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.

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.

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.

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.

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.

Antisense DNA and RNA agents against picornaviruses.

Anti-picornaviral antisense agents are part of a broader group of nucleic acid-based molecules developed for sequence-specific inhibition of translation and/or transcription of the target sequence through induced nuclease activity or physical hindrance. Three types of nucleic acid-based gene silencing molecules can be distinguished, including DNA-base antisense oligonucleotides (ASO), nucleic acid enzymes (ribozyme and DNAzyme) and double-stranded small interfering RNA (siRNA or microRNA). These antisense DNA and RNA molecules have been widely studied for gene functional studies and therapeutic purposes. In this review, we focus on drug development using ASO and siRNA strategies to inhibit picornavirus infections. The picornavirus genome organization and life cycle is described, followed by discussion of design considerations, chemical modifications and drug delivery approaches. Recent studies using antisense against picornavirus are reviewed. Finally, we compare the advantages and disadvantages of the antisense agents with those of other therapeutics, taking into consideration their limitations which need to be overcome to achieve the final goal of clinical application.

Antisense targeting of perlecan blocks tumor growth and angiogenesis in vivo.

Perlecan, a ubiquitous heparan sulfate proteoglycan, possesses angiogenic and growth-promoting attributes primarily by acting as a coreceptor for basic fibroblast growth factor (FGF-2). In this report we blocked perlecan expression by using either constitutive CMV-driven or doxycycline- inducible antisense constructs. Growth of colon carcinoma cells was markedly attenuated upon obliteration of perlecan gene expression and these effects correlated with reduced responsiveness to and affinity for mitogenic keratinocyte growth factor (FGF-7). Exogenous perlecan effectively reconstituted the activity of FGF-7 in the perlecan-deficient cells. Moreover, soluble FGF-7 specifically bound immobilized perlecan in a heparan sulfate-independent manner. In both tumor xenografts induced by human colon carcinoma cells and tumor allografts induced by highly invasive mouse melanoma cells, perlecan suppression caused substantial inhibition of tumor growth and neovascularization. Thus, perlecan is a potent inducer of tumor growth and angiogenesis in vivo and therapeutic interventions targeting this key modulator of tumor progression may improve cancer treatment.

Novel therapies blocking the renin-angiotensin-aldosterone system in the management of hypertension and related disorders.

Although significant advances have been made in the therapeutic blockade of the renin-angiotensin-aldosterone system (RAAS) using angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers and non-selective aldosterone receptor antagonists, there is a clear need for both additional blocking strategies and enhancements of current therapeutic approaches. Vasopeptidase inhibition may still find a role despite the small incremental value of this approach and the obvious issue of kinin-mediated adverse effects still to be fully addressed. Blockade of the RAAS upstream using renin inhibitors as well as the greater selectivity of aldosterone blockade using selective aldosterone blockers such as eplerenone are also novel approaches. Not yet in clinical use but certainly an attractive therapeutic target is angiotensin II growth factor receptor transactivation, with selective inhibitors having been developed for various specific kinase pathways. Finally, ACE2 augmentation, antisense gene strategies, and vaccination against the renin-angiotensin system should still be considered experimental, but have significant appeal as additional approaches to the blockade of this system.

Specific apoptosis induction in human papillomavirus-positive cervical carcinoma cells by photodynamic antisense regulation.

Human papillomavirus type 18 (HPV18) is frequently detected in cervical cancer cells. The viral proteins E6 and E7 are expressed consistently and have oncogenic activities. The E7 protein binds to a tumor suppressor, the retinoblastoma gene product (pRB), however, leading to the stabilization of tumor suppressor, p53 protein. On the other hand, another viral product, E6, forms complexes with p53 and abrogates its function, resulting in tumor progression. These facts imply that the E6 oncogene is one of the ideal targets for directed gene therapy in HPV-positive cervical cancer. In this study, we tried photodynamic antisense regulation of the antiapoptotic E6 expression using a photocross-linking reagent, 4,5',8-trimethylpsoralen, conjugated oligo(nucleoside phosphorothioate) (Ps-S-Oligo). This photodynamic antisense strategy effectively elicited the apoptotic death of HPV18-positive cervical cancer cells through the selective repression of E6 mRNA and consequent stabilization of p53 protein. E7-mediated signals potentially activated the p53 function and mobilized the p53 pathway to deliver pro-apoptotic signals to the cancer cells, leading to the suppression of in vivo tumorigenesis. An extremely low concentration of cisplatin in addition to Ps-S-Oligos further up-regulated p53 activity, provoking massive apoptotic induction. These results suggest that the photodynamic antisense strategy has the great therapeutic potential in HPV-positive cervical cancers.

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.

Therapy of "SHIV" infected macaques with liposomes delivering antisense interleukin-4 DNA.

