A selection strategy in plant transformation based on antisense oligodeoxynucleotide inhibition

Antisense oligodeoxynucleotide (asODN) inhibition was developed in the 1970s, and since then has been widely used in animal research. However, in plant biology, the method has had limited application because plant cell walls significantly block efficient uptake of asODN to plant cells. Recently, we have found that asODN uptake is enhanced in a sugar solution. The method has promise for many applications, such as a rapid alternative to time‐consuming transgenic studies, and high potential for studying gene functionality in intact plants and multiple plant species, with particular advantages in evaluating the roles of multiple gene family members. Generation of transgenic plants relies on the ability to select transformed cells. This screening process is based on co‐introduction of marker genes into the plant cell together with a gene of interest. Currently, the most common marker genes are those that confer antibiotic or herbicide resistance. The possibility that traits introduced by selectable marker genes in transgenic field crops may be transferred horizontally is of major public concern. Marker genes that increase use of antibiotics and herbicides may increase development of antibiotic‐resistant bacterial strains or contribute to weed resistance. Here, we describe a method for selection of transformed plant cells based on asODN inhibition. The method enables selective and high‐throughput screening for transformed cells without conferring new traits or functions to the transgenic plants. Due to their high binding specificity, asODNs may also find applications as plant‐specific DNA herbicides.     

Nanogels for oligonucleotide delivery to the brain

Systemic delivery of oligonucleotides (ODN) to the central nervous system is needed for development of therapeutic and diagnostic modalities for treatment of neurodegenerative disorders. Macromolecules injected in blood are poorly transported across the blood-brain barrier (BBB) and rapidly cleared from circulation. In this work we propose a novel system for ODN delivery to the brain based on nanoscale network of cross-linked poly(ethylene glycol) and polyethylenimine ("nanogel"). The methods of synthesis of nanogel and its modification with specific targeting molecules are described. Nanogels can bind and encapsulate spontaneously negatively charged ODN, resulting in formation of stable aqueous dispersion of polyelectrolyte complex with particle sizes less than 100 nm. Using polarized monolayers of bovine brain microvessel endothelial cells as an in vitro model this study demonstrates that ODN incorporated in nanogel formulations can be effectively transported across the BBB. The transport efficacy is further increased when the surface of the nanogel is modified with transferrin or insulin. Importantly the ODN is transported across the brain microvessel cells through the transcellular pathway; after transport, ODN remains mostly incorporated in the nanogel and ODN displays little degradation compared to the free ODN. Using mouse model for biodistribution studies in vivo, this work demonstrated that as a result of incorporation into nanogel 1 h after intravenous injection the accumulation of a phosphorothioate ODN in the brain increases by over 15 fold while in liver and spleen decreases by 2-fold compared to the free ODN. Overall, this study suggests that nanogel is a promising system for delivery of ODN to the brain.     

硫代磷酸反义寡脱氧核苷酸抗HBV细胞的实验研究

针对ＨＢＶ的５个基因位点作为靶序列,设计合成硫代反义寡聚核苷酸（Ｓ－ａｓＯＤＮ）．应用ＥＬＩＳＡ方法、ＭＴＴ比色法、电子显微镜等手段,观察Ｓ－ａｓＯＤＮ对ＨｅｐＧ２２２１５细胞ＨＢｓＡｇ、ＨＢｅＡｇ抗原的表达,及细胞的毒性、细胞形态和超微结构的影响．结果显示不同靶位点的Ｓ－ａｓＯＤＮ对ＨＢｓＡｇ、ＨＢｅＡｇ表达都有显著的抑制作用,且表现为序列特异性、剂量相关性、联合协同性和一定的抗核酸酶性,在浓度为２０μｍｏｌ／Ｌ时,对细胞无明显杀伤作用,对细胞的超微结构无显著的改变．结果提示Ｓ－ａｓＯＤＮ有望发展为抗ＨＢＶ的有效药物,但靶序列的选择、透细胞膜性及联合用药配伍等仍值得进一步研究和解决．     

Specific inhibition of Stat5a/b promotes apoptosis of IL-2-responsive primary and tumor-derived lymphoid cells

