中性胞苷脂材包载部分磷硫代反义核酸的生物活性评价及作用机制初探

目的反义核酸是一段与靶基因互补的单链寡核苷酸序列,通过与细胞内核酸杂交成双链结构抑制靶基因转录和翻译过程,从而调控基因表达。然而,无载体递送反义核酸跨膜能力较差且寡核苷酸磷酸二酯键易被核酶水解。为提高反义核酸入胞效率,并对活性作用的发挥进行进一步优化,本课题主要从寻求高效低毒的载体系统及优化修饰策略入手对其进行研究。方法本研究利用中性胞苷脂材(DNCA)混合阳离子脂质体(CLD)对反义核酸G3139包载递送,结合部分位点磷硫代(PS)修饰方式,考察不同修饰位点的序列活性与作用机制。结果与结论采用混合脂材包载能显著提高反义核酸G3139的抗MCF7/ADR细胞增殖能力,并发现9～16位(5′-端计算)区域内多位点(≥4)修饰物抗肿瘤活性较高。此外,G3139部分位点PS修饰物有效激活RNase H降解靶mRNA,并降低杂交双链Tm(DNA的熔解温度)值。结合转录组学与蛋白组学研究,确证减少PS修饰位点数可以降低反义核酸与非特异性靶标结合,从而降低毒副作用。     

阳离子脂质体增加bcl—2反义寡脱氧核苷酸G3139在HL—60细胞中的摄取及活性

目的:探讨阳离于脂质体DOSPER对bcl-2反义寡脱氧核苷酸G3139在HL-60细胞中的摄取和活性的影响。方法:流式细胞仪测定细胞结合的平均荧光强度和Bcl-2蛋白阳性细胞的百分数;荧光显微镜观察细胞内FTTC荧光素标记G3139的分布;RT-RCR测定bcl-2 mRNA的表达。结果:①DOSPER增加细胞对G3139的摄取,当DOSPER/G3139(μg:μg)为2:1时,细胞对G3139的摄取在2h左右达到高峰,为G3139直接作用时的20倍,细胞核和细胞浆中的G3139为明亮的点状聚集;而G3139直接作用时,G3139则弥漫分布于细胞浆中;②细胞内的G3139可向胞外转运。DOSPER介导转染G3139 4h后,细胞内的G3139符合方程C(t)=68.2e~(-0.60t) 31.8e~(-0.02t)(初值为100％),t_(1/2)约为1.1h,而G3139直接作用后,细胞内的G3139符合方程C(t)=64.8e~(-2.27t) 35.2e~(-0.04t),t_(1/2)约为18min;③在DOSPER介导转染下,G3139在浓度1μmol·L~(-1)时,特异性减少bcl-2 mRNA的表达,同时使Bcl-2蛋白阳性率从97％±4％下降到70.6％±2.1％。结论:DOSPER增加细胞对G3139的摄取和改变G3139在细胞内的分布可能是其增强G3139活性的重要机制。     

脂质体转染bcl-2反义寡核苷酸G3139在HL-60细胞中的动力学模式

目的　应用阳离子脂质体介导或直接转染时 ,建立HL 6 0细胞摄入由 5 FITC标记的bcl 2反义寡核苷酸G3139的动力学模式及观察G 3139在细胞内的分布。方法　流式细胞仪测定细胞内的相对平均荧光强度 ,荧光显微镜观察细胞内的荧光分布。结果　①脂质体介导转染法显著提高HL 6 0细胞对G 3139的摄入 ,当DOSPER/G 3139(μg/μg)为 2 2 / 1时 ,细胞内相对平均荧光强度可达G 3139直接作用时的 40倍 ,而且HL 6 0细胞对G 3139的摄入跟G3139的浓度和作用时间有关。②细胞内的G 3139可向胞外转运 ,在DOSPER介导转染后 ,胞内荧光强度衰减缓慢 ,t1/ 2 约为 4h ,而G 3139直接作用后 ,t1/ 2 约为 0 5h ;③DOSPER介导转染 6h后 ,细胞内荧光物质主要以明亮的点状聚集在细胞核和部分细胞浆的细胞器中 ,而G 3139直接作用时 ,荧光物质则弥漫分布于胞浆中。结论　DOSPER可以增加G 3139的细胞摄入并改变其胞内分布 ,可能是阳离子脂质体增加bcl 2反义寡核苷酸生物活性的重要原因。     

