THE USE OF (OLIGONUCLEOTIDE-MALEIC ANHYDRIDE COPOLYMER) CONJUGATES IN NUCLEIC ACID DIAGNOSTIC ASSAYS: EFFECT OF THE NUMBER OF OLIGONUCLEOTIDES PER POLYMER ON TEST SENSITIVITY IMPROVEMENT

(Polymer-oligonucleotide) conjugates were obtained via direct ON synthesis from the poly(ethylene-alt-maleic anhydride: PEMA) grafted onto glass bead surfaces. The effect of the ON number per polymer chain on binding was evaluated through Tm experiments. Results were correlated with ELOSA (Enzyme Linked Oligosorbent Assay) tests run with conjugates in the capture step.     

Cholesterol conjugated oligonucleotide and LNA: A comparison of cellular and nuclear uptake by Hep2 cells enhanced by Streptolysin-O

Antisense and antigene oligonucleotides (ONs) are attractive drugs for gene therapy, but major limiting factors for their routine use are inefficient cellular uptake and low accessibility to the target sites. Adding various lipophilic conjugates to the ON improves intracellular delivery as has been previously reported.We studied the cellular delivery of various ON modifications, as well as their cytosolic and nuclear distribution in mammalian Hep2-EGFP-NLS cell line. We compared uptake efficacy of ON and LNA, both conjugated with cholesterol at the 5′ end. All ONs were 3′ labeled with fluorescent Cy5 dye. We made a comparison of the ONs uptake efficacy and the kinetics, both adding ONs to the culture medium, and using streptolysin-O (SL-O) permeabilization.The cellular uptake of each ON used in this study was visualized by fluorescent microscopy. We confirmed the results by FACS analysis. We determined the ratio between initial ON-chol concentration (0.4 μM) and the final amount in nucleus.SL-O can highly improve kinetics of ON delivery; not only into the cytoplasm but also to the nucleus, the presumed site of antigene ON action. The most effective nuclear uptake was observed when ON conjugated with cholesterol (ON-chol) and SL-O was used. Nuclear distribution of ON was reached within few minutes. In contrast, ON simply added to the medium reached cytoplasm only and the process of delivery took several hours. (Mol Cell Biochem 276: 61–69, 2005)     

Arginine-rich cell penetrating peptides: design, structure-activity, and applications to alter pre-mRNA splicing by steric-block oligonucleotides.

Rerouting the splicing machinery with steric-block oligonucleotides (ON) might lead to new therapeutic strategies in the treatment of diseases such as beta-thalassemia, Duchenne muscular dystrophy, or cancers. Interfering with splicing requires the sequence-specific and stable hybridization of RNase H-incompetent ON as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligomers (PMO). Unfortunately, these uncharged DNA mimics are poorly taken up by most cell types and conventional delivery strategies that rely on electrostatic interaction do not apply. Likewise, conjugation to cell penetrating peptides (CPPs) as Tat, Arg9, Lys8, or Pen leads to poor splicing correction efficiency at low concentration essentially because PNA- and PMO-CPP conjugates remain entrapped within endocytotic vesicles. Recently, we have designed an arginine-rich peptide (R-Ahx-R)4 (with Ahx for aminohexanoic acid) and an arginine-tailed Penetratin derivative which allow sequence-specific and efficient splicing correction at low concentration in the absence of endosomolytic agents. Both CPPs are undergoing structure-activity relationship studies for further optimization as steric-block ON delivery vectors.     

LNA guanine and 2,6-diaminopurine. Synthesis, characterization and hybridization properties of LNA 2,6-diaminopurine containing oligonucleotides

LNA guanine and 2,6-diaminopurine (D) phosphoramidites have been synthesized as building blocks for antisense oligonucleotides (ON). The effects of incorporating LNA D into ON were investigated. As expected, LNA D containing ON showed increased affinity towards complementary DNA (Delta Tm +1.6 to +3.0 degrees C) and RNA (Delta Tm +2.6 to +4.6 degrees C) ON. To evaluate if LNA D containing ON have an enhanced mismatch sensitivity compared to their complementary LNA A containing ON thermal denaturation experiments towards singly mismatched DNA and RNA ON were undertaken. Replacing one LNA A residue with LNA D, in fully LNA modified ON, resulted in higher mismatch sensitivity towards DNA ON (Delta Delta Tm -4 to >-17 degrees C). The same trend was observed towards singly mismatched RNA ON (Delta Delta Tm D-a = -8.7 degrees C and D-g = -4.5 degrees C) however, the effect was less clearcut and LNA A showed a better mismatch sensitivity than LNA D towards cytosine (Delta Tm +5.5 degrees C).     

