Acid‐activated nonviral peptide vector for gene delivery

Nonviral vector–based gene therapy is a promising strategy for treating a myriad of diseases. Cell‐penetrating peptides are gaining increasing attention as vectors for nucleic acid delivery. However, most studies have focused more on the transfection efficiency of these vectors than on their specificity and toxicity. To obtain ideal vectors with high efficiency and safety, we constructed the vector stearyl‐TH by attaching a stearyl moiety to the N‐terminus of the acid‐activated cell penetrating peptide TH in this study. Under acidic conditions, stearyl‐TH could bind to and condense plasmids into nanoparticle complexes, which displayed significantly enhanced cellular uptake and transfection efficiencies. In contrast, stearyl‐TH lost the capacities of DNA binding and transfection at physiological pH. More importantly, stearyl‐TH and the complexes formed by stearyl‐TH and plasmids displayed no obvious toxicity at physiological pH. Consequently, the high transfection efficiency under acidic conditions and low toxicity make stearyl‐TH a potential nucleic acid delivery vector for gene therapy.     

Chemically modified siRNA prolonged RNA interference in renal disease

BACKGROUND: We previously demonstrated that transfection of synthetic short interfering RNAs (siRNAs) targeting against TGF-beta1 could be effective and therapeutic in silencing TGF-beta1 expression in glomerulus, thereby ameliorated the progression of matrix expansion in anti-Thy-1 model of glomerulonephritis. However, a major concern in applying RNAi to gene therapy is the prolonged existence of silencing potential in vivo. METHOD: We examined the duration of siRNA stability in kidney and muscle, and checked the tissue distribution of siRNase, eri-1. Thereafter, we tested the effect of chemically modified siRNA called siSTABLE on progressive glomerulosclerosis model. RESULTS: A single introduction of siRNA for EGFP (siEGFP) or its expression vector into kidney resulted in the reduction of masangial EGFP expression only for up to two weeks, while transfection of siEGFP into the pretibial muscle silenced EGFP expression unexpectedly for more than 90 days. These observations could be explained by the different expression of eri-1 between kidney and muscle. In addition, transfection of ERI-1-resistant siSTABLE for TGF-beta1 significantly reduced glomerular matrix deposition in progressive glomerulosclerosis model.Conclusion: Treatment with siRNA resistant to eri-1 may be effective and promising strategy for progressive renal disease.     

共表达siRNA和hIL-12的新型乙肝多表位DNA疫苗的研究

目的 构建含有靶向乙肝表面抗原(HBsAg)基因的siRNA、乙肝复合多表位抗原基因和hIL-12共质粒表达的新型DNA疫苗,并在HepG2细胞中检测siRNA的效果以及各基因的表达。方法 设计并合成复合多表位HBV抗原基因,将其与增强型绿色荧光蛋白（EGFP）基因融合克隆进真核表达载体pVAX1的多克隆位点中,同时将带CMV启动子的完整hIL-12表达单元克隆进载体的BspH I位点之间,再设计并合成乙肝siRNA表达单元,将其克隆进载体的Mlu I位点之间,得到真核三元共表达重组质粒pVAX1-siHB-HB-EGFP-hIL12。以该重组质粒瞬时转染人肝癌细胞系HepG2,通过EGFP的荧光标记观察多表位抗原的表达,以ELISA测定培养细胞上清中hIL-12的表达,以rtPCR检测siRNA对HBsAg基因的沉默效果。结果 经酶切鉴定和测序证实共表达siRNA、hIL-12的HBV 多表位DNA疫苗构建成功。转染细胞中检测到绿色荧光,证实抗原表达;转染后48 h hIL-12的检出量为1 289 pg/mL细胞上清,72 h检出量为1 712 pg/mL细胞上清;转染后HBsAg表达量明显降低,证实siRNA效果良好。结论 成功构建乙肝复合多表位抗原基因与siRNA、hIL-12共质粒表达的DNA疫苗,并能在真核细胞中有效表达抗原与hIL-12基因,而且siRNA对HBsAg显示出明显的沉默效果。我们的工作为进一步研究该复合型DNA疫苗抗HBV的治疗效果打下基础。     