第二代反义寡核苷酸的研究

第一代反应核酸寡核苷酸硫代磷酸酯，是目前研究最广泛，最成熟的反义核酸药物，已进入临床试验阶段，但其具有一定的毒副作用；第二代反义核酸，包括嵌合寡核苷酸，杂合寡核苷酸等，是在硫代寡核苷酸的基础上，再引入其它的修复基团，从而提高其生物活性，减少毒副作用。本文着重介绍了第二代反应核酸的生物物理学性质，药动学及礤与第一代反应核酸毒性的比较。     

针对HBV S基因的反义锁核酸抗乙肝病毒表达的初探

目的 探讨针对HBVS基因翻译起始区的反义锁核酸（LNA）片断体外抗乙型肝炎病毒表达的作用。方法 合成三段均互补于HBVS区同一位点的反义核酸片断：锁核酸、反义寡核苷酸、全硫代反义寡核苷酸及无关对照序列,作用于HepG22．2．15细胞,采用ELISA法动态检测细胞上清中HBsAg含量的变化并比较其抗HBV抗原表达作用,以四甲基偶氮唑兰（MTT）法检测LNA对细胞的毒性。结果 三段反义寡核苷酸均能抑制HBsAg的表达,作用7d后抑制率分别为52％、42％、45％,其中LNA抗病毒活性最强且对细胞代谢无影响．无关序列无明显抑制作用。结论 针对HBVS区的锁核酸体外能有效抑制乙型肝炎病毒的表达．为乙肝的分子治疗开辟了新的前景。     

Livin mRNA的反义核酸诱导MCF-7乳癌细胞凋亡作用

目的：以livin mRNA为靶点设计反义硫代脱氧寡核苷酸(PS—ODNs)，探讨其体外抗肿瘤效应及其机制。方法：设计以livin mRNA为靶点的反义核酸并作用于MCF-7乳癌细胞，用MTT、RT—PCR和流式细胞仪检测等方法对其进行评价研究。结果：在设计的一些反义核酸中，YMZ05能够有效地抑制livin基因的表达，增加caspase-3的活性，诱导MCF-7细胞凋亡，明显抑制其生长，结论：通过抑制livin基因的表达能够抑制MCF-7乳癌细胞生长、诱导其凋亡，livin可能会成为抗肿瘤的新靶点。     

靶向Bcl-2、Bcl-xl、Mcl-1、Bcl-w、A1反义核酸抑制消化系肿瘤细胞增殖效果的差异

目的比较靶向Bcl-2、Bcl-xl、Mcl-1、Bcl-w、A1反义核酸(antisense oligodeoxynucleotide,ASO)对消化系统肿瘤细胞(肝癌HepG2细胞、胃癌MGC-803细胞、结肠癌Lovo细胞)的增殖抑制作用和致凋亡作用的差异。方法分别合成靶向Bcl-2、Bcl-xl、Mcl-1、Bcl-w、A1的反义核酸和随机序列反义核酸(randomolig odeoxynucleotide,RODN),采用脂质体Li-pofectamineTM 2000转染细胞,WST法检测相同浓度的5种ASOs对肝癌HepG2细胞、胃癌MGC-803细胞、结肠癌Lovo细胞的增殖抑制作用和致凋亡作用。结果 Bcl-2、Bcl-xl、Mcl-1、Bcl-w、A1等5种ASOs中,不论是对细胞增殖抑制还是致凋亡作用,均以Bcl-xlASO、Mcl-1ASO的作用效果较好。结论在Bcl-2、Bcl-xl、Mcl-1、Bcl-w、A1等5种ASOs中,Bcl-xlASO、Mcl-1ASO可明显抑制消化系肿瘤细胞增殖,诱导细胞凋亡。     

反义核酸药物硫代磷酸酯寡核苷酸

对一类颇有应用前景的反义核酸药物硫磷酸酯寡核苷酸合成技术，反义特性、作用原理及药物动力学等几方面的最新研究进展，作了较为详尽的论述。     

PTEN及Bcl-2蛋白在膀胱移行细胞癌中的表达及其意义

目的：探讨PTEN及Bcl-2蛋白在膀胱移行细胞癌的表达规律及其临床意义。方法：应用免疫组织化学链亲和素-生物素-霉复合物（SP）方法检测43例膀胱移行细胞癌和7例正常黏膜组织中PTEN及Bcl-2蛋白的表达．分析二者的表达与膀胱癌病理参数的关系。结果：（1）G1、G2、G3肿瘤PTEN表达阳性率分别为85．7％、80．4％、73．3％，浸润性肿瘤和表浅性肿瘤表达阳性率分别为70．4％和93．8％，说明PTEN表达阳性率与肿瘤病理分级、临床分期有关。（2）G。、G2、G3肿瘤Bcl-2表达阳性率分别为14．2％、57．14％、66．67％，浸润性肿瘤和表浅性肿瘤表达阳性率分别为59．3％和43．8％，说明Bcl-2表达阳性率与肿瘤病理分级、临床分期有关。（3）随着肿瘤恶性程度的增高和临床分期进展，PTEN蛋白阳性率呈下降，Bcl-2表达呈增高趋势，二者表达呈负相关关系。结论：PTEN的抑癌作用可能与Bcl-2有关．二者的异常表达在膀胱移行细胞癌的发生、发展过程中起重要作用。二者同时检测有助于判断预后。     

