Cell therapy for transient hematopoietic recovery

之前对犬模型的研究表明，及时给予单一细胞因子G-CSF可以将狗从400cGy体照射(TBI)中解救出来，否则就是致命的剂量。这种保护作用在500cGy时消失。同样，不能长期植入的MHC不匹配的骨髓细胞也提供了一种保护作用，允许在450cGyTBI后进行自体重建。在这种情况下，最大耐受辐射剂量是未知的，防护机制也是未知的。假设，不匹配的细胞可以提供短暂的血细胞群体，这将缓解细胞减少症，直到发生自体重建。或者， 通过同种异体反应，它们可能引发一场类似于注射G-CSF的“细胞因子风暴”。如果前者被证明是真的，那么将生长因子与非移植细胞产品相结合可能会产生额外的保护作用，增加受害者可以获救的辐射剂量阈值。因此，建议的研究旨在确定在有和没有添加生长因子的情况下，通过输注不匹配的造血细胞群体，通过自体造血重建可以实现存活的最大辐射剂量。研究将使用犬类照射模型，该模型对人类患者的结果具有很高的预测性。提出了三个具体目标：目标1；假设“最佳”细胞产品主要由定向的髓系祖细胞组成，将开发体外培养这些细胞的小规模方法。这包括为扩增细胞产品的质量控制和冷冻保存技术的优化开发体外检测方法。然后，将使用良好制造规范(GMP)指南扩大最优的体外扩增方案。在目标2中，将使用目标1中定义的最佳细胞产物 以确定在提供一过性造血支持时允许自体恢复的最大辐射剂量。使用VNTR分析将供体细胞与自体细胞区分开来。然后将确定是否需要免疫抑制方案来防止宿主对移植物的反应，否则可能会中和“救援效果”。在目标3中，细胞因子将与髓系祖细胞产品相结合，旨在优化“救援效果”。除G-CSF外，还需检测的细胞因子将通过单独的支持进行鉴定(RO1 AI66498-01)。还将确定辐射暴露后的“机会之窗”，在此期间，祖细胞产品的输注是有效的。该项目的总体目标是提供“现成”产品，在确定合适的干细胞来源(在项目5和6中涉及)的同时，能够促进自体重建或至少提供一段时间的支持。

Previous studies in the canine model have shown that a single cytokine, G-CSF, given in a timely manner can rescue dogs from 400cGy total body irradiation (TBI), an otherwise lethal dose. This protective effect is lost at 500 cGy. Similarly, MHC-mismatched marrow cells that do not engraft long-term also provide a protective effect that allows for autologous reconstitution following 450 cGy TBI. In this case, the maximum tolerated radiation dose is not known, nor is the protective mechanism. Hypothetically, the mismatched cells could provide a transient population of blood cells that would mitigate the cytopenia until autologous reconstitution occurs. Alternatively, they could, through alloreactivity, trigger a 'cytokine storm' that mimics the injection of G-CSF. If the former proves true, combining growth factors with a non-engrafting cell product might have additive protective effects, increasing the radiation dose threshold from which victims can be rescued. Therefore, the proposed studies are designed to determine the maximum radiation dose at which survival, with autologous hematopoietic reconstitution, can be achieved by infusing a non-matched population of hematopoietic cells, with and without added growth factors. Studies will use the canine irradiation model that has been highly predictive for outcomes in human patients. Three Specific Aims are proposed: Aim 1; given the assumption that the "optimal" cell product consists primarily of committed myeloid progenitors, small-scale methods for ex vivo generation of these cells will be developed. This includes development of in vitro assays for quality control of expanded cell products and optimization of cryopreservation techniques. The optimal ex vivo expansion protocol will then be scaled-up using Good Manufacturing Practice (GMP) guidelines. In Aim 2, the optimal cell product as defined under Aim 1 will be used to determine the maximum radiation dose that allows for autologous recovery when transient hematopoietic support is provided. Donor cells will be distinguished from autologous cells using VNTR analysis. It will then be determined whether immunosuppressive regimens are needed to prevent host-versus-graft reactions that might otherwise neutralize the "rescue effect". In Aim 3, cytokines will be combined with the myeloid progenitor cell product aimed at optimizing the "rescue effect". The cytokines to be tested in addition to G-CSF will be identified through separate support (RO1 AI66498-01). The "window of opportunity" after radiation exposure, during which infusion of the progenitor cell product is effective, will also be determined. The overall goal of this project is to provide 'off-the-shelf' products that can facilitate autologous reconstitution or at least provide a period of support while an appropriate stem cell source (addressed in Projects 5 and 6) is identified.