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Nucleic acid-based therapeutics hold great promise for the treatment of numerous diseases, including neuromuscular disorders, such as Duchenne muscular dystrophy (DMD). Some antisense oligonucleotide (ASO) drugs have already been approved by the US FDA for DMD, but the potential of this therapy is still limited by several challenges, including the poor distribution of ASOs to target tissues, but also the entrapment of ASO in the endosomal compartment. Endosomal escape is a well recognized limitation that prevents ASO from reaching their target pre-mRNA in the nucleus. Small molecules named oligonucleotide-enhancing compounds (OEC) have been shown to release ASO from endosomal entrapment, thus increasing ASO nuclear concentration and ultimately correcting more pre-mRNA targets. In this study, we evaluated the impact of a therapy combining ASO and OEC on dystrophin restoration in mdx mice. Analysis of exon-skipping levels at different time points after the co-treatment revealed improved efficacy, particularly at early time points, reaching up to 4.4-fold increase at 72 h post treatment in the heart compared to treatment with ASO alone. Significantly higher levels of dystrophin restoration were detected two weeks after the end of the combined therapy, reaching up to 2.7-fold increase in the heart compared to mice treated with ASO alone. Moreover, we demonstrated a normalization of cardiac function in mdx mice after a 12-week-long treatment with the combined ASO + OEC therapy. Altogether, these findings indicate that compounds facilitating endosomal escape can significantly improve the therapeutic potential of exon-skipping approaches offering promising perspectives for the treatment of DMD.

Cell-penetrating peptides (CPPs) are routinely used for the delivery of macromolecules into live human cells. To enter the cytosolic space of cells, CPPs typically permeabilize the membrane of endosomes. In turn, several approaches have been developed to increase the endosomal membrane permeation activity of CPPs so as to improve delivery efficiencies. The endocytic pathway is, however, important in maintaining cellular homeostasis, and understanding how endosomal permeation impacts cells is now critical to define the general utility of CPPs. Herein, we investigate how CPP-based delivery protocols affect the endocytic network. We detect that, in some cases, cell penetration induces the activation of Chmp1b, Galectin-3, and TFEB, which are components of endosomal repair, organelle clearance, and biogenesis pathways, respectively. We also detect that cellular delivery modulates endocytosis and endocytic proteolysis. Remarkably, a multimeric analogue of the prototypical CPP TAT permeabilizes endosomes efficiently without inducing membrane damage responses. These results challenge the notion that reagents that make endosomes leaky are generally toxic. Instead, our data indicates that it is possible to enter cells with minimal deleterious effects.

Ineffective cellular delivery is a common problem in numerous biological applications. Developing delivery reagents that work robustly in a variety of experimental settings remains a challenge. Herein, we report how peptides derived from the prototypical cell penetrating peptide TAT can be used in combination with a small molecule, UNC7938, to deliver macromolecules into the cytosol of cells by a simple co-incubation protocol. We establish successful delivery of peptides, DNA plasmids, and a single-chain variable fragment antibody. We also demonstrate that delivery works in hard-to-transfect mammalian cells and under conditions typically inhibitory to cell-penetrating peptides. Mechanistically, UNC7938 destabilizes the membrane of endosomes. This, in turn, enhances the endosome-leakage activity of cell-penetrating peptides and facilitates the endosomal escape of macromolecules initially internalized by mammalian cells via endocytosis. This combined selective membrane-destabilization represents a new chemical space for delivery tools and provides a novel solution to the problem of endosomal entrapment that often limits the effectiveness of reagent-based delivery approaches.

Signal transduction pathways play key roles in the initiation, progression and dissemination of cancer. Thus, signaling molecules are attractive targets for cancer therapeutics and enormous efforts have gone into the development of small molecule inhibitors of these pathways. However, regrettably, there has been only moderate progress to date, primarily in connection with the RAS signaling pathway. Oligonucleotide-based drugs potentially offer several advantages for addressing signaling pathways, including their exquisite selectivity and their ability to exploit both enzymatic and nonenzymatic targets. Nonetheless, there are problems inherent in the oligonucleotide approach, not the least being the challenge of effectively delivering these complex molecules to intracellular sites within tumors. This survey article will provide a selective review of recent studies where oligonucleotides were used to address cancer signaling and will discuss both positive aspects and limitations of those studies. This will be set in the context of an overview of various cancer signaling pathways and small molecule approaches to regulate those pathways. The survey will also evaluate the challenges and opportunities implicit in the oligonucleotide-based approach to cancer signaling and will point out several possibilities for future research.

