Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II

Frog virus 3 (FV3) is a large DNA virus that encodes approximately 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication. We designed asMOs directed against the major capsid protein (MCP), an 18 kDa immediate-early protein (18K) that was thought to be a viral regulatory protein, and the viral homologue of the largest subunit of RNA polymerase II (vPol-IIalpha). All three asMOs successfully inhibited translation of the targeted protein, and two of the three asMOs resulted in marked phenotypic changes. Knock-down of the MCP resulted in a marked reduction in viral titer without a corresponding drop in the synthesis of other late viral proteins. Transmission electron microscopy (TEM) showed that in cells treated with the anti-MCP MO assembly sites were devoid of viral particles and contained numerous aberrant structures. In contrast, inhibition of 18K synthesis did not block virion formation, suggesting that the 18K protein was not essential for replication of FV3 in fathead minnow (FHM) cells. Finally, consistent with the view that late viral gene expression is catalyzed by a virus-encoded or virus-modified Pol-II-like protein, knock-down of vPol-IIalpha triggered a global decline in late gene expression and virus yields without affecting the synthesis of early viral genes. Collectively, these results demonstrate the utility of using asMOs to elucidate the function of FV3 proteins.     

Pharmacological treatment options for severe hypertriglyceridemia and familial chylomicronemia syndrome

Introduction: A spectrum of disorders, ranging from rare severe cases of homozygous null lipoprotein lipase deficiency (LPLD)–familial chylomicronemia syndrome (FCS) to heterozygous missense LPLD or polygenic causes, result in hypertriglyceridemia and pancreatitis. The effects of mutations are exacerbated by environmental factors such as diet, pregnancy, and insulin resistance.

Areas covered: In this review, authors discuss chronic treatment of FCS by ultra-low fat diets allied with the use of fibrates, omega-3 fatty acids, niacin, statins, and insulin-sensitizing therapies depending on the extent of residual lipoprotein lipase (LPL) activity; novel therapies in development target triglyceride (TG)-rich lipoprotein particle clearance. Previously, a gene therapy approach to LPL-alipogene tiparvovec showed that direct targeting of LPL function reduced pancreatitis events. An antisense oligonucleotide to apolipoprotein-C3, volanesorsen has been shown to decrease TGs by 70–80% and possibly to reduce rates of pancreatitis admissions. Studies are underway to validate its long-term efficacy and safety. Other approaches investigating the role of LPL modulating proteins such as angiopoietin-like petide-3 (ANGPTL3) are under consideration.

Expert opinion: Current therapeutic options are not sufficient for management of many cases of FCS. The availability of antisense anti-apoC3 therapies and, in the future, ANGPTL3 therapies may remedy this.     

乙酰肝素酶反义寡核苷酸对人肝癌细胞株乙酰肝素酶表达的影响

目的:探讨乙酰肝素酶(Hpa)反义寡脱氧核苷酸对人肝癌细胞株Hpa mRNA和蛋白表达的影响。方法:设计合成互补于Hpa mRNA起始密码区的硫代反义寡脱氧核苷酸AS-ODN及相应的无义对照寡脱氧核苷酸NS-ODN,以阳离子脂质体Oligofectamine~(TM) Reagent包埋后转染SMMC-7721、BEL-7402细胞,以RT-PCR和Western-blot检测转染前后细胞Hpa mRNA以及蛋白表达的变化。结果:与正常对照组和NS-ODN相应浓度组相比,转染AS-ODN组肝癌细胞Hp amRNA和蛋白的表达均下降。结论:Hpa反义寡脱氧核苷酸下调Hpa mRNA和蛋白的表达,可能抑制肝癌的侵袭转移。     

Changing Blue Fluorescent Protein to Green Fluorescent Protein Using Chemical RNA Editing as a Novel Strategy in Genetic Restoration

