Design of nuclease resistant protein kinase calpha DNA enzymes with potential therapeutic application

For the therapeutic application of catalytic nucleic acids it is desirable to have small, stable and inexpensive compounds that are active at physiological Mg(2+) concentrations. We have explored the possibility of using the versatile 10-23 DNA catalytic core to suppress the expression of the protein kinase Calpha (PKCalpha) isoform in malignant cells. By introducing either a 3'-3'-inverted thymidine nucleotide or site-specific phosphorothioate modification into a PKCalpha DNA enzyme, we have designed stable catalysts that retained a significant in vitro cleavage activity. In particular, a DNA enzyme containing phosphorothioate analogues in the antisense arms and in the pyrimidine residues of the catalytic core was found to be remarkably stable in 50 % human serum (t(1/2)>90 hours) and inhibited in vitro cell growth by up to 90 % at nanomolar concentrations. The inhibition of PKCalpha gene expression is sequence-specific, as a DNA enzyme with reversed antisense arms was found to be ineffective. Epifluorescence microscopic analysis of cells transfected with a 5' fluorescein isothiocyanate-conjugated DNA enzyme showed that the DNA enzyme molecules are mainly localised in the nuclei. Most of the DNA enzyme-treated cells were killed by apoptosis. The ability of the described PKCalpha DNA enzymes to trigger apoptosis (apoptozymes) in malignant cells illustrates their therapeutic potential. Furthermore, such agents can be a valuable tool for probing gene function.     

3'-End conjugates of minimally phosphorothioate-protected oligonucleotides with 1-O-hexadecylglycerol: synthesis and anti-ras activity in radiation-resistant cells

Activation of the ras oncogene has been implicated in many types of human tumors. It has been shown that downmodulation of ras expression can lead to the reversion of the transformed phenotype of these tumor cells. Antisense oligodeoxyribonucleotides (ODNs) can inhibit gene expression by hybridization to complementary mRNA sequences. To minimize toxicity associated with all-phosphorothioated ODNs and improve cellular uptake, we used partially phosphorothioate (PPS)-modified ODNs having an additional hydrophobic tail at the 3'-end (PPS-C(16)). The PPS ODNs are protected against degradation by PS internucleotide linkages at both the 3'- and 5'-ends and additionally stabilized at internal pyrimidine sites, which are the major sites of endonuclease cleavage. Here we show that anti-ras PPS-C(16) ODN retains the high sequence-specificity of PPS ODNs and provides maximal inhibition of Ras p21 synthesis with minimal toxicity even without the use of a cellular uptake enhancer. Moreover, treatment of T24, a radiation-resistant human tumor cell line that carries a mutant ras gene, with anti-ras PPS-C(16) ODN resulted in a reduction in the radiation resistance of the cells in vitro. We also demonstrate that the growth of RS504 (a human c-Ha-ras transformed NIH/3T3 cell line) mouse tumors was significantly inhibited by the combination of intratumoral injection of anti-ras PPS-C(16) ODN and radiation treatment. These findings indicate the potential of this combination of antisense and conventional radiation therapy as a highly effective cancer treatment modality.     

Bcr3/abl2和VEGF基因反义寡核苷酸联用增强对K562细胞的生长抑制作用

目的:研究BCRABL和VEGF反义寡核苷酸联用对K562细胞株的作用及其相互作用的影响。方法:设计针对bcr3/abl2和VEGF的反义寡核苷酸(ASODNs),应用脂质体Oligofectamine作为转染载体。在转染后72h进行台盼蓝染色细胞计数;建立裸鼠K562移植瘤动物模型,瘤内注射ASODNs,观察肿瘤体积生长变化,组织学检测肿瘤血管密度和肿瘤细胞凋亡情况。结果:转染后72h,各实验组与空白组相比,细胞增殖抑制率分别为13.47%(ASOB3/A2组),12.79%(ASOVEGF组)和41.55%(半量联合治疗组)。经过4次治疗后,与对照组相比,肿瘤生长抑制率分别为23.18%(ASOB3/A2组),17.28%(ASOVEGF组)和57.83%(半量联合治疗组)。联合治疗组肿瘤生长速率显著低于单一治疗组,伴随明显的肿瘤细胞凋亡增加和肿瘤血管密度减少。结论:双基因反义寡核苷酸联合应用协同抑制K562细胞增殖,抗肿瘤作用明显优于单一治疗组,可为CML基因治疗提供一项新策略。     