Stat5a/b exhibits 96% homology and are required for normal immune function. The present studies examined Stat5a/b function in lymphoid cells by specific and simultaneous disruption of both proteins using novel phosphorothioate-2'-O-methoxyethyl antisense oligodeoxynucleotides (asODN). Efficient delivery was confirmed by the presence of fluorescent TAMRA-labeled ODN in >or=55 and 95% in human primary and tumor cell lines, respectively. Acute asODN administration reduced levels of Stat5a (90%) in 6 h, whereas Stat5b required nearly 48 h to attain the same inhibition, suggesting that the apparent turnover rate for Stat5a was 8-fold higher than that for Stat5b. Expression of the closely related Stat3 protein was unchanged after asODN treatment, however. Molecular ablation of Stat5a/b promoted apoptotic cell death in a significant population of primary PHA-activated T cells (72%) and lymphoid tumor cell line (e.g., YT; 74%) within 24 h, as assessed by 1) visualization of karyolytic nuclear degeneration and other generalized cytoarchitectural alterations, 2) enzymatic detection of TdT-positive DNA degradation, and 3) automated cytometric detection of annexin V translocation. Contrary to findings from Stat5a/b-null mice, cell cycle progression did not appear to be significantly affected. Interestingly, IL-2-insensitive and unprimed T cells and Jurkat cells remained mostly unaffected. Finally, evidence is provided that the cytotoxicity associated with Stat5a/b ablation may derive from activation of caspase-8, an initiator protease that contributes to apoptotic cell commitment. We propose that in lymphoid cells competent to activate Stat5a and Stat5b, both proteins preferentially mediate an antiapoptotic survival influence.     

Slow release and delivery of antisense oligonucleotide drug by self-assembled peptide amphiphile nanofibers

Antisense oligonucleotides provide a promising therapeutic approach for several disorders including cancer. Chemical stability, controlled release, and intracellular delivery are crucial factors determining their efficacy. Gels composed of nanofibrous peptide network have been previously suggested as carriers for controlled delivery of drugs to improve stability and to provide controlled release, but have not been used for oligonucleotide delivery. In this work, a self-assembled peptide nanofibrous system is formed by mixing a cationic peptide amphiphile (PA) with Bcl-2 antisense oligodeoxynucleotide (ODN), G3139, through electrostatic interactions. The self-assembly of PA-ODN gel was characterized by circular dichroism, rheology, atomic force microscopy (AFM) and scanning electron microscopy (SEM). AFM and SEM images revealed establishment of the nanofibrous PA-ODN network. Due to the electrostatic interactions between PA and ODN, ODN release can be controlled by changing PA and ODN concentrations in the PA-ODN gel. Cellular delivery of the ODN by PA-ODN nanofiber complex was observed by using fluorescently labeled ODN molecule. Cells incubated with PA-ODN complex had enhanced cellular uptake compared to cells incubated with naked ODN. Furthermore, Bcl-2 mRNA amounts were lower in MCF-7 human breast cancer cells in the presence of PA-ODN complex compared to naked ODN and mismatch ODN evidenced by quantitative RT-PCR studies. These results suggest that PA molecules can control ODN release, enhance cellular uptake and present a novel efficient approach for gene therapy studies and oligonucleotide based drug delivery.     

Stimulatory effect of an indirectly attached RNA helicase-recruiting sequence on the suppression of gene expression by antisense oligonucleotides

Antisense oligonucleotides (ODNs) are powerful tools with which to determine the consequences of the reduced expression of a selected target gene, and they may have important therapeutic applications. Methods for predicting optimum antisense sites are not always effective because various factors, such as RNA-binding proteins, influence the secondary and tertiary structures of RNAs in vivo. To overcome this obstacle, we have attempted to engineer an antisense system that can unravel secondary and tertiary RNA structures. To create such an antisense system, we connected the constitutive transport element (CTE), an RNA motif that has the ability to interact with intracellular RNA helicases, to an antisense sequence so that helicase-binding hybrid antisense ODN would be produced in cells. We postulated that this modification would enhance antisense activity in vivo, with more frequent hybridization of the antisense ODN with its targeting site. Western blotting analysis demonstrated that a hybrid antisense ODN targeted to the bcl-2 gene suppressed the expression of this gene more effectively than did the antisense ODN alone. Our results suggest that the effects of antisense ODNs can be enhanced when their actions are combined with those of RNA helicases.     

10-23型DNA酶作为鉴定mRNA靶点有效性的新工具

10-23DNA酶是能主动切割mRNA的一类反义寡核苷酸.利用10-23DNA酶的直接切割作用验证mRNA结构靶点的有效性.对筛选的绿色荧光蛋白(GFP)基因mRNA的4个靶点平行设计了4条反义寡核苷酸和4条10-23DNA酶,对照组反义寡核苷酸将最佳靶点——靶点2的反义寡核苷酸突变2个碱基,对照组10-23DNA酶将靶点2的10-23DNA酶结合臂中央突变2个碱基.体外4条10-23DNA酶切割mRNA的结果和相应的4条反义寡核苷酸依赖的RNaseH降解结果完全相似,细胞内4条10-23DNA酶对绿色荧光蛋白的表达抑制作用与相应的4条反义寡核苷酸相似,表明10-23DNA酶显示的最佳作用靶点同样是最佳作用效果的反义寡核苷酸结合靶.10-23DNA酶可以作为评价mRNA结构靶点有效性的新工具.     