Using the transition from cytosine of BFP (blue fluorescent protein) gene to uridine of GFP (green fluorescent protein) gene at position 199 as a model, we successfully controlled photochemical RNA editing to effect site‐directed deamination of cytidine (C) to uridine (U). Oligodeoxynucleotides (ODNs) containing 5′‐carboxyvinyl‐2′‐deoxyuridine (^CVU) were used for reversible photoligation, and single‐stranded 100‐nt BFP DNA and in vitro‐transcribed full‐length BFP mRNA were the targets. Photo‐cross‐linking with the responsive ODNs was performed using UV (366 nm) irradiation, which was followed by heat treatment, and the cross‐linked nucleotide was cleaved through photosplitting (UV, 312 nm). The products were analyzed using restriction fragment length polymorphism (RFLP) and fluorescence measurements. Western blotting and fluorescence‐analysis results revealed that in vitro‐translated proteins were synthesized from mRNAs after site‐directed RNA editing. We detected substantial amounts of the target‐base‐substituted fragment using RFLP and observed highly reproducible spectra of the transition‐GFP signal using fluorescence spectroscopy, which indicated protein stability. ODNc restored approximately 10% of the C‐to‐U transition. Thus, we successfully used non‐enzymatic site‐directed deamination for genetic restoration in vitro. In the near future, in vivo studies that include cultured cells and model animals will be conducted to treat genetic disorders.     

Dissecting human cytomegalovirus gene function and capsid maturation by ribozyme targeting and electron cryomicroscopy

Human CMV (HCMV) is the leading viral cause of birth defects and causes one of the most common opportunistic infections among transplant recipients and AIDS patients. Cleavage of internal scaffolding proteins by the viral protease (Pr) occurs during HCMV capsid assembly. To gain insight into the mechanism of HCMV capsid maturation and the roles of the Pr in viral replication, an RNase P ribozyme was engineered to target the Pr mRNA and down-regulate its expression by >99%, generating premature Pr-minus capsids. Furthermore, scaffolding protein processing and DNA encapsidation were inhibited by 99%, and viral growth was reduced by 10,000-fold. 3D structural comparison of the Pr-minus and wild-type B capsids by electron cryomicroscopy, at an unprecedented 12.5-angstroms resolution, unexpectedly revealed that the structures are identical in their overall shape and organization. However, the Pr-minus capsid contains tenuous connections between the scaffold and the capsid shell, whereas the wild-type B capsid has extra densities in its core that may represent the viral Pr. Our findings indicate that cleavage of the scaffolding protein is not associated with the morphological changes that occur during capsid maturation. Instead, the protease appears to be required for DNA encapsidation and the subsequent maturation steps leading to infectious progeny. These results therefore provide key insights into an essential step of HCMV infection using an RNase P ribozyme-based inhibition strategy.     

BCR-ABL influences the antileukaemic efficacy of alkylphosphocholines

We have compared the antileukaemic efficacy of a series of new i.v. injectable alkylphosphocholines (APC) with their clinically used congeners miltefosine and perifosine. The test system consisted of four leukaemic cell lines carrying the bcr-abl rearrangement (K-562, LAMA-84, CML-T1 and BV-173) and two other leukaemic cell lines (HL-60 and SKW-3) without this genetic alteration. The prototype of i.v. injectable APC, erucylphosphocholine, was more active against BCR-ABL-positive cell lines than the two reference APC. It induced programmed cell death in HL-60 and SKW-3 cells after exposure for 24 h, and in bcr-abl expressing cells after a prolonged incubation period (48 h). LAMA-84 cells responded to i.v. injectable APC with increased conversion to an adherent, fibroblast-like phenotype. Experiments with a cell-free system showed that the target structures of APC are localized within the cytoplasmic compartment. Blockade of ceramide synthase by fumonisin B1 was insufficient to prevent oligonucleosomal DNA fragmentation. Using RT-PCR we confirmed that K-562 and LAMA-84 cells carry the b3a2 fusion type, and CML-T1 and BV-173 the b2a2 variant. BV-173 cells had the lowest level of bcr-abl mRNA which correlated with their increased sensitivity. Transfection of K-562 cells with antisense oligonucleotides directed against bcr-abl caused a specific suppression of K-562 clonogenicity. Our data indicated that i.v. injectable alkylphosphocholines are potent inducers of apoptosis and display increased antileukaemic efficacy against BCR-ABL-positive blasts as compared with miltefosine and perifosine. The expression of BCR-ABL cannot prevent apoptosis but delays erucylphosphocholine-induced programmed cell death. Transfection with bcr-abl directed antisense oligonucleotides reduces the clonogenicity of K-562 cells.     