Stearylated octaarginine and artificial virus-like particles for transfection of siRNA into primary rat neurons

RNA interference (RNAi) provides a powerful experimental tool for sequence-specific gene silencing, allowing efficient analysis of gene function in a multitude of cell types. However, application of RNAi in primary mammalian neurons has been limited by low-transfection efficiency and considerable toxicity of conventional transfection methods. In this study, we evaluated a peptide-mediated and a polymer/lipid-based cellular delivery method for siRNA into rat primary neurons and compared the results with a commonly used liposomal transfection reagent. Stearylated octaarginine (Stearyl-R8) was used as polypeptide and artificial virus-like particles (AVPs) were used as a combined liposomal-polymeric vector, since both reagents have been previously shown to successfully transfect DNA into cell lines. Stearyl-R8 and AVPs both promoted siRNA transfection into primary hippocampal neurons via the endosomal pathway. SiRNA-mediated gene silencing could be effectively induced in primary neuron cultures. In comparison with the commonly used cationic liposome transfection agent, both novel reagents were less detrimental to cell metabolic activity. We conclude that these novel transfection methods yield performances comparable to cationic liposome-mediated transfection for siRNA, while being less cytotoxic in primary neurons. Stearyl-R8 and AVPs may therefore represent novel and more cost-efficient alternatives to conventional siRNA-transfection reagents.     

P27特异性siRNA重组腺病毒载体的构建及功能鉴定

目的构建针对P27的siRNA腺病毒载体及相应的对照病毒载体,并鉴定重组腺病毒在小鼠胰岛中对内源性皿7基因表达的影响。方法合成针对p27的siRNA的靶DNA序列及相应的阴性对照序列,连接至pShuttle-H1质粒中,然后用NotI和HindHI限制性内切酶将H1-siRNA片段从pShuttle—H1-siRNA质粒上双酶切下来,克隆至空载pAdTrack穿梭质粒上,与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中进行同源重组,转染QBI-293A细胞,包装得到pAd—P27-siRNA和pAd—NC重组腺病毒。用病毒体外感染小鼠胰岛,Western印迹法检测P27蛋白表达水平。结果重组腺病毒载体经测序鉴定正确,制备的病毒感染效率高,能有效抑制小鼠胰岛中P27的表达。结论成功构建了针对P27的siRNA重组腺病毒载体,为进一步研究P27在胰岛β细胞生长中的作用提供了基础。     

siRNA表达载体的构建及其表达

本研究用限制性内切酶消化质粒pCMV-tag-2B,除去巨细胞病毒(Cytomegalovirus,CMV)启动子核苷酸序列,剩下的核苷酸序列作为构建表达siRNA(Small interfering RNA,siRNA)载体的前体。依据文献提供的扩增H1RNA启动子核苷酸序列的引物序列合成一对引物,以带有H1RNA启动子序列的质粒DNA为模板扩增HIRNA启动子序列,插入前体,构建SiRNA的表达载体pCH1。另外将H1RNA启动子插入pGEM．1lfz相应位点,构建瞬时表达载体pGHl。依据EGFP的有效SiRNA抑制位点,合成两条分别为64bp的核苷酸链,通过体外退火,形成双链,然后插入已构建的两个表达载体。将这两个载体分别与表达EGFP蛋白的质粒pEGFP．N3共转染Bel．7402细胞,观察siRNA对EGFP的抑制效应。研究结果表明构建的载体有效表达了siRNA,这些载体可以用于与siRNA相关抗病毒治疗性试验研究。     