Expression of PirB Protein in Intact and Injured Optic Nerve and Retina of Mice

The aim of this study was to investigate the expression of PirB protein in intact mice ON (optic nerve) and retina, and to evaluate its change after ON injury. The mouse ON crush model was established. The immunohistochemistry and western blot were used to detect PirB expression. We discovered PirB signals were located as beaded arrangement along the ON long axis in intact ON, disordered in injured ON, and distributed mainly in ganglion cell layer in intact and injured retina. Both PirB expression in injured ON and retina were significantly increased at 1-day post injury (1-dpi), nearly peaked at 7-dpi, but thereafter there was no significant change of them till at least 28-dpi. We concluded the expression of PirB was positive in intact ON and retina, and significantly increased after ON injury. These findings, coupled with previous studies, may imply that PirB is probably a critical molecule in inhibition of axonal regeneration by myelin inhibitors after ON injury.     

Immunohistochemical Localization of Intermediate Filament Proteins of Neuronal and Nonneuronal Origin in the Goldfish Optic Nerve: Specific Molecular Markers for Optic Nerve Structures

The predominant proteins (58K) of the intermediate filament complex in the goldfish visual pathway consist of a series of isoelectric variants. Previous biochemical studies have shown that proteins ON1 and ON2 are of neuronal origin, whereas ON3 and ON4 are of nonneuronal origin. Polyclonal antibodies, purified by affinity chromatography, that are specific for ON1 and ON2 or ON3 and ON4 have been used to localize histologically the ON proteins within the normal and crushed optic nerve. Anti-ON1/ON2 antiserum presented a pattern consistent with intraaxonal staining. A nonneuronal staining pattern was observed with anti-ON3/ON4 antiserum. The two patterns were distinct from and complementary to each other. The data suggest that ON3 and ON4 represent a novel glial fibrillary acidic protein. The results are discussed in terms of the function of these proteins in development, plasticity, and regeneration.     

Endogenous oligonucleotides of human milk and their possible biological function.

Oligonucleotides (ON) 4 to 60 nucleotides in length were isolated by ion-exchange chromatography on a column with Fractogel TSK DEAE-650 (M) from human milk which was hydrolyzed with proteinase K. ON from 60 to 16 nucleotides were degraded by RNase A but were resistant to DNase I, and, thus, they were ribooligonucleotides. In the presence of [gamma-32P]ATP, ON and heparin inhibited the phosphorylation of 38- and 20-kD milk proteins and failed to affect the phosphorylation of a 76-kD protein. Human milk is believed to contain polyanion-dependent and polyanion-independent protein kinases. The influence of the ON on the activity of the cytotoxic fraction of human milk alpha-lactalbumin towards human mammary gland carcinoma MCF-7 cells was studied. The ON inhibited the cytostatic and cytotoxic effects of alpha-lactalbumin. Synthetic oligonucleotides and heparin had similar effects. The endogenous ON are suggested to be involved in the regulation of cytotoxic activity of human milk.     

Change of Th17 Lymphocytes and Treg/Th17 in Typical and Atypical Optic Neuritis

Background

Typical and atypical optic neuritis (ON) are two clinical types of autoimmune inflammatory diseases of the optic nerve that causes acute vision loss, and are difficult to distinguish in their early stages. The disturbance in the balance of Th17 and Treg lymphocytes is thought to play an essential role in these autoimmune inflammatory diseases.

Objectives

To detect the clinical relevance of Th17 and Treg in peripheral blood and the ratio of Treg/Th17 in patients with typical and atypical ON. To determine whether analysis of Th17 and Treg lymphocytes will provides insights into the different disease phenotypes of typical and atypical ON.