用IE—HPLC、PAGE分析合成的硫代寡核苷酸相关物质“n—1”、“n—2”和“n—3”

目的:分析合成的n=20-mer硫代寡核苷酸中相关物质“n-1”、“ n-2”和“ n-3”杂质失败序列。方法:离子交换高效液相色谱(IE-HPLC)法和聚丙烯酰胺凝胶电泳法(PAGE)。色谱柱为Gen-Pak~(TM)FAX离子交换柱(4.6 mm×100mm),流动相A为62.5mmol·L~(-1)三羟甲基氨基甲烷盐酸(Tris·Cl),pH 8.15,流动相B为62.5mmol·L~(-1) Tris·Cl,pH 8.15,2.5 mol·L~(-1) LiCl,流动相C为100％乙腈;梯度洗脱条件为B:30％→50％ 30 min,C:恒为20％;流速为0.75mL·min~(-1);检测波长为260nm。PAGE分析采用20％变性聚丙烯酰胺凝胶,恒定功率25W进行电泳。结果:IE-HPLC分离纯化出3种相关物质,通过PAGE印证了它们是由于合成偶联不完全而产生的n-1、n-2和n-3的杂质失败序列。结论:所用离子交换高效液相色谱法能将合成的硫代寡核苷酸中相关物质“n-1”、“n-2”和“n-3”杂质失败序列与全序列n一一分离出来,对硫代寡核苷酸的纯化和分析具有重要的参考价值。     

Lipid Nanoparticles Loaded with an Antisense Oligonucleotide Gapmer Against Bcl-2 for Treatment of Lung Cancer

Purpose

Bcl-2 is an anti-apoptotic gene that is frequently overexpressed in human cancers. G3139 is an antisense oligonucleotide against bcl-2 that has shown limited efficacy in clinical trials. Here, we report the synthesis of a new antisense oligonucleotide containing additional chemical modifications and its delivery using nanoparticles.

Methods

An oligonucleotide G3139-GAP was synthesized, which has 2’-O-methyl nucleotides at the 5’ and 3’ ends based on a “gapmer” design. Furthermore, G3139-GAP was incorporated into lipid nanoparticles (LNPs) composed of DOTAP/egg PC/cholesterol/Tween 80. The LNP-loaded G3139-GAP was evaluated in A549 lung cancer cells both in vitro and in a murine xenograft model for biological activity and therapeutic efficacy.

Results

The LNPs showed excellent colloidal and serum stability, and high encapsulation efficiency for G3139-GAP. They have a mean particle diameter and zeta potential of 134 nm and 9.59 mV, respectively. G3139-GAP-LNPs efficiently downregulated bcl-2 expression in A549 cells, as shown by 40% and 83% reduction in mRNA and protein levels, respectively. Furthermore, G3139-GAP-LNPs were shown to inhibit tumor growth, prolong survival, and downregulate tumor bcl-2 expression in an A549 murine xenograft tumor model. These data indicate that G3139-GAP-LNPs have excellent anti-tumor efficacy and warrant further evaluation.

     

Bcl-2 antisense in the treatment of human malignancies: a delusion in targeted therapy

Regulation of cell death (apoptosis) is frequently affected in the development of malignant diseases, and all molecular steps from extracellular signalling receptors through intracellular pathways, cell death rheostats and cell death executioners may be involved. Bcl-2 is an anti-apoptotic member of a family of anti- and pro-apoptotic proteins that is upregulated in a variety of cancers and specifically overexpressed through chromosomal translocation in some non-Hodgkin lymphomas. Experimental attenuation of Bcl-2 lowers the threshold for undergoing chemotherapy-induced apoptosis. Therefore, therapeutic targeting of Bcl-2 appears as an attractive approach currently intensely explored using mRNA degradation strategies and small inhibitory molecules. One phosphorothioate oligodeoxynucleotide antisense against Bcl-2 mRNA, oblimersen (Genasense, G3139), has been used in a substantial number of clinical trials. In this review we will discuss the current developments of G3139, and scrutinize its proposed mechanism of action. Several studies indicate that G3139 involves various intracellular mechanisms and modulation of the immune system. To this date G3139 has not been justified in cancer therapy due to modest or absent effects. But, surprisingly, some of its off-target effects may represent useful therapeutic principles. Therefore, antisense uptake improvements and new design of the oligonucleotide may provide us with useful therapeutics, including both the targeted gene and new anticancer mechanisms. This may be another example of how targeted therapy molecules evolve into multimodality drugs when moved from laboratory bench to bedside use, and illustrate our limited ability for target prediction and scant understanding of biological systems when designing therapeutic strategies.     

