Gene therapy for chronic granulomatous disease in combination with selective cell amplification and utilizing hematopoietic microenvironment

为了提高造血干细胞基因治疗的疗效，对选择性放大基因（sag）进行了改良。sag是一种人工基因，其编码的嵌合受体包括生长因子受体和促红细胞生成素（Epo）受体的细胞外结构域。这种融合分子在被Epo激活时产生生长信号，所有成分均来源于人类蛋白质。在证实了精制的SAG在体外具有细胞生长开关的功能后，我们选择了最好的SAG，进一步明确其在x连锁慢性肉芽肿病（X-CGD）小鼠模型中的体内功效。用携带SAG和gp91-phox基因（X-CGD的治疗基因）的逆转录病毒载体转导供体X-CGD小鼠的骨髓细胞，并将其输入X-CGD受体。第一轮Epo给药后，功能校正中性粒细胞显著增加（从1.0±0.1%增加到3.9±0.8%,p=0.006），第二轮Epo给药后，功能校正中性粒细胞显著增加（从1.5±1.1%增加到3.9±0.8%,p=0.065）。结果表明，epo反应性SAG产物增强了干细胞基因治疗的疗效。针对非人类灵长类动物的SAG系统的临床前研究即将启动。在X-CGD小鼠模型上研究骨髓内移植（iBMT）的疗效。这种方法允许极少数量的造血干细胞移植，否则会产生排斥反应。在干细胞移植和X-CGD的基因治疗中，减少强度预处理是可取的，以尽量减少手术相关的风险。当通过静脉输注或iBMT移植超过10^6个野生型骨髓细胞时，给予低剂量（4 Gy）照射将供体细胞植入X-CGD受体。然而，当供体细胞数小于10^6时，只有iBMT允许野生型细胞移植。间充质干细胞（MSCs）可分化为多种细胞系，包括具有造血生态位的骨髓基质细胞。我们研究了mscs样10T1/2细胞与其前脂肪细胞样衍生物A54细胞之间的差异。A54细胞过度表达细胞因子，如Steel因子、CXCL12和Ang-1，这些细胞因子在髓细胞源性细胞因子处理后进一步上调。根据这种上调，造血干细胞/祖细胞共培养的鹅卵石和集落形成增强。提示间充质干细胞的共移植可支持造血干细胞的移植。少

To improve the efficacy of hematopoietic stem cell gene therapy, selective amplifier genes (SAGs) were refined. SAGs are artificial genes encoding chimeric receptors comprising growth factor receptors and the extracellular domain of erythropoietin (Epo) receptor. Such fusion molecules generate growth signal when activated by Epo, and all the components were derived from human proteins. After confirming the fact that the refined SAGs functioned as cell growth switch in vitro, we chose the best SAG to further clarify its in vivo efficacy in the mouse model of X-linked chronic granulomatous disease (X-CGD). Bone marrow cells from donor X-CGD mice were transduced with a retrovirus vector carrying the SAG and the gp91-phox gene, the therapeutic gene for X-CGD, and transfused to X-CGD recipients. The first round Epo administration resulted in a significant increase of functionally corrected neutrophils in the recipients (from 1.0±0.1% to 3.9±0.8%; p=0.006), and the second round Epo administr … More ation lead brought up a similar increase (from 1.5±1.1% to 3.9±0.8%; p=0.065). The result demonstrated that the Epo-responsive SAG product reinforced the efficacy of stem cell gene therapy. Preclinical studies of the SAG system with non-human primates are to be launched.The efficacy of intra-bone marrow transplantation (iBMT) was investigated in the X-CGD mouse model. This method allows very small number of hematopoietic stem cells to engraft otherwise rejected. Reduced intensity preconditioning is preferable in stem cell transplantation and gene therapy for X-CGD, to minimize procedure-associated risks. When more than 10^6 wild type bone marrow cells were transplanted through either intravenous infusion or iBMT, donor cells engrafted in X-CGD recipients given a low-dose irradiation (4 Gy). However only iBMT allowed wild type cells to engraft when the number of donor cells were smaller than 10^6.Mesnchimal stem cells (MSCs) give rise to various lineages of cells including bone marrow stromal cells displaying the hematopoietic niche. We investigated the difference between MSC-like 10T1/2 cells and their preadipocyte-like derivative A54 cells. A54 cells overexpressed cytokines such as Steel factor, CXCL12 and Ang-1 which were further upregulated upon treatment of a myeloid cell-derived cytokine. In accordance with this upregulation, cobblestone and colony formation by cocultured hematopoietic stem/progenitor cells was augmented. The result suggested that cotransplantation of MSCs would support the engraftment of hematopoic stem cells. Less