三重表达肺结核DNA疫苗在细胞水平的检测

目的 检测共表达siRNA、抗原基因与hIL-12的新型肺结核DNA疫苗在人胚肾293细胞中表达。方法 在已构建含有靶向抗调亡基因Mcl-1L的siRNA、结核分枝杆菌抗原基因Ag85B-ESAT6 (PVAE)、hIL-12的新型肺结核DNA疫苗pVAX-siRNA-PVAE-IL-12基础之上,将抗原基因与增强型绿色荧光蛋白（EGFP）基因融合,得到真核表达重组质粒pVAX1-siRNA-PVAE-EGFP-hIL12。并将siRNA单元用靶向EGFP基因的siEGFP代替,得到pVAX1-siEGFP-PVAE-EGFP-hIL12重组表达载体。用重组质粒转染人胚肾细胞293,分别观察融合抗原基因与siEGFP的表达。以ELISA测定培养细胞上清中hIL-12的表达。结果 经酶切鉴定和测序证实肺结核DNA疫苗改造成功。转染细胞中检测到绿色荧光,证实抗原表达。对照组检测到siEGFP的表达。转染48 h后检测出细胞上清中hIL-12的量为1571.63 pg/mL;72 h后检测出细胞上清中hIL-12的量为2392.25pg/mL。结论 已构建的肺结核质粒DNA疫苗能在真核细胞中有效表达siRNA、抗原基因与hIL-12。为进一步研究该DNA疫苗抗肺结核的免疫治疗和基因治疗效果打下基础。     

Suppression of RNA interference pathway <Emphasis Type="Italic">in vitro</Emphasis> by Grass carp reovirus

The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined.The present study aimed to investigate the effect of Grass carp reovirus...     

绿色荧光蛋白标记的D-氨基酸氧化酶基因在人宫颈癌细胞中的表达研究

为了探讨绿色荧光蛋白标记的红色酵母D 氨基酸氧化酶 (DAAO)基因在人宫颈癌细胞 (HeLa细胞 )中的表达及其功能 ,采用基因重组技术构建了含有CMV启动子和EGFP、DAAO基因开放阅读框 (ORF)的真核表达载体 pIRES DAAO。脂质体法转染HeLa细胞 ,荧光显微镜下观察转染细胞中绿色荧光蛋白的表达 ,流式细胞术分析转染效率并筛选荧光阳性细胞 ,命名为HeLa D。以不同浓度的前药D Ala处理HeLa D细胞 ,MTT法检测细胞存活率。结果显示 ,荧光显微镜下可见绿色荧光蛋白在HeLa D细胞中表达 ,流式细胞术成功筛选出HeLa D细胞。前药D Ala能明显杀伤HeLa D细胞。结果表明 ,EGFP可作为报告基因快速筛选DAAO表达载体转染的细胞 ,DAAO/D Ala自杀基因系统可进一步用于肿瘤的基因治疗研究     

siRNA binding proteins of microglial cells: PKR is an unanticipated ligand

Small interfering RNA (siRNA), double-stranded RNA (dsRNA) 21-23 nucleotides (nt) long with two nt 3' overhangs, has been shown to mediate powerful sequence-specific gene silence in mammalian cells through RNA interference (RNAi). Due to its high efficiency and high specificity siRNA has been used as a powerful post genomic tool and a potent therapeutic candidate. However, there is still a lot to learn about the mobility of siRNA inside cells and the cellular factors that might interfere with the specificity and activity of siRNA. Microglia are the brain's effector cells of the innate immune system and suitable targets in the development of novel therapeutic strategies. Here, we show the cellular uptake and intracellular distribution of siRNA in murine microglial N9 cells. siRNA was internalized by microglial N9 cells without transfection reagent and mainly localized to the endosomes However, no significant gene silencing effects were observed. Its cellular uptake and cellular distribution pattern were similar with that of a same length single stranded DNA (ssDNA). Further, cellular binding proteins of siRNA were purified and identified by mass spectrometry. Negative control siRNA and siRNA targeted to beta-actin were used in this part of experiment. Most of the siRNA binding proteins for negative control siRNA and siRNA targeted to beta-actin were dsRNA-binding proteins, such as dsRNA-dependent protein kinase R (PKR). Furthermore, both control siRNA and siRNA targeted to beta-actin activated PKR in N9 cells, which suggest that siRNA might cause off-target effects through activation of PKR.     

