反义寡核苷酸治疗的分子影像技术进展

反义寡核苷酸治疗在许多疾病的治疗中具有巨大的潜力。同时,反义寡核苷酸治疗药物在生物体内转运至靶点发挥作用过程中也面临许多障碍,并存在一些不可预期的不良反应。分子影像技术可以对反义寡核苷酸治疗的生物进程进行成像,并对治疗效果进行监测,对于优化反义寡核苷酸治疗至关重要。目前,多种分子影像模式已经应用在反义寡核苷酸治疗相关的研究上,每一个成像方式都有其独特的优势,但同样也有其固有的缺陷,多模态成像成为未来发展的趋势。

β1整合素反义寡核苷酸对人胰腺癌裸鼠皮下移植瘤的治疗作用

目的探讨β1整合素反义寡核苷酸(ASODN)对裸鼠人胰腺癌移植瘤生长和基因表达的影响。方法建立裸鼠人胰腺癌皮下移植瘤模型,随机分为3组:对照组(皮下注射脂质体和转染液)、非特异序列(RODN)组(皮下注射RODN、脂质体和转染液)和ASODN组(皮下注射ASODN、脂质体和转染液)。治疗后计算抑瘤率和肿瘤缩小率,检测肿瘤中β1整合素mRNA和蛋白表达。结果RODN组、ASODN组抑瘤率分别为4.7 5%和7 2.7 0%,ASODN组肿瘤缩小1 0.9 1%。与其他两组比较,ASODN组裸鼠肿瘤中β1整合素mRNA和蛋白表达水平明显降低。结论靶向β1整合素的反义寡核苷酸对人胰腺癌裸鼠皮下移植瘤的生长具有一定的抑制作用,可能为胰腺癌治疗提供新的方法。

BCL-2反义寡核苷酸对膀胱癌EJ细胞的影响

目的探讨BCL-2反义寡核苷酸对膀胱癌EJ细胞致瘤能力和对荷瘤裸鼠肿瘤的影响。方法以膀胱癌EJ细胞中加入BCL-2PS-ASO、BCL-2PS-SO、培养液共同孵育作为实验组、阴性对照组和空白对照组,处理72 h的三组细胞做集落形成及裸鼠移植瘤形成实验;向荷瘤裸鼠的肿瘤内注射上述三组药物,观察肿瘤生长情况。结果体外实验显示,上述三组克隆数分别为8.5±1.5、22±2.2、20±2.1,肿瘤重量(g)分别为1.3±0.3、3.3±0.4、3.0±0.2 g,实验组明显低于其他两组(P<0.05);体内实验显示,实验组、阴性对照组肿瘤抑制率分别为(47.6±5.41)%、(3.03±0.23)%,实验组明显高于阴性对照组(P<0.05)。结论BCL-2PS-ASO能降低膀胱癌EJ细胞致瘤能力,对荷瘤裸鼠的肿瘤有抑制作用。

高脂蛋白(a)血症和冠心病的关系及治疗进展

脂蛋白(a)是脂蛋白的一种,可致动脉粥样硬化并影响纤维蛋白代谢,在冠心病一级预防和二级预防中占有重要位置。荟萃分析、孟德尔随机遗传研究和全基因组关联研究都显示脂蛋白(a)水平升高是冠心病的独立危险因素。此外,在低密度脂蛋白胆固醇控制达标的情况下,脂蛋白(a)水平升高仍然和冠心病不良预后相关。近年研究显示前蛋白转化酶枯草杆菌蛋白酶9抑制剂、反义寡核苷酸以及血脂净化均可降低脂蛋白(a)水平,部分药物可以改善冠心病的预后。然而,单独降低脂蛋白(a)水平的临床获益还不明确,需要进一步研究。

LC-MS法在治疗性寡核苷酸药物检测中的应用

治疗性寡核苷酸(oligonucleotides,OGNs)药物是人工化学合成的短单链或双链核酸,长度通常为15~30个碱基对。OGNs作为一种新的治疗药物正在迅速发展,并在各种疾病领域的药物发现和开发中受到越来越多的关注。与欧美相比,除Spinraza(nusinersen)作为孤儿药在中国获批上市外,暂无其他OGNs药物在国内上市。国内OGNs药物开发起步较晚,仍然处于发展初期,但由于国内患者群体基数较大,需求较多,未来伴随OGNs药物开发的持续推进,以及国内企业相应技术的逐步成熟,我国OGNs药物市场有望迎来快速发展。OGNs药物由于其独特的理化性质,生物分析方法开发难度大,目前,国内关于OGNs药物定量分析方法的报道还很少。开发一种灵敏可靠的方法来表征生物样品中的OGNs是研究其药代动力学和药效学性质的关键。相对于传统的ELISA法,LC-MS法可以同时定量完整OGNs及其代谢物,特别是高分辨质谱的广泛应用不仅可以提供目标OGNs的定量信息,还可以对代谢物进行鉴定,提供碱基组成、序列结构等信息,目前已成为OGNs定量分析的首选方法。本文主要描述了LC-MS在治疗性OGNs药物检测中的应用,并阐述了其优点和局限性,最后探讨了LC-MS用于检测OGNs的发展趋势,即更低的检测水平和潜在的通用方法。

