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.     

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.     

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.     

Extending the Coding Potential of Viral Genomes with Overlapping Antisense ORFs: A Case for the De Novo Creation of the Gene Encoding the Antisense Protein ASP of HIV-1

Gene overprinting occurs when point mutations within a genomic region with an existing coding sequence create a new one in another reading frame. This process is quite frequent in viral genomes either to maximize the amount of information that they encode or in response to strong selective pressure. The most frequent scenario involves two different reading frames in the same DNA strand (sense overlap). Much less frequent are cases of overlapping genes that are encoded on opposite DNA strands (antisense overlap). One such example is the antisense ORF, asp in the minus strand of the HIV-1 genome overlapping the env gene. The asp gene is highly conserved in pandemic HIV-1 strains of group M, and it is absent in non-pandemic HIV-1 groups, HIV-2, and lentiviruses infecting non-human primates, suggesting that the ~190-amino acid protein that is expressed from this gene (ASP) may play a role in virus spread. While the function of ASP in the virus life cycle remains to be elucidated, mounting evidence from several research groups indicates that ASP is expressed in vivo. There are two alternative hypotheses that could be envisioned to explain the origin of the asp ORF. On one hand, asp may have originally been present in the ancestor of contemporary lentiviruses, and subsequently lost in all descendants except for most HIV-1 strains of group M due to selective advantage. Alternatively, the asp ORF may have originated very recently with the emergence of group M HIV-1 strains from SIVcpz. Here, we used a combination of computational and statistical approaches to study the genomic region of env in primate lentiviruses to shed light on the origin, structure, and sequence evolution of the asp ORF. The results emerging from our studies support the hypothesis of a recent de novo addition of the antisense ORF to the HIV-1 genome through a process that entailed progressive removal of existing internal stop codons from SIV strains to HIV-1 strains of group M, and fine tuning of the codon sequence in env that reduced the chances of new stop codons occurring in asp. Altogether, the study supports the notion that the HIV-1 asp gene encodes an accessory protein, providing a selective advantage to the virus.     

大鼠gp130反义寡核苷酸阻断rhIL-6对大鼠急性髓系白血病细胞系R2体外增殖的抑制效应

IL 6受体 (IL 6R) β链是分子量为 130kD的膜结合糖蛋白 (gp130 ) ,它是介导IL 6生物效应的信号转导子。我们的实验已经证实了重组人IL 6 (rhIL 6 )可作用于大鼠急性髓系白血病细胞系R2 ,rhIL 6与R2细胞表达的hIL 6R结合并与大鼠 gp130缔合而转导IL 6的信号 ,产生其生物效应。本文在体外液体培养中试验观察到 ,大鼠 gp130的反义寡核苷酸片段被摄入R2细胞后 ,对rhIL 6生物效应产生明显的影响。我们的研究结果表明 ,所合成的大鼠gp130反义寡核苷酸片段在细胞体外液体培养中可部分阻断rhIL 6对R2细胞的增殖抑制效应 ,在最适的终浓度 ,其阻断率可达 (45± 7) %。     

Effect of antisense oligonucleotide targeting bFGF on apoptosis of hepatoma cells

Objective： To investigate the cell cycle changes of hepatoma cells and the role of antisense oligonucleotide targeting bFGF. Methods： Inhibition of bFGF protein expression was investigated by confocal microscopy analysis and Western blot in the best condition of transfecting antisense oligonucleotide targeting bFGF. Cell cycle and apoptosis were detected with flow cytometry analysis. Results： Treatment with antisense oligonucleotide of bFGF not only reduced the expression of bFGF by confocal microscopy and Western blot analysises, but also increased the apoptosis of HepG2 cells （ P 〈 0.01）. Conclusion： bFGF may take part in apoptosis regulation of hepatoma cells and be used as a target of hepatocellular carcinoma therapy.     

Ki67反义核酸对裸鼠人肾癌细胞移植瘤生长及凋亡的影响

目的 观察Ki67基因反义寡核苷酸(ASODNs)对裸鼠人肾癌细胞移植瘤生长及凋亡的影响。方法 BALB／C-nu裸鼠接种入肾癌786-0细胞,治疗组瘤体注射ASODNs(10 mmol／g),连续4 d。对照组注射RPMI 1640培养基。药物处理后第3、6、12天每次处死小鼠4只,取瘤组织检测肿瘤体积;免疫组织化学、Western blot检测Ki67表达;脱氧核苷酸末端转移酶介导的缺口末端标记(TUNEL)法检测凋亡。结果ASODNs处理组小鼠肿瘤体积缩小,与对照组比较差异有显著性(P<0.01);肿瘤Ki-67抗原表达率、Ki-67蛋白定量均降低,与对照组比较差异均有显著性(P<0.01);肿瘤细胞凋亡率增加,与对照组比较差异均有显著性(P<0.01)。结论Ki67 ASODNs能有效抑制裸鼠入肾癌细胞移植瘤生长并促进其凋亡。