Kang E, Giri N, Wu T, Sellers S, Kirby M, Hanazono Y, Tisdale J, Dunbar C E
Molecular and Clinical Hematology Branch, National Institute of Diabetes and Digestive and Kidney Disorders/NIH, 9000 Rockville Pike, Bethesda, MD 20892, USA.
Hum Gene Ther. 2001 Sep 1;12(13):1663-72. doi: 10.1089/10430340152528156.
Many nonmalignant hematologic disorders could potentially be treated by genetic correction of as few as 5-10% of target lineage cells. However, immune system clearance of cells expressing gene products perceived as foreign could be limiting. There is evidence that tolerance to foreign proteins can result when myeloablative conditioning is used, but this limits the overall applicability of such techniques. Therefore, we sought to evaluate the engraftment of hematopoietic stem cells carrying a foreign transgene after low-dose irradiation by comparing in vivo survival of murine long-term repopulating cells (LTRC) transduced with either a retroviral vector expressing the bacterial neomycin phosphotransferase gene (neo) or a vector containing neo gene sequences but modified to prevent protein expression (nonexpression). First, marrow cells from congenic donors were transduced with either vector and transplanted into recipients treated with standard dose irradiation of 800 rads. High-level engraftment and gene marking resulted, without differences in the marking levels or pattern of persistence of the cells between cells transduced with either vector. Low-dose irradiation at 100 rads was tested using higher cell doses. Marking levels as high as 10% overall were obtained, again with no differences between mice receiving cells transduced with the neo versus the nonexpression vectors. To investigate a potentially more immunogenic protein, marrow cells were transduced with a vector containing the green fluorescent protein (GFP) gene, and their persistence was studied in recipient mice receiving 100 rads. Stable GFP expression in 5-10% of circulating cells was observed long term. We conclude that even with very low dose conditioning, engraftment by genetically modified LTRC cells at clinically significant levels can be achieved without evidence for clearance of cells known to be expressing immunogenic proteins.
许多非恶性血液系统疾病有可能通过对仅5%-10%的靶谱系细胞进行基因校正来治疗。然而，免疫系统对表达被视为外来基因产物的细胞的清除可能会成为限制因素。有证据表明，使用清髓性预处理时可产生对外来蛋白质的耐受性，但这限制了此类技术的整体适用性。因此，我们试图通过比较用表达细菌新霉素磷酸转移酶基因（neo）的逆转录病毒载体或含有neo基因序列但经修饰以防止蛋白质表达（无表达）的载体转导的小鼠长期重建造血细胞（LTRC）的体内存活率，来评估低剂量照射后携带外源转基因的造血干细胞的植入情况。首先，将同基因供体的骨髓细胞用任一载体转导，并移植到接受800拉德标准剂量照射的受体中。获得了高水平的植入和基因标记，两种载体转导的细胞之间在标记水平或细胞持续存在模式上没有差异。使用更高的细胞剂量测试了100拉德的低剂量照射。总体标记水平高达10%，接受neo载体转导细胞的小鼠与接受无表达载体转导细胞的小鼠之间同样没有差异。为了研究一种潜在免疫原性更强的蛋白质，用含有绿色荧光蛋白（GFP）基因的载体转导骨髓细胞，并在接受100拉德照射的受体小鼠中研究其持久性。长期观察到5%-10%的循环细胞中稳定表达GFP。我们得出结论，即使采用非常低剂量的预处理，经基因修饰的LTRC细胞也能实现具有临床意义水平的植入，且没有证据表明表达免疫原性蛋白质的细胞被清除。