The pharmacological effects of antisense and siRNA oligonucleotides are hindered by the tendency of these molecules to become entrapped in endomembrane compartments thus failing to reach their targets in the cytosol or nucleus. We have previously used high throughput screening to identify small molecules that enhance the escape of oligonucleotides from intracellular membrane compartments and have termed such molecules OECs (oligonucleotide enhancing compounds). Here, we report on the structure–activity relationships of a family of OECs that are analogs of a hit that emerged from our original screen. These studies demonstrate key roles for the lipophilic aromatic groups, the tertiary nitrogen, and the carbamate moiety of the parent compound. We have also investigated the intracellular site of action of the OECs and have shown that activity is due to the release of oligonucleotides from intermediate endosomal compartments rather than from early endosomes or from highly acidic downstream compartments. At high concentrations of OECs toxicity occurs in a manner that is independent of caspases or of lysosomal cathepsins but instead involves increased plasma membrane permeability. Thus, in addition to describing specific characteristics of this family of OECs, the current study provides insights into basic mechanisms of oligonucleotide trafficking and their implications for oligonucleotide delivery.

The pharmacological effectiveness of oligonucleotides has been hampered by their tendency to remain entrapped in endosomes, thus limiting their access to cytosolic or nuclear targets. We have previously reported a group of small molecules that enhance the effects of oligonucleotides by causing their release from endosomes. Here, we describe a second novel family of oligonucleotide enhancing compounds (OECs) that is chemically distinct from the compounds reported previously. We demonstrate that these molecules substantially augment the actions of splice switching oligonucleotides (SSOs) and antisense oligonucleotides (ASOs) in cell culture. We also find enhancement of SSO effects in a murine model. These new compounds act by increasing endosome permeability and causing partial release of entrapped oligonucleotides. While they also affect the permeability of lysosomes, they are clearly different from typical lysosomotropic agents. Current members of this compound family display a relatively narrow window between effective dose and toxic dose. Thus, further improvements are necessary before these agents can become suitable for therapeutic use.

The oligonucleotide therapeutics field has seen remarkable progress over the last few years with the approval of the first antisense drug and with promising developments in late stage clinical trials using siRNA or splice switching oligonucleotides. However, effective delivery of oligonucleotides to their intracellular sites of action remains a major issue. This review will describe the biological basis of oligonucleotide delivery including the nature of various tissue barriers and the mechanisms of cellular uptake and intracellular trafficking of oligonucleotides. It will then examine a variety of current approaches for enhancing the delivery of oligonucleotides. This includes molecular scale targeted ligand-oligonucleotide conjugates, lipid- and polymer-based nanoparticles, antibody conjugates and small molecules that improve oligonucleotide delivery. The merits and liabilities of these approaches will be discussed in the context of the underlying basic biology.

Addition of small molecule Retro-1 has been described to enhance antisense and splice switching oligonucleotides. With the aim of assessing the effect of covalently linking Retro-1 to the biologically active oligonucleotide, three different derivatives of Retro-1 were prepared that incorporated a phosphoramidite group, a thiol or a 1,3-diene, respectively. Retro-1&ndash, oligonucleotide conjugates were assembled both on-resin (coupling of the phosphoramidite) and from reactions in solution (Michael-type thiol-maleimide reaction and Diels-Alder cycloaddition). Splice switching assays with the resulting conjugates showed that they were active but that they provided little advantage over the unconjugated oligonucleotide in the well-known HeLa Luc705 reporter system.

The major research-intensive pharmaceutical companies (big pharma), are facing a profound crisis. Their R&D expenditures have grown at a staggering rate, while the creation of drugs that represent truly novel advances in therapy, so-called new molecular entities (NMEs), has stagnated. Here I present a comprehensive analysis of the NME crisis and discuss possible solutions. This includes proposals to fix the problem by increasing the efficiency of corporate R&D, as well as several alternative strategies for pharmaceutical research, including academic drug discovery centers and the public--private partnerships that have been so successful in attacking neglected diseases. Finally, a new policy initiative, the creation of non-profit drug development corporations (NPDDCs) designed to bring key aspects of early-stage drug development into the pre-competitive public arena is proposed. The creation of effective NPDDCs will be challenging, but promises to provide the health care system with less costly and more effective medicines. Copyright The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com, Oxford University Press.

The nascent field of nanomedicine has evoked enormous interest among physical and biological scientists and has already attracted hundreds of millions of dollars of research funding. The great appeal of nanomedicine lies in its promise of using the unique properties of nanoscale materials to address some of the most challenging problems of medical diagnosis and therapy. While some questions have been raised about the possible toxicities of nanomaterials, and about the ethical implications of applying these advanced, but undoubtedly expensive, technologies to medical practice, for the most part nanomedicine has enjoyed unalloyed enthusiasm from both its proponents in the research community and from policy makers. However, will the promise of nanomedicine actually be fulfilled? This paper discusses the internal intellectual culture of nanomedicine and how that gives rise to problems and contradictions that may slow its evolution and introduction into clinical practice. Copyright The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com, Oxford University Press.

