Systemic Delivery Of Antisense Oligonucleotides Research Articles

The Potential Of Antisense Oligonucleotides (ASO) Through Inhalation Based On Gold Nanoparticle (AuNP) Delivery System In Inhibiting SARS-CoV-2 Replication And Transcription

Coronavirus disease 2019 (COVID-19) is an emerging infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). This virus infects the respiratory, digestive, and nervous systems with a rapid transmission and a fairly high mortality rate. However, there has been no specific therapy to treat COVID-19. Previous studies have shown that antisense oligonucleotide (ASO) has good efficacy in DNA and RNA viral infections. This literature review aims to investigate the potential of inhaled ASO based on gold nanoparticles (AuNp) delivery system in inhibiting the replication and transcription of SARS-CoV-2. Literature searching using several databases, such as Google Scholar, Science Direct, ResearchGate, and NCBI. Inclusion and exclusion criteria are used to eliminate the journals that does not match the criteria, thus 28 journals are obtained. The results show that ASO has the potential to inhibit the replication and transcription of the SARS-CoV-2 virus through different mechanisms by binding to the target RNA and modulating the viral protein synthesis. One form of ASO modification that is often used is LNA GapmeR. LNA GapmeR stimulates viral RNA cleavage and can be administered by inhalation with nebulized ASO solution. AuNP as an ASO delivery system through inhalation can reduce toxicity and increase ASO concentrations in reaching target cells. Therefore, ASO therapy with AuNP through inhalation needs to be considered for COVID-19 treatment. Further clinical study about the ideal delivery system and optimal dosage of ASO based AuNP via inhalation for COVID-19 are needed to investigate soon.

FSHD: EP.165 Systemic delivery of antisense oligonucleotides targeting DUX4; a promising therapeutic strategy for facioscapulohumeral muscular dystrophy

FSHD: EP.165 Systemic delivery of antisense oligonucleotides targeting DUX4; a promising therapeutic strategy for facioscapulohumeral muscular dystrophy

Targeting RyR Activity Boosts Antisense Exon 44 and 45 Skipping in Human DMD Skeletal or Cardiac Muscle Culture Models

Systemic delivery of antisense oligonucleotides (AO) for DMD exon skipping has proven effective for reframing DMD mRNA, rescuing dystrophin expression, and slowing disease progression in animal models. In humans with Duchenne muscular dystrophy treated with AOs, low levels of dystrophin have been induced, and modest slowing of disease progression has been observed, highlighting the need for improved efficiency of human skipping drugs. Here, we demonstrate that dantrolene and Rycals S107 and ARM210 potentiate AO-mediated exon skipping of exon 44 or exon 45 in patient-derived myotube cultures with appropriate mutations. Further, dantrolene is shown to boost AO-mediated exon skipping in patient-derived, induced cardiomyocyte cultures. Our findings further validate the ryanodine receptors (RyR) as the likely target responsible for exon skip boosting and demonstrate potential applicability beyond exon 51 skipping. These data provide preclinical support of dantrolene trial as an adjuvant to AO-mediated exon-skipping therapy in humans and identify a novel Rycal, ARM210, for development as a potential exon-skipping booster. Further, they highlight the value of mutation-specific DMD culture models for basic discovery, preclinical drug screening and translation of personalized genetic medicines.

Delivery of antisense oligonucleotide using polyethylenimine-based lipid nanoparticle modified with cell penetrating peptide

Efficient and stable delivery system of antisense oligonucleotide (ASO) is important and urgently needed. Here, an ASO delivery system, Lp-PPRP, which contains a cationic polymer based on PEI (branched, 25 kDa), named PEI-PC and a palmitic acid modified R8 (R8-PA) was prepared to deliver a kind of ASO, LOR-2501. The characteristics of the nanoparticles and the cellular uptake of LOR-2501 in HeLa cells and A549 cells were studied. Lp-PPRP showed suitable particle size and zeta potential to combine with LOR-2501; the particle size and zeta potential of Lp-PPRP/LOR were 276.87 ± 5.63 nm and 18.03 ± 0.25 mV. In vitro experiments suggested that Lp-PPRP had lower cytotoxic and higher transfection efficiency for delivering LOR-2501 compared with PEI. The addition of PEI-PC and R8-PA contributed to enhance the transfection efficiency of the nanoparticles. In HeLa cells and A549 cells, Lp-PPRP could transport LOR-2501 and down-regulate the level of R1 protein efficiently, and the R1 down regulations were 64.56% and 66.34%, respectively. Results suggested potential utility of Lp-PPRP in the development of ASO in tumor therapy.

Elusive sources of variability of dystrophin rescue by exon skipping.

BackgroundSystemic delivery of anti-sense oligonucleotides to Duchenne muscular dystrophy (DMD) patients to induce de novo dystrophin protein expression in muscle (exon skipping) is a promising therapy. Treatment with Phosphorodiamidate morpholino oligomers (PMO) lead to shorter de novo dystrophin protein in both animal models and DMD boys who otherwise lack dystrophin; however, restoration of dystrophin has been observed to be highly variable. Understanding the factors causing highly variable induction of dystrophin expression in pre-clinical models would likely lead to more effective means of exon skipping in both pre-clinical studies and human clinical trials.MethodsIn the present study, we investigated possible factors that might lead to the variable success of exon skipping using morpholino drugs in the mdx mouse model. We tested whether specific muscle groups or fiber types showed better success than others and also correlated residual PMO concentration in muscle with the amount of de novo dystrophin protein 1 month after a single high-dose morpholino injection (800 mg/kg). We compared the results from six muscle groups using three different methods of dystrophin quantification: immunostaining, immunoblotting, and mass spectrometry assays.ResultsThe triceps muscle showed the greatest degree of rescue (average 38±28 % by immunostaining). All three dystrophin detection methods were generally concordant for all muscles. We show that dystrophin rescue occurs in a sporadic patchy pattern with high geographic variability across muscle sections. We did not find a correlation between residual morpholino drug in muscle tissue and the degree of dystrophin expression.ConclusionsWhile we found some evidence of muscle group enhancement and successful rescue, our data also suggest that other yet-undefined factors may underlie the observed variability in the success of exon skipping. Our study highlights the challenges associated with quantifying dystrophin in clinical trials where a single small muscle biopsy is taken from a DMD patient.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0070-6) contains supplementary material, which is available to authorized users.

