Antibacterial performance of graphene oxide/alginate-based antisense hydrogel for potential therapeutic application in Staphylococcus aureus infection.

Staphylococcus aureus (S. aureus) is an opportunistic bacterium that causes several infections in humans. However, chronic biofilms remain a major challenge associated with recalcitrance toward traditional treatments. Herein, an antibacterial hydrogel composed of antisense DNA oligonucleotides, graphene oxide and alginate is construed for biofilm management and infection care. The hydrogel is established through noncovalent binding and possesses injectability and degradability properties. Furthermore, hydrogels present controllable release of cargoes, genetic targeting antibacterial effects and stem cell supporting capabilities. Our in vivo results reveal a high antibiofilm performance and good biocompatibility, which significantly improve tissue regeneration. The hydrogel inhibits biofilm formation by decreasing the expression of YycFG with antisense and viability of strains by graphene oxide. Thus, antisense hydrogels can be a promising antibacterial bioactive material for potential therapeutic S. aureus infection.

Regulation of Expression of Hyperalgesic Priming by Estrogen Receptor α in the Rat.

Hyperalgesic priming, a sexually dimorphic model of transition to chronic pain, is expressed as prolongation of prostaglandin E2-induced hyperalgesia by the activation of an additional pathway including an autocrine mechanism at the plasma membrane. The autocrine mechanism involves the transport of cyclic adenosine monophosphate (AMP) to the extracellular space, and its conversion to AMP and adenosine, by ecto-5'phosphodiesterase and ecto-5'nucleotidase, respectively. The end product, adenosine, activates A1 receptors, producing delayed onset prolongation of prostaglandin E2 hyperalgesia. We tested the hypothesis that the previously reported, estrogen-dependent, sexual dimorphism observed in the induction of priming is present in the mechanisms involved in its expression, as a regulatory effect on ecto-5'nucleotidase by estrogen receptor α (EsRα), in female rats. In the primed paw AMP hyperalgesia was dependent on conversion to adenosine, being prevented by ecto-5'nucleotidase inhibitor α,ß-methyleneadenosine 5'-diphosphate sodium salt and A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. To investigate an interaction between EsRα and ecto-5'nucleotidase, we treated primed female rats with oligodeoxynucleotide antisense or mismatch against EsRα messenger RNA. Whereas in rats treated with antisense AMP-induced hyperalgesia was abolished, the A1 receptor agonist N6-cyclopentiladenosine still produced hyperalgesia. Thus, EsRα interacts with this autocrine pathway at the level of ecto-5'nucleotidase. These results demonstrate a sexually dimorphic mechanism for the expression of priming. PERSPECTIVE: This study presents evidence of an estrogen-dependent mechanism of expression of chronic pain in female rats, supporting the suggestion that differential targets must be considered when establishing protocols for the treatment of painful conditions in men and women.

Therapeutic Potential of Tricyclo-DNA antisense oligonucleotides.

Oligonucleotide therapeutics hold great promise for the treatment of various diseases and the antisense field is constantly gaining interest due to the development of more potent and nuclease resistant chemistries. Despite a rather low success rate with only three antisense drugs being clinically approved, the frontiers of AON therapeutic applications have increased over the past three decades and continue to expand thanks to a steady increase in understanding the mechanisms of action of these molecules, progress in chemical modification and delivery.In this review, we will examine the recent advances obtained with the tricyclo-DNA chemistry which displays unique pharmacological properties and unprecedented uptake in many tissues after systemic administration. We will review their specific properties and their therapeutic applications mainly for neuromuscular disorders, including exon-skipping for Duchenne muscular dystrophy and exon-inclusion for spinal muscular atrophy, but also aberrant splicing correction for Pompe disease. Finally, we will discuss their advantages and potential limitations, with a focus on the need for careful toxicological screen early in the process of AON drug development.

Fluorescence Characterization of Gold Modified Liposomes with Antisense N-myc DNA Bound to the Magnetisable Particles with Encapsulated Anticancer Drugs (Doxorubicin, Ellipticine and Etoposide).

Liposome-based drug delivery systems hold great potential for cancer therapy. The aim of this study was to design a nanodevice for targeted anchoring of liposomes (with and without cholesterol) with encapsulated anticancer drugs and antisense N-myc gene oligonucleotide attached to its surface. To meet this main aim, liposomes with encapsulated doxorubicin, ellipticine and etoposide were prepared. They were further characterized by measuring their fluorescence intensity, whereas the encapsulation efficiency was estimated to be 16%. The hybridization process of individual oligonucleotides forming the nanoconstruct was investigated spectrophotometrically and electrochemically. The concentrations of ellipticine, doxorubicin and etoposide attached to the nanoconstruct in gold nanoparticle-modified liposomes were found to be 14, 5 and 2 µg·mL(-1), respectively. The study succeeded in demonstrating that liposomes are suitable for the transport of anticancer drugs and the antisense oligonucleotide, which can block the expression of the N-myc gene.

