已获批准的寡核苷酸疗法的化学、结构和功能（三）

《已获批准的寡核苷酸疗法的化学、结构和功能（一）》

接上篇文章《已获批准的寡核苷酸疗法的化学、结构和功能(二）》

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SIMPLE BEGINNINGS: PHOSPHOROTHIOATES AND VITRAVENE 简单的起点：磷酸硫酸酯和 VITRAVENE

VITRAVENE (fomivirsen) was the first antisense therapeutic approved by the FDA, and the only antiviral oligonucleotide-based drug brought to market to date. Several ongoing clinical trials in various phases are assessing the efficacy of oligonucleotides against HBV and HCV targets and there is continued interest in the antimicrobial antisense therapeutic platform. VITRAVENE was a 21mer phosphorothioate oligo-2′-deoxynucleotide (PS-DNA; Figures 4, 6) that binds to the complementary mRNA sequence of major immediate-early region (IE2) proteins of human cytomegalovirus (CMV), thereby inhibiting replication of the virus. The drug was administered intravitreally to delay progression of CMV retinitis in AIDS patients. In immunocompromised individuals this disease leads to loss of retinal cells and eventually vision. In cell culture, the ASO was demonstrated to reduce immediate-early protein synthesis in CMV-infected dermal fibroblasts in a dose-dependent fashion. Fomivirsen also showed activity against drug-resistant mutants of the virus in vitro and combinations of fomivirsen with the antiviral drugs ganciclovir or foscarnet resulted in additive anti-CMV activity. Studies of the pharmacokinetic parameters of fomivirsen in non-human primates established half-lives of its elimination from the vitreous and retina of 22 and 78 h, respectively. This clearance occurred because of the distribution of the drug to other ocular tissues and some chain shortening was most consistent with exonuclease degradation. Fomivirsen was not detected in the plasma of either animals or patients, indicating that there was no appreciable systemic exposure to the oligonucleotide.

VITRAVENE（fomivirsen）是首个获得 FDA 批准的反义治疗药物，也是迄今为止唯一一种上市的基于寡核苷酸的抗病毒药物。目前有多项不同阶段的临床试验正在评估寡核苷酸针对乙型肝炎病毒（HBV）和丙型肝炎病毒（HCV）靶点的疗效，并且对抗微生物的反义治疗平台仍然存在着持续的兴趣。VITRAVENE 是一种 21 碱基磷酸硫酯寡聚-2'-脱氧核苷酸（PS-DNA；见图 4、6），它与人巨细胞病毒（CMV）主要即刻早期区（IE2）蛋白的互补 mRNA 序列结合，从而抑制病毒的复制。该药物通过玻璃体内给药以延缓 AIDS 患者 CMV 视网膜炎的进展。在免疫功能受损的个体中，该疾病会导致视网膜细胞丧失，最终影响视力。在细胞培养中，该反义寡核苷酸被证明可以剂量依赖性地降低 CMV 感染皮肤成纤维细胞中即刻早期蛋白的合成。Fomivirsen 还显示出对病毒耐药突变株的体外活性，并且与抗病毒药物甘西奇洛韦或福斯卡尼特的联合应用具有协同的抗 CMV 活性。对非人灵长类动物的 Fomivirsen 药代动力学参数的研究确定了其从玻璃体和视网膜中消除的半衰期分别为 22 小时和 78 小时。这种清除是由于药物分布到其他眼部组织以及某种链缩短，最符合外切酶降解的情况。Fomivirsen 在动物和患者的血浆中均未检测到，表明该寡核苷酸几乎没有体内系统暴露。

图 6.

At the time VITRAVENE was approved, antisense drug discovery and the utility of ASOs as pharmaceutical agents were viewed with skepticism by many . The 21mer PS-DNA shares none of the features of a typical small-molecule drug; it carries 20 negative charges and is actually a mixture of 220Rp- and Sp-PS diastereoisomers (Figures and 7A). The main mechanism of action is by RNase H-mediated degradation (Figure 6); at higher concentration of the oligo binding to viral coat proteins was suggested, although this non-antisense mode of action did not contribute to its activity. Similarly, there is no evidence that VITRAVENE is cleared by oxidative metabolic pathways (i.e. P450 enzymes). Oligo PS-DNAs bind to plasma proteins (mainly albumin and α2-macroglobulin), and are cleared from plasma by dose-dependent distribution to various tissues and nuclease metabolism with only minor excretion in urine or feces.