17β-estradiol-containing liposomes as a novel delivery system for the antisense therapy of ER-positive breast cancer: An in vitro study on the MCF-7 cell line.

The present study suggests and describes the application of a delivery system for antisense oligonucleotides against mRNA encoding estrogen receptor proteins α and β. The delivery system is composed of a cationic liposome envelope containing 17β-estradiol (E2) in its structure. Cationic liposomes protect cargo against the extracellular matrix, and E2 can increase its shuttling efficiency into cells. Using MCF-7 cells derived from estrogen receptor-positive ductal carcinoma, treatment with liposomes against ERα was found to decrease MCF-7 proliferation, and importantly the application of both the antisense against ERα and β exhibited an antiproliferative effect expressed as cell viability. Using qRT-PCR, it was shown that MT1A, NF-κB1 and K-ras genes, but not TFF1, were downregulated using E2-based liposomes (evaluated at P=0.05). Further indicators of oxidative stress were employed to assess the effect on treatment efficiency. Glutathione (GSH/GSSG redox ratio), metallothionein (MT) and malondialdehyde (MDA) confirmed a positive effect of antisense therapy resulting in their decreased levels in the MCF-7 cells. Based on these data, we suggest that E2-based liposomes offer sufficient transfer efficiency and moreover, due to the effect on NF-κB1, MT and GSH, tumor cells can be chemosensitized to increase treatment effectiveness.

Systemic Delivery of a Peptide-Linked Morpholino Oligonucleotide Neutralizes Mutant RNA Toxicity in a Mouse Model of Myotonic Dystrophy

Expansions of CUG trinucleotide sequences in RNA transcripts provide the basis for toxic RNA gain-of-function that leads to detrimental changes in RNA metabolism. A CTG repeat element normally resides in the 3' untranslated region of the dystrophia myotonica-protein kinase (DMPK) gene, but when expanded it is the genetic lesion of myotonic dystrophy type 1 (DM1), a hereditary neuromuscular disease. The pathogenic DMPK transcript containing the CUG expansion is retained in ribonuclear foci as part of a complex with RNA-binding proteins such as muscleblind-like 1 (MBNL1), resulting in aberrant splicing of numerous RNA transcripts and consequent physiological abnormalities including myotonia. Herein, we demonstrate molecular and physiological amelioration of the toxic effects of mutant RNA in the HSA(LR) mouse model of DM1 by systemic administration of peptide-linked morpholino (PPMO) antisense oligonucleotides bearing a CAG repeat sequence. Intravenous administration of PPMO conjugates to HSA(LR) mice led to redistribution of Mbnl1 protein in myonuclei and corrections in abnormal RNA splicing. Additionally, myotonia was completely eliminated in PPMO-treated HSA(LR) mice. These studies provide proof of concept that neutralization of RNA toxicity by systemic delivery of antisense oligonucleotides that target the CUG repeat is an effective therapeutic approach for treating the skeletal muscle aspects of DM1 pathology.

Carbosilane Dendrimers are a Non-Viral Delivery System for Antisense Oligonucleotides: Characterization of Dendriplexes

The success of gene therapy depends on the development of suitable carriers, and because of their architecture dendrimers are promising tools for gene delivery. This research concerns the use of second generation carbosilane dendrimers as carriers for anti-HIV oligodeoxynucleotides (ODNs). The aim was to characterize complexes formed by positively charged dendrimers and negatively charged oligonucleotides using a fluorescence method, laser Doppler electrophoresis, dynamic light scattering (DLS), atomic force microscopy (AFM), transmission electron microscopy (TEM) and molecular modeling. The zeta-potential of ODNs increased from -25 mV to positive values after the addition of dendrimers. DLS and TEM revealed that the diameters of dendriplexes ranged from 75 to 240 nm and from 50 to 260 nm, respectively, and this was dependent on the type of dendrimer and the molar ratios of the complexes formed; complexes were stable for between 100 and 300 minutes. AFM measurements and molecular modeling studies were carried out to determine the structure and size of dendriplexes. The physicochemical properties of the dendriplexes studied and data from previous research suggest that carbosilane dendrimers are good candidates for nucleic acid delivery.

