和 这些载体在transgemc镰状小鼠中的长期表达。3)确定这些载体的基因转移和功效 在人SCA祖细胞的RBC后代中，使用从正常和非正常细胞产生人RBC的独特模型， SCA祖细胞在我们的实验室开发。这些目标共同构成了一项重点研究， 和人SCA RBC中γ-珠蛋白的治疗水平，并形成未来临床前研究的基础。

Sickle cell anemia (SCA) is characterized by repeated vascular occlusions and hemolysis, primarily resulting from the sickle-shaped RBCs formed under hypoxia. Treatment for the disease is mainly symptomatic. Bone marro_ transplantation, a curative modality, is limited to a few with matched donors and has potential side effects. Increasing expression of the "anti-sickling' 7-globin in the RBCs by long-term administration of hydroxyurea reduces the frequency of sickling events. Gene therapy using the "/-globin gene in hematopoietic stem cells (HSCs) can improve the survival of RBCs derived from the genetically modified HSCs permanently. Gene therapy for hemoglobinopathies with oncoretroviral vectors has suffered from problems of vector instability, low titers and variable expression. With the advent of better vectors, improved gene transfer techniques and a better understanding of stem cell and vector biology, gene therapy is going from the bench to the bedside, in disorders like SCID and hemophilia B. The recently developed, lentiviral vectors transduce the non-dividing HSCs and stably export large genomic fragments required for high-level regulated "globin' gene expression. Self-inactivating (SIN) lentiviral vectors are even more advantageous: the viral long terminal repeat is deleted upon integration into cells, completely inactivating viral transcription, a feature ideal for the expression of a highly lineage-restricted gene, and additionally improves the bio-safety. We have recently shoven 1:- remarkably lineage-specific and long-term expression of GFP and a therapeutic correction of the murine erythropoietic porphyria in primary and secondary mice with SIN-lentiviral vectors. We would like to extend these results and examine the properties of SIN-lentiviral vectors in carrying the human y-globin gene and erythroid regulatory elements for gene transfer into HSCs, resulting in high-level, stable and sustained expression of ,/-globin in RBCs. The aims of the study are: 1) Develop SIN-lentiviral vectors carrying the human y-globin gene and erythroid regulatory elements, and screen them in MEL cells for stable transmission and high-level expression. 2) Determine the efficacy, lineage-specificity and long term expression of these vectors in transgemc sickle mice. 3) Determine gene transfer and efficacy of these vectors in the RBC progeny of human SCA progenitor cells, using the unique model of human RBC production from normal and SCA progenitors developed in our laboratory. Together, these aims comprise a focussed research to produce sustained and therapeutic levels ofy-globin in human SCA RBCs, and form the basis of future preclinical studies.