A Specific Picomolar Hybridization-Based ELISA Assay for the Determination of Phosphorothioate Oligonucleotides in Plasma and Cellular Matrices

Purpose To develop and validate an ultrasensitive and specific hybridization-based enzyme-linked immunosorbent assay method for quantification of two phosphorothioate oligonucleotides (PS ODNs) (G3139 and GTI-2040) in biological fluids. Methods This assay was based on hybridization of analytes to the biotin-labeled capture ODNs followed by ligation with digoxigenin-labeled detection ODN. The bound duplex was then detected by anti-digoxigenin-alkaline phosphatase using Attophos^? (Promega, Madison, WI, USA) as substrate. S1 nuclease and major factors such as the hybridization temperature, concentration of capture probe, and the use of detergent were evaluated toward assay sensitivity, selectivity, and accuracy. Results The method is selective to the parent drugs with minimal cross-reactivity (<6%) with 3′-end deletion oligomers for both G3139 and GTI-2040. A linear range of 0.05 to 10 nM (r² > 0.99) was observed for GTI-2040 in a variety of biological matrices. For both G3139 and GTI-2040, the within-day precision and accuracy values were found to be <20% and 90–110%, respectively; the between-day precision and accuracy were determined to be <20% and 90–120%. Addition of S1 nuclease combined with washing step greatly improved the assay linearity and selectivity. The utility of this assay was demonstrated by simultaneous determination of GTI-2040 in plasma and its intracellular levels in treated acute myeloid leukemia patients. Conclusions The validated hybridization enzyme-linked immunosorbent assay method is specific for quantitation of PS ODNs in biological samples to picomolar level. This method provides a powerful technique to evaluate plasma pharmacokinetics and intracellular uptake of PS ODNs in patients and shows its utility in clinical evaluations.     

The role of the G protein gamma(2) subunit in opioid antinociception in mice

We examined the role of the gamma(2) subunit of G proteins (Ggamma(2)) in the antinociception produced by c[D-Pen(2), D-Pen(5)]enkephalin (DPDPE) in mice. DPDPE produced 84.0+/-9.0% antinociception in vehicle-treated mice. After intracerebroventricular (i.c.v.) treatment with an antisense phosphorothioate oligodeoxynucleotide to the Ggamma(2) subunit, DPDPE-mediated antinociception decreased to 24.4+/-7.4%. The mismatch phosphorothioate oligodeoxynucleotide-treated mice showed 65.1+/-10.3% antinociception, while the missense phosphorothioate oligodeoxynucleotide-treated mice showed 76.4+/-23.6% antinociception by DPDPE. The reduction of analgesia in antisense phosphorothioate oligodeoxynucleotide-treated mice was significant in comparison with vehicle-treated (P<0.001), mismatch phosphorothioate oligodeoxynucleotide-treated (P<0.01) and missense phosphorothioate oligodeoxynucleotide-treated (P<0.05) mice. These results suggest that the G protein gamma(2) subunit is involved in the transduction pathway leading to antinociception by DPDPE.     

Transferrin Receptor Targeted Lipopolyplexes for Delivery of Antisense Oligonucleotide G3139 in a Murine K562 Xenograft Model