Oral delivery of nucleic acid-based therapeutics

Therapeutic DeliveryVol. 3, No. 2 EditorialFree AccessOral delivery of nucleic acid-based therapeuticsGregory E HardeeGregory E Hardee12951 Caminito En Flor Del Mar, CA 92014, USA. Search for more papers by this authorEmail the corresponding author at gehardee@oligodevelopment.comPublished Online:2 Feb 2012https://doi.org/10.4155/tde.11.143AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: cost of therapynucleic acid-based therapeuticsoligonucleotideoral bioavailabilityoral deliverypenetration enhancersCan nucleic acid-based therapeutics (NABTs) be delivered orally? The simple answer is ‘yes,’ when properly formulated. As evidenced by an increasing number of preclinical reports, the appearance of intact NABTs in the systemic circulation after per os (p.o.) dosing can lead to expression of pharmacology [1–3]. Additionally, a few clinical pharmacokinetic reports have appeared that include descriptions of measured pharmacologic effect as well as target reduction after repeated dosing [4,5].Therefore, today’s relevant question is ‘can NABTs delivered by the p.o. route be developed into drug products?’ While technical challenges remain to further develop p.o. delivery (as detailed below), the primary obstacle to administering NABTs by this route is the absolute and relative cost of therapy. In this regard, it is useful to examine the current economics of NABT therapy, specifically: ▪ Dose/potency;▪ Cost of goods;▪ Efficiency of delivery.Dose/potencyHistorically, NABTs that accessed their targets via the systemic circulation required doses of greater than 1 mg/kg/day. However, with the investment into more metabolically stable and higher affinity chemistries, and constructs with different modes of action, the effective potency is increasing dramatically. We now see potencies described in terms of less than 0.1 mg/kg/day [6].Cost of goodsA fair number of single- and double-stranded NABTs are currently under development, and while the cost may vary depending upon the chemistry, they utilize similar synthesis technologies, which we can roughly speak of as a group. To date, it could be expected that the small quantities required for discovery, preclinical and clinical work, would cost more than US$1000/g. However, with recent advancements in the processing and purification of NABTs, combined with the economies of scale for both raw materials and finished goods, we can expect to see these prices fall to the $100/g range for metric ton/year quantities.Efficiency of deliveryIn considering this part of the equation, it is important to understand the interplay between a route’s efficiency and the cost of convenience. For example, on the surface, with 10% absolute p.o. bioavailability, a p.o. dose would cost ten-times as much as an intravenous dose. Assuming an approximate intravenous dose cost of $100, the choice between convenience and cost seems clear. However, if economies of scale in drug production continue to drop and drug potencies continue to increase, the argument for the convenience of orally administered therapies becomes much stronger. If a therapeutic dose costs closer to $1.00, the convenience of a p.o. form with less than 100% efficiency becomes attractive. Given these trends, it seems as if this is the appropriate time to be investing in the research and development necessary to bring p.o. dosage forms of NABTs to market.Oral bioavailability enhancement of NABTsThe p.o. absorption of hydrophilic macromolecules such as soluble NABTs, is limited due to poor permeability across the gastrointestinal membranes [7]. As an example, single-strand oligonucleotides, constructed with a variety of chemical modifications to impart presystemic stability and of varying lengths from 14 to 22 nucleotides, show an unaided p.o. bioavailability of much less than 1% [8,9]. However, it is well known that certain permeation enhancers can transiently open the epithelial tight junctions at the apical side of the enterocytes, thus, significantly increasing paracellular absorption of a variety of hydrophilic and charged molecules [10]. One such enhancer, the fatty acid sodium caprate (C10), has been extensively studied for this application and was found suitable for p.o. delivery of oligonucleotides in both animals and man [11]. Permeation data from in vivo/in situ rodent studies of oligonucleotides calculate tight junction opening after C10 dosing to be consistent with the cross-sectional diameter of the single-strand oligonucleotides [12]. It remains to be seen if double-stranded constructs will show the same degree of increased permeation when paracellular junctions are opened to the same degree.Creating solid dosage formsBased on promising bioavailability data from solution presentations of oligonucleotides with penetration enhancers, efforts to create solid dosage forms for delivery of oligonucleotides have been reported in literature [13]. Initially, these efforts showed that when solution formulations of C10 are formulated as immediate-release solid dosage forms, much, if not all, of the bioavailability seen with solution formulations was lost. Analysis of these data show that, when presented as an immediate-release dosage form to the stomach, the C10 enhancer is both diluted in the intestinal milieu and rapidly absorbed, decreasing the effect on downstream paracellular junctions, ultimately leading to a reduction in the amount of oligonucleotide absorbed. Considering the dynamics of gut absorption, it is easy to imagine that the oligonucleotide moves beyond the mucosal site made more permeable by the transient enhancer. This can be demonstrated rather easily using rodent models that contrast a static GI tract with a flowing GI tract, as well as with a controlled-release dosage form having synchronous release of C10 and drug [14].Expanding upon this simple observation, a variety of innovative studies have been reported that provide insight into the pharmacodynamics of sodium caprate [15]. The most useful of these data come from animal models and human trials using solution presentations of formulations, typically through the use of ports or catheter-intubations, wherein the timing and the amount of enhancer released (relative to the drug and GI tract location) were directly controlled. From the understanding of sodium caprate’s pharmacodynamics, criteria for engineering of an effective controlled-release solid dosage form were established. The three key criteria determined were: ▪ A threshold amount of Caprate is required to open the paracellular junctions;▪ The effect has a limited duration;▪ In oligonucleotide dose ranges >1 mg/kg, no dose dependency is noted (i.e., a first-order absorption process) [16].Using these insights, dosage forms were prototyped and characterized in vitro and in vivo in dogs. Attention was paid to both the release characteristics of the drug and the penetration enhancer; either of which do not necessarily need to occur at the same time, place or rate. Selected dosage forms (having different release profiles) were studied clinically in normal, healthy human volunteers. p.o. absorption of the 2´-O-(2-methoxyethyl) modified antisense oligonucleotide, was demonstrated to have an average 9.5% plasma bioavailability across four formulations tested. The greatest average performance achieved for a single formulation was 12% bioavailability, with an individual dose and subject range of 2–28% [5]. Fortunately in the case of most NABTs, the dose-to-dose and patient-to-patient variability is acceptable because of the broad therapeutic window and the long pharmacodynamic half-lives. Nonetheless, we would expect these variations to decrease as the absolute bioavailability for advanced formulations increase.Questions of immediate interest & where do we go next?The question of the safety of using a penetration enhancer as a formulation adjunct is fair and appropriate as this adjuvant/excipient is intended to have pharmacological activity. One of the most advanced penetration enhancers, indeed registered in certain regions of the world, is capric acid also known as C10. Excellent review articles have been published, which collect valuable information for the safe and effective use of this enhancer [11,17]. Directly in conjunction with NABT p.o. dosing, caprate was generally well tolerated in clinical studies with up to 3 months of daily dosing [4]. Based upon this body of data, it would be prudent for anyone exploring this route of dosing a NABT to start with this enhancer.The next question may well be, ‘Is there a better formulation, one which achieves a higher oral bioavailability?’ There are certainly a number of other penetration enhancing agents, which accomplish the same objective of opening the paracellular junction [18]. It is conceivable that an alternative permeation enhancer would offer greater formulatability, permeation enhancer action, improved safety profile and bioavailabilities approaching 25%.Also unanswered at this time, is the question of the impact of a greatly decreased dose. Is there a point where alternate mechanisms become important to the p.o. uptake of a NABT? In relation to this point it has been recently reported that miRNA can be taken up into the circulation at very low levels, and in at least one example, influence upon circulating macrophages has been reported [2]. These reports are consistent with the GI tract sampling of the gut-associated lymph tissue system but, at this time, do not seem to offer a therapeutic opportunity for most NABTs.ConclusionConvergent increases in absolute bioavailability resulting from formulation efforts and the decreased cost of therapy arising from increased potencies and lower cost of goods would point to the ultimate success of orally delivering future NABTs. However, the most current development programs are still emerging from low-potency and high-cost-of-goods programs, so it may be a while before we see real reports of clinical development of p.o. dosage forms of NABTs.Financial & competing interests disclosureThe author is a consultant offering advice in the subject matter discussed in the article. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.References1 Hardee GE, Lindholm MW, Hansen HF et al. Pharmacology, target reduction and drug accumulation after oral feeding of an LNA antisense oligonucleotide directed against apoB-100. Presented at: Arteriosclerosis, Thrombosis and Vascular Biology. San Francisco, CA, USA, April 2010.Google Scholar2 Aouadi M, Tesz GJ, Nicoloro SM et al. Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation. Nature458,1180–1185 (2009).Crossref, Medline, CAS, Google Scholar3 Bennett FC, Swayze E, Geary RS et al.Antisense Oligonucleotide-Based Therapeutics, Gene and Cell Therapy (Second Edition). Marcel Decker Inc., NY, USA, 347–374 (2004).Google Scholar4 Hardee GE, Tillman LG, Geary RS. Routes and formulations for delivery of antisense oligonucleotides. Stanley TC(Ed). Antisense Drug Technology, Chapter 8. CRC Press, Taylor Francis Group, UK, 217–236 (2006).Google Scholar5 Tillman L, Geary R, Hardee GE. Oral delivery of antisense oligonucleotides: plasma bioavailability of ISIS 104838 in man. J. Pharm. Sci.97(1),225–236 (2008).Crossref, Medline, CAS, Google Scholar6 Patil SD, Rhodes DG, Burgess DJ. DNA-based therapeutics and DNA delivery systems: a comprehensive review. J. Am. Pharm. Sci.7(1),E61–E77 (2005).CAS, Google Scholar7 Hayashi M, Sakai T, Hasegawa Y et al. Physiological mechanism for enhancement of paracellular drug transport. J. Control. Release Soc.62,141–148 (1999).Crossref, Medline, CAS, Google Scholar8 Geary RS, Khatsenko O, Bunker K et al. Absolute bioavailability of 2´-O-(2-methoxyethyl)-modified antisense oligonucleotides following intraduodenal instillation in rats. J. Pharmacol. Exp. Ther.296(3),898–904 (2001).Medline, CAS, Google Scholar9 Nicklin PL, Bayley D, Giddings J et al. Pulmonary bioavailability of a phosphorothioate oligonucleotide (CGP 64128A): comparison with other delivery routes. Pharm. Res.15(4),583–591 (1998).Crossref, Medline, CAS, Google Scholar10 Salama NN, Eddington ND, Fasano A. Tight junction modulation and its relationship to drug delivery. Adv. Drug Deliv. Rev.58,15–28 (2006)Crossref, Medline, CAS, Google Scholar11 Maher S, Leonard TW, Jacobsen J et al. Safety and efficacy of sodium caprate in promoting drug absorption: from in vitro to the clinic. Adv. Drug Deliv. Rev.61,1427–1449 (2009).Crossref, Medline, CAS, Google Scholar12 Tsutsumi K, Li SK, Hymas RV et al. Systematic studies on the paracellular permeation of model permeants and oligonucleotides in the rat small intestine with chenodeoxycholate as enhancer. J. Pharm. Sci.97(1),350–367 (2008).Crossref, Medline, CAS, Google Scholar13 Raoof AA, Chiu P, Ramtoola, Z et al. Oral bioavailability and multiple dose tolerability of an antisense oligonucleotide tablet formulated with sodium caprate. J. Pharm. Sci.93(6),1431–1439 (2004).Crossref, Medline, CAS, Google Scholar14 Baluom M, Friedman M, Assaf P et al. Synchronized release of sulpiride and sodium decanoate from HPMC matrices: a rational approach to enhance sulpiride absorption in the rat intestine. Pharm. Res.17,1071–1076 (2000)Crossref, Medline, CAS, Google Scholar15 Cano-Cebrian MJ, Zornoza T, Granero L et al. Intestinal absorption enhancement via the paracellular route by fatty acids, chitosans and others: a target for drug delivery. Curr. Drug Deliv.2,9–22 (2005).Crossref, Medline, CAS, Google Scholar16 Geary RS, Teng CL, Raoof AA et al. Cross-species comparison of the tolerability and effect of absorption enhancer on systemic delivery of antisense oligonucleotides following intra-intestinal administration. Presented at: AAPS Annual Meeting. Indianapolis, IN, USA, 29 October–2 November 2000.Google Scholar17 Cox AB, Rawlinson LA, Baird AW et al.In vitro interactions between the oral absorption promoter, sodium caprate (C10) and S. typhimurium in rat intestinal ileal mucosae. Pharm. Res.25(1),114–122 (2008).Crossref, Medline, CAS, Google Scholar18 Whitehead K, Karr N, Mitragotri S. Safe and effective permeation enhancers for oral drug delivery. Pharm. Res.25,1782–1788 (2007).Crossref, Medline, Google ScholarFiguresReferencesRelatedDetailsCited ByCan colorectal delivery technology provide a platform for enteral oligonucleotide-based therapeutics?Drug Discoveries & Therapeutics, Vol. 10, No. 5Influence of diverse chemical modifications on the ADME characteristics and toxicology of antisense oligonucleotides2 March 2013 | Expert Opinion on Biological Therapy, Vol. 13, No. 6 Vol. 3, No. 2 Follow us on social media for the latest updates Metrics History Published online 2 February 2012 Published in print February 2012 Information© Future Science LtdKeywordscost of therapynucleic acid-based therapeuticsoligonucleotideoral bioavailabilityoral deliverypenetration enhancersFinancial & competing interests disclosureThe author is a consultant offering advice in the subject matter discussed in the article. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download

