Stable shRNA-mediated gene silencing of the beta 7 integrin to ameliorate graft-v

描述（由申请人提供）：同种异体血液和骨髓移植（BMT）形式的免疫性T细胞治疗已被证明是血液恶性肿瘤的少数治愈性治疗之一。供体接种物中的成熟供体T细胞在介导针对预处理方案后持续存在的残留肿瘤细胞的移植物抗肿瘤（GVT）应答以及促进供体免疫重建中发挥核心作用。然而，移植物抗宿主病（GVHD）的发生极大地限制了这种临床干预的充分利用，GVHD仍然是BMT的主要并发症之一。急性GVHD的特征在于对移植患者的皮肤、肝脏、胃肠道和淋巴组织的严重且潜在致命的组织损伤，其由响应于宿主同种异体抗原的供体T细胞介导。因此，T细胞归巢至GVHD靶器官及其通过整合素、选择素和趋化因子受体的调节已被认为是用于干预以改善或预防GVHD同时仍允许GVT效应的潜在新位点。T细胞运输和归巢涉及一系列事件，这些事件需要特异性粘附分子和趋化因子受体在T细胞表面上的表达，沿着它们的配体对应物在发炎组织的高内皮小静脉（HEV）上的空间和时间表现。事实上，许多研究集中在α 4 β 7整联蛋白（在T细胞表面上表达的肠归巢受体）的β 7链表达的操纵上，发现供体T细胞向宿主肠上皮的浸润减少，这反过来与各种GVHD鼠模型中肠组织损伤的减少相关。然而，这些研究的临床转化由于缺乏可以安全有效地操纵供体造血干细胞接种物中的T细胞以下调其α 4 β 7整联蛋白表达的可用方法而受到阻碍。因此，拟议的研究将集中在使用小发夹RNA（shRNA）来测试以下假设：在移植前通过慢病毒感染稳定敲低供体T细胞的7整合素链表达，可以长期降低GVHD严重程度，而不影响这些供体T细胞的有益GVT潜力。还将评估7-shRNA转导的人T细胞的免疫功能和细胞粘附能力，作为将所提出的方法转化为临床环境的第一步。 公共卫生相关性：移植物抗宿主病（GVHD）仍然是骨髓移植（BMT）的主要并发症之一，骨髓移植是一种广泛接受的治疗许多血液恶性肿瘤的方法。在目前的研究中，我们将研究新的遗传技术的实施，以重定向供体T细胞（GVHD的因果实体），使其远离对患者器官的损害（GVHD的标志）。这种方法将为BMT的新的和潜在的侵略性较低的治疗策略开辟许多可能性。更重要的是，拟议研究的结果可能会增加BMT的供体数量，因为移植的T细胞将被设计成较少渗透到器官中，当它们遇到自身和宿主细胞之间的差异时，它们可能会造成不必要的伤害;这种情况特别是当供体不能完全匹配时。

DESCRIPTION (provided by applicant): Adoptive T cell therapy in the form of allogeneic blood and marrow transplantation (BMT) has proven to be one of the few curative treatments for hematological malignancies. Mature donor T cells in the donor inoculum play a central role in mediating graft-versus-tumor (GVT) responses against residual tumor cells that persist after conditioning regimens, and also in facilitating donor immune reconstitution. However the full exploitation of this clinical intervention is greatly limited by the occurrence of graft-versus-host disease (GVHD), which remains one of the main complications of BMT. Acute GVHD is characterized by severe, and potentially lethal, tissue damage to the skin, liver, gastrointestinal tract and lymphoid tissues of transplanted patients, mediated by donor T cells responding to host alloantigens. The homing of T cells to GVHD target organs and their regulation via integrins, selectins and chemokine receptors has therefore been recognized as potential novel sites for intervention to ameliorate or prevent GVHD while still allowing GVT effects. T cell trafficking and homing involves a compendium of events, that require the expression of specific adhesion molecules and chemokine receptors on the T cell surface, along with the spatial and temporal manifestation of their ligand counterparts on high endothelial venules (HEV) of inflamed tissues. Indeed, a number of studies focusing on the manipulation of the ¿7 chain expression of the a4¿7 integrin (an intestinal homing receptor expressed on the surface of T cells) found decreased infiltration of donor T cells to the host gut epithelium, which in turn was associated with decreased intestinal tissue damage in various GVHD murine models. The clinical translation of these studies is however hindered by the lack of an available approach that could safely and efficaciously manipulate T cells in the donor hematopoietic stem cell inoculum, to downregulate their expression of a4¿7 integrin. The proposed studies will thus focus on the use of small-hairpin RNA (shRNA) to test the hypothesis that stable knockdown expression of the ¿7 integrin chain of donor T cells via lentiviral infection, prior to transplant, can provide long-term reduction of GVHD severity without affecting the beneficial GVT potential of those donor T cells. The immune functionality and cell adhesion capabilities of ¿7-shRNA-transduced human T cells will also be assessed, as a first step towards translation of the proposed methodology to the clinical setting. PUBLIC HEALTH RELEVANCE: Graft-versus-host disease (GVHD) still remains one of the main complications associated with bone marrow transplantation (BMT), a well accepted treatment for a number of blood malignancies. In the current study we will investigate the implementation of novel genetic techniques to redirect donor T cells (the causal entities of GVHD) away from inflicting damage to the patient's organs (the hallmark of GVHD). This approach will open up a number of possibilities for new and potentially less aggressive therapeutic strategies for BMT. More importantly, the results from the proposed study could increase the number of donors for BMT, because transplanted T cells would be engineered to infiltrate less into the organs where they can cause undesirable harm upon encountering differences between themselves and the host cells; a situation that occurs specially when the donor cannot be fully matched.