Dendronized chitosan derivative as a biocompatible gene delivery carrier

To improve the transfection efficiency of chitosan as a nonviral gene delivery vector, a dendronized chitosan derivative was prepared by a copper-catalyzed azide alkyne cyclization reaction of propargyl focal point poly(amidoamine) dendron with 6-azido-6-deoxy-chitosan. Its structure was characterized by (1)H NMR and FTIR analyses and its buffering capacity was evaluated by acid-base titration. In particular, its complexation with plasmid DNA was investigated by agarose gel electrophoresis, zeta potential, and particle size analyses as well as transmission electron microscopy observation. Compared to unmodified chitosan, such a chitosan derivative has better water solubility and buffering capacity. Compared to commonly used polyethyleneimine (PEI, 25 kDa), it could exhibit enhanced transfection efficiency in some cases and lower cell toxicity, as confirmed by in vitro transfection and cytotoxicity tests in human kidney 293T and human nasopharyngeal carcinoma CNE2 cell lines. In addition, the effect of serum on its transfection efficiency was also studied.     

新型PEI 衍生物在COS-7 细胞中的转染与毒性研究

目的:寻找一种新型的转染效率高,毒性低的非病毒基因载体.方法:通过化学方法合成Polyimine-MPEI,然后以不同质量比包裹绿色荧光蛋白质粒,检测在COS-7细胞中的转染效率和毒性.结果:在比例从5到100之间,转染效率均比较理想,能达到1.00E+07以上,Polyimine-MPEI的毒性也很小,细胞的生长率均在80％以上,明显高于PEI25KDa对照组.结论:Polyimine-MPEI是一个很有研究前景的聚合物载体,具有高转染效率低毒性的特点,可以通过延长反应时间,增加分子量,增大转染能力.     

siRNA干扰绵羊胚胎成纤维细胞Lig4基因增加同源重组载体重连修复效率

在动物细胞中,抑制非同源末端连接(Non-homologous end joining, NHEJ)修复途径,可以提高同源重组(Homologous recombination, HR)修复基因组双链断裂(Double-strand brakes, DSBs)的发生概率。为了提高绵羊胚胎成纤维细胞的HR效率,针对NHEJ修复途径中的关键因子Lig4(DNA ligase 4)基因,本文设计合成4个具有靶向性的siRNA(Small interfering RNA)。绵羊胚胎成纤维细胞经电转染导入siRNA,通过实时荧光定量PCR(qRT-PCR)和Western blotting检测,筛选出有效抑制Lig4基因表达的2个siRNA。应用质粒重连法检测HR修复效率,将I-SceⅠ酶线性化的HR质粒和siRNA共转染绵羊胚胎成纤维细胞,经72 h培养及流式细胞仪检测,与对照组细胞比较,结果表明HR质粒重连效率提高了3~4倍。瞬间干扰Lig4基因的表达可提高HR质粒重连效率,为改善绵羊胚胎成纤维细胞基因打靶效率提供理论基础。     

Linear cationic click polymers/DNA nanoparticles: in vitro structure-activity relationship and in vivo evaluation for gene delivery

The aim of this work was to explore the structure--activity relationships (SAR) of a series of novel linear cationic click polymers with various structures for in vitro gene delivery and in vivo gene transfer. The experimental results revealed that the minimal structure variation could result in a crucial effect on DNA-binding ability, buffering capacity, and the cellular delivery capacity of polymer, all of which brought about the obvious effects on their transfection efficiencies. The polymer synthesized from diazide monomer containing bis-ethylenediamine unit and dialykene monomer containing bis-ethylene glycol unit (B(2)) could effectively condense DNA into complex nanoparticles (B(2)Ns), which showed the highest in vitro transfection efficiency. The biodistribution and transfection efficiency of B(2)Ns in nude mice bearing tumor demonstrated the ability of effectively delivering DNA into tumor tissue. These results implied that this gene vector based on linear cationic click polymer could be a promising gene delivery system for tumor gene therapy.     

