携双基因HSV1载体靶向治疗HSK病毒潜伏及复发的实验研究

In clinical, herpes simplex virus 1 (HSV1) caused herpes simplex keratitis (HSK) always be recurrent and persistent, which is also the dominant disorder in keratopathy for its leading incidence and blindness rate. However, no specific methods inhibiting viral latency and recurrence have been exploited. On the basis of many years’ research foundation, and by the aid of the nerve hobby characteristic of HSV, the replication-defective HSV1 vectors were established. This vector carries IFNγ gene, which plays an important role in anti-inflammatory and anti-virus, and also carries siRNA, which could clear the latent virus related LAT gene. The double gene targeting vector targets the trigeminal ganglion, and mediates the function of the IFNγ and siRNA. The real-time PCR, cornea confocal microscopy, flow cytometry assay and other technologies were employed to evaluate the therapeutic effects in vivo and in vitro, so as to explore the mechanism of the therapeutic gene. We have succeed in constructing replication-defective HSV1 vector carried IFNγ (rdHSV1-IFNγ) and confirming its function of targeting and antivirus. This study would provide the novel materials for clarifying the mechanism of HSV latency and HSK recurrence, and provide the new methods for inhibiting the virus latency and therapy for clinical. Totally, the present study could lay foundations for the double gene targeting vector clinical application and HSK prevention.

由单纯疱疹病毒1型（HSV1）引起的单纯疱疹病毒性角膜炎（HSK）因病毒潜伏感染导致反复发作，使该病在角膜病中发病率及致盲率居首位，目前病毒潜伏和复发的机制不明，针对性的治疗手段甚少。在前期多年研究基础上，本项目利用HSV病毒嗜神经特性,构建HSV1病毒载体并携带具有抗病毒、抗炎作用的干扰素γ（IFNγ）基因以及促使潜伏感染相关基因（LAT）沉默的siRNA双基因，靶向定位到病毒潜伏的三叉神经节，采用实时定量 PCR、角膜共聚焦、流式细胞术等方法,通过细胞实验和动物实验评价双基因靶向定位治疗的效果，探索其作用机制。目前，我们已成功构建携带IFNγ基因的HSV1重组病毒载体，并通过实验证实其具有抗病毒及靶向定位作用。本项目的深入研究将为明确病毒潜伏机制提供新的理论依据，为有效抑制病毒潜伏感染，解决HSK复发瓶颈提供新思路，新方法，为进一步临床应用、防控HSK奠定基础。

由单纯疱疹病毒1型（HSV1）引起的单纯疱疹病毒性角膜炎（HSK）因病毒潜伏感染导致反复发作，使该病在角膜病中发病率及致盲率居首位。但目前HSV1病毒潜伏和复发的机制不明，针对性的治疗手段甚少。本项目利用HSV病毒嗜神经特性，将HSV病毒改造为HSV复制缺陷型病毒载体，并使其携带具有抗病毒、抗炎作用的干扰素γ（IFNγ）基因以及能够沉默潜伏感染相关基因（LAT）的siRNA。采用实时定量 PCR、角膜共聚焦、流式细胞术等方法，通过细胞实验和动物实验评价IFNγ基因的表达、LAT基因的沉默、治疗载体的靶向定位效果及其对HSK的治疗效果；同时，探索rdHSV-IFNγ-siRNA抗炎、抗角膜损伤的作用机制。本项目成功构建了rdHSV-IFNγ-siRNA双基因靶向治疗系统，该系统可以靶向定位于三叉神经节、高效表达IFNγ、沉默LAT基因；体内体外实验均证实rdHSV-IFNγ-siRNA具有安全性、有效性。rdHSV-IFNγ-siRNA系统可以显著减轻大鼠HSK的临床症状。rdHSV-IFNγ-siRNA系统激活TLR4/NF-κB信号传导途径，并级联活化TLR4/NF-κB途径的下游TNF-α、IL-6、IL-1β及MCP-1等炎性因子。因此，双基因靶向治疗系统（rdHSV1-IFNγ-siRNA）可以靶向定位于三叉神经节、有效的抑制HSV病毒潜伏，并通过激活TLR4/NF-κB信号传导途径发挥治疗作用。本研究将为有效抑制HSV病毒潜伏感染、解决HSK复发瓶颈提供了新思路、新方法，具有重要的理论创新意义和良好的临床应用前景。