BACKGROUND/OBJECTIVES: To explore the effects of antisense (AS) interleukin (IL)-4 on virus replication and CD8+ T-cell responses in lymph nodes and blood of macaques infected with simian human immunodeficiency virus, SHIV(89.6)P. METHODS: Six macaques were inoculated with simian human immunodeficiency virus (SHIV(89.6)P). Seven days later, four of the animals were given 1 mg AS IL-4 plasmid complexed with Megafectin liposome, intravenously, and two of these received a second injection of the same material on day 9. All six macaques were killed at 2 weeks post infection (pi) and monitored for viral RNA and CD8+ T cells in blood and lymph nodes by real-time reverse transcriptase-polymerase chain reaction, flow cytometry and immunohistochemistry. RESULTS: In contrast to the lymph nodes from virus control animals, the lymph nodes of AS IL-4-treated animals had a significant reduction in viral loads and reduced depletion of cells from the nodes. There was an increase in CD8+ T cells in the nodes, and many of the cells expressed granzyme B, suggesting functional activation. This trend of virus reduction and increased CD8+ T cell numbers was also reflected in blood. CONCLUSIONS: The therapeutic effect of the AS IL-4 suggests indirectly that the acute immunosuppressive disease caused by SHIVs is mediated, in part, by IL-4 that causes enhanced virus replication by suppressing anti-viral CD8+ T-cell responses, and that this effect was reduced by treatment of the animals with AS IL-4.

Long-term inhibition of the central alpha(2B)-adrenergic receptor gene via recombinant AAV-delivered antisense in hypertensive rats.

BACKGROUND: Salt-induced hypertension is mediated via the alpha(2B)-adrenergic receptor (AR) subtype. In alpha(2B)-AR gene knockout mice, blood pressure (BP) does not rise with salt loading, and in rats with salt-induced hypertension, BP decreases transiently with antisense (AS) treatment targeting the alpha(2B)-AR gene. The present experiments were designed to explore the possibility of gene transfection in the brain by intracerebroventricular (ICV) delivery of AS-DNA via adeno-associated virus (AAV) to prolong alpha(2B)-AR inhibition and hence reversal of salt-dependent hypertension. METHODS: A recombinant AAV (rAAV) vector preparation encoding the alpha(2B)-AS fragment (previously tested in vitro for inhibition of alpha(2B)-AR protein production in cells) and containing green fluorescence protein (GFP) for visualization was injected ICV into subtotally nephrectomized, salt-fed rats. Control rats received rAAV-GFP (n = 8 per group). RESULTS: We observed that BP rose from a baseline of 120 +/- 10 to 184 +/- 12 mm Hg. Injection of rAAV-alpha(2B)-AS produced a 35 +/- 12 mm Hg fall in BP, lasting without evidence of diminishing for at least 16 days, whereas rAAV-GFP-injected rats showed a continued rise in BP. Rats treated with rAAV-alpha(2B)-AS treated had a 45% to 65% decrease in alpha(2B)-AR protein levels in key regulatory regions of the brain. Neither group had signs of immunologic response to the virus injection. CONCLUSIONS: These results indicate that our construct, when given by ICV means, could reach multiple sites of the central nervous system relevant to BP regulation and could safely inhibit the central alpha(2B)-adrenergic receptor, thereby achieving prolonged reversal of salt-induced hypertension.

Permanent cardiovascular protection from hypertension by the AT(1) receptor antisense gene therapy in hypertensive rat offspring.

Our previous studies have demonstrated that the introduction of angiotensin II type I receptor antisense (AT(1)R-AS) cDNA by a retrovirally mediated delivery system prevents the development of hypertension in the spontaneously hypertensive rat (SHR), an animal model for primary hypertension in humans. These results have led us to propose the hypothesis that an interruption of the renin-angiotensin system (RAS) activity at a genetic level would prevent hypertension on a permanent basis. F(1) and F(2) generations of offspring from a retroviral vector, LNSV- and LNSV-AT(1)R-AS-treated SHR, were generated, and various physiological parameters indicative of hypertension were studied and compared with those of their parents to investigate this hypothesis. Both F(1) and F(2) generations of LNSV-AT(1)R-AS-treated SHR expressed a persistently lower blood pressure, decreased cardiac hypertrophy and fibrosis, decreased medial thickness, and normalization of renal artery excitation-contraction coupling, Ca(2+) current, and [Ca(2+)](i) when compared with offspring derived from the LNSV-treated SHR. In fact, the magnitude of the prevention of these pathophysiological alterations was similar to that observed in the LNSV-AT(1)R-AS-treated SHR parent. The prevention of cardiovascular pathophysiology and expression of normotensive phenotypes are, at least in part, a result of integration and subsequent transmission of AT(1)R-AS from the SHR parents to offspring. These data demonstrate that a single intracardiac injection of LNSV-AT(1)R-AS causes a permanent cardiovascular protection against hypertension as a result of a genomic integration and germ line transmission of the AT(1)R-AS in the SHR offspring.

Antisense angiopoietin-1 inhibits tumorigenesis and angiogenesis of gastric cancer.