Antisense vimentin cDNA combined with chondroitinase ABC promotes axon regeneration and functional recovery following spinal cord injury in rats.

The formation of glial scar restricts axon regeneration after spinal cord injury (SCI) in adult mammalian. Chondroitin sulfate proteoglycans (CSPGs) are mostly secreted by reactive astrocytes, which form dense scar tissues after SCI. Chondroitinase ABC (ChABC), which can digest CSPGs, is a promising therapeutic strategy for SCI. However, to date ChABC has exhibited only limited success in the treatment of chronic SCI. The intermediate filament protein vimentin underpins the cytoskeleton of reactive astrocytes. We targeted glial scar in injured spinal cord by sustained infusion of ChABC and antisense vimentin cDNA. Using anterograde tracing, BBB scoring and hind limb placing response, we found that this combined treatment promoted axon regeneration and functional recovery after SCI in rats. Our results indicate that axon regeneration may be promoted by modified physical and biochemical characteristics of intra- and extracellular architecture in glial scar tissues. Theses findings could potentially help us to understand better the composition of glial scar in central nervous system injury.

The effect of C-reactive protein reduction with a highly specific antisense oligonucleotide on atrial fibrillation assessed using beat-to-beat pacemaker Holter follow-up.

PURPOSE: C-reactive protein (CRP) is known to be strongly associated with atrial fibrillation (AF). However, it is not clear if CRP is a causal factor for AF. ISIS-CRPRx is a novel antisense oligonucleotide that reduces CRP production by specifically inhibiting mRNA translation. The effect of ISIS-CRPRx on AF was evaluated. METHODS: A double-blind phase II trial of ISIS-CRPRx in patients with paroxysmal AF and DDDRP permanent pacemakers (PPMs) with advanced atrial and ventricular Holters allowing beat-to-beat arrhythmia follow-up. RESULTS: Twenty six patients were screened and seven patients dosed with ISIS-CRPRx. After 4 weeks of baseline assessment, patients were randomly assigned to two treatment periods of either placebo then ISIS-CRPRx or ISIS-CRPRx then placebo. All patients were followed up for 8 weeks after the active treatment period. There was a 63.7 % (95 % CI 38.4 to 78.6 %, p = 0.003) relative reduction in CRP on treatment with ISIS-CRPRx versus baseline. Sensitivity analyses demonstrated a consistent treatment effect. The primary end-point was change in AF burden assessed by PPM. There was no significant difference in AF burden on treatment with ISIS-CRPRx versus baseline (OR 1.6, 95 % CI -2.42 to 5.62, p = 0.37). ISIS CRPRx was safe and well tolerated and there were no serious adverse events. CONCLUSIONS: Treatment with ISIS-CRPRx did not reduce AF burden in patients with paroxysmal AF and PPMs, despite a large relative reduction in CRP. In this population, highly specific CRP reduction had no clinically discernable effect upon paroxysmal AF. However, average levels of CRP at baseline were relatively low, so it remains possible that AF patients with higher levels of CRP may benefit from CRP-directed therapy.

Spinal muscular atrophy: diagnosis and management in a new therapeutic era.

Spinal muscular atrophy (SMA) describes a group of disorders associated with spinal motor neuron loss. In this review we provide an update regarding the most common form of SMA, proximal or 5q-SMA, and discuss the contemporary approach to diagnosis and treatment. Electromyography and muscle biopsy features of denervation were once the basis for diagnosis, but molecular testing for homozygous deletion or mutation of the SMN1 gene allows efficient and specific diagnosis. In combination with loss of SMN1, patients retain variable numbers of copies of a second similar gene, SMN2, which produces reduced levels of the survival motor neuron (SMN) protein that are insufficient for normal motor neuron function. Despite the fact that understanding of how ubiquitous reduction of SMN protein leads to motor neuron loss remains incomplete, several promising therapeutics are now being tested in early-phase clinical trials.

[Growth inhibition effection of perlecan anti-sense cDNA on human laryngeal carcinoma xnograft in nude mice].

