Gene therapy of chronic granulomatous disease with GFP-tagged retrovirus vectors

为了改进x连锁慢性肉芽肿病（X-CGD）的基因治疗载体，研究了逆转录病毒顺式元件的高效基因转移和表达。检测治疗性gp91基因和绿色荧光蛋白（GFP）标记基因的表达水平。我们发现MSCV的长末端重复序列和引物结合位点在基因转移和长期表达中是有效的，MFG的剪接信号有利于转基因的强表达。因此，我们将MSCV和MFG中的顺式元件连接在一起，构建了一个新的逆转录病毒主干MGK。mgk衍生的双链载体可以有效地将基因转移到X-CGD骨髓细胞中，并具有良好的转基因表达，从而纠正了治疗动物的CGD表型。我们还研究了基因修饰的造血细胞在体内扩增的可行性。为此，我们开发了“选择性放大基因”，该基因编码粒细胞集落刺激因子受体（GCSFR）和类固醇受体的配体结合域（lbd）之间的融合蛋白。在本研究中，LBD特异性结合4-羟基他莫昔芬（4-HT），作为分子开关将GCSFR转化为依赖配体的生长信号发生器。用含有选择性扩增基因和GFP基因的双胞逆转录病毒转导骨髓细胞重建小鼠造血后，用4-HT刺激受体。攻毒小鼠的GFP+白细胞比例明显高于对照组。扩增后的细胞比淋巴细胞含有更多的粒细胞/单核细胞，而淋巴细胞是CGD基因治疗的靶细胞。我们正计划构建含gp91基因和选择性扩增基因的mgg双链逆转录病毒载体，用于X-CGD小鼠的临床前研究。

To improve gene therapy vectors for X-linked chronic granulomatous disease (X-CGD), retroviral cis-elements were investigated for efficient gene transfer and expression. The vectors were evaluated for exprssion levels of the therapeutic gp91 gene and the green fluorescent protein (GFP) marker gene. We found the long terminal repeats and the primer binding site from MSCV were efficient in gene transfer and long-term expression, and the splicing signals from MFG were beneficial for strong transgene expression. Therefore we hooked up these cis-elements from MSCV and MFG to construct a new retroviral backbone, MGK.An MGK-derived bicistronic vector gave efficient gene transfer into X-CGD bone marrow cells and good transgene expression, which corrected the CGD phenotype in the treated animals. We have also investigated the feasibility of in vivo expansion of gene-modified hematopoietic cells. For this purpose, we have developed "selective amplifier genes", which encode fusion proteins between the granulocyte colony-stimulating factor receptor (GCSFR) and the ligand-binding domains (LBDs) of steroid receptors. The LBD, in our case specifically binds to 4-hydroxytamoxifen (4-HT), functions as a molecular switch to convert GCSFR into a ligand-dependent growth signal generator. After reconstituting murine hematopoiesis with the bone marrow cells transduced by a bicistronic retrovirus containing the selective amplifier gene and the GFP gene, the recipients were challenged with 4-HT.The challenged mice had significantly greater proportion of GFP+ leukocytes than the controls. The expanded cells contained more granulocytes/monocytes than lymphocytes, the target lineage cells of CGD gene therapy. We are planning to construct MGK-based bicistronic retroviral vectors containing the gp91 gene and the selective amplifier gene, for preclinical studies with X-CGD mice.