Increased brain content of the endogenous benzodiazepine receptor ligand, octadecaneuropeptide (ODN), following portacaval anastomosis in the rat.

Evidence suggests that endogenous benzodiazepine receptor ligands such as diazepam binding inhibitor (DBI) and its metabolite octadecaneuropeptide (ODN) may be implicated in the pathogenesis of hepatic encephalopathy. Using an immunocytochemical technique and an antibody of high specific activity to synthetic ODN, we studied the effects of portacaval anastomosis (PCA) on ODN distribution in rat brain. Four weeks after PCA, ODN immunolabeling was increased in several brain regions including cerebral cortex, hippocampus, hypothalamus and thalamus. Increased ODN immunolabeling was confined to nonneuronal elements such as astrocytes and ependymal cells. Neuropathological evaluation of brain following PCA reveals astrocytic rather than neuronal changes. These results are consistent with a role for endogenous neuropeptide ligands for astrocytic benzodiazepine receptors in the pathogenesis of hepatic encephalopathy.     

CpG-A-induced monocyte IFN-gamma-inducible protein-10 production is regulated by plasmacytoid dendritic cell-derived IFN-alpha

Unmethylated CpG motifs in bacterial DNA or synthetic oligodeoxynucleotides (ODN) are known for inducing a Th1 cytokine/chemokine environment, but the mechanisms regulating this have been unclear. Recent studies have defined two classes of CpG ODN, CpG-A ODN that induce plasmacytoid dendritic cells (pDC) to secrete very high levels of IFN-alpha, and CpG-B ODN that induce only low levels of IFN-alpha production, but strongly activate B cells. We now demonstrate that a CpG-A ODN directly activates pDC secretion of IFN-alpha and other soluble factors that secondarily induce purified monocytes to secrete high levels of the Th1-promoting chemokine IFN-gamma-inducible protein-10 (IP-10). Cell contact between the monocytes and pDC is not required for this interaction. IFN-alpha is necessary, but only partially sufficient, for this indirect CpG-induced monocyte IP-10 production. Although CpG ODN induce human PBMC to make only very slight amounts of IFN-gamma, we find that these low concentrations synergize with IFN-alpha for inducing monocyte production of IP-10. These studies provide a better understanding of the mechanisms through which CpG ODN create a Th1-like environment.     

Novel immunostimulatory agent based on CpG oligodeoxynucleotide linked to the nontoxic B subunit of cholera toxin

In this study, we report the development of a novel, rationally designed immunostimulatory adjuvant based on chemical conjugation of CpG oligodeoxynucleotide (ODN) to the nontoxic B subunit of cholera toxin (CTB). We demonstrate that the immunostimulatory effects of CpG can be dramatically enhanced by conjugation to CTB. Thus, CpG ODN linked to CTB (CTB-CpG) was shown to be a more potent stimulator of proinflammatory cytokine and chemokine responses in murine splenocytes and human PBMCs than those of CpG ODN alone in vitro. The presence of CpG motif, but not modified phosphorothioate ODN backbone, was found to be critical for the enhanced immunostimulatory effects of CTB-CpG. Our mode-of-action studies, including studies on cells from specifically gene knockout mice suggest that similar to CpG, CTB-CpG exerts its immunostimulatory effects through a TLR9/MyD88- and NF-kappaB-dependent pathway. Surprisingly, and as opposed to CpG ODN, CTB-CpG-induced immunity was shown to be independent of endosomal acidification and resistant to inhibitory ODN. Furthermore, preincubation of CTB-CpG with GM1 ganglioside reduced the immunostimulatory effects of CTB-CpG to those of CpG ODN alone. Interestingly, conjugation of CpG ODN to CTB confers an enhanced cross-species activity to CpG ODN. Furthermore, using tetanus toxoid as a vaccine Ag for s.c. immunization, CTB-CpG markedly enhanced the Ag-specific IgG Ab response and altered the specific pattern of Ab isotypes toward a Th1 type response. To our knowledge, CTB is the first nontoxic derivative of microbial toxins discovered that when chemically linked to CpG remarkably augments the CpG-mediated immune responses.     

