ROLE OF ENDOTHELIN-1 INSICKLEACUTE CHEST SYNDROME

镰状细胞性贫血(SCA)的特征是反复血管闭塞和溶血，主要原因是 低氧条件下形成镰刀状红细胞。该病的治疗主要是对症治疗。骨灰石_ 移植是一种治疗方式，仅限于少数匹配捐赠者，并有潜在的副作用。渐增 长期服用羟基脲的红细胞表达“抗镰状”7-珠蛋白的频率降低 令人恶心的事件。在造血干细胞(HSCs)中使用“/-珠蛋白基因”进行基因治疗可以提高小鼠的存活 从转基因的HSCs中永久衍生的红细胞。用肿瘤逆转录病毒基因治疗血红蛋白病 载体一直存在载体不稳定、低滴度和可变表达等问题。随着……的到来 更好的载体，改进的基因转移技术，以及对干细胞和载体生物学、基因 对于SCID和血友病B等疾病，治疗正在从板凳到床边进行。最近开发的， 慢病毒载体转导未分裂的HSCs并稳定输出高水平表达所需的大基因组片段 受调控的“珠蛋白”基因表达。自失活(SIN)慢病毒载体更具优势：病毒长 末端重复序列在整合到细胞后被删除，完全使病毒转录失效，这是一种理想的特征 表达了高度血统限制的基因，并额外提高了生物安全性。我们最近推出了Shoven 1：- GFP的显著谱系特异性和长期表达及对小鼠红细胞生成素的治疗纠正 携带SIN慢病毒载体的原发和继发小鼠中的卟啉症。我们想要扩展这些结果并检查 携带人γ-珠蛋白基因和红系基因调控元件的SIN慢病毒载体的特性 转移到HSCs中，在红细胞中高水平、稳定和持续地表达/-珠蛋白。这项研究的目的 1)研制携带人γ-珠蛋白基因和红系调节元件的慢病毒载体，并筛选 它们在MEL细胞中稳定传播和高水平表达。2)确定疗效、谱系特异性和 这些载体在易位镰刀鼠体内的长期表达。3)检测这些载体的基因转移和效果。 在人类SCA祖细胞的RBC后代中，使用正常和 本实验室研制了SCA前体细胞。总而言之，这些目标包括一项集中研究，以产生可持续的 和治疗水平的人SCA红细胞，并形成了未来的临床前研究的基础。

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.