在 VITRAVENE 获得批准时，许多人对反义药物的发现以及 ASO 作为药物的实用性持怀疑态度。这种 21mer PS-DNA 没有典型小分子药物的特征；它带有 20 个负电荷，实际上是 220 个Rp-和Sp-PS 对映异构体的混合物。其主要作用机制是通过 RNase H 介导的降解（图 6）；在较高浓度下，该寡核苷酸与病毒外壳蛋白结合的机制也被提出，尽管这种非反义作用方式并未对其活性产生贡献。类似地，没有证据表明 VITRAVENE 通过氧化代谢途径（即 P450 酶）清除。寡核苷酸 PS-DNA 与血浆蛋白（主要是白蛋白和α2-巨球蛋白）结合，通过剂量依赖的分布到各种组织和核酸酶代谢从血浆中清除，只有少量通过尿液或粪便排出。

图 7.

One of the attractive features of the PS modification compared to the parent phosphodiester-based oligonucleotides is the enhanced nuclease resistance. The susceptibility to attack by nucleases differs for the two stereoisomers. Thus, snake venom phosphodiesterase (SVPD, 3′-exonuclease) degrades the Rp PS isomer more rapidly than the Sp isomer. Crystal structures of DNA polymerase I Klenow fragment exonuclease in complex with short PS-modified oligos demonstrated that the Sp-PS modification affords better protection. Accordingly, the sulfur atom of that isomer intrudes into the coordination sphere of one of the two metal ions bound at the active site. By comparison, the endonucleases DNase I, S1 and P1 all prefer the Sp- over the Rp-PS isomer.

与磷酸二酯基寡核苷酸相比，PS 修饰的一个吸引人的特点是增强的核酸酶抗性。两个立体异构体对核酸酶的敏感性不同。例如，蛇毒磷酸二酯酶（SVPD，3'-外切酶）比较快速地降解Rp-PS 异构体，而比较慢地降解Sp-PS 异构体。晶体结构显示，DNA 聚合酶 I Klenow 片段外切酶与短的 PS 修饰寡核苷酸结合时，Sp-PS 修饰提供了更好的保护效果。因此，该异构体的硫原子侵入到活性位点结合的两个金属离子的配位球中。相比之下，内切酶 DNase I、S1 和 P1 都更倾向于Sp-PS 异构体而非Rp-PS 异构体。

Unfortunately, in light of the fact that Sp PS-DNA is more resistant to attack by 3′-exonuclease, this isomer forms thermodynamically less stable duplexes with complementary RNA than the Rp isomer. Incorporation of PS modifications with mixed stereochemistry into a DNA reduces the melting temperature Tm of the duplex with RNA by about 1°C per modification relative to the parent hybrid. Since the reduction in stability is greater for PS-modified A:T pairs than G:C pairs, a high GC content of PS-DNA oligos may be desirable for biological applications. However, the demonstration that RNase H activity with PS-DNA:RNA hybrids exceeds considerably that vis-à-vis all-phosphodiester hybrid duplexes is perhaps more important in this regard.

不幸的是，考虑到Sp-PS-DNA 更不易受到 3'-外切酶的攻击，与Rp 异构体相比，该异构体与互补的 RNA 形成的双链结构在热力学上更不稳定。将混合立体化学结构的 PS 修饰引入到 DNA 中，相对于纯正的杂交结构，每个修饰会使与 RNA 形成的双链的解链温度 Tm 降低约 1°C。由于 PS 修饰对 A:T 碱基对的稳定性降低大于 G:C 碱基对，因此在生物应用中，PS-DNA 寡核苷酸具有较高的 GC 含量可能是可取的。然而，与所有磷酸二酯杂交双链相比，显示出 PS-DNA:RNA 杂交双链的 RNase H 活性明显增强，这方面可能更为重要。

PS oligonucleotide-associated toxicities and immune-stimulation have been analyzed in detail. In the case of the VITRAVENE 21mer PS-ASO it is clear that the antiviral activity did not arise as result of immune stimulation by the two CpG motifs (Figure 6) in the sequence. Thus, methylation of the cytosines in the CpG dimers did not lead to a reduction in the antiviral activity and an ASO mechanism remains the most plausible mode of action by VITRAVENE . Ultimately, the drug was taken off the US market in 2006 as the development of highly active antiretroviral therapies led to a reduced number of CMV cases.

PS 寡核苷酸相关的毒性和免疫刺激已经被详细分析。对于 VITRAVENE 的 21mer PS-ASO，明确的是其抗病毒活性并非由序列中的两个 CpG 结构（图 6）引起的免疫刺激导致。因此，在 CpG 二聚体中的胞嘧啶进行甲基化并未导致抗病毒活性的降低，因此 ASO 机制仍然是 VITRAVENE 最合理的作用方式。最终，该药物在 2006 年从美国市场撤出，因为高效抗逆转录病毒治疗的发展导致 CMV 病例数量减少。