DNA transfer into vascular smooth muscle using fusigenic Sendai virus (HJV)-liposomes

Manipulation of the genetic machinery of cells both in vitro and in vivo is becoming an ever more important means of elucidating pathways of molecular and cellular biochemistry. In addition, gene therapy has been proposed as a novel and potentially powerful treatment for both inherited and acquired diseases. Successful gene transfer and gene blockade generally depend on high efficiency delivery of exogenous DNA or RNA into living cells, and much effort has therefore been focused on the development of methods for achieving this delivery in a safe and effective manner. We describe here our application of fusigenic Sendai virus (HVJ)-liposome technology toward the effective delivery of DNA into vascular smooth muscle cells (VSMC) in cell culture. Cellular uptake and intracellular distribution of oligodeoxynucleotide (ODN) after transfection with HVJ-liposome complexes was characterized using fluorescent (FITC)-labeled ODN, and the biologic effect of HVJ-liposome mediated transfection was demonstrated via inhibition of DNA synthesis in cultured VSMC using antisense ODN against basic fibroblast growth factor.     

Antisense therapy in clinical oncology

Nucleic acid molecules have emerged as versatile tools with promising utility as therapeutics for human diseases. The specificity of hybridization of an antisense oligonucleotide (AS ODN) to the target mRNA makes the AS strategy attractive to selectively modulate the expression of genes involved in the pathogenesis of malignant or non-malignant diseases. One AS drug has been approved for local therapy of cytomegalovirus retinitis, and a number of AS ODN are currently tested in clinical trials including ODN that target bcl-2, survivin, and DNA methyltransferase. The clinical studies indicate that AS ODN are well tolerated and may have therapeutic activity. In this overview, we summarize therapeutic concepts, clinical studies, and new promising molecular targets to treat human cancer with AS ODN.     

Progress in the delivery of therapeutic oligonucleotides: organ/cellular distribution and targeted delivery of oligonucleotides in vivo

Oligonucleotide (ODN) therapy is a powerful tool for modulation of gene expression in vivo. With advances in ODN chemistry and progress in formulation development, ODNs are becoming widely acceptable drugs. This review summarizes the current status and future trend of the in vivo application of ODN therapeutics, especially antisense ODNs. Here, we review the current understanding of the tissue/organ distribution and cellular uptake of ODN drugs administered parenterally or nonparenterally to intact animals. The problems and advantages inherent in the use of different delivery methods for the treatment of particular diseases are discussed in detail. Emphasis is placed on the most widely studied ODN analogs, the phosphorothioates (PS). Lessons learned from antisense PS studies have broad implications for ODN therapeutics in general.     

Monitoring antisense oligodeoxynucleotide activity in hematopoietic cells

Traditionally, methods designed to impair translation through direct interactions with target messenger RNA (mRNA) have been designated as "antisense" strategies because of their reliance on the formation of reverse complementary (antisense) Watson-Crick base pairs between the targeting oligodeoxynucleotide (ODN) and the mRNA whose function is to be disrupted. Proof of putative "antisense effects," and other mechanistic studies, would be greatly facilitated by the ability to directly demonstrate hybridization between an antisense (AS) ODN and its mRNA target in vivo. In addition, evidence of AS activity by demonstrating reduced levels of RNA or protein or by showing cleaved target molecules would lend proof of the concept. In this article we discuss how AS ODN may be used to down-regulate target gene expression with an emphasis on those targets chosen for our investigations, and we summarize the methods employed for this type of study.     

A theoretical approach to select effective antisense oligodeoxyribonucleotides at high statistical probability.

Up to now, out of approximately 20 antisense oligodeoxyribonucleotides (as ODN) selected and tested against a given target gene, only one species shows substantial suppression of target gene expression. In part, this seems to be related to the general assumption that the structures of local target sequences or antisense nucleic acids are unfavorable for efficient annealing. Experimental approaches to find effective as ODN are extremely expensive when including a large number of antisense species and when considering their moderate success. Here, we make use of a systematic alignment of computer-predicted secondary structures of local sequence stretches of the target RNA and of semi-empirical rules to identify favorable local target sequences and, hence, to design more effective as ODN. The intercellular adhesion molecule 1 (ICAM-1) gene was chosen as a target because it had been shown earlier to be sensitive to antisense-mediated gene suppression. By applying the protocol described here, 10 ICAM-1-directed as ODN species were found that showed substantially improved inhibition of target gene expression in the endothelial cell line ECV304 when compared with the most effective published as ODN. Further, 17 out of 34 antisense species (50%) selected on the theoretical basis described here showed significant (>50%) inhibition of ICAM-1 expression in mammalian cells.     