LOX-1 Inhibition in ApoE KO Mice Using a Schizophyllan-based Antisense Oligonucleotide Therapy

Takedatsu et al.1 in their article titled “A new therapeutic approach using a schizophyllan-based drug delivery system for inflammatory bowel disease” recently reported a novel therapeutic approach for inflammatory bowel disease using an innovative delivery system for antisense oligonucleotides (ODNs) that is based on Schizophyllan (SPG), a polysaccharide that belongs to the β-(1-3) glucan family.1 These authors demonstrated that this system has several advantages over the effective suppression of targeted RNA. The SPG complex is stable in vivo and is not a substrate for deoxyribonuclease; furthermore, it is efficiently taken up by macrophages through the dectin-1 receptor. In particular, Takedatsu et al.1 administered an antisense migration inhibitory factor/SPG complex that significantly ameliorated intestinal inflammation in a mouse model.1 The development of this new delivery system is very important because therapeutic applications of antisense ODNs have recently been shown to be effective against many different types of disease, including inflammatory, oncological, and genetic disorders. In particular, the use of antisense ODNs exploiting antisense-mediated exon skipping, has been successfully developed to modulate splicing of the dystrophin gene in Duchenne muscular dystrophy.2 Here, we utilized SPG to inhibit the lectin-like oxidized low-density lipoprotein (LDL) receptor 1 (LOX-1) gene in vivo as a possible treatment for atherosclerosis. LOX-1, which is encoded by the oxidized LDL receptor 1 (OLR1) gene,3 is the major receptor for ox-LDL in endothelial cells. As such, LOX-1 is responsible for the binding, uptake, and degradation of oxLDLs.4 LOX-1 is also expressed in macrophages, vascular smooth muscle cells, platelets, and cardiomyocytes.4 Multiple lines of evidence have implicated LOX-1 in the pathogenesis of atherosclerosis,5 and LOX-1 has emerged as a promising therapeutic target for atherosclerosis and related diseases within the last several years.6 Specific antisense ODNs targeting LOX-1 mRNA have previously been used for in vitro experiments to study the pathophysiological relevance of LOX-1.7 Here, we extend these findings, showing for the first time that LOX-1 antisense ODNs are effective as a therapeutic strategy in vivo. In collaboration with NapaJen (NapaJen Pharma, Koganei, Japan) and as described by Takedatsu et al.,1 we created a new SPG complex consisting of an antisense phosphorothioate ODN targeting the coding sequence (exon 7) of the murine Olr1 mRNA and two single SPG chains (SPG/Olr1AS). The inhibitory effect of the SPG/Olr1AS complex was first assessed in vitro in RAW 264.7 murine macrophages. RAW 264.7 cells were transfected with increasing concentrations of SPG/Olr1AS complex (from 25 to 800 nmol/l) or a scramble control ODN/SPG complex (SPG/CTRL). At 24 hours after transfection, RNA and proteins were extracted, and LOX-1 expression was assessed. We observed a decrease in LOX-1 mRNA and protein levels at all SPG/Olr1AS concentrations tested, but LOX-1 downregulation was most significant at 25 nmol/l SPG/Olr1AS (P < 0.05) (Figure 1). At this concentration, mRNA and protein expression levels were reduced to ~60 and 80%, respectively, compared with nontransfected cells. Notably, the SPG/CTRL complex had no effect (Figure 1). Figure 1 LOX-1 downregulation in RAW 264.7 cells. The incubation of RAW264.7 cells with SPG/ASOlr1 (25 nmol/l) for 24 hours decreased the basal expression of LOX-1 mRNA and protein, as determined by (a) qRT-PCR and (b) western blot analysis. LOX-1 mRNA ... These in vitro data, demonstrating that even the lowest concentration of SPG/Olr1AS tested was able to inhibit the expression of the LOX-1 receptor, prompted us to investigate its activity in vivo in a mouse model of atherosclerosis, the apolipoprotein E knock out (ApoE−/−) mouse. We evaluated the efficacy of two different doses of the SPG complex (0.05 and 0.005 mg/kg body weight); both dosages were lower than those used for inflammatory bowel disease.1 This investigation conformed to the European Commission Directive 86/609/EEC and was approved by the local ethical committee (Progetto di Ricerca 2009/3). At 8 weeks of age, ApoE−/− mice were fed ad libitum with a western type diet (21% fat, 0.15% cholesterol, and 19.5% casein; Harlan, Bresso, Italy) for 8 weeks. Then, SPG complexes were administrated once a day, by intraperitoneal injection, for three consecutive days. The mice were euthanized with an overdose of Avertin 2.5% (Sigma Aldrich, St Louis, MO), followed by cervical dislocation on the fourth day.8 The heart and the arterial tree were perfused with saline solution under physiological pressure. The LOX-1 mRNA and protein expression in the aorta (from the last part of the ascending up to the thoracic aorta) was analyzed by quantitative reverse transcription-PCR and by western blot. Each mouse was weighed before and after treatments, and there was no significant change in total body weight in any of the groups. We observed a significant downregulation of LOX-1 mRNA and protein in the aorta of mice treated with 0.05 mg/kg SPG/Olr1AS (Figure 2a–c). In particular, we found a 63% reduction in the LOX-1 protein level in aortas of mice treated with SPG/Olr1AS compared with phosphate-buffered saline and CTRL (Figure 2b). Neither phosphate-buffered saline nor a scramble ODN SPG complex (SPG/CTRL) had any effect on LOX-1 expression (Figure 2a–c). Interestingly, we also observed a significant decrease in LOX-1 mRNA and protein at a lower dosage of SPG/Olr1AS (0.005 mg/kg) (Figure 2d–f). However, at this concentration of SPG/Olr1AS complex, the LOX-1 protein level in the mice aortas was decreased by 32% compared with controls (Figure 2f). To assess the possible proinflammatory effect of SPG complex both in vitro and in vivo, we also investigated tumor necrosis factor-α and nuclear factor-κB expression in RAW 264.7 cells and aortic tissues. Consistent with Takedatsu et al.,1 we did not observe any increase in these proinflammatory molecules (data not shown). Furthermore, no significant differences in mean triacylglycerol level, total cholesterol, and cholesterol fractions were observed in the different treatment groups. This result was not unexpected because we used an acute treatment. Figure 2 LOX-1 downregulation in ApoE−/− mice. The effects of the two different dosages of SPG/Olr1AS on the expression of (a,d) LOX-1 mRNA and (b,c,e,f) protein in ApoE−/− aortas. β-Actin was used as the loading control. ... In summary, our data indicate that the inhibition of LOX-1 can be beneficial in atherosclerotic disease. Moreover, this study exploits a novel delivery system for antisense ODNs with good in vivo efficacy at very low concentrations. The efficacy of low SPG complex concentrations, as shown by our study, was confirmed by in vivo experiments using SPG/migration inhibitory factor complex at 0.007–0.0007 mg/kg body weight (Y. Koyama, personal communication). These data suggest that humans, like mice, will benefit from lowering LOX-1 activity. Thus, SPG should be considered as an innovative and useful delivery system to reduce the inflammation process in atherosclerosis and cardiovascular diseases.

