Abstract:
In previous animal model studies, we demonstrated the potential of rAAV2-sVEGFRv-1, which encodes a truncated variant of the alternatively spliced soluble version of VEGF receptor-1 (VEGFR1), as a human gene therapy for age-related macular degeneration (AMD) and diabetic retinopathy (DR). Here, we elucidate in vitro some of the mechanisms by which rAAV2-sVEGFRv-1 exerts its therapeutic effects. Human umbilical vein endothelial cells (HUVECs) were infected with rAAV2-sVEGFRv-1 or a control virus vector in the presence of members of the VEGF family to identify potential binding partners via ELISA, which showed that VEGF-A, VEGF-B, and placental growth factor (PlGF) are all ligands of its transgene product. In order to determine the effects of rAAV2-sVEGFRv-1 on cell proliferation and permeability, processes that are important to the progression AMD and DR, HUVECs were infected with the therapeutic virus vector under the stimulation of VEGF-A, the major driver of the neovascularization that characterizes the forms of these conditions most associated with vision loss. rAAV2-sVEGFRv-1 treatment, as a result, markedly reduced the extent to which these processes occurred, with the latter determined by measuring zonula occludens 1 expression. Finally, the human microglial HMC3 cell line was used to show the effects of the therapeutic virus vector upon inflammatory processes, another major contributor to angiogenic eye disease pathophysiology, with rAAV2-sVEGFRv-1 reducing therein the secretion of pro-inflammatory cytokines interleukin (IL)-1β and IL-6. Combined with our previously published in vivo data, the in vitro activity of the expressed transgene here further demonstrates the great promise of rAAV2-sVEGFRv-1 as a potential human gene therapeutic for addressing angiogenic ocular conditions.
摘要:
在以前的动物模型研究中,我们证明了rAAV2-sVEGFRv-1的潜力,它编码可变剪接的可溶性VEGF受体-1(VEGFR1)的截短变体,作为人类年龄相关性黄斑变性(AMD)和糖尿病视网膜病变(DR)的基因疗法。这里,我们在体外阐明了rAAV2-sVEGFRv-1发挥其治疗作用的一些机制。在存在VEGF家族成员的情况下,用rAAV2-sVEGFRv-1或对照病毒载体感染人脐静脉内皮细胞(HUVECs),以通过ELISA鉴定潜在的结合伴侣,这表明VEGF-A,VEGF-B,和胎盘生长因子(PlGF)都是其转基因产物的配体。为了确定rAAV2-sVEGFRv-1对细胞增殖和通透性的影响,对进展AMD和DR很重要的过程,在VEGF-A的刺激下,用治疗性病毒载体感染HUVECs,新血管形成的主要驱动因素是这些与视力丧失最相关的疾病的特征。rAAV2-sVEGFRv-1治疗,因此,显著降低了这些过程发生的程度,后者通过测量小带闭塞1的表达来确定。最后,人类小胶质细胞HMC3细胞系用于显示治疗性病毒载体对炎症过程的影响,血管生成性眼病病理生理学的另一个主要贡献者,其中rAAV2-sVEGFRv-1减少促炎细胞因子白细胞介素(IL)-1β和IL-6的分泌。结合我们先前发表的体内数据,表达的转基因的体外活性进一步证明了rAAV2-sVEGFRv-1作为解决血管生成眼部疾病的潜在人类基因治疗的巨大前景。