Enhanced activity of an antisense oligonucleotide targeting murine protein kinase C-alpha by the incorporation of 2'-O-propyl modifications.

We have previously described the characterization of a 20mer phosphorothioate oligodeoxynucleotide (ISIS 4189) which inhibits murine protein kinase C-alpha (PKC-alpha) gene expression, both in vitro and in vivo. In an effort to increase the antisense activity of this oligonucleotide, 2'-O-propyl modifications have been incorporated into the 5'- and 3'-ends of the oligonucleotide, with the eight central bases left as phosphorothioate oligodeoxynucleotides. Hybridization analysis demonstrated that these modifications increased affinity by approximately 8 and 6 degrees C per oligonucleotide for the phosphodiester (ISIS 7815) and phosphorothioate (ISIS 7817) respectively when hybridized to an RNA complement. In addition, 2'-O-propyl incorporation greatly enhanced the nuclease resistance of the oligonucleotides to snake venom phosphodiesterase or intracellular nucleases in vivo. The increase in affinity and nuclease stability of ISIS 7817 resulted in a 5-fold increase in the ability of the oligonucleotide to inhibit PKC-alpha gene expression in murine C127 cells, as compared with the parent phosphorothioate oligodeoxynucleotide. Thus an RNase H-dependent phosphorothioate oligodeoxynucleotide can be modified as a 2'-O-propyl 'chimeric' oligonucleotide to provide a significant increase in antisense activity in cell culture.     

Immunization with murine breast cancer cells treated with antisense oligodeoxynucleotides to type I insulin-like growth factor receptor induced an antitumoral effect mediated by a CD8+ response involving Fas/Fas ligand cytotoxic pathway

We have demonstrated that in vivo administration of phosphorothioate antisense oligodeoxynucleotides (AS[S]ODNs) to type I insulin-like growth factor receptor (IGF-IR) mRNA resulted in inhibition of C4HD breast cancer growth in BALB/c mice. The present study focused on whether in vivo administration of C4HD tumor cells pretreated with IGF-IR AS[S]ODN and irradiated could provide protection against C4HD wild-type tumor challenge and also on elucidating the mechanism mediating this effect. Our results showed that mice immunized with IGF-IR AS[S]ODN-treated C4HD cells experienced a growth inhibition of 53.4%, 61.6%, and 60.2% when compared with PBS-treated mice, wild-type C4HD cell-injected mice, or phosphorothioate sense oligodeoxynucleotide-treated C4HD cell-injected mice, respectively. The protective effect was C4HD-specific, because no cross-protection was observed against other syngeneic mammary tumor lines. The lack of protection against tumor formation in nude mice indicated that T cells were involved in the antitumoral response. Furthermore, cytotoxicity and splenocyte proliferation assays demonstrated that a cellular CD8(+)-dependent immune response, acting through the Fas/Fas ligand death pathway, could be mediating the antitumor effect induced by immunization with AS[S]ODN-treated cells. Immunization also induced splenocytes to produce Ag-dependent IFN-gamma, indicating the presence of a type 1 response. We demonstrated for the first time that IGF-IR AS[S]ODN treatment of breast cancer cells induced expression of CD86 and heat shock protein 70 molecules, both involved in the induction of the immunogenic phenotype. Immunization with these tumor immunogens imparted protection against parental tumor growth through activation of a specific immune response.     