Methods

We studied a consecutive series of patients aged 14–70 years who presented to our neurological department with typical ON (n = 30) or atypical ON (n = 33) within 4 weeks of their acute attacks. Routine clinical tests and ophthalmological examination were performed in all patients. Blood samples were collected from untreated patients and from gender- and age-matched healthy controls (n = 30). The proportion of peripheral blood Th17 cells and Treg cells was determined by flow cytometry.

Results

Patients with atypical ON had a higher proportion of Th17 cells than patients with typical ON (3.61±1.56 vs 2.55±1.74, P<0.01) or controls (1.45±0.86, P<0.01). The proportion of Th17 cells in patients with typical ON was also markedly higher than in controls (P<0.01). The mean percentage of Treg cells in atypical ON (6.31±2.11) and typical ON (6.80±2.00) were significantly lower when compared to controls (8.29±2.32, both P<0.01). No significant difference in Treg frequency was observed between typical ON and atypical ON (p>0.05).

Conclusions

The frequency of Th17 cells is higher in atypical ON than typical ON, and patients with atypical ON have a greater imbalance of pro-inflammatory and regulatory cells than patients with typical ON when compared with controls. These changes are indicative of distinct pathological mechanisms and may provide useful information to distinguish typical and atypical ON.     

Long-term release and improved intracellular penetration of oligonucleotide-polyethylenimine complexes entrapped in biodegradable microspheres

The aim of this work was to design a biodegradable delivery system for oligonucleotides providing both a sustained release and an improved intracellular penetration. To this purpose oligonucleotide/polyethylenimine (ON/PEI) complexes at nitrogen to phosphate (N/P) molar ratios of about 15 or 40 were encapsulated into poly(lactide-co-glycolide) microspheres by the multiple emulsion-solvent evaporation technique. ON/PEI complexes were efficiently entrapped inside microspheres. The introduction of salts within the external aqueous phase allowed an improvement of microsphere characteristics. In particular, the use of sodium chloride led to a reduced microsphere porosity and a more homogeneous ON distribution inside the polymeric matrix. These effects were attributed to the reduced flux of water from the external aqueous phase toward the internal aqueous droplets, due to the osmotic effect of sodium chloride. Both, the reduced porosity and the improved ON distribution inside the matrix, were considered responsible for the lower burst effect and the slower ON release rate from microsphere prepared with sodium chloride. ON/PEI complexes encapsulated inside microspheres were also protected toward enzymatic degradation in fetal calf serum. Interestingly, ON/PEI complexes slowly released from microspheres efficiently penetrated inside HeLa cells and oligonucleotides were preferentially located in the nucleus.     

Potentiation of the antitumor effect of tamoxifen by combination with the antiprogestin onapristone

The present study was undertaken to investigate the antitumor effect of a combination of the antiestrogen tamoxifen (TAM) and either the antiprogestin onapristone (ON) or the progestogen megestrol acetate (MEG) in experimental mammary tumor models. Rats bearing DMBA- or NMU-induced mammary tumors were treated with ON or MEG either alone or in combination with TAM for four weeks. In the DMBA-tumor model, treatment with ON or TAM alone caused tumor remissions, whereas the combination of ON and TAM was almost as effective as ovariectomy (100% remission) and led to a remission of 86–100%. The combination of TAM and ON was distinctly more effective than that of TAM and MEG. A similar potentiation of the antitumor effect of TAM and ON was observed in the NMU-tumor model. In DMBA-tumors, the concentration of progesterone receptors was found to increase after treatment for three days with TAM and ON. In hosts bearing DMBA-tumors, treatment with the combination of TAM and ON caused a reduction in ovarian and uterine weights. In the same animals, the basal level of progesterone was decreased in spite of a slight increase in the LH level. These findings suggest that the high antitumor effect of the combination of TAM and ON compared to the corresponding monotherapies can be related not only to the interaction of antihormones and receptors, but also to the up-regulation of PR and to a decrease in progesterone production. These data clearly suggest the sense of a combination of TAM with an antiprogestin in breast cancer treatment.     

