Cationic lipids as gene transfer agents: a patent review

Background: Cationic lipids have been among the more efficient synthetic gene delivery reagents in vitro. They condense nucleic acids into cationic particles when the components are mixed together. Since the first studies were conducted, hundreds of cationic lipids have been synthesized as candidates for non-viral gene delivery and a few entered clinical trials. Objective: This paper reviews the lipid structure of cationic lipids, their structure–activity relationship, toxicity and transfection efficacy. A revision of the more recent patents is also included. Methods: Bibliographic research was carried out using the PubMed database. Patent literature was searched using the Derwent Innovations Index^SM. Results/conclusions: There is a wide range of scientific literature and patents about new cationic lipids and at present many research groups focus their investigation on the development of new molecules. Advances in the development of new technologies to better understand the in vitro and in vivo behavior of the lipoplexes will help to redesign new cationic lipids. The results obtained in clinical trials confirm gene therapy as a very promising therapeutic tool in the future.     

Modulation of GDP-fucose level for generating proteins with reduced rate of fucosylation (WO2010141855)

Background: The application (WO2010141855) is in the field of glycobiology, and involves the control of the rate of fucosylation of proteins by exogenous factors.

Objective: It aims at controlling the rate of protein fucosylation with inhibitors (drugs or nucleic acid antagonists) of enzymes involved in the synthesis of GDP-fucose.

Methods: Mammalian cell lines were cultured in the presence of inhibitors, for example, siRNA. The rates of GDP-fucose in cells and during protein fucosylation were characterized.

Results: The level of protein fucosylation decreases rapidly in response to a decrease in GDP-fucose level.

Conclusion: The relationship between the rate of fucosylation of proteins and the level of GDP-fucose in a cell is non-linear. Reduction in the rate of protein fucosylation can be achieved with a minimal reduction of the level of GDP-fucose in cells. The paradigm may be used to synthesize proteins and antibodies, with a reduced rate of fucosylation. The application claims that the use of drugs or nucleic acid antagonists that inhibit the enzymes involved in GDP-fucose biosynthesis optimizes the level of GDP-fucose present in cells, and reduces the rate of fucosylation of glycoproteins.     

Lipoplexes versus nanoparticles: pDNA/siRNA delivery

Abstract

Small interfering RNA (siRNA) has been widely used as potential therapeutic for treatment of various genetic disorders. However, rapid degradation, poor cellular uptake and limited stability in blood limit the effectiveness of the systemic delivery of siRNA. Therefore, an efficient delivery system is required to enhance its transfection and duration of therapeutics. In the present study, plasmid DNA (pEGFPN3) expressing green fluorescent protein (GFP) was used as a reporter gene. Chitosan nanoparticles/polyplexes and cationic liposomes/lipoplexes were developed and compared for their transfectivity and therapeutic activity in mammalian cell line (HEK 293). The nanoparticulates were first characterized by assessing the surface charge (zeta potential), size (dynamic light scattering) and morphology (transmission electron microscope) followed by evaluation for their DNA retardation ability, transfection efficiency and cytotoxicity on HEK 293 cell line. The chitosan nanoparticles/plasmid DNA (pDNA) complex and liposomes/pDNA complex were co-transfected with GFP-specific siRNA into HEK 293 cells and it was found that both are efficient delivery vehicles for siRNA transfection, resulting in ～57% and ～70% suppression of the targeted gene (GFP), respectively, as compared with the mock control (cells transfected with nanocarrier/pDNA complexes alone). This strong inhibition of GFP expression indicated that cationic liposomes are better than chitosan nanoparticles and can be used as an effective carrier of siRNA in mammalian cells.     

重组腺病毒CXCR7-siRNA对人胃癌细胞增殖及迁移的影响

目的 体外考察趋化因子受体7 （chemokine receptor 7,CXCR7）在胃癌细胞MGC-803中的表达和CXCR7-siRNA重组腺病毒表达载体靶向抑制CXCR7的表达对人胃癌细胞MGC-803增殖、迁移活性的影响.方法 将MGC-803细胞分为实验组、空载体组及空白对照组,实验组加入CXCR7-siRNA重组腺病毒,空载体组加入空载腺病毒,空白对照组加入等量容积细胞培养基.噻唑蓝（MTT）法测各组细胞增殖活性;Hoechst 33258 染色法观察各组细胞凋亡情况;Western blot 免疫印迹法检测各组MGC-803细胞中CXCR7的表达情况及凋亡执行蛋白Caspase-3 表达情况; Transwell细胞迁移实验检测各组细胞趋化活性.结果 实验组细胞增殖受到抑制,转染3天后,实验组细胞MTT值为1.22±0.01,与空载体转染组（1.48±0.05）及空白对照组（1.61±0.03）比较,差异有统计学意义（F=31.2,P＜0.05）.实验组细胞凋亡比率为（37.66±1.31）%,与空载体组（3.39±0.76）%及空白对照组（13.12±0.85）%比较差异具有统计学意义（F=391.9,P〈0.05）.实验组CXCR7表达水平低于空载体组和空白对照组;实验组Caspase-3剪切激活,空载体组见少许Caspase-3剪切激活,空白对照组未见Caspase-3剪切激活;转染3天后,Transwell细胞迁移实验显示实验组穿透滤过膜细胞数量（15.00±1.73）,较空载体组（35±2.89）及空白对照组（37±4.04）显著减少,差异均有统计学意义（F=12.51,P〈0.05）.结论 靶向沉默CXCR7的表达抑制胃癌细胞MGC-803的增殖和迁移活性.     