Potent and selective gene inhibition using antisense oligodeoxynucleotides

The development of antisense technology as a generally useful tool relies on the use of potent agents and the utilization of many controls in experiments. Here we describe our experience using oligodeoxynucleotides (ODNs) containing C-5 propynyl pyrimidine and phosphorothioate modifications as broadly applicable gene inhibition agents in cell culture. Methods include selection of antisense sequences, synthesis and purification of ODNs, choice of controls, delivery methods (microinjection, cationic lipid transfection, and electroporation), and analysis of gene inhibition.     

Short modified antisense oligonucleotides directed against Ha-ras point mutation induce selective cleavage of the mRNA and inhibit T24 cells proliferation.

We have used derivatized antisense oligodeoxynucleotides both in vitro and in vivo specifically to inhibit translation of the activated human oncogene Ha-ras. The oligonucleotides (5'-CCACACCGA-3') were targeted to a region of Ha-ras mRNA including the point mutation G----T at the 12th codon which leads to a Gly----Val substitution in the ras p21 protein. They were linked to an intercalating agent and/or to a hydrophobic tail, both to increase their affinity for their mRNA target and to enhance their uptake by tumor cells. A cell-free translation system was used to demonstrate an RNase H-dependent specific inhibition of activated ras protein synthesis. 50% inhibition was observed at a concentration of 0.5 microM of the most efficient oligonucleotide (5'-substitution with an acridine derivative and 3'-substitution by a dodecanol chain). This inhibitory effect stems from a point mutation-sensitive cleavage of the mRNA and it mirrors the growth inhibition obtained with T24 bladder carcinoma cells, which carry activated Ha-ras. The proliferation of HBL100 cells (non tumorigenic human mammary cell line) which carry two copies of normal Ha-ras was unaffected. This study shows that it is possible to design antisense agents that will inactivate the mutated oncogene but not the protooncogene which is generally essential to cell survival.     

Oligodeoxynucleotides enhance lipopolysaccharide-stimulated synthesis of tumor necrosis factor: dependence on phosphorothioate modification and reversal by heparin.

BACKGROUND: Specific inhibition of target proteins by antisense oligodeoxynucleotides is an extensively studied experimental approach. This technique is currently being tested in clinical trials applying phosphorothioate-modified oligonucleotides as therapeutic agents. These polyanionic molecules, however, may also exert non-antisense-mediated effects. MATERIALS AND METHODS: We examined the influence of oligonucleotides on lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNF alpha) synthesis in freshly isolated human peripheral blood mononuclear cells. Oligonucleotides (18 mer) with different degrees of phosphorothioate modification were studied. RESULTS: The addition of phosphorothioate oligonucleotides (5 microM) caused amplification of TNF synthesis of up to 410% compared with the control with LPS alone. Without LPS stimulation, phosphorothioate oligonucleotides did not induce TNF production. We demonstrate that the enhancement of LPS-stimulated TNF production by phosphorothioate oligonucleotides does not rely on the intracellular presence of oligonucleotides and is not mediated by LPS contamination. Partially phosphorothioate-modified oligonucleotides and unmodified oligonucleotides did not increase TNF synthesis. High concentrations of the polyanion heparin reversed the oligonucleotide-induced enhancement of TNF synthesis. CONCLUSIONS: The data suggest that amplification of TNF synthesis may be caused by binding of the polyanionic phosphorothioate oligonucleotide to cationic sites on the cell surface. Such binding sites have been proposed for polyanionic glycoaminoglycans of the extracellular matrix, which have also been described to augment LPS-stimulated TNF synthesis. The present results are relevant to all in vitro studies attempting to influence protein synthesis in monocytes by using phosphorothioate oligonucleotides. The significance of our findings for in vivo applications of phosphorothioates in situations where there is a stimulus for TNF synthesis, such as in sepsis, should be elucidated.     

