Lentiviral vectors for gene therapy for beta-thalassemia

描述(申请人提供)：最常见的是B-地中海贫血 人类缺乏或降低B-珠蛋白所致的单基因缺陷 合成，导致严重贫血。患有重型B地中海贫血的患者有 接受终生输血治疗。骨髓移植可以 治愈，但仅限于少数匹配捐赠者，并有可能 严重的并发症。将正常的B-珠蛋白基因替换为造血细胞 干细胞(HSCs)有可能永久纠正这种疾病，避免 与移植相关的并发症。随着更好的未来的到来 载体、改进的基因转移技术和对干细胞的更好理解 细胞和媒介生物学，基因治疗正在从板凳上走到床边， 在SCID和血友病B等疾病中。“金球蛋白”基因疗法遭受了 载体不稳定、低滴度和可变表达的问题。这个 新近发展的慢病毒载体转导未分裂的HSCs和 通过独特的RNA输出机制稳定地输出大的基因组片段， 赋予珠蛋白载体稳定性。自灭活慢病毒 载体甚至更有优势：整合后病毒LTR被删除 进入细胞，使病毒转录完全失活。此功能非常理想 对于高度血统限制的基因，如珠蛋白的表达，以及 此外，还提高了它们的生物安全性。我们最近表现出了显著的 SIN中GFP和γ-珠蛋白的谱系特异性和长期表达 慢病毒载体在小鼠红白血病(MEL)细胞、原代小鼠和 人类细胞。我们建议利用这些研究结果，研究 SIN慢病毒载体携带人B-珠蛋白基因和 红系调节元件的基因转移到HSCs导致 B-珠蛋白在红细胞中稳定、谱系特异性和持续表达。目标 本研究的主要目的是：1)研制携带人SIN的慢病毒载体 红系调控元件调控的B-珠蛋白基因及其筛选 在MEL细胞中稳定传播和高水平表达。2)确定 B-珠蛋白SIN的有效性、谱系特异性和长期表达 慢病毒载体体内，在地中海贫血小鼠。3)确定基因转移 慢病毒载体B-珠蛋白在小鼠红细胞子代中的免疫效果 使用独特的人红细胞模型的人地中海贫血祖细胞 我们实验室从造血祖细胞开发的产品。 这些目标共同构成了一个有重点的研究计划，以产生 治疗和维持人地中海贫血红细胞中的B-珠蛋白水平，以及 形成了未来临床前研究的基础。

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.