JAZF1基因抑制对3T3-L1脂肪细胞糖、脂代谢的影响

目的 探讨JAZF1基因抑制对3T3-L1脂肪细胞糖、脂代谢相关基因的影响.方法 构建JAZF1小发夹RNA (shRNA)表达载体并转染3T3-L1细胞,实时荧光定量PCR(RT-QPCR)和蛋白印迹法检测JAZF1 mRNA和蛋白水平的表达;氢三放射示踪法检测3T3-L1细胞糖摄取率;蛋白印记法检测糖、脂代谢相关基因蛋白水平;油红O染色检测脂肪细胞甘油三酯(TG)含量变化.结果 成功构建JAZF1-shRNA;转染脂肪细胞48 h后,JAZF1 mRNA和蛋白水平明显低于对照组(P＜0.05);氢3放射性示踪法显示转染组葡萄糖摄取率明显降低(P＜0.05);PPAR-γ蛋白表达升高(P＜0.05),激素敏感脂肪酶(HSL)、内脏脂肪素(Visfatin)、胰岛素诱导基囚-2 (Insig-2)蛋白表达降低(均P＜0.05);油红O染色显示JAZF1转染组细胞内脂质积聚明显,比对照组升高约25％(P＜0.05).结论 JAZF1基因抑制可减少基础糖转运,增加脂质与胆固醇合成,减少脂质分解并减少相关脂肪细胞因子的表达.     

MT2-MMP基因沉默对缺氧条件下培养的胰腺癌AsPC-1细胞增殖、凋亡及侵袭力的影响

目的 利用特异性siRNA沉默人胰腺癌AsPC-1细胞的膜型金属蛋白酶2（MT2-MMP）基因表达,观察其对缺氧条件下培养的细胞增殖、凋亡和侵袭力的影响.方法 采用脂质体转染法将插入MT2-MMP特异性siRNA的真核表达质粒转染至人胰腺癌AsPC-1细胞株（siRNA组）,以转染过表达MT2-MMP质粒（过表达组）或阴性对照质粒（空载体组）的AsPC-1作为对照.实时定量PCR法和蛋白质印迹法检测转染细胞的MT2-MMP mRNA和蛋白的表达.在缺氧条件（37℃、1％O2、5％ CO2、饱和湿度的三气培养箱）下培养后,应用CCK-8法检测转染细胞的增殖,流式细胞仪检测细胞的凋亡,Transwells小室检测细胞的侵袭能力.结果 成功获取MT2-MMP基因沉默的AsPC-1细胞株和过表达的细胞株.缺氧条件下培养24h后,空载体组、过表达组、siRNA组细胞增殖的吸光度值（A490值）分别为0.68±0.08、0.80 ±0.08、0.52±0.07;细胞凋亡率分别为（6.2±1.5）％、（2.8±1.1）％、（21.4±3.9）％;每个视野（200倍）的穿膜细胞数分别为（115.8 ±23.2）、（256.4±38.6）、（45.8±18.2）个.siRNA组较空载体组的细胞增殖显著被抑制,穿膜细胞数显著减少,而细胞凋亡率显著增加（P值均＜0.05）.过表达组较空载体组的细胞增殖显著增强,穿膜细胞数显著增加,而细胞凋亡率显著降低（P值均＜0.05）.结论 MT2-MMP基因沉默的AsPC-1细胞在缺氧条件下培养后细胞凋亡增加,增殖被抑制,侵袭力减弱.     

Targeted genetic dependency screen facilitates identification of actionable mutations in FGFR4, MAP3K9, and PAK5 in lung cancer

Approximately 70% of patients with non–small-cell lung cancer present with late-stage disease and have limited treatment options, so there is a pressing need to develop efficacious targeted therapies for these patients. This remains a major challenge as the underlying genetic causes of ∼50% of non–small-cell lung cancers remain unknown. Here we demonstrate that a targeted genetic dependency screen is an efficient approach to identify somatic cancer alterations that are functionally important. By using this approach, we have identified three kinases with gain-of-function mutations in lung cancer, namely FGFR4, MAP3K9, and PAK5. Mutations in these kinases are activating toward the ERK pathway, and targeted depletion of the mutated kinases inhibits proliferation, suppresses constitutive activation of downstream signaling pathways, and results in specific killing of the lung cancer cells. Genomic profiling of patients with lung cancer is ushering in an era of personalized medicine; however, lack of actionable mutations presents a significant hurdle. Our study indicates that targeted genetic dependency screens will be an effective strategy to elucidate somatic variants that are essential for lung cancer cell viability.     

Endoglin is necessary for angiogenesis in human ovarian carcinoma-derived primary endothelial cells

Endoglin (CD105, END) is upregulated in proliferating endothelial cells, suggesting potential therapeutic properties. However, it is not clear whether endoglin mediates an enhanced proliferative rate or may be upregulated as part of a negative feedback loop. To gain insights into context-dependent and cell type-dependent regulatory effects of endoglin, we studied its role properties in human ovarian carcinoma-derived endothelial cells (ODMECs). We isolated and cultured primary ODMECs from epithelial ovarian carcinoma tissue. ODMECs had higher expression of endoglin and VEGFR-2, and also exhibited enhanced spontaneous formation of vessel-like structures in vitro. Transfection of siRNA targeting endoglin in ODMECs cells resulted in the reduction of the proliferation and tube formation. These results indicate that a subset of ODMECs display abnormal angiogenic properties and this phenotype was blocked by decreasing endoglin levels, suggesting endoglin is essential for stimulating angiogenesis, and targeting it may be an attractive approach to anti-angiogenesis therapy for ovarian carcinoma.