Abstract LB-302: Inhibition of STAT3 following systemic delivery of STAT3 antisense oligonucleotide results in target inhibition in tumor and tumor-associated stromal cells and dramatic reduction in circulating pro-tumorigenic cytokines

Abstract Constitutive activation of STAT3 has been documented in a broad range of neoplastic tissues. STAT3 is a transcription factor that regulates the expression of numerous genes involved in the proliferation, survival, and metastasis of tumor cells. In addition, STAT3 contributes to tumorigenesis by modulating immune responses mediated by tumor stromal cells in the tumor microenvironment. The immuno-suppressive, pro-tumorigenic effects of STAT3 are regulated by induction or inhibition of several key cytokines. However STAT3 remains a difficult target to drug with conventional approaches. Here, we have studied the effects of selective downregulation of STAT3 in various cancer settings by optimized antisense oligonucleotides (ASOs). We assessed the effects of STAT3 downregulation on the expression of cytokines and the progression of cancer. Initially, the safety of inhibiting STAT3 was demonstrated in tolerability studies, where no notable toxicities were observed despite near complete abrogation of STAT3 expression in the liver following systemic delivery of ASO in both rodents and in non-human primate. ASO treatment led to STAT3 inhibition in tumor cells and several non-tumor stromal cell populations of the tumor. In addition, dramatic reduction in key pro-tumorigenic cytokines including IL-6 and IL-1b was observed in C26 colon cancer-bearing animals, which correlated with the decrease of STAT3 level in tumor and stromal cells. This result suggests that STAT3 might not only transmit the IL-6 signal from upstream pathway, but regulates IL-6 level possibly in an auto-feedback manner. Downregulation of STAT3 also led to the inhibition of IL-6-induced C-reactive protein (CRP) expression, which was recently reported to play a key role in the progression of multiple myeloma (MM). Moreover, inhibition of STAT3 resulted in a significant delay in tumor growth in STAT3-dependent MDA-MB-231 breast cancer and U251 glioblastoma xenograft models following systematic ASO treatment. The results from this study collectively suggest that STAT3 expression can be specifically modulated by ASOs in vivo tumor models and that in addition to effects in tumor cells STAT3 ASOs regulate the balance of tumorigenic cytokines in the tumor microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-302. doi:10.1158/1538-7445.AM2011-LB-302

Survivin antisense oligonucleotide mediated by polyamidoamine dendrimer induces apoptosis of colorectal cancer cells

Objective To investigate the possibility of polyamidoamine dendrimer for Survivin Antisense Oligonucleotide delivery system and explore the effects of polyamidoamine dendrimer-Survivin antisense oligonucleotide on the Survivin expression and apoptosis of human colorectal cancer cell line SW620. Methods The PAMAM anti-gene transfection complex and the liposome anti-gene transfection complex was manufactured. The shape of the complex were observed by transmission electron microscope, the size was determined by laser particle size analysator and the zeta potential was measured by analytical tool. The encapsulating efficiency, DNA loading level, release progress in vitro were determined by ultravio- let spectrophotometer in centrifuging method. The transfection efficiency of Survivin-asODN in SW620 cells was mearsured. The protein expression of Survivin was measured by Western blot analysis and the apoptosis rate of SW620 cells was assessed by flow cytometry. Results The diameter of PAMAM-Survivin-asODN complex was less than that of liPosome-Survivin-asODN complex(P < 0.01 ), the zeta potential was high than that for liposome-Survivin-asODN complex(P < 0.05 ). There was no significant difference between two groups for envelopment ratio and loaded dosage. The PAMAM' s release progress in vitro lasted 14 days and 5 days for the liposome. The transfection efficiency for PAMAM-Survivin-asODN was higher than that for liposome-Survivin-asODN (P < 0.05 ). The expression of Survivin protein for PAMAM-Survivin-asODN was less than that for liposome-Survivin-asODN(P <0. 05)and the cell apoptosis rate was higher than that for liposome-Survivin-asODN (P < 0. 01 ). Conclusion The polyamidoamine dendrimer can delivery Sur-vivin-asODN into SW620 cells effectively and induce SW620 cells apoptosis. Key words: Polyamidoamine dendrimer;  Antisenze oligonucleotides (aSODN) ;  Colorectal

Chitosan-coated PLGA nanoparticles for DNA/RNA delivery: effect of the formulation parameters on complexation and transfection of antisense oligonucleotides

Cationically modified poly( d, l-lactide- co-glycolide) (PLGA) nanoparticles have recently been introduced as novel carriers for DNA/RNA delivery. The colloidal characteristics of the nanoparticles—particle size and surface charge—are considered the most significant determinants in the cellular uptake and trafficking of the nanoparticles. Therefore, our aim was to introduce chitosan-coated PLGA nanoparticles, whose size and charge are tunable to adapt for a specific task. The results showed that biodegradable nanoparticles as small as 130 nm and adjustable surface charge can be tailored controlling the process parameters. As a proof of concept, the overall potential of these particulate carriers to bind the antisense oligonucleotides, 2′- O-methyl-RNA, and improve their cellular uptake was demonstrated. The study proved the efficacy of chitosan-coated PLGA nanoparticles as a flexible and efficient delivery system for antisense oligonucleotides to lung cancer cells.