OBJECTIVE: To observe growth inhibition effect of perlecan anti-sense cDNA (pAP) on human laryngeal carcinoma xnografted in nude mice. To vertify its antitumor effect and mechanism in vivo, and it may be useful as a biomarker in carcinoma of larynx cancer. METHOD: Created the model of human laryngeal carcinoma xnograft in nude mice. To observe growth of those xnografts in nude mice and draw growth curve of xnografted. The expression of perlecan mRNA and portein in xnografts were examined by RT-PCR and immunohistochemistry. RESULT: Volume of xnografts in the group transfected by the plasmids of pAP were significant small as compared with other two groups made by the wild type cells and phpApr-neol cells (P < 0.05). It was showed that the expression of perlecan mRNA and protein were significantly reduced in the tumor of pAP transfected Hep-2 cells as compared with the tumors transfected by the wild type cells and phßApr-neol cells (P < 0.01). CONCLUSION: These data raise the possibility that pAP many play key roles in the growth of those xnografts in nude mice.

Development and antitumor activity of a BCL-2 targeted single-stranded DNA oligonucleotide.

PNT100 is a 24-base, chemically unmodified DNA oligonucleotide sequence that is complementary to a region upstream of the BCL-2 gene. Exposure of tumor cells to PNT100 results in suppression of proliferation and cell death by a process called DNA interference. PNT2258 is PNT100 that is encapsulated in protective amphoteric liposomes developed to efficiently encapsulate the PNT100 oligonucleotide, provide enhanced serum stability, optimized pharmacokinetic properties and antitumor activity of the nanoparticle both in vivo and in vitro. PNT2258 demonstrates broad antitumor activity against BCL-2-driven WSU-DLCL2 lymphoma, highly resistant A375 melanoma, PC-3 prostate, and Daudi-Burkitt's lymphoma xenografts. The sequence specificity of PNT100 was demonstrated against three control sequences (scrambled, mismatched, and reverse complement) all encapsulated in a lipid formulation with identical particle characteristics, and control sequences did not demonstrate antiproliferative activity in vivo or in vitro. PNT2258 is currently undergoing clinical testing to evaluate safety and antitumor activity in patients with recurrent or refractory non-Hodgkin's lymphoma and additional studies are planned.

Efficacy of antisense monocyte chemoattractant protein-1 (MCP-1) in a rat model of mesangial proliferative glomerulonephritis.

OBJECTIVE: The effects of inhibition of monocyte chemoattractant protein-1 (MCP-1) on a rat model of mesangial proliferative glomerulonephritis (MsPGN) were evaluated. METHODS: The anti-Thy-1 MsPGN model was developed by intravenously injecting anti-Thy-1 monoclonal antibodies into rats, followed by an injection of mesangial cells transfected with antisense MCP-1 into the renal artery. Exogenous cells were detected by in situ hybridization. Rats (40 total) were randomly divided into five groups: SO (sham operation), TG (Thy-1 glomerulonephritis model), MC (non-transfected normal rat mesangial cell), BC (pLXSN empty vector or blank control), and AM (antisense MCP-1 transfection) groups. Effects of exogenous MCP-1 on urinary protein excretion rate, biochemical parameters, and pathological changes were evaluated. Expression of MCP-1 and transforming growth factor-ß1 (TGF-ß1) were detected by immunohistochemistry. mRNA expression of MCP-1, TGF-ß1, and CC chemokine receptor 2 (CCR2) were detected by RT-PCR. RESULTS: Exogenous MCP-1 cDNA was successfully transfected into mesangial cells. Exogenous mesangial cells were detected in glomeruli by in situ hybridization. Glomerular mesangial cell proliferation, 24-h urinary protein excretion rate, mRNA expression of MCP-1, TGF-ß1, and CCR2, and protein expression of MCP-1 all decreased in the AM group as compared to the control group (p < 0.05), but there was no significant difference in the expression level of TGF-ß1 protein. CONCLUSIONS: (1) Mesangial cells can be used as a vector to transfect exogenous genes into kidneys; (2) antisense MCP-1 decreases mesangial cell proliferation and pathological injury in MsPGN model rats by decreasing expression of MCP-1 and CCR2; and (3) antisense MCP-1 suppressed mesangial cell proliferation and matrix accumulation in anti-Thy-1 MsPGN model rats, which did not entirely depend on TGF-ß1.

Bioinformatic and functional optimization of antisense phosphorodiamidate morpholino oligomers (PMOs) for therapeutic modulation of RNA splicing in muscle.