AIM: To investigate the effect of angiopoietin-1 (Ang-1) on biological behaviors in vitro and tumorigenesis and angiogenesis in vitro of human gastric cancer cells. METHODS: Human full-length Ang-1 gene was cloned from human placental tissues by RT-PCR method. Recombinant human Ang-1 antisense eukaryotic expression vector was constructed by directional cloning, and transfected by lipofectin method into human gastric cancer line SGC7901 with high Ang-1 expression level. Inhibition efficiency was confirmed by semi- quantitative PCR and Western blot method. Cell growth curve and cell cycle were observed with MTT assays and flow cytometry, respectively. Nude mice tumorigenicity test was employed to compare in vitro tumorigenesis of cells with Ang-1 suppression. Microvessel density (MVD) of implanted tumor tissues was analyzed by immunohistochemistry for factor VIII staining. RESULTS: Full-length Ang-1 gene was successfully cloned and stable transfectants were established, namely 7Ang1- for antisenseìand 7901P for empty vector transfected. 7Ang1- cells showed down-regulated Ang-1 expression, while its in vitro proliferation and cell cycle distribution were not significantly changed. In contrast, xenograft of 7Ang1- cells in nude mice had lower volume and weight than those of 7901P after 30 days' implantation (P<0.01, 293.00+/-95.54 mg vs. 624.00+/-77.78 mg) accompanied with less vessel formation with MVD 6.00+/-1.73 compared to 7901P group 8.44+/-1.33 (P<0.01). CONCLUSION: Ang-1 may play an important role in tumorigenesis and angiogenesis of gastric cancer, and targeting its expression may be beneficial for the therapy of gastric cancer.

Effects of survivin antagonists on growth of established tumors and B7-1 immunogene therapy.

BACKGROUND: Survivin, a member of the inhibitor of apoptosis (IAP) protein family, is detectable in most types of cancer, and its presence is associated with a poor prognosis. We determined the effects of gene-based therapies that inhibit survivin function in a mouse tumor model. METHODS: Using five to six mice per treatment group, we injected tumors derived from mouse EL-4 thymic lymphoma cells with plasmids encoding antisense survivin, a dominant-negative mutant survivin, and the T-cell costimulator B7-1. Expression of endogenous survivin and the proteins encoded by the injected plasmids were examined by immunohistochemical staining of tumor sections and by western blot and flow cytometry analyses of isolated tumor cells. Tumor growth, the generation of antitumor cytotoxic T-lymphocyte (CTL) activity, apoptosis, and the contribution of leukocyte subsets to antitumor activity were measured. All statistical tests were two-sided. RESULTS: Large (1.0-cm diameter) tumors had approximately 10-fold more survivin than small (0.2-cm diameter) tumors. At 28 days after injection, antisense and dominant-negative mutant survivin plasmids statistically significantly inhibited the growth of both small (P =.006 and P =.0018, respectively) and large (P<.001 for both plasmids) EL-4 tumors compared with tumors injected with empty plasmid. The growth of large tumors was further inhibited by intratumoral injection with antisense survivin and B7-1 (P =.004); thus, inhibition of survivin expression renders large tumors susceptible to B7-1-mediated immunotherapy. Mice whose tumors were completely eradicated by injection of B7-1 remained tumor free for 26 days after re-injection with EL-4 cells (when the experiment ended). Compared with tumors injected with empty plasmid, tumors injected with survivin-based plasmids had increased apoptosis, and animals bearing such tumors generated more antitumor CTLs. CONCLUSION: Intratumoral injection of plasmids that block survivin expression and stimulate the generation of tumor-specific CTLs may be beneficial for the treatment of large lymphomas.

Circulating plasma vascular endothelial growth factor in mice bearing human ovarian carcinoma xenograft correlates with tumor progression and response to therapy.

Vascular endothelial growth factor (VEGF) performs as an angiogenic and permeability factor in ovarian cancer, and its overexpression has been associated with poor prognosis. However, models to study its role as a marker of tumor progression are lacking. We generated xenograft variants derived from the A2780 human ovarian carcinoma (1A9), stably transfected with VEGF(121) in sense (1A9-VS-1) and antisense orientation (1A9-VAS-3). 1A9, 1A9-VS-1, and 1A9-VAS-3 disseminated in the peritoneal cavity of nude mice, but only 1A9-VS-1, the VEGF(121)-overexpressing tumor variant, produced ascites. Tumor biopsies from 1A9-VS-1 showed alterations in the vascular pattern and caused an angiogenic response in the chorioallantoic membrane assay. A significant level of soluble VEGF was detectable in the plasma of mice bearing 1A9-VS-1 even at an early stage of tumor growth. Plasma VEGF correlated positively with tumor burden in the peritoneal cavity and ascites accumulation. Cisplatin reduced the tumor burden and ascites in mice bearing 1A9-VS-1; the response was associated with a significant decrease of VEGF in plasma. This 1A9-VS-1 xenograft model reproduces the behavior of human ovarian cancer by growing in the peritoneal cavity, being highly malignant, and producing ascites. Plasma VEGF as a marker of tumor progression offers a valuable means of detecting early tumor response and following up treatments in an animal model.

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.