Albumin–protamine–oligonucleotide nanoparticles as a new antisense delivery system. Part 1: Physicochemical characterization

In this paper, a ternary system of albumin–protamine–oligonucleotide nanoparticles (AlPrO-NP) recently developed by Vogel et al. [V. Vogel, D. Lochmann, J. Weyermann, G. Mayer, C. Tziatzios, J.A. van den Broek, W. Haase, D. Wouters, U.S. Schubert, J. Kreuter, A. Zimmer, D. Schubert, Oliganucleotide–protamin–albumin nanoparticles: preparation, physical properties and intracellular processing, J. Controlled Rel. (in press)] [1] which could serve as a potential drug delivery system for antisense oligonucleotides. Former studies of binary protamine-oligonucleotide nanoparticles showed two main disadvantages: (i) aggregation of the particles within a few minutes in the presence of salt; (ii) low intracellular dissociation between protamine and oligonucleotide, especially phosphorothioates. To overcome these problems, human serum albumin (HSA) as a non-toxic, biodegradable macromolecule was introduced as protective colloid. The assembly process of AlPrO-NP was investigated by small angle X-ray scattering (SAXS), fluorescence correlation spectroscopy (FCS), photon correlation spectroscopy (PCS) measurements and scanning electron microscopy (SEM). ‘Initial complexes’ of HSA and protamine sulphate with a mean hydrodynamic diameter ( d h) of about 10–14 nm were found. After adding oligonucleotides (unmodified, phosphorothioate DNA and small interfering RNA), nanoparticles (NPs) were assembled in water and in isotonic media with a d h in a range of 230–320 nm for most preparations. The chemical composition of the particles was investigated by high performance liquid chromatography and fluorescence spectrometry. The whole amount of oligonucleotides (30 μg) was entrapped into the particles at a 1:2 mass ratio (oligonucleotide/protamine). Approximately 7–10% (w/w) of the HSA was bound to the particles. The surface charge of the particles ranged from about +12 to −60 mV depending on the protamine concentration and the ionic conditions. The size and the molecular weight of the components, initial complexes and two model NP preparations were calculated from FCS data. These data verified the PCS, SEM and SAXS measurements.

Albumin nanoparticles improved the stability, nuclear accumulation and anticytomegaloviral activity of a phosphodiester oligonucleotide

The goal of this study was to evaluate the potential of albumin nanoparticles as a delivery system for antisense oligonucleotides. Nanoparticles were prepared by a coacervation process and cross-linkage with glutaraldehyde. Phosphodiester (PO) and phosphorotioate (PS) oligonucleotides were either adsorbed on the surface of nanoparticles (PO-NPA and PS-NPA) or incorporated in the nanoparticle matrix (PO-NPB and PS-NPB). When PO-loaded nanoparticles were incubated with phosphodiesterase, only NPB was able to keep the oligonucleotide hybridization capability for at least 60 min. The antiviral activity was evaluated in MRC-5 fibroblasts infected with human cytomegalovirus at a MOI of 0.0035. Both PO nanoparticle formulations significantly increased the antiviral activity of free PO ( P<0.001) and NPB showed slightly higher efficacies than NPA ( P<0.05). On the other hand, PS exhibited significant higher activity than free PO ( P<0.001), however, no significant differences were found between PS-nanoparticle and PO-nanoparticle formulations. These findings were well correlated with the intracellular distribution observed for fluorescent oligonucleotide-loaded albumin nanoparticles. Even these carriers delayed and decreased the uptake of PO by MRC-5 cells, they finally induced a diffused cytoplasmic distribution and major nuclear accumulation. In summary, albumin nanoparticles partially protected a PO against enzymatic degradation and improved their presence in the nucleus and thus, increased its efficiency.

Targeted delivery system for antisense oligonucleotides: a novel experimental strategy for neuroblastoma treatment

Neuroblastoma (NB) is the most common neuroectoderma derived solid tumour of paediatric age. Since conventional treatments are often inefficient, novel therapeutic interventions are required. Among these, the use of antisense oligonucleotides (asODNs) as therapeutic antineoplastic agents has been recently investigated. Oligonucleotide in vivo applicability is impaired from their high sensitivity to cellular nuclease degradation. Encapsulating them within liposomes could nevertheless increase their stability. C-myb gene expression has been reported in several solid tumours of different embryonic origin, including NB, where it is linked to cell proliferation and/or differentiation. We performed a new technique to encapsulate c-myb antisense oligonucleotides within lipid particles. Liposomes resulting from this technique were called coated cationic liposomes (CCLs), since they were made up of a central core of a cationic phospholipid bound to myb-asODNs, and an outer shell of neutral lipids. A monoclonal antibody (mAb) specific for the neuroectoderma antigen disialoganglioside GD 2, has been covalently coupled to their external surface. The resulting anti-GD 2-targeted CCLs showed high loading efficiency for the asODNs, small particle size and good stability. In vitro, they were able to deliver myb-asODNs selectively to GD 2-positive NB cell lines more efficiently than non-targeted liposomes or free asODNs. Consequently, targeted formulations showed greater inhibition of cell proliferation than non-targeted formulations or free asODNs. Furthermore, we demonstrated that the inhibition of cell proliferation was dependent on the down-modulation of c-myb protein expression. Pharmacokinetic studies showed that these targeted liposomal formulations were long circulating in blood. Biodistribution studies presented differences between the free and the encapsulated myb-as ODN profiles, as well. While free myb-as ODNs are widely distributed (mainly liver, kidney and spleen) even after 30 min post-injection, myb-as ODN entrapped into CCL or anti-GD 2-CCL presents only an accumulation in the spleen after 24 h. Future studies will be performed to evaluate the antitumour efficacy of the above formulations in animal models.

Regulators of serine/threonine protein phosphatases at the dawn of a clinical era?

Reversible phosphorylation is a key mechanism for regulating the biological activity of many human proteins that affect a diverse array of cellular processes, including protein-protein interactions, gene transcription, cell-cycle progression and apoptosis. Once viewed as simple house keeping enzymes, recent studies have made it eminently clear that, like their kinase counterparts, protein phosphatases are dynamic and highly regulated enzymes. Therefore, the development of compounds that alter the activity of specific phosphatases is rapidly emerging as an important area in drug discovery. Because >98% of protein phosphorylation occurs on serine and threonine residues, the identification of agents that alter the activity of specific serine/threonine phosphatases seems especially promising for drug development in the future. This review is focused on the enzymes encoded by the PPP-gene family, which includes PP1, PP2A, PP2B, PP4, PP5, PP6 and PP7. The structure/functions of human phosphatases will be addressed briefly, as will the natural product inhibitors of PP1-PP6 (e.g. okadaic acid, microcystins, nodularin, cantharidin, calyculin A, tautomycin, and fostriecin). The development of chimeric antisense oligonucleotides that support RNAase H mediated degradation of the targeted mRNA has resulted in compounds capable of specifically suppressing the expression of PP5 (ISIS 15534) and PP1gamma 1 (ISIS 14435) in human cells. Such compounds have already proven useful for the validation of drug targets, and if difficulties associated with systemic delivery of antisense oligonucleotides can be overcome, antisense is poised to have a major impact on the clinical management of many human disorders.