RNAi in primary human chondrocytes: Efficiencies, kinetics, and non-specific effects of siRNA-mediated gene suppression

RNAi-mediated gene silencing is a recent, powerful tool to investigate gene function. Controlling for experimental factors such as transfection efficiencies, siRNA concentration, gene suppression levels, gene suppression kinetics, or non-specific effects is key to robust results. In this methods paper, we compare the efficiencies of different transfection reagents in primary human chondrocytes (PHCs). We investigated TAK1 gene suppression efficiencies and kinetics on the mRNA and protein level depending on the siRNA concentration used. Furthermore, we evaluated PKR, IL-6, and TNF-alpha induction, as well as IkappaB degradation and NFkappaB activation as control parameters of non-specific siRNA effects. PKR and IL-6 proved to be appropriate markers of cellular inflammatory responses resulting from siRNA transfection. In addition, we compared different siRNAs (silencing, non-silencing, classic 21-mer, and 25-mer stealth siRNA) with respect to their capacity to induce cellular inflammatory responses. We found the occurrence of cellular responses in PHCs to be a function of the specific siRNA sequence in use. Hence, it is essential to analyze and to compare gene silencing siRNAs and control siRNAs with respect to their off-target effects prior to any functional gene validation.     

Inducible H1 promoter-driven lentiviral siRNA expression by Stuffer reporter deletion

Introduction of 19-23-bp small interfering RNA (siRNA) into mammalian cells has become a standard procedure to downregulate mRNA with high efficacy. siRNAs can be introduced into cells either as synthetic duplexes or as hairpin structures produced by Pol III promoter-driven vectors. Pol III promoter-expressed small hairpin RNAs (shRNAs) offer a great possibility for the production of endogenous siRNA, which can be used for stable siRNA production in vivo. A major drawback of this strategy is the incapability of detecting rapidly occurring cellular responses. Here, we present a lentiviral shRNA-producing vector system, which can be induced by CRE recombinase enzyme to overcome these limitations. Following the addition of CRE, the pLIND (LentiINDucible) will activate siRNA production by deleting EGFP and a stop cassette between the promoter and siRNA oligo. Target gene downregulation capacity was comparable to that of a noninducible siRNA system.     

Two-wavelength fluorescence assay for DNA repair

A simple and reliable quantitative assay for measuring cellular DNA repair capacity has been developed. It is based on the host cell reactivation of the UV-irradiated plasmid pEGFP carrying the marker gene for the enhanced green fluorescent protein (EGFP). As a reference we used the plasmid pEYFP carrying the gene for a red-shifted fluorescent protein (EYFP). Both proteins can be excited by visible light with a maximum at 488 nm, but EGFP emits with a maximum at 509 nm, while EYFP emits with a maximum at 527 nm. This makes it possible to monitor the expression of the two genes simultaneously by measuring the fluorescence at two wavelengths. HEK293 cells were cotransfected with a mixture of UV-irradiated pEGFP and undamaged pEYFP. At different time intervals after transfection the fluorescence of EGFP was determined relative to the fluorescence of EYFP to compensate for any differences in the transfection efficiency or other experimental variables. It was used to calculate the number of UV lesions in DNA and hence the repair capacity of the host cells. It was found that HEK293 cells were able to repair approximately 1.4 UV lesions per 1000 nucleotides DNA for 12 h on the average.