Targeting host E-selectin expression by antisense oligodeoxynucleotides as potential antiendotoxin therapy in vivo

This study sought to determine if antisense oligodeoxynucleotides would inhibit E-selectin expression, which mediates leukocyte adhesion on endothelial cells, otherwise induced by in vivo endotoxin challenge. Six antisense phosphorothioate oligodeoxynucleotides calculated to bind porcine E-selectin mRNA were tested in porcine aortic endothelial cells. One, ISIS9481, exerted significant inhibition of E-selectin expression induced by tumor necrosis factor-α?+?endotoxin [lipopolysaccharide (LPS)]. Pigs were challenged with LPS (10?μg/kg) and treated with ISIS9481 (10?mg/kg) (n?=?6). Two control groups were used, an antisense inactive in porcine aortic endothelial cells (n?=?6) and saline (n?=?5), and were combined as control (C?=?11). Control pigs challenged with LPS expressed E-selectin in heart, lung, kidneys, and liver, whereas antisense-treated pigs expressed little E-selectin in these tissues. Cardiovascular data indicated that antisense treatment attenuated pathophysiological alterations induced by LPS. Specifically, in control pigs, LPS reduced cardiac output 32% from baseline, increased pulmonary (+116%) and systemic vascular resistances (+16%), and generated neutropenia (from 51,000 at basal to 18,000 polymorphonuclear neutrophils (PMN)/μL after LPS). In antisense-treated pigs, cardiac output decreased only 18%, pulmonary vascular resistance remained unchanged, whereas systemic vascular resistance decreased compared with basal values (-37%). PMN counts remained at 45,000-54,000/μL at 3-4 hours after LPS. These data demonstrate that antisense oligodeoxynucleotides, designed and tested in vitro to interact with 1 gene product, can be developed as either therapeutics or experimental tools in vivo.     

In Vivo Delivery of Antisense Oligodeoxynucleotides into Rat Kupffer Cells

Abstract

Kupffer cells play a key role in the pathogenesis of liver diseases. Liver injury is believed to result from an excessive release of cytokines and prostanoids from these cells. A targeted delivery of antisense oligonucleotides into Kupffer cells might reduce or prevent liver injury. In this report, we describe a method in which anionic liposome-encapsulated antisense phosphorothioate oligodeoxynucleotides (S-Oligos) are delivered to Kupffer cells in vivo. Delivery was assessed using an antisense S-Oligo (TJU-2749) targeted against the 3’ untranslated region of rat tumor necrosis factor-α mRNA. At 90 min post-intravenous injection, 90% of the S-Oligo was absorbed from circulation. Of this, 40% was found in the liver and 10% in spleen. Other organs, including lungs, kidneys, muscle, stomach, brain, testes and small intestine, showed only minor incorporation (<5%). Greater than 65% of the liver-associated S-Oligo was found in Kupffer cells. Relative accumulation of S-Oligo in Kupffer cells was 200-fold that of the combined body tissues. For an average injected dose of 1.2 mg antisense/Kg body weight, the intracellular concentration of the S-Oligo attained in Kupffer cells was 65 μM. These studies suggest that liposome-encapsulated delivery provides an efficient means of targeting antisense molecules to Kupffer cells in vivo.     

Antisense phosphorothioate oligodeoxynucleotides targeted to the vpr gene inhibit human immunodeficiency virus type 1 replication in primary human macrophages.

The replication of human immunodeficiency viruses (HIV) in human macrophages is influenced by genetic determinants which have been mapped predominantly to the viral envelope. However, in HIV-2, the vpr gene has also been suggested as an important modulator of viral expression in human macrophages. We synthesized five antisense phosphorothioate oligodeoxynucleotides complementary to the vpr mRNA of HIV-1Ba-L, a highly macrophage-tropic viral strain, and measured their effect on HIV-1Ba-L replication in primary human macrophages. All of the oligodeoxynucleotides displayed some level of non-sequence-specific inhibition of viral replication; however, only the antisense one had an additional effect on viral production in primary macrophages. Of the five antisense oligodeoxynucleotides tested, only one did not show any additional effect on viral production, whereas all the others inhibited viral replication to a similar degree (70 to 100%). Variation in the degree of inhibition was observed by using five different donors of human primary macrophages. The phosphorothioate oligonucleotides, targeted to the initiating methionine of the Vpr protein, had an inhibitory effect at both 20 and 10 microM only when the size was increased from 24 to 27 bases. Thus, HIV-1 replication in human macrophages is modulated by the expression of the vpr gene, and it is conceivable that vpr antisense oligodeoxynucleotides could be used in combination with antisense oligodeoxynucleotides against other HIV-1 regulatory genes to better control viral expression in human macrophages.     