A Lipid-based Delivery System for Antisense Oligonucleotides Derived from a Hydrophobic Complex

Antisense oligodeoxynucleotides (ASOs) prevent expression of proteins by binding to specific regions of mRNA. This report investigates a potential lipid-based delivery system for ASO. A hydrophobic complex was recovered following addition of cationic lipids to ASOs in a Bligh and Dyer monophase [chloroform/methanol/water (1:2.1:1, v/v/v)]. The addition of monovalent cationic lipids (dioleyldimethylammonium chloride, dimethyldioctadecylammonium bromide, dioleoyltrimethylammonium propane), resulted in >95% recovery of the ASOs from the organic phase when ASO phosphate charge was neutralized. Cholesteryldimethylaminoethylcarbamate mediated efficient extraction at a charge ratio (+/ −) >5.2. ASOs could not be extracted into the organic phase by the polyvalent lipids, dioctadecylamidoglycyl spermine and 2,3-dioleyloxy- N -[2(sperminecarboxamido)ethyl]- N, N -dimethyl-1-propaminium trifluoroacetate, even at a charge ratio (+/ −) >5. Dioleoylphosphatidylethanolamine, but not dioleoylphosphatidylcholine, prevented formation and destabilized the hydrophobic complexes. The characterization of the hydrophobic complex led to the development of lipid-ASO particles containing dioleyldimethylammonium chloride, dioleoylphosphatidylethanolamine and poly(ethylene glycol)-conjugated phosphatidylethanolamine (LAPs). When FITC-labeled ASOs in LAPs were added to B-cell lymphoma cells (DoHH2) in vitro, cell-associated ASO decreased as poly(ethylene glycol)-conjugated phosphatidylethanolamine incorporation increased. Western Blot analysis demonstrated that no significant downregulation of Bcl-2 protein was observed when using LAPs. The results suggest that the use of stabilized PEG-conjugated lipids may be detrimental for cationic lipid-based ASO delivery.

Delivery to cancer cells of antisense L‐myc oligonucleotides incorporated in fusogenic, cationic‐lipid‐reconstituted influenza‐virus envelopes (cationic virosomes)

Antisense oligodeoxy-nucleoside phosphorothioates (OPTs) of L-myc were encapsulated into reconstituted influenza-virus-A envelopes (virosomes). The envelopes of the virosomes consisted of a single positively charged (cationic) lipid bilayer. Binding of cationic virosomes to cellular receptors that are membrane glycoproteins or glycolipids containing terminal sialic acid is mediated by the hemagglutinin glycoprotein (HA) of the influenza virus. After internalization through receptor-mediated endocytosis, cationic virosomes fuse efficiently with the membranes of the endosomal-cell compartment, and as a consequence the encapsulated OPT are delivered to the cell cytoplasma. Examination by fluorescence microscopy of the cellular uptake of cationic virosomes containing fluorescein-labeled OPT showed rapid and efficient incorporation of virosomes. Addition of cationic virosomes (75–150 μl) containing antisense L-myc OPT in the picomolar range to small-cell-lung-cancer (SCLC) cell cultures that expressed highly the L-myc oncogene led to strong inhibition of thymidine incorporation in a concentration-dependent manner. Virosome-entrapped sense L-myc OPT and random-order OPT had only minimal effects on the thymidine uptake. Cells of SCLC cell line NCI-H82 expressing a very low level of L-myc were not affected by antisense-L-myc virosomes. In Western-blot analysis, expression of L-myc protein was suppressed in the antisense-virosome-treated NCI-H209 cells but not in untreated control NCI-H209 cells. These results suggest that cationic virosomes may have great potential as an efficient delivery system for antisense oligonucleotides in cancer therapy. Int. J. Cancer 77:728–733, 1998. © 1998 Wiley-Liss, Inc.

Delivery to cancer cells of antisense L-myc oligonucleotides incorporated in fusogenic, cationic-lipid-reconstituted influenza-virus envelopes (cationic virosomes).

Antisense oligodeoxy-nucleoside phosphorothioates (OPTs) of L-myc were encapsulated into reconstituted influenza-virus-A envelopes (virosomes). The envelopes of the virosomes consisted of a single positively charged (cationic) lipid bilayer. Binding of cationic virosomes to cellular receptors that are membrane glycoproteins or glycolipids containing terminal sialic acid is mediated by the hemagglutinin glycoprotein (HA) of the influenza virus. After internalization through receptor-mediated endocytosis, cationic virosomes fuse efficiently with the membranes of the endosomal-cell compartment, and as a consequence the encapsulated OPT are delivered to the cell cytoplasma. Examination by fluorescence microscopy of the cellular uptake of cationic virosomes containing fluorescein-labeled OPT showed rapid and efficient incorporation of virosomes. Addition of cationic virosomes (75-150 microl) containing antisense L-myc OPT in the picomolar range to small-cell-lung-cancer (SCLC) cell cultures that expressed highly the L-myc oncogene led to strong inhibition of thymidine incorporation in a concentration-dependent manner. Virosome-entrapped sense L-myc OPT and random-order OPT had only minimal effects on the thymidine uptake. Cells of SCLC cell line NCI-H82 expressing a very low level of L-myc were not affected by antisense-L-myc virosomes. In Western-blot analysis, expression of L-myc protein was suppressed in the antisense-virosome-treated NCI-H209 cells but not in untreated control NCI-H209 cells. These results suggest that cationic virosomes may have great potential as an efficient delivery system for antisense oligonucleotides in cancer therapy.