LHRH neuronal migration: heterotypic transplantation analysis of guidance cues

During embryonic development, the olfactory placode (OP) differentiates into the olfactory epithelium (OE). Luteinizing hormone-releasing hormone (LHRH) neurons migrate out of the OE in close association with the olfactory nerve (ON) to the telencephalon. LHRH neuronal migration and ON extension to the telencephalon may be independent events which are correlated but do not represent a causal relationship. However, we hypothesize that LHRH neurons are dependent on ON axons to migrate to the brain. To test this hypothesis, we ablated the right trigeminal placode and replaced it with an OP from another chick embryo. After several days' additional incubation, the embryos were fixed, sectioned, and immunostained with antibodies against LHRH or N-CAM. The ectopic OPs were well integrated into the host and developed into relatively normal appearing OEs. The ONs extended from the OE to several different sites: the lateral rectus of the eye, the ciliary ganglion, and the trigeminal ganglion. In all cases, LHRH neurons were found in the OE and ON, regardless of where the ON terminated. When the ON extended to the trigeminal ganglion, LHRH neurons could clearly be seen entering the metencephalon. Our results support the idea that LHRH neurons are dependent on the ON for guidance as they appear to follow the nerve even when it extends away from the brain. The cues which direct the ON and LHRH neurons to the telencephalon do not appear to be unique to this brain region.     

Kinetics of intramembrane charge movement and conductance activation of batrachotoxin-modified sodium channels in frog node of Ranvier

Sodium current and intramembrane gating charge movement (Q) were monitored in voltage-clamped frog node of Ranvier after modification of all sodium channels by batrachotoxin (BTX). Sodium current activation followed a single-exponential time course, provided a delay was interposed between the onset of the step ON depolarization and that of the current change. The delay decreased with increased ON depolarization and, for a constant ON depolarization, increased with prehyperpolarization. ON charge movement followed a single-exponential time course with time constants tau Q,ON slightly larger than tau Na, ON. For pulses between -70 and -50 mV, tau Q,ON/tau Na,ON = 1.14 +/- 0.08. The OFF charge movement and OFF sodium current tails after a depolarizing pulse followed single-exponential time courses, with tau Q, OFF larger than tau Na, OFF. tau Q,OFF/tau Na,OFF increased with OFF voltage from 1 near -100 mV to 2 near -160 mV. At a set OFF potential (-120 mV), both tau Q,OFF and tau Na,OFF increased with ON pulse duration. The delay in INa activation and the effect of ON pulse duration on tau Q,OFF and tau Na,OFF are inconsistent with a simple two-state, single-transition model for the gating of batrachotoxin-modified sodium channels.     

Differential effects of intravitreal optic nerve and sciatic nerve grafts on the survival of retinal ganglion cells and the regeneration of their axons

We have investigated the effects of intravitreal sciatic nerve (SN) and/or optic nerve (ON) grafts on the survival and the axonal regeneration of retinal ganglion cells (RGCs). Following transection of the ON, approximately 40% RGCs survived at 7 days post-axotomy (dpa). Results showed that the intravitreal ON graft significantly promoted the survival of RGCs at 7 dpa (39,063 vs 28,246). Intravitreal SN graft, however, did not rescue axotomized RGCs at 5, 7 or 14 dpa. Axotomized RGCs could be induced to regenerate axons along a segment of SN graft attached to the proximal stump of ON. On average, 608 axotomized RGCs were induced to regenerate axons along the attached SN graft. The presence of intravitreal SN graft promoted about 100% increase in the number of regenerating RGCs (1,227) relative to the control groups. The intravitreal ON graft, surprisingly, also induced about 100% more regenerating RGCs (1220) than in the control group. When SN and ON grafts were co-transplanted into the vitreous, about 200% more regenerating RGCs (1916) were observed than in the control group. These findings illustrated that the intravitreal ON graft rescued axotomized RGCs and enhanced the regeneration of retinal axons. This is the first report to show that ON promotes RGC axonal regeneration. The intravitreal SN graft did not rescue RGCs but promoted axonal regeneration. The differential effects of intravitreal ON and SN grafts on the survival and the RGC regeneration suggest that these might be two independently operating events.     