Translation efficiency of zein mRNA is reduced by hybrid formation between the 5'- and 3'-untranslated region

The secondary structure of zein mRNA affects its translation potential. Here we show that in a cell-free system the translation efficiency of zein mRNA containing inverted repeats in the 5'- and 3'-untranslated regions is reduced. This translational block is released after deletion of the 3'-inverted repeat. We conclude that the translational block is caused by hybrid formation between the two inverted repeats. The translational efficiency of zein mRNAs, is also affected by varying the length or the primary structure of the 5'-untranslated region.     

Midkine binds to 37-kDa laminin binding protein precursor, leading to nuclear transport of the complex

Midkine (MK) is a heparin binding multifunctional protein that promotes cell survival and cell migration. MK was found to bind to 37-kDa laminin binding protein precursor (LBP), a precursor of 67-kDa laminin receptor, with K(d) of 1.1 nM between MK and LBP-glutathione-S-transferase fusion protein. The binding was inhibited by laminin, anti-LBP, amyloid beta-peptide, and heparin; the latter two are known to bind to MK. In CMT-93 mouse rectal carcinoma cells, LBP was mostly located in the cytoplasm as revealed by immunostaining with anti-LBP antibody. That a portion of LBP or 67-kDa laminin receptor was located at the surface of these cells was verified by inhibition of cell attachment to laminin-coated dishes by anti-LBP antibody. When MK was added to culture medium of these cells, a part of LBP migrated to the nucleus. The movement occurred concomitantly with nuclear transport of biotin-labeled MK. These findings suggested that the binding of MK to LBP caused nuclear translocation of the molecular complex.     

Sequence confirmation of synthetic phosphorothioate oligodeoxynucleotides using Sanger sequencing reactions in combination with mass spectrometry

A protocol relying on Sanger sequencing reactions in combination with mass spectrometry (MS) for sequence confirmation of antisense phosphorothioate oligodeoxynucleotides is described. In this procedure, synthetic phosphorothioate oligodeoxynucleotides are used as reverse primers for extension of matched templates with enough length (approximately 150-300 bp) for well-established Sanger sequencing. Because the complementary strand of modified primer is used directly for sequencing primer extension, the base order shown in the sequencing result is reversely complementary to phosphorothioate oligodeoxynucleotide. This sequencing method can be applied not only to phosphorothioate oligodeoxynucleotides with different lengths (13-21 mer) and base composition but also to sequences with bases' switch, deletion, or insertion. In addition, modified primers incorporate the 5' end of polymerase chain reaction (PCR) products conveying the characters of phosphorothioate modification. The method requires only common reagents and instruments and so is better suited to routine sequence analysis in quality control of phosphorothioate antisense drugs.     

Intracellular availability of unmodified, phosphorothioated and liposomally encapsulated oligodeoxynucleotides for antisense activity.

