TRANSPLANT BIOLOGY, GENE TRANSFER, AND STEM CELL SOURCES

描述(改编自申请人的摘要)长期目标 本研究的目的是确定造血干细胞的最佳来源 移植、基因转移和移植物预防的细胞(HSC) 对抗宿主病(GVHD)。虽然大多数移植是 使用骨髓(BM)作为干细胞来源进行的研究， 对化疗和/或生长的兴趣与日俱增 因子动员的外周血祖/干细胞(PB)和 脐带血(CB)作为替代干细胞来源。在 同种异体外周血更适合供者，而脐带血有很多 优势，包括创造冷冻保存的脐带血的潜力 BANK与CB可能降低的免疫反应性 淋巴细胞数量减少，GVHD也随之降低。在自体设置中 CB和动员的PB可能包含更多的循环HSC， 因此，为逆转录病毒介导的基因构建更好的靶点 调职。移植这两种替代方案的主要问题之一 干细胞的来源和基因转移效率的研究进展 临床应用是缺乏最佳的人类干细胞检测系统。目标 1将干细胞含量和生长因子与基质细胞进行比较 骨髓高度浓缩原始静息G0细胞的反应性， Cb和Pb，以检验Cb和Pb富含这些元素的假设 细胞。这一目标将通过使用新的方法来丰富 并通过在体外和体内进行异种移植试验 培养系统，努力优化人类长期存活的检测方法 重新填充干细胞。目标2将检测干细胞的能力 从这些不同的来源通过传统的基因改造 逆转录病毒载体或假型表达生长因子的新型载体 基因取代了逆转录病毒的包膜基因。GVHD仍然是一个来源 异基因骨髓移植后的主要发病率和死亡率 移植(BMT)。可以假设Pb和Cb不同于 BM与其Th1或Th2 T细胞亚群的功能有关 可以解释细胞移植中观察到的移植物抗宿主病的减少 从这些来源获得，目标3将研究替代人类 HSC来源被牵连的细胞的数量和功能 在GVHD中。在小鼠GVHD模型中，初步数据显示减少 供体T细胞培养产生干扰素-γ的研究 骨髓移植前的白介素4(IL-4)可阻止这种炎性级联反应； G-CSF似乎具有与IL-4类似的作用。目标3将探索 假设用G-CSF治疗小鼠供体细胞可防止 GVHD的炎性细胞因子级联反应。通过这些手段，人们希望 为移植和基因选择干细胞的基本原理 在不同的临床情况下转移将被放置在更安全的 科学基础，通过了解和预防GVHD将 最终有可能更成功地延长移植 跨越了人类白细胞抗原的障碍。

DESCRIPTION (Adapted from the applicant's abstract) The long-term goal of this study is to determine the optimal source of hematopoietic stem cells (HSC) for transplantation, gene transfer, and prevention of graft versus host disease (GVHD). While the majority of transplants are carried out using bone marrow (BM) as the source of stem cells, there is increasing interest in the use of both chemotherapy and/or growth factor-mobilized peripheral blood progenitor/stem cells (PB) and umbilical cord blood (CB) as alternative stem cell sources. In the allogeneic setting PB would be preferable for the donor, and CB has many advantages, including the potential to create cryopreserved cord blood banks and the possible reduced immunological reactivity of CB lymphocytes with consequent decrease in GVHD. In the autologous setting CB and mobilized PB may contain increased numbers of cycling HSC and, therefore, constitute better targets for retrovirally mediated gene transfer. One of the major problems in transplanting both alternative sources of stem cells and advances in gene transfer efficiency to clinical use is the lack of optimal human stem cell assay systems. Aim 1 will compare the stem cell content and growth factor and stromal cell responsiveness of highly enriched primitive resting G0 cells from BM, CB, and PB, to test the hypothesis that CB and PB are enriched for these cells. This aim will be achieved by using novel means to enrich for these cells and by testing them in in vitro and xenogeneic in vivo culture systems, in an effort to optimize an assay for human long-term repopulating stem cells. Aim 2 will examine the capacity of stem cells from these different sources to be genetically modified by conventional retroviral vectors or novel vectors pseudotyped to express growth factor genes in place of the retroviral envelope gene. GVHD remains a source of major morbidity and mortality after allogeneic bone marrow transplantation (BMT). One can hypothesize that PB and CB differ from BM with respect to the function of their Th1 or Th2 T cell subsets that could account for the reduced GVHD observed in transplants with cells obtained from these sources, and Aim 3 will examine alternative human HSC sources for the number and function of the cells that are implicated in GVHD. In a murine GVHD model, preliminary data show that reduction of interferon-gamma (IFN- gamma) production by culturing donor T cells in interleukin 4 (IL-4) before BMT prevents this inflammatory cascade; G-CSF appears to have a similar effect to IL-4. Aim 3 will explore the hypothesis that treatment of murine donor cells with G-CSF prevents the inflammatory cytokine cascade of GVHD. By these means it is hoped that the rationale for selecting stem cells for transplantation and for gene transfer in different clinical contexts will be placed on a more secure scientific basis, and that by understanding and preventing GVHD it will eventually be possible to extend transplantation more successfully across HLA barriers.