Lentiviral vectors for gene therapy for beta-thalassemia

描述（由申请人提供）：B-地中海贫血是最常见的 人类单基因缺陷，由B-珠蛋白缺失或减少引起 合成，导致严重贫血。重型B型地中海贫血患者 终生输血治疗。骨髓移植可以 治愈，但仅限于少数匹配的捐赠者，并有可能 严重并发症。将正常B-珠蛋白基因置换成造血细胞 干细胞（HSC）可以永久纠正这种疾病， 与移植相关的并发症随着更好的 载体，改进的基因转移技术和更好地了解茎 细胞和载体生物学，基因治疗正在从实验室走向临床， 在像联合免疫缺陷和血友病B这样的疾病中。“珠蛋白”基因疗法遭受了 载体不稳定、低滴度和可变表达的问题。的 最近开发的慢病毒载体将非分裂的HSC和 通过独特的RNA输出机制稳定输出大的基因组片段， 赋予珠蛋白载体稳定性。自失活（SIN）慢病毒 载体甚至更有利：病毒LTR在整合时被删除 进入细胞，完全灭活病毒转录。这个功能是理想的 用于表达高度谱系限制的基因如珠蛋白，和 进一步提高了生物安全性。我们最近的研究显示 SIN中GFP和γ-珠蛋白的谱系特异性和长期表达 小鼠红白血病（MEL）细胞中的慢病毒载体，原代鼠和 人体细胞我们建议利用这些研究结果， SIN慢病毒载体携带人B-珠蛋白基因的能力， 用于将基因转移到HSC中的红系调节元件， B-珠蛋白在RBC中稳定、谱系特异性和持续表达。目标 本研究的主要目的是：1）开发携带人源性慢病毒的SIN-慢病毒载体， 红系调节元件控制下的B-珠蛋白基因，并筛选它们 在MEL细胞中稳定传递和高水平表达。2)确定 B-珠蛋白SIN的功效、谱系特异性和长期表达 在地中海贫血小鼠体内的慢病毒载体。3)确定基因转移 B-珠蛋白SIN慢病毒载体在红细胞子代中的能力和功效 人地中海贫血祖细胞，使用独特的人红细胞模型 在我们的实验室里从造血祖细胞中发展出来的。 这些目标共同构成了一个重点研究计划， 人地中海贫血RBC中的B-珠蛋白的治疗和持续水平，和 为未来的临床前研究奠定基础。

DESCRIPTION (provided by applicant): The B-thalassemias are the most common single gene defect in humans and result from absent or decreased B-globin synthesis, leading to severe anemia. Patients with B-thalassemia major are treated with life-long transfusions. Bone marrow transplantation can be curative, but is limited to a few with matched donors, and has potentially serious complications. Replacement of a normal B-globin gene into hematopoietic stem cells (HSCs) can potentially correct the disorder permanently, avoiding the complications associated with a transplant. 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 diseases like SCID and hemophilia B. 'Globin' gene therapy has suffered from problems of vector instability, low titers and variable expression. The recently developed lentiviral vectors transduce the non-dividing HSCs and stably export large genomic fragments by unique RNA export mechanisms, imparting stability to globin vectors. Self-inactivating (SIN) lentiviral vectors are even more advantageous: the viral LTR is deleted upon integration into cells, completely inactivating viral transcription. This feature is ideal for the expression of a highly lineage-restricted gene such as globin, and additionally improves their bio-safety. We have recently shown remarkably lineage-specific and long-term expression of GFP and gamma-globin from SIN lentiviral vectors in mouse erythroleukemia (MEL) cells, primary murine and human cells. We propose to capitalize on these findings by examining the capabilities of SIN lentiviral vectors to carry the human B-globin gene and erythroid regulatory elements for gene transfer into HSCs that results in stable, lineage-specific and sustained expression of B-globin in RBCs. The aims of the study are to: 1) Develop SIN-lentiviral vectors carrying the human B-globin gene under control of 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 B-globin SIN lentiviral vectors in vivo, in thalassemic mice. 3) Determine the gene transfer capacity and efficacy of B-globin SIN lentiviral vectors in the RBC progeny of human thalassemia progenitor cells, using a unique model of human RBC production developed in our laboratory from hematopoietic progenitor cells. Together, these aims comprise a focussed research program to produce therapeutic and sustained levels of B-globin in human thalassemia RBCs, and form the basis for future preclinical studies.