Pigpen is a cellular binding protein of therapeutic oligonucleotides

BACKGROUND: Understanding the mechanism of oligonucleotide (ON) uptake and cellular distribution is important for rational design of ON-based therapeutic strategies. The aim of this study was to investigate the possible relationship between cellular distribution of ON and the protein pigpen. METHODS: In vitro interaction of ON with the protein pigpen was detected using mass spectrometry. Cellular distribution of pigpen and co-localization of pigpen with ON was studied by fluorescence microscopy in endothelial YPEN and microglial N9 cells. RESULTS: Pigpen had similar distribution patterns in endothelial YPEN and microglial N9 cells. Pigpen was localized to the cytoplasm of both cell types. In addition, pigpen distributed to nuclei, excluding the nucleoli, and concentrated along the nuclear membrane and plasma membrane. Intensely stained foci were only observed in the nucleus and cytoplasm of YPEN cells. Although co-localization of pigpen with phosphorothioate (PS) ON was not observed for the first hour after ON uptake, co-localization was observed 8 h later. DISCUSSION: These data suggest that pigpen binds therapeutic ON and thus might contribute to ON cellular distribution.     

Homology and Diversity Between Intermediate Filament Proteins of Neuronal and Nonneuronal Origin in Goldfish Optic Nerve

The predominant intermediate filament proteins of the goldfish optic nerve have molecular weights of 58K. They can be separated into a series of four major isoelectric variants of neuronal (ON1 and ON2) and nonneuronal (ON3 and ON4) origin. The extent of homology between the goldfish 58K intermediate filament proteins themselves and to rat optic nerve vimentin and glial fibrillary acidic protein (GFAP) was investigated. Unlabeled and [32P]orthophosphate-labeled proteins were subjected to partial hydrolysis by V8 protease, chymotrypsin, and CNBr. The results show that the goldfish intermediate filament proteins share with vimentin and GFAP a 40K chymotrypsin-resistant core fragment. Phosphorylated moieties appear to be located outside the core region since they are preferentially cleaved off by chymotrypsin and not found associated with the 40K core. In addition, the goldfish ON proteins contain the antigenic site within the core that is common to most intermediate filaments. V8 or CNBr digestion indicates that many fragments that are common to ON1 and ON2 are clearly distinct from fragments that are common to ON3 and ON4. In addition, structural variability is observed between the goldfish intermediate filament proteins and vimentin and GFAP. The results are discussed in terms of intermediate filament structure and their possible role in nerve growth.     

Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structure-activity studies

Redirecting the splicing machinery through the hybridization of high affinity, RNase H- incompetent oligonucleotide analogs such as phosphoramidate morpholino oligonucleotides (PMO) might lead to important clinical applications. Chemical conjugation of PMO to arginine-rich cell penetrating peptides (CPP) such as (R-Ahx-R)₄ (with Ahx standing for 6-aminohexanoic acid) leads to sequence-specific splicing correction in the absence of endosomolytic agents in cell culture at variance with most conventional CPPs. Importantly, (R-Ahx-R)₄–PMO conjugates are effective in mouse models of various viral infections and Duchenne muscular dystrophy. Unfortunately, active doses in some applications might be close to cytotoxic ones thus presenting challenge for systemic administration of the conjugates in those clinical settings. Structure–activity relationship studies have thus been undertaken to unravel CPP structural features important for the efficient nuclear delivery of the conjugated PMO and limiting steps in their internalization pathway. Affinity for heparin (taken as a model heparan sulfate), hydrophobicity, cellular uptake, intracellular distribution and splicing correction have been monitored. Spacing between the charges, hydrophobicity of the linker between the Arg-groups and Arg-stereochemistry influence splicing correction efficiency. A significant correlation between splicing correction efficiency, affinity for heparin and ability to destabilize model synthetic vesicles has been observed but no correlation with cellular uptake has been found. Efforts will have to focus on endosomal escape since it appears to remain the limiting factor for the delivery of these splice-redirecting ON analogs.     

Involvement of the adrenal glands in the prolactin rise induced in the female rat by an antiprogestin, onapristone

The present study was undertaken to investigate our recent finding that the peripheral levels of prolactin are elevated after the treatment of intact tumor-bearing rats with antiprogestins, like ONAPRISTONE (ON) and MIFEPRISTONE (MI). In ovariectomized rats, s.c. administration of ON (10 mg/kg/day for 5 days) induced a significant increase in the peripheral levels of prolactin without stimulating uterine growth or suppressing LH secretion. Additionally, treatment with ON enhanced the estradiol-induced increase in the serum prolactin levels, suggesting different mechanism(s) for the effects of ON and estradiol on prolactin secretion. In the castrated animals treated with ON we also found a significant increase in the serum levels of aldosterone and corticosterone, but no measurable amount of estradiol and no significant change in the levels of serum androstenedione.