Purpose  Transferrin (Tf) conjugated lipopolyplexes (LPs) carrying G3139, an antisense oligonucleotide for Bcl-2, were synthesized and evaluated in Tf receptor positive K562 erythroleukemia cells and then in a murine K562 xenograft model. Materials and Methods  Particle size and Zeta potentials of transferrin conjugated lipopolyplexs containing G3139 (Tf-LP-G3139) were measured by Dynamic Light Scattering and ZetaPALS. In vitro and in vivo sample’s Bcl-2 downregulation was analyzed using Western blot and tumor tissue samples also exhibited by immunohistochemistry method. For athymic mice bearing with K562 xenograft tumors, tumor growth inhibition and survival rate were investigated. Nanoparticle distribution in 3-D cell cluster was observed by Laser scan confocal microscopy. IL-12 production in the plasma was measured by ELISA kit. Results   In vitro, Tf-LP-G3139 was more effective in inducing down regulation of Bcl-2 in K562 cells than non-targeted LP-G3139, free G3139 and mismatched control ODN-G4126 in the same formulation. In vivo Tf-LP-G3139 was less effective than free G3139 in Bcl-2 down regulation. 3-D cell cluster model diffusion results indeed indicated limited penetration of the LPs into the cell cluster. Finally, the therapeutic efficacies of Tf-LP-G3139 and free G3139 were determined in the K562 xenograft model. Tf-LP-G3139 showed slower plasma clearance, higher AUC, and greater accumulation in the tumor compared to free G3139. In addition, Tf-LP-G3139 was found to be more effective in tumor growth inhibition and prolonging mouse survival than free G3139. This was associated with increased spleen weight and IL-12 production in the plasma. Conclusion  The role of the immune system in the therapeutic response obtained with the Tf-LPs is necessary and in vitro 3-D cell cluster model can be a potential tool to evaluate the nanoparticle distribution.     

Modulation of tumor radiation response with G3139, a bcl-2 antisense oligonucleotide

Summary Overexpression of anti-apoptotic bcl-2 protein has been found in hematological and solid tumors and has been associated with increased resistance against cytotoxic therapy. While bcl-2 antisense (AS) treatment combined with chemotherapy has been successfully tested in clinical trials, trials evaluating the combination of bcl-2 AS with radiotherapy have not yet been performed. The aim of this study was to investigate in vivo anti-tumor effects of a combined modality treatment scheme consisting of radiation and the bcl-2 targeted AS oligonucleotide (ODN) G3139 (Oblimersen Sodium). Two human colon carcinoma cell lines, SW620, bcl-2 positive and HT-29, bcl-2 negative, were grown as xenografts and compared in their response to combined bcl-2 AS/radiation treatment. G3139 potentiated the radiation response of bcl-2 positive SW620 tumors, but had no significant effect on bcl-2 negative HT-29 tumors assayed by tumor growth delay. The profound enhancement of SW620 tumor growth delay by G3139 did not translate into effects on tumor cure, as no significant effect of G3139 was found on SW620 radiocurability (TCD₅₀ assay). The control ODN G3622 had no effect on SW620 radiation response, indicating an ODN sequence specific effect.     

Pharmacokinetic evaluation of oblimersen sodium for the treatment of chronic lymphocytic leukemia

INTRODUCTION: Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the western hemisphere. Developing new therapies remains a priority as present treatment options do not offer a cure. BCL-2 overexpression in CLL is associated with aggressive disease features and resists chemotherapy. Oblimersen sodium (G3139) is a phosphorothioate oligonucleotide antisense drug targeting the BCL-2 mRNA and is the first antisense to reach advanced clinical testing in oncology. Preclinical evaluation has demonstrated good antineoplastic effect in B-cell cancers; several clinical trials have confirmed its safety and efficacy both alone and in combination with other therapeutics. AREAS COVERED: This review focuses on the chemistry, pharmacodynamics, pharmacokinetics and clinical evaluation of oblimersen in CLL. PubMed and MEDLINE searches assisted in data collection. EXPERT OPINION: Bcl-2 is an important target in CLL. Antisense therapy is a novel approach to target oncoproteins; this can be beneficial in the clinical setting. Oblimersen sodium demonstrates the clinical safety of the antisense therapeutic approach and, with chemotherapy, shows survival advantage in a subset of CLL patients. However, future approval of oblimersen sodium in CLL remains uncertain. Nevertheless, BCL-2 remains a critical target in drug development and is an area of high-priority research.     

联合新型Bcl-2反义核酸与足叶乙苷抑制人小细胞肺癌细胞的增殖

目的　研究一种新型Bcl 2反义寡核苷酸 F95 1在与足叶乙苷 (VP16 )联合使用时 ,对人小细胞肺癌细胞增殖的影响。方法　分别单独及联合应用F95 1、VP16于人小细胞肺癌细胞株NCI H4 4 6 ,通过MTT法检测细胞存活率 ,通过集落形成法检测集落生存率 ,按Welander法计算联合应用组的预测值 ,通过实测值与预测值的比较进行效应分析。结果　细胞存活率 (MTT法 )和集落生存率 (集落形成法 )均显示联合应用F95 1、VP16组的实测值明显低于预测值。结论　联合应用F95 1与VP16对人小细胞肺癌细胞的增殖有协同的抑制作用。