Anti-nucleolin aptamer AS1411: Applications in Kaposi's Sarcoma Associated Herpes Virus (KSHV) biology

卡波西肉瘤相关疱疹病毒是人类γ疱疹病毒家族的成员，也被称为 为人类疱疹病毒8型(HHV-8)。KSHV是一种名为Kaposi‘s的内皮肿瘤的病原体 肉瘤(KS)和一种称为原发性渗出性淋巴瘤的高度侵袭性淋巴增生性B细胞淋巴瘤 (贝利)。KS是最常见的血管恶性肿瘤，在HIV感染患者中具有很高的发病率和死亡率。 PEL是一种罕见的造血和淋巴组织肿瘤，预后不良。PEL案件 自从联合抗逆转录病毒疗法(CART)的广泛使用以来，在美国已经大大减少了 但KSHV仍在发展中国家造成重大死亡。在KSHV目标方面有迫切的需要 特定的抗病毒药物，耐受性很好，没有免疫功能受损的严重不良事件 KSHV相关恶性肿瘤患者。KSHV在其生命周期中表现出两个不同的阶段。在延迟期间， 最小数量的延迟基因，如vFlIP(ORF71)、vCyclin(ORF72)、延迟相关核 抗原-1(LANA-1；ORF73)和Kaposin被表达。相反，在裂解阶段，KSHV表达 广泛的即早(IE)、延迟早(DE)和晚基因；经历活跃的复制和产生 病毒的后代。KSHV已被证明利用多种宿主生长因子、细胞因子、血管生成因子和 细胞信号和代谢相关蛋白，为其复制，生存， 和延迟。我们令人兴奋的研究发现：1)KS皮损表达非常强健的 核仁素与健康皮肤组织的比较；2)KSHV新生感染原代内皮细胞诱导高表达 核仁素和磷酸核仁素水平，更重要的是，3)KSHV感染细胞与G- 四链形成抗核仁适配子AS1411减少KSHV潜伏期(ORF73)和增加溶血素(ORF50) 基因表达，我们在核仁素沉默的KSHV感染的PEL细胞中获得了类似的结果。4)AS1411 治疗对KSHV感染的PEL细胞系有明显的诱导细胞死亡作用。根据我们的初步结果， 我们假设KSHV诱导宿主因子核仁来支持其潜伏期、生命周期和靶向性 含有AS1411的核仁素对KSHV相关的恶性肿瘤有治疗潜力。为了测试这一点 假设，我们已经制定了两个具体目标，在这两个目标中，我们将1)确定调控机制 核仁蛋白在KSHV感染中的表达及其在KSHV潜伏期中的作用，以及2)评估 AS1411治疗KS和PEL的化疗潜力我们的研究意义重大，并将对 在KSHV生物学中推进未探索的和新的靶向核酸适体的治疗领域的积极影响 了解它们的抗病毒和抗癌潜力也可以应用于其他病毒恶性肿瘤。

Kaposi’s sarcoma-associated herpesvirus (KSHV), a member of the human γ-herpesvirus family is also termed as human herpesvirus type 8 (HHV-8). KSHV is an etiological agent of an endothelial tumor called Kaposi’s sarcoma (KS) and a highly aggressive lymphoproliferative B cell lymphoma called primary effusion lymphoma (PEL). KS is the most common vascular malignancy causing high morbidity and mortality in HIV-infected patients. PEL is a rare tumor of hematopoietic and lymphoid tissues and is associated with a poor prognosis. PEL cases have been dramatically reduced in the US since the widespread use of combined antiretroviral therapy (cART) but KSHV still causes significant mortality in the developing world. There is a critical need in KSHV targeting specific antiviral drugs, which are very well tolerated with no severe adverse events for immunocompromised patients with KSHV associated malignancies. KSHV exhibits two distinct phases in its life cycle. During latency, a minimal number of latency genes such as vFLIP (ORF71), vCyclin (ORF72), latency-associated nuclear antigen-1 (LANA-1; ORF73), and Kaposin are expressed. By contrast, during the lytic phase, KSHV expresses a wide array of immediate-early (IE), delayed-early (DE), and late genes; undergo active replication and produce virion progeny. KSHV has been shown to utilize multiple host growth factors, cytokines, angiogenic factors, and cell signaling and metabolism-related proteins for creating a beneficial environment for its replication, survival, and latency. Our exciting studies discovered that: 1) KS skin lesions express remarkably robust expression of nucleolin compared to healthy skin tissue; 2) KSHV de novo infection in primary endothelial cells induced a high level of nucleolin and phospho-nucleolin, and more importantly, 3) Incubation of KSHV infected cells with G- quadruplex forming anti-nucleolin aptamer AS1411 reduced KSHV latent (ORF73) and increased lytic (ORF50) gene expression and we obtained similar results in nucleolin silenced KSHV infected PEL cells. 4) AS1411 treatment was efficacious in inducing cell death in KSHV infected PEL cell lines. Based on our preliminary results, we hypothesize that KSHV induces host factor nucleolin to support its latency and life cycle and targeting nucleolin with AS1411 would have therapeutic potential in KSHV associated malignancies. To test this hypothesis, we have formulated two specific aims in which we will 1) To determine the regulatory mechanisms of nucleolin expression upon KSHV infection and its role in KSHV latency, and 2) To evaluate the chemotherapeutic potential of using AS1411 to treat KS and PEL. Our studies are significant and will have a positive impact by advancing the unexplored and novel targeted theranostic field of aptamers in KSHV biology and understanding their antiviral and anticancer potential can also be applied to other viral malignancies.