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

描述(申请人提供)：同种异体血液和骨髓移植(BMT)形式的过继T细胞疗法已被证明是治疗血液系统恶性肿瘤的为数不多的治疗方法之一。供体接种中的成熟供体T细胞在介导移植物抗肿瘤(GVT)反应和促进供体免疫重建方面发挥重要作用。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措施的充分利用，移植物抗宿主病仍然是骨髓移植的主要并发症之一。急性GVHD的特点是移植患者的皮肤、肝脏、胃肠道和淋巴组织受到严重的、潜在的致命性组织损伤，由宿主同种异体抗原的供者T细胞介导。因此，T细胞归巢到GVHD靶器官，并通过整合素、选择素和趋化因子受体对其进行调节，被认为是在仍然允许GVT效应的情况下改善或预防GVHD的潜在的新的干预部位。T细胞的运输和归巢涉及一系列事件，这些事件需要T细胞表面表达特定的黏附分子和趋化因子受体，以及它们的配体对应物在炎症组织的高内皮微静脉(HEV)上的时空表现。事实上，许多专注于操纵A4？7整合素(一种表达在T细胞表面的肠道归巢受体)链表达的研究发现，供体T细胞对宿主肠道上皮细胞的渗透减少，这反过来又与各种GVHD小鼠模型的肠道组织损伤减少有关。然而，由于缺乏一种有效的方法来安全有效地操作捐赠者造血干细胞接种中的T细胞，以下调其A4？7整合素的表达，这些研究的临床翻译受到了阻碍。因此，拟议的研究将集中在使用小发夹RNA(ShRNA)来检验这样的假设，即在移植前通过慢病毒感染稳定地下调供者T细胞整合素链的表达，可以在不影响这些捐赠者T细胞有益的GVT潜力的情况下提供长期的GVHD严重程度的降低。7-shRNA转导的人类T细胞的免疫功能和细胞黏附能力也将被评估，作为将拟议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，骨髓移植是一种被广泛接受的治疗血液恶性肿瘤的方法。在目前的研究中，我们将研究新的基因技术的实施，以使供体T细胞(GVHD的原因实体)远离对患者器官的损害(GVHD的标志)。这种方法将为骨髓移植的新的和可能不那么激进的治疗策略开辟许多可能性。更重要的是，这项拟议研究的结果可能会增加骨髓移植的捐赠者数量，因为移植的T细胞将被改造成较少渗透到器官中，因为它们在遇到自身与宿主细胞之间的差异时可能会造成不良伤害；这种情况尤其发生在捐赠者不能完全匹配的情况下。

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.