Duchenne muscular dystrophy (DMD) is caused by mutations that disrupt the reading frame of the human DMD gene. Selective removal of exons flanking an out-of-frame DMD mutation can result in an in-frame mRNA transcript that may be translated into an internally deleted, Becker muscular dystrophy (BMD)-like, but functionally active dystrophin protein with therapeutic activity. Antisense oligonucleotides (AOs) can be designed to bind to complementary sequences in the targeted mRNA and modify pre-mRNA splicing to correct the reading frame of a mutated transcript so that gene expression is restored. AO-induced exon skipping producing functional truncated dystrophin exon has been demonstrated in animal models of DMD both in vitro and in vivo, and in DMD patient cells in vitro in culture, and in DMD muscle explants. More recently, AO-mediated exon skipping has been confirmed in DMD patients in Phase I clinical trials. However, it should be noted that personalized molecular medicine may be necessary, since the various reading frame-disrupting mutations are spread across the DMD gene. The different deletions that cause DMD would require skipping of different exons, which would require the optimization and clinical trial workup of many specific AOs. This chapter describes the methodologies available for the optimization of AOs, and in particular phosphorodiamidate morpholino oligomers (PMOs), for the targeted skipping of specific exons on the DMD gene.

Present and future of antisense therapy for splicing modulation in inherited metabolic disease.

The number of mutations identified deep in introns which activate or create novel splice sites resulting in pathogenic pseudoexon inclusion in mRNA continues to grow for inherited metabolic disease (IMD) and other human genetic diseases. A common characteristic is that the native splice sites remain intact thus retaining the potential for normal splicing. Antisense oligonucleotides (AO) have been shown to modulate the splicing pattern by steric hindrance of the recognition and binding of the splicing apparatus to the selected sequences. In the case of pseudoexons, AO force the use of the natural splice sites, recovering normally spliced transcripts encoding functional protein. This review summarizes the present knowledge of antisense splicing modulation as a molecular therapy approach for pseudoexon-activating mutations, with a focus in IMD. Although the feasibility of treatment for patients with IMD has yet to be proven, it appears to be clinically promising, as positive results have been reported in cellular and animal models of disease, and antisense therapy for splicing modulation is currently in the clinical trials phase for Duchenne muscular dystrophy patients. Here, we review the most recent advances in AO stability, targeting and delivery, and other issues to be considered for an effective treatment in the clinical setting. Although the number of patients who can be potentially treated is low for each IMD, it represents an excellent therapeutical option as a type of personalized molecular medicine which is especially relevant for diseases for which there is, to date, no efficient treatment.

Therapeutic exon skipping for dysferlinopathies?

Antisense-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy (DMD) currently tested in clinical trials. The aim is to reframe dystrophin transcripts using antisense oligonucleotides (AONs). These hide an exon from the splicing machinery to induce exon skipping, restoration of the reading frame and generation of internally deleted, but partially functional proteins. It thus relies on the characteristic of the dystrophin protein, which has essential N- and C-terminal domains, whereas the central rod domain is largely redundant. This approach may also be applicable to limb-girdle muscular dystrophy type 2B (LGMD2B), Myoshi myopathy (MM) and distal myopathy with anterior tibial onset (DMAT), which are caused by mutations in the dysferlin-encoding DYSF gene. Dysferlin has a function in repairing muscle membrane damage. Dysferlin contains calcium-dependent C2 lipid binding (C2) domains and an essential transmembrane domain. However, mildly affected patients in whom one or a large number of DYSF exons were missing have been described, suggesting that internally deleted dysferlin proteins can be functional. Thus, exon skipping might also be applicable as a LGMD2B, MM and DMAT therapy. In this study we have analyzed the dysferlin protein domains and DYSF mutations and have described what exons are promising targets with regard to applicability and feasibility. We also show that DYSF exon skipping seems to be as straightforward as DMD exon skipping, as AONs to induce efficient skipping of four DYSF exons were readily identified.

Overview of the role of macrophage migration inhibitory factor (MIF) in inflammatory bowel disease.

An array of investigations has revealed that macrophage migration inhibitory factor (MIF) plays an important role in the exacerbation of a wide range of inflammatory diseases. For the past two decades, we have extensively studied MIF's pathophysiological roles in human diseases, and have accumulated evidence elucidating its molecular mechanisms in the pathogenesis of immune disorders and inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel diseases (IBD). In a study of IBD, we demonstrated for the first time that anti-MIF antibody suppressed the degree of dextran-sulfate sodium (DSS)-induced colitis, indicating its potential therapeutic use for IBD patients. Following that report, a number of researchers, including us, clarified that MIF was profoundly involved in various gastrointestinal disorders, such as hepatitis and pancreatitis. We recently revealed that a MIF-deficient mouse was resistant to a challenge of DSS, and showed few clinical and pathological signs. Currently, we are developing new therapeutic approaches targeting MIF in inflammatory disorders, particularly IBD. We here overview MIF's pathophysiological function, mainly in IBD, and introduce two therapeutic approaches, anti-MIF antibody treatment and MIF-antisense therapy, via a drug delivery system using 1,3-beta glucan.