Accordingly, we supposed that the effect of ON on prolactin secretion may be induced by suppression of the known activity of adrenal corticosteroids in inhibiting the prolactin secretion. In a further study using ovariectomized and adrenalectomized rats we, in fact, found no appreciable effect of ON on the serum prolactin levels at all. By contrast, dexamethasone (DEX) (0.15 mg/kg for 5 days, s.c.) significantly decreased the prolactin levels which were elevated after adrenalectomy. This effect of DEX was partially reversed by a simultaneous application of ON. From the present observations, it is anticipated that the increase in the peripheral prolactin levels found after treatment with ON is partly due to the antiglucocorticoid effect of the compound.     

Chemically modified oligonucleotides with efficient RNase H response

Ten different chemically modified nucleosides were incorporated into short DNA strands (chimeric oligonucleotides ON3-ON12 and ON15-ON24) and then tested for their capacity to mediate RNAse H cleavage of the complementary RNA strand. The modifications were placed at two central positions directly in the RNase H cleaving region. The RNA strand of duplexes with ON3, ON5 and ON12 were cleaved more efficiently than the RNA strand of the DNA:RNA control duplex. There seems to be no correlation between the thermal stability between the duplexes and RNase H cleavage.     

Characterization of oligonucleotide/lipid interactions in submicron cationic emulsions: influence of the cationic lipid structure and the presence of PEG-lipids

We have recently described how oligonucleotide (ON) stability and release from O/W cationic emulsions are governed by the lipid composition. The aim of the present paper was to investigate the properties of the ON/lipid complexes through fluorescence resonance energy transfer (FRET), size, surface tension measurements and cryomicroscopy. Starting from a typical emulsion containing stearylamine as a cationic lipid, the influence of the lipid structure (monocationic molecules bearing mono or diacyl chains, or polycations) as well as of the presence of PEGylated lipids, were studied. The presence of a positive charge on the droplet surface clearly contributed to enhance the ON interaction with lipid monolayers and to bring the ON molecules closer to the interface. Hydrophobic interactions through the acyl chains were shown to further enhance the anchorage of the ON/lipid complexes. In contrast, the incorporation of PEGylated lipids acted as a barrier against the establishment of electrostatic bindings, the polyethyleneglycol chains acting themselves as interaction sites for the ON leading to hydrophilic complexes. Similar features were observed for the polycationic lipid, and cryomicroscopy revealed the existence of bridges of various intensities between the droplets of the emulsion containing either PEG or the polycation, probably because of the configuration of the ON at the interface.     

Comparison of basic peptides- and lipid-based strategies for the delivery of splice correcting oligonucleotides

Expression of alternatively spliced mRNA variants at specific stages of development or in specific cells and tissues contributes to the functional diversity of the human genome. Aberrations in alternative splicing were found as a cause or a contributing factor to the development, progression, or maintenance of numerous diseases. The use of antisense oligonucleotides (ON) to modify aberrant expression patterns of alternatively spliced mRNAs is a novel means of potentially controlling such diseases. Oligonucleotides can be designed to repair genetic mutations, to modify genomic sequences in order to compensate for gene deletions, or to modify RNA processing in order to improve the effects of the underlying gene mutation. Steric block ON approach have proven to be effective in experimental model for various diseases. Here, we describe our experience in investigating two strategies for ON delivery: ON conjugation with basic peptides and lipid-based particulate system (lipoplex). Basic peptides or Cell Penetrating Peptides (CPP) such as the TAT-derived peptide appear to circumvent many problems associated with ON and drug delivery. This strategy may represent the next paradigm in our ability to modulate cell function and offers a unique avenue for the treatment of disease. Lipoplexes result from the intimate interaction of ON with cationic lipids leading to ON carrying particles able to be taken up by cells and to release ON in the cytoplasm. We have used as an experimental model the correction of a splicing alteration of the mutated beta-globin intron causing thalassemia. Data on cell penetration and efficacy of correction of specific steric block ON delivered either by basic peptides or lipoplex are described. A comparison of the properties of both delivery systems is made respective to the use of this new class of therapeutic molecules.