治疗用单链寡核苷酸药物的非临床研究评价概述

单链寡核苷酸(single-stranded oligonucleotide,SSO)是一种人工合成寡核苷酸,可按碱基互补配对原则主要通过与靶RNA杂交,诱导断裂、调节剪切、抑制翻译等作用抑制靶RNA功能。未经修饰的SSO易被核酸酶降解,经化学修饰的SSO(如经锁核酸修饰后的SSO)其稳定性、杂交亲和力及酶切效率大大提高,成药性显著增强。目前,已有4个治疗用SSO药物在美国或欧洲上市,更多的候选药物处于研发阶段。本文从监管、非临床特点、非临床安全性试验一般原则等方面阐述治疗用SSO药物的非临床研究评价要点。

VEGF和PCNA反义寡核苷酸联合治疗裸鼠骨肉瘤的实验研究

目的探讨血管内皮生长因子（VEGF）反义寡核苷酸和增殖细胞核抗原（PCNA）反义寡核苷酸单用或联用对裸鼠骨肉瘤生长的抑制作用。方法制备Balb／c裸鼠皮下骨肉瘤模型24只，随机分为4组：VEGF反义寡核苷酸（ASODN）治疗组、PCNA反义寡核苷酸治疗，组、联合治疗组和对照组，6只／组。接种MG-63人成骨肉瘤细胞后24h内分别用VEGF反义寡核苷酸或（和）PCNA反义寡核苷酸皮下注射进行治疗，对照组只注射生理盐水，2次／周，连续4周；观察各组裸鼠肿瘤的生长情况、裸鼠的一般情况与生存期。断颈处死裸鼠，游标卡尺测量肿瘤体积大小，天平称量肿瘤的重量。结果VEGF-ASODN治疗组、PCNA—ASODN治疗组和联合治疗组裸鼠骨肉瘤的生长均受到不同程度的抑制，抑瘤率分别为44．56％、46．53％和72．71％。结论VEGF和PCNA两种反义寡核苷酸均对骨肉瘤的生长具有抑制作用，两种反义寡核苷酸联合应用比单用的疗效更为显著。

从转化角度谈表观遗传学与变态反应性疾病

由于表观遗传修饰具有调节环境因素在变态反应性疾病发病或疾病保护方面的作用,最近应用表观遗传修饰研究变态反应性疾病得到了广泛的关注。此外,近期研究证据表明,很多疾病相关细胞的表观基因组发生了改变。尽管目前仍为早期阶段,但是表观遗传修饰,尤其是DNA甲基化和微小RNA (miRNAs)可能具有患者治疗分型和补充甚至替代未来生化或临床检测的潜能。成功免疫治疗相关的表观遗传生物标志物已有第一个报道,即使不考虑个体化治疗方案,表观遗传修饰将在监测甚至预测个体化治疗应答方面扮演重要角色。然而,在表观遗传学应用之前,还需要在表型明确的大规模队列并且特殊的细胞类型进行进一步研究。随着miRNAs类似物、抑制剂和反义寡核苷酸在其他疾病患者中的临床研究,表观遗传组为治疗干预提供了有意义的靶点。尽管需要进一步的技术改进,胞外膜泡和表观遗传编辑的选择和工程改造技术代表着调节细胞表型和应答的新型工具。此外,对宿主表观基因组和微生物组之间相互作用的进一步认识,使通过微生物组干预调节表观基因组预防变态反应性疾病的目标提供了可能性。