Nano-MK-ASODN对人肝癌裸鼠原位移植模型的影响

目的 探讨中期因子反义寡核苷酸纳米脂质体（Nano-MK-ASODN）对人肝癌裸鼠原位移植模型的抑制作用。方法 利用人肝癌裸鼠原位移植模型,观察受试药品中期因子反义寡核苷酸（MK-ASODN）和Nano-MK-ASODN对荷瘤裸鼠体重、瘤重、以及肿瘤生长的抑制率的影响。检测荷瘤裸鼠血常规和血浆甲胎蛋白水平。结果 MK-ASODN高剂量组25 mg.kg-1组的瘤重抑制率为53.72%,12.5 mg.kg-1组的瘤重抑制率为48.76%,6.25 mg.kg-1组的瘤重抑制率为42.98%,Nano-MK-ASODN 25 mg.kg-1剂量组抑制率为66.94%,12.5 mg.kg-1组的瘤重抑制率为56.20%,6.25 mg.kg-1组的瘤重抑制率为52.07%,所有试验组肿瘤重量与溶剂对照组比较均有显著差异,P<0.01。Nano-MK-ASODN组肿瘤重量明显小于同等剂量MK-ASODN,P<0.01。Nano-MK-ASODN治疗组肿瘤体积以及AFP水平明显小于对照组,P<0.01。治疗组瘤重明显小于生理盐水对照组,P<0.05。Nano-MK-ASODN各治疗组及用药后体重、血常规与对照组比较指标差异均无显著性意义,P>0.05。同时,解剖学和病理组织学检查显示,Nano-MK-ASODN治疗组荷瘤裸鼠的肝内肿瘤体积缩小,并可见液化,能使部分肝癌细胞变性坏死。结论 Nano-MK-ASODN对人肝癌裸鼠原位移植瘤具有显著的抑制作用,且效果显著强于MK-ASODN。     

联合VEGF反义寡核苷酸和PDGF三链形成寡核苷酸抑制大鼠脑胶质瘤生长

目的:观察血小板源生长因子(PDGF) B链基因(PDGF-B)的三链形成寡核苷酸(triplex forming oligonucleotide,TFO)PDGF-TFO和血管内皮生长因子(VEGF)反义寡核苷酸(antisense oligonucleotide,AON)VEGF-AON对大鼠脑胶质瘤生长的抑制作用.方法:36只雄性SD大鼠,分为4组,所有大鼠均在立体定向导引下行右尾状核区微量灌注含1×106 C6胶质瘤细胞的生理盐水20 μl.在细胞接种后第8天,实验Ⅰ组6只大鼠原位注射含PDGF-TFO 1.5 mg的 20 μl生理盐水,实验Ⅱ组12只和实验Ⅲ组12只大鼠则分别原位注射含PDGF-TFO 1.5 mg VEGF-AON 0.125 mg和PDGF-TFO 1.5 mg VEGF-AON 0.25 mg的20 μl生理盐水.以后每隔72 h原位注射相同剂量的药物1次,共注射3次.对照组6只大鼠仅在相同时间原位注射20 μl生理盐水.实验3周时处死所有大鼠,观察肿瘤的生长情况,定性和定量观察肿瘤PDGF-B、VEGF和肿瘤核增殖抗原(PCNA)表达.结果:实验Ⅰ组的成瘤抑制率为53.1 %,实验Ⅱ组为81.4 %,实验Ⅲ组为93.1 %,3组比较有明显差异(P<0.01).PDGF-TFO对C6胶质瘤细胞PDGF-B、VEGF、PCNA表达有明显的抑制作用;联合应用PDGF-TFO和VEGF-AON能更好地抑制PDGF-B、VEGF、PCNA表达.结论:联合应用PDGF-TFO和VEGF-AON 比单用PFGF-TFO能更有效地抑制肿瘤生长.