Synthesis of light-activated antisense oligodeoxynucleotide

The activity of a 20-mer antisense oligodeoxynucleotide (asODN) is transiently blocked by attaching a partially complementary sense strand (sODN) via a heterobifunctional photocleavable linker (PL). The asODN-PL-sODN conjugate forms a DNA hairpin-like structure that is considerably more stable than the corresponding asODN/sODN duplex. In conjugate form, the asODN is prevented from hybridizing to exogenous RNA or DNA molecules. Activity is restored after modest exposure to UV light (lambda approximately 365 nm). Here, we provide a detailed procedure for synthesizing photoactive asODNs in good yields. Synthesis, purification and analysis of the light-activated asODN can be completed within 1-2 weeks.     

Supramolecular assemblies for the cytoplasmic delivery of antisense oligodeoxynucleotide: polyion complex (PIC) micelles based on poly(ethylene glycol)-SS-oligodeoxynucleotide conjugate

A novel cytoplasmic delivery system of antisense oligodeoxynucleotide (asODN) was developed by assembling a PEG-asODN conjugate with disulfide linkage (smart linkage) (PEG-SS-asODN) into polyion complex (PIC) micelles through the complexation with branched poly(ethylenimine) (B-PEI). The PIC micelle thus prepared showed a significant antisense effect against luciferase gene expression in HuH-7 cells, far more efficient than nonmicelle systems (asODN and PEG-SS-asODN in free form) and PIC micelle encapsulating the conjugate without the disulfide linkage. Use of poly(l-lysine) (PLL) instead of the B-PEI for PIC micellization led to a substantial decrease in the antisense effect. These results indicate that the PIC micelles formulated from PEG-SS-asODN conjugate and B-PEI is successfully transported from the endosomal compartment into the cytoplasm by the buffering effect of the B-PEI, releasing hundreds of active asODN molecules via cleavage of the disulfide linkage into the cellular interior, responding to a high glutathione concentration in the cytoplasmic compartment. Furthermore, the type of smart linkage (glutathione-sensitive SS linkage vs pH-sensitive linkage) in the conjugates substantially affected the antisense effect of the PIC micelles, depending on the nature of the counter polycation (B-PEI vs PLL).     

Antisense oligodeoxynucleotide-conjugated hyaluronic acid/protamine nanocomplexes for intracellular gene inhibition

Green fluorescent protein (GFP) antisense oligodeoxynucleotide (ODN) was covalently conjugated to hyaluronic acid (HA) via a reducible disulfide linkage, and the HA-ODN conjugate was complexed with protamine to increase the extent of cellular uptake and enhance the gene inhibition efficiency of GFP expression. The HA-ODN conjugate formed more stable polyelectrolyte complexes with protamine as compared to naked ODN, probably because of its increased charge density. The higher cellular uptake of protamine/HA-ODN complexes than that of protamine/naked ODN complexes was attributed to the formation of more compact nanosized complexes (approximately 200 nm in diameter) in aqueous solution. Protamine/HA-ODN complexes also showed a comparable level of GFP gene inhibition to that of cytotoxic polyethylenimine (PEI)/ODN complexes. Since both HA and protamine are naturally occurring biocompatible materials, the current formulation based on a cleavable conjugation strategy of ODN to HA could be potentially applied as safe and effective nonviral carriers for ODN and siRNA nucleic acid therapeutics.     

Improved intracellular delivery of oligonucleotides by square wave electroporation

Prior studies have shown that electroporation is a simple and effective method for the introduction of oligonucleotides (ODN) into cells. In ex vivo bone marrow purging models, electroporation of ODN into cells has been associated with selective killing of human neoplastic cells while sparing hematopoietic stem cells. Prior studies used conventional electroporation methods (i.e., exponential decay) to introduce ODN into cells. Square wave electroporation allows the delivery of a more defined and regulated electrical pulse and is associated with high transfection efficiencies in a variety of systems. The current study was undertaken to determine whether square wave electroporation was more effective than exponential decay electroporation for the delivery of ODN into hematopoietic cells. Using fluorescein-tagged ODN and K562, chronic myelogenous leukemia (CML) cells, higher transfection rates were observed after square wave electroporation. In addition, c-myc antisense ODN were more effective in reducing c-myc protein when introduced by square wave electroporation, as compared with introduction by exponential decay electroporation. Square wave electroporation is thus identified as the optimal method for delivering ODN into hematopoietic cells.