Subang Maria C, Fatah Rewas, Wu Ying, Hannaman Drew, Rice Jason, Evans Claire F, Chernajovsky Yuti, Gould David
Bone & Joint Research Unit, Queen Mary University of London, William Harvey Research Institute, Charterhouse Square, London EC1M 6BQ, UK.
Curr Gene Ther. 2015;15(1):3-14. doi: 10.2174/1566523214666141114204943.
Immune responses to expressed foreign transgenes continue to hamper progress of gene therapy development. Translated foreign proteins with intracellular location are generally less accessible to the immune system, nevertheless they can be presented to the immune system through both MHC Class I and Class II pathways. When the foreign protein luciferase was expressed following intramuscular delivery of plasmid DNA in outbred mice, expression rapidly declined over 4 weeks. Through modifications to the expression plasmid and the luciferase transgene we examined the effect of detargeting expression away from antigen-presenting cells (APCs), targeting expression to skeletal muscle and fusion with glycine-alanine repeats (GAr) that block MHC-Class I presentation on the duration of luciferase expression. De-targeting expression from APCs with miR142-3p target sequences incorporated into the luciferase 3'UTR reduced the humoral immune response to both native and luciferase modified with a short GAr sequence but did not prolong the duration of expression. When a skeletal muscle specific promoter was combined with the miR target sequences the humoral immune response was dampened and luciferase expression persisted at higher levels for longer. Interestingly, fusion of luciferase with a longer GAr sequence promoted the decline in luciferase expression and increased the humoral immune response to luciferase. These studies demonstrate that expression elements and transgene modifications can alter the duration of transgene expression but other factors will need to overcome before foreign transgenes expressed in skeletal muscle are immunologically silent.
对表达的外源转基因的免疫反应持续阻碍着基因治疗的发展进程。细胞内定位的翻译后外源蛋白通常较难被免疫系统识别，不过它们可通过MHC I类和II类途径呈递给免疫系统。当在远交系小鼠中通过肌肉注射质粒DNA表达外源蛋白荧光素酶时，其表达在4周内迅速下降。通过对表达质粒和荧光素酶转基因进行修饰，我们研究了将表达从抗原呈递细胞（APC）上脱靶、将表达靶向骨骼肌以及与阻止MHC I类呈递的甘氨酸 - 丙氨酸重复序列（GAr）融合对荧光素酶表达持续时间的影响。将含miR142 - 3p靶序列的荧光素酶3'UTR导入以从APC上脱靶表达，可降低对天然荧光素酶和经短GAr序列修饰的荧光素酶的体液免疫反应，但并未延长表达持续时间。当将骨骼肌特异性启动子与miR靶序列结合时，体液免疫反应受到抑制，荧光素酶表达在更长时间内维持在较高水平。有趣的是，荧光素酶与更长的GAr序列融合会促使荧光素酶表达下降，并增加对荧光素酶的体液免疫反应。这些研究表明，表达元件和转基因修饰可改变转基因表达的持续时间，但在骨骼肌中表达的外源转基因实现免疫沉默之前，还需要克服其他因素。
