Ginn Samantha L, Fleming Jane, Rowe Peter B, Alexander Ian E
Gene Therapy Research Unit, The Children's Hospital at Westmead and Children's Medical Research Institute, Westmead, NSW, Australia 2145.
Hum Gene Ther. 2003 Aug 10;14(12):1127-37. doi: 10.1089/104303403322167975.
In a previous study using an early-generation VSV-G-pseudotyped lentivirus vector encoding enhanced green fluorescent protein (EGFP) under the transcriptional control of a human cytomegalovirus (CMV) immediate-early promoter, we examined transduction efficiency in dissociated dorsal root ganglia (DRG) cultures. In cultures of murine origin, transgene expression was observed solely in the sensory neurons with the stromal cell population failing to show evidence of transduction. In contrast, efficient and sustained transduction of both sensory neurons and the stromal cell population was observed in cultures of human origin. Given the widespread use of murine models in preclinical gene therapy studies, in the current study we investigated the basis of this apparent neuron specificity of lentivirus-mediated transduction in murine DRG cultures. The interspecies differences persisted at high multiplicities of infection, and irrespective of whether lentiviral vector stocks were packaged in the presence or absence of human immunodeficiency virus type 1 (HIV-1) accessory proteins. Cell-type specificity of CMV promoter expression, tropism of the VSV-G envelope, and blocks to molecular transduction were also precluded as possible mechanisms, thereby implicating transcriptional repression of the internal heterologous promoter. This promoter interference effect was found to be mediated by cis-acting sequences upstream of the core promoter elements located in the U3 region of the proviral long terminal repeats (LTRs). Deletion of this region, as in late-generation self-inactivating (SIN) lentivirus vectors, relieves this effect. This provides a basis for reevaluating data produced using early-generation U3-bearing lentivirus vectors and for reconciling these with results obtained using more contemporary SIN lentivirus vectors carrying a U3 deletion.
在先前的一项研究中，我们使用了一种早期一代的水疱性口炎病毒糖蛋白（VSV-G）假型慢病毒载体，该载体在人巨细胞病毒（CMV）立即早期启动子的转录控制下编码增强型绿色荧光蛋白（EGFP），我们检测了其在解离的背根神经节（DRG）培养物中的转导效率。在源自小鼠的培养物中，仅在感觉神经元中观察到转基因表达，而基质细胞群体未显示出转导迹象。相比之下，在源自人类的培养物中，观察到感觉神经元和基质细胞群体均有高效且持续的转导。鉴于小鼠模型在临床前基因治疗研究中的广泛应用，在本研究中，我们调查了慢病毒介导的转导在小鼠DRG培养物中这种明显的神经元特异性的基础。种间差异在高感染复数时仍然存在，且与慢病毒载体储备是否在存在或不存在1型人类免疫缺陷病毒（HIV-1）辅助蛋白的情况下包装无关。CMV启动子表达的细胞类型特异性、VSV-G包膜的嗜性以及对分子转导的阻断也被排除为可能的机制，从而暗示内部异源启动子的转录抑制。发现这种启动子干扰效应是由位于前病毒长末端重复序列（LTRs）U3区域的核心启动子元件上游的顺式作用序列介导的。如在晚期一代自我失活（SIN）慢病毒载体中那样删除该区域可缓解这种效应。这为重新评估使用早期一代含U3慢病毒载体产生的数据以及将这些数据与使用携带U3缺失的更现代的SIN慢病毒载体获得的结果相协调提供了基础。