c—fos反义寡核苷酸对兔实验性增生性玻璃体视网膜病变的抑制作用

目的评价c—fos反义寡核苷酸（AS-ON）对兔实验性增生性玻璃体视网膜病变（PVR）的抑制作用。方法将9只兔（18只眼）分为c—fos—AS-ON治疗组和对照组（每组9只眼）。c—fos—AS—ON治疗组以平衡盐溶液（BSS）配制2pgc—fos—AS—ON和10μg脂质体的混合液0．1ml、对照组以0．1mlBSS分别与2．5×10^5个／ml人视网膜色素上皮（RPE）细胞稀释液混合后同时注入玻璃体腔，观察两组在玻璃体腔注射后第7、14、21、28天PVR发生的程度。结果随着观察时间的延长，两组PVR程度逐渐加重。第7天两组PVR程度无统计学差异（P=0．05），第14、21、28天，c—fos—AS-ON治疗组的PVR程度较对照组轻，差异有统计学意义（P〈0．05）；第28天，c—fos—AS—ON治疗组牵拉性视网膜脱离发生率为25．0％，对照组为62．5％。结论c—fos—AS—ON对兔实验性PVR的发生有一定的抑制作用。

Anti-sense oligonucleotide labeled with technetium-99m using hydrazinonictinamide derivative and N-hydroxysuccinimidyl S-acetylmercaptoacetyltriglycline: A comparison of radiochemical behaviors and biological properties

AIM:To explore and compare the radiochemical behavior and biological property of anti-sense oligonuc-leotide (ASON) labeled with technetium-99m using N-hydroxysuccinimidyl S-acetylmercaptoacetyltriglycl ine (NHS-MAG3) and hydrazinonictinamide derivative (HYNIC). METHODS:After HYNIC and NHS-MAG3 were synthesized, ASON was labeled with technetium-99m using HYNIC and NHS-MAG3 as a bifunctional chelator. The in vivo and in vitro stability, binding rates of labeled compounds to serum albumen, biodistribution of 99mTc-MAG3-ASON and 99mTc-HYNIC-ASON in BALB/C mouse and its HT29 tumor cellular uptake were compared. RESULTS:The labeling efficiency and stability of 99mTc-MAG3-ASON were significantly higher than those of 99mTc-HYNIC-ASON (P = 0.02, and P = 0.03, respectively). 99mTc-MAG3-ASON had a significantly lower rate of binding to serum albumen than 99mTc-HYNIC-ASON (P < 0.05). In contrast to 99mTc-HYNIC-ASON, the biodistribution of 99mTc-MAG3-ASON was significantly lower in blood, heart, liver and stomach (P < 0.05), slightly lower in intestines and spleen (P > 0.05) and significantly higher in lung and kidney (P < 0.05). The HT29 tumor cellular uptake rate of 99mTc-MAG3-ASON was significantly higher than that of 99mTc-HYNIC-ASON (P < 0.05). CONCLUSION:99mTc-MAG3-ASON shows superior radiochemical behaviors and biological properties than 99mTc-HYNIC-ASON. 99mTc-MAG3-ASON is a potential radiopharmaceutical agent for in vivo application.

New therapeutic opportunities for Hepatitis C based on small RNA

Hepatitis C virus （HCV） infection is one of the major causes of chronic liver disease, including cirrhosis and liver cancer and is therefore, the most common indication for liver transplantation. Conventional antiviral drugs such as pegylated interferon-alpha, taken in combination with ribavirin, represent a milestone in the therapy of this disease. However, due to different viral and host factors, clinical success can be achieved only in approximately half of patients, making urgent the requirement of exploiting alternative approaches for HCV therapy. Fortunately, recent advances in the understanding of HCV viral replication and host cell interactions have opened new possibilities for therapeutic intervention. The most recent technologies, such as small interference RNA mediated gene-silencing, antisense oligonucleotides （ASO）, or viral vector based gene delivery systems, have paved the way to develop novel therapeutic modalities for HCV. In this review, we outline the application of these technologies in the context of HCV therapy. In particular, we will focus on the newly defined role of cellular microRNA （miR-222） in viral replication and discuss its potential for HCV molecular therapy.

The Role of β3-adrenergic Receptor Gene in Neuropeptide Y Y5Receptor Antisense Gene Therapy of Diet-induced Obese Rats