We have studied factors which may effect the intracellular availability of oligonucleotides to achieve antisense activity. 15-20 mer unmodified, phosphorothioate modified and liposomally encapsulated oligodeoxynucleotides have been tested in leukemia MOLT-3 cells. Phosphorothioate analogs penetrated and accumulated intact in cells in contrast to unmodified oligomers, which showed a high instability in cell culture medium. A slow decrease of intracellular concentration of undegraded phosphorothioate oligodeoxynucleotides was observed after cell treatment and could be predominantly explained by a significant efflux transport. Using laser-assisted confocal microscopy we have observed that fluorescein 5-end-labeled phosphorothioate derivatives predominantly distributed in intracytoplasmic endocytic vesicles following cell treatment. The end-capped version of phosphorothioate oligodeoxynucleotides exhibited greater cellular uptake than fully modified analogues while exhibiting similar biological stability. Liposome encapsulation made possible oligomer protection in serum-containing medium and substantially improved cellular accumulation. Furthermore, the efflux rate of oligomer initially introduced within liposomes is 2-fold lower than that observed in cells which have been incubated with free oligonucleotides. Liposomal preparations of oligodeoxynucleotides facilitate release from endocytic vesicles, and thus, cytoplasmic and nuclear localization are observed following cell treatment. Furthermore, intracellular distribution studies demonstrate that intracellular transport of unmodified oligomers is effectively achieved using the liposomal carrier.     

Effects of β1-integrin antisense phosphorothioate-modified oligonucleotide on myoblast behaviour in vitro

Myoblasts gene-engineered in vitro and then injected in vivo are safe, efficient options for gene therapy. While isolation of satellite cells is routinely achieved, their proliferation potential in vitro remains a limiting factor for cell transplantation under clinical conditions. We have studied the role of reversible inhibition of gene expression by antisense oligonucleotides on the proliferation of the myogenic cells. Addition of antisense oligonucleotides to myoblast cultures has been used to inhibit specifically the expression of the β1-integrin subunit gene. Here we show that the effects of multiple pulses of a phosphorothioate oligodeoxinucleotide antisense on the attachment to substrata and on the proliferation of myoblasts are dose-dependent. The addition of antisense to rat myoblasts caused rounding up of the cells and most of the cells became detached after several days in culture. A single pulse did not show any consistent effect, while in the presence of continously administered antisense, the relative numbers of myoblasts in the treated muscle culture increased. We have no evidence of inhibition of myoblast fusion under these conditions. On the other hand, [³H]-TdR incorporation, total DNA and total number of cells decreased in antisense-treated cultures thus demonstrating an inhibitory effect of the phosphorothioate oligonucleotides on DNA synthesis. These side-effects could be overcome by substituting the phosphorothioate by unmodified oligonucleotides, so decreasing the half-life of the antisense, but also its toxicity. The overall results suggest a potential role of integrin antisense strategy in modulating the potential of myoblasts to proliferate.     

Inhibition of the androgen receptor by antisense oligonucleotides regulates the biological activity of androgens in SZ95 sebocytes.

One novel strategy for the blockade of the androgen receptor could be the selective inhibition of androgen receptor by antisense oligonucleotides or small interfering RNA molecules. Here we describe the down regulation of the androgen receptor in cultured human SZ95 sebocytes with antisense oligonucleotides modified with phosphorothioates and 2'- O-methylribosyl residues. The ability of antisense oligonucleotides to cross the cellular membrane was enhanced by establishing a transient transfection system based on cationic lipid vesicles. Both antisense oligonucleotide types administered caused assumedly translational arrest. Dose-dependent inhibition of androgen receptor protein expression was observed after SZ95 sebocyte transfection with modified phosphorothioate oligonucleotides and modified 2'- O-methylribonucleotides which were directed against the translational start of the androgen receptor mRNA. The strongest transient inhibition of androgen receptor expression was detected after 14 hours with 1.0 muM antisense 2'- O-methylribonucleotides (88+/-1.3%, p<0.001). With longer recovery times than 24 hours, androgen receptor protein expression returned to the native control levels. Inhibition of the expression of androgen receptor by antisense oligonucleotides, reduced the enhanced proliferation of SZ95 sebocytes challenged by testosterone and 5alpha-dihydrotestosterone. This administration opens new therapeutic possibilities in androgen-associated skin diseases, since we could also show androgen inhibition with these antisense oligonucleotides in a reconstituted human epidermis model (Horm Metab Res 2007; 39:157-165).