Objective: To study the role of β3-adrenergic receptor gene in neuropeptide Y(NPY) Y5 receptor antisense gene therapy of diet-induced obese rats.Methods: The diet-induced obese rats were prepared by feeding a high-nutrition diet. Lateral ventricular was cannulated in obese rats which then received an intraventricular injection of either 5 μg/μl NPY Y5 receptor antisense or 10 μl missense oligodeoxynucleotide or saline of 10 μl respectively in every rat. When the rats were killed, the wet weight of abdominal adipose tissue, the level of serum lipid and lipoprotein were measured. Total RNA from the retroperitoneal adipose tissue was extracted and the level of β3-adrenergic receptor gene mRNA expression was evaluated by RT-PCR.Results: ①The wet weight of abdominal adipose tissue, the levels of serum lipids were greatly higher in diet-induced obese rats than those in normal rats. However, there were much lower β3-adrenergic receptor gene mRNA expression levels in retroperitoneal adipose tissue in diet-induced obese rats as compared with those in normal rats. ②After the diet-induced obese rats were intraventricularly administered with NPY Y5 receptor antisense oligodeoxynucleotide, the levels of β3-adrenergic receptor gene mRNA expression in retroperitoneal adipose tissue of diet-induced obese rats were strikingly up-regulated, whereas the wet weight of abdominal adipose tissue, the levels of serum lipids were markedly reduced.Conclusion: Intraventricular administration of antisense oligodeoxynucleotide to NPY Y5 receptor could significantly reduce the abdominal adipose tissue and the levels of serum lipids in diet-induced obese rats by up-regulating the level of β3-adrenergic receptor gene mRNA expression in retroperitoneal adipose tissue.

ROLE OF ANTISENSE AND DECOY OLIGONUCLEOTIDE OF NF-κB IN VESSEL STENOSIS AND NEOINTIMA FORMATION IN BALLOON-INJURED RAT ARTERY

Objective To examine antisense and decoy oligonucleotides of nuclear factor kappa B in vivo effects on intima proliferation and balloon-injured monocytes chemotactic protein-1 （ MCP-1 ） and extraceUular signal regulated kinase-2 （ERK2） κexpression in the carotid artery of rats. Methods Sprague-Dawley rats underwent balloon-dilation injury of the left carotid artery. Rats are divided into 7 groups （ n = 18 ） and each group includes6 time points （6 h, 1, 3, 5, 7, 14 d） （n =3）. Uninjured right carotid artery of the same rat was used as controls. Results In model group, sense group and scramble group, vessel intima area , media area and intima/media ratio increased after 5 d and reached the maximum after 7 d. The effect of antisense plus decoy group on intimal hyperplasia was more obvious than that of antisense group and decoy group alone. MCP-1 mRNA expression was increased expression continuously at 3, 5 and 7 d and decreased at 14 d. Compared with model group, sense group and scramble group, antisense group, decoy group and antisense plus decoy group had lowered MCP-1 mRNA expression in each time point （ P 〈 0. 05 ）. NF-KB p65 was dispersed positive stain 6 h after injury and increased after 1 d and peaked at 7 d, but the protein expression was weak at 14 d. ERK2 protein synthesis increased at I d and reached the peak at 7 d, while protein expression after 14 d was similar to that at 7 d. Treatment of antisense group, decoy group and antisense plus decoy group inhibited protein synthesis more significantly than those of model group, sense group and scramble group（ P 〈 0. 05）. Conclusion NF-KB modulates genes expression and protein synthesis of MCP-1 and ERK2. Celluar proliferation in vessel wall was dynamically changed after balloon angioplasty injury. Antisense and decoy oligonucleotide of NF-KB by local lipofectaraine transfer inhibit NF-KB activating gene modulation and neointimal hyperplasia.

Survivin ASODN targeted therapy in XWLC-05 cell transplanted nude mice

Objective:The aim of this study was to study the inhibiting effect of survivin mRNA on transplanted XWLC-05 tumor on nude mice.Methods:We established XWLC-05 transplanted nude mice model.44 mice would be divided randomly into 4 groups:control group (blank),Lip group (simple liposome),survivin SODN group (transfected by sense oligonucleotide) and survivin ASODN group (transfected by antisense oligonucleotide).We would study general activities of nude mice in these 4 groups,measure the size of tumor and calculate the tumor inhabiting rate also.Pathological methods were applied in the analysis of the effect of different treatment on heart,kidney and liver of nude mice in these 4 groups.Results:Tumor grew slowly and size,weight of tumor was lower in survivin ASODN group when compared with that of others.Nude mice of survivin ASODN group showed lower growth index and tumor inhabiting rate was significantly higher than that of other groups (P < 0.05).Transplanted tumor on nude mice in control group (blank),Lip group,and survivin SODN group grew bigger as time passed and there was no significance among them (P > 0.05).We found a great deal of tumor cell necrosis in survivin ASODN group.No death of nude mice was observed in all 4 groups and we did not found obvious lesion in vital organs.Conclusion:Survivin ASDON could be used for the inhibitation of subcutaneously transplanted tumor in nude mice without obvious lesion in vital organs.