We report the preparation and physical and biological characterization of human serum albumin-based micelles of approximately 30 nanometers diameter for the delivery of amphipathic drugs, represented by doxorubicin. The micelles were surface conjugated with cyclic RGD peptides to guide selective delivery to cells expressing the alpha v/beta 3 integrin. Multiple poly(ethylene glycol)s (PEGs) with molecular weight of 3400 daltons were used to form a hydrophilic outer layer, with the inner core formed by albumin conjugated with doxorubicin via disulfide bonds. Additional doxorubicin was physically adsorbed into this core to attain a high drug loading capacity, where each albumin was associated with about 50 doxorubicin molecules. The formed micelles were stable in serum but continuously released doxorubicin when incubated with free thiols at concentrations mimicking the intracellular environment. When incubated with human melanoma cells (M21+) that express the alpha v/beta 3 integrin, higher uptake and longer retention of doxorubicin was observed with the RGD-targeted micelles, than in the case of untargeted control micelles, or free doxorubicin. Consequently the RGD-targeted micelles manifested cytotoxicity at lower doses of drug than control micelles or free drug.

Gastrin-releasing peptide receptor (GRPR), a member of the G protein-coupled receptor superfamily, has been utilized for receptor-mediated targeting of imaging and therapeutic agents; here we extend its use to oligonucleotide delivery. A splice-shifting antisense oligonucleotide was conjugated to a bombesin (BBN) peptide, and its intracellular delivery was tested in GRPR expressing PC3 cells stably transfected with a luciferase gene interrupted by an abnormally spliced intron. The BBN-conjugate produced significantly higher luciferase expression compared to unmodified oligonucleotide, and this increase was reversed by excess BBN peptide. Kinetic studies revealed a combination of saturable, receptor-mediated endocytosis and non-saturable pinocytosis for uptake of the conjugate. The K(m) value for saturable uptake was similar to the EC(50) value for the pharmacological response, indicating that receptor-mediated endocytosis was a primary contributor to the response. Use of pharmacological and molecular inhibitors of endocytosis showed that the conjugate utilized a clathrin-, actin- and dynamin-dependent pathway to enter PC3 cells. The BBN-conjugate partially localized in endomembrane vesicles that were associated with Rab7 or Rab9, demonstrating that it was transported to late endosomes and the trans-golgi network. These observations suggest that the BBN-oligonucleotide conjugate enters cells via a process of GRPR mediated endocytosis followed by trafficking to deep endomembrane compartments.

Oligonucleotides manifest much promise as potential therapeutic agents. However, understanding of how oligonucleotides function within living organisms is still rather limited. A major concern in this regard is the mechanisms of cellular uptake and intracellular trafficking of both ‘free’ oligonucleotides and oligonucleotides associated with various polymeric or nanocarrier delivery systems. Here we review basic aspects of the mechanisms of endocytosis and intracellular trafficking and how insights from these processes can be used to understand oligonucleotide delivery. In particular we discuss opportunities for escape of oligonucleotides from endomembrane compartments and describe recent studies using small molecules to enhance oligonucleotide effects.

Cholesterol modified mono-, di-, and tetrameric oligonucleotides were synthesized and hybridized with antisense oligonucleotides to study their incorporation in cationic liposomes together with the influence of this dendrimeric delivery system on biological activity. Electrostatic interactions seem to play the most important role during complexation with cationic lipids. This oligonucleotide formulation gives a small but significant increase in the inhibition of P-glycoprotein expression in a cellular system.

The construction of nanomaterials from oligonucleotides by modular assembly invariably requires the use of branched nucleic acid architectures such as three- and four-way junctions (3WJ and 4WJ). We describe the stabilization of DNA 3WJ by using non-nucleotide lipophilic spacers to create a hydrophobic pocket within the junction space. Stabilization of nucleic acid junctions is of particular importance when constructing nanostructures in the "ultra-nano" size range (

The spliceosome machinery is composed of several proteins and multiple small RNA molecules that are involved in gene regulation through the removal of introns from pre-mRNAs in order to assemble exon-based mRNA containing protein-coding sequences. Splice-switching oligo-nucleotides (SSOs) are genetic control elements that can be used to specifically control the expression of genes through correction of aberrant splicing pathways. A current limitation with SSO methodologies is the inability to achieve conditional control of their function paired with high spatial and temporal resolution. We addressed this limitation through site-specific installation of light-removable nucleobase-caging groups as well as photocleavable backbone linkers into synthetic SSOs. This enables optochemical OFF → ON and ON → OFF switching of their activity and thus precise control of alternative splicing. The use of light as a regulatory element allows for tight spatial and temporal control of splice switching in mammalian cells and animals.

Antisense oligonucleotides are potentially powerful tools for selective control of cellular and viral gene expression. Crucial to successful application of this approach is the specificity of the oligonucleotide for the chosen RNA target. Here we apply DNA array technology to examine the specificity of antisense oligonucleotide treatments. The molecules used in these studies consisted of phosphorothioate oligomers linked to the Antennapedia (Ant) delivery peptide. The antisense oligonucleotide component was complementary to a site flanking the AUG of the MDR1 message, which codes for P-glycoprotein, a membrane ATPase associated with multidrug resistance in tumor cells. Using a DNA array of 2059 genes, we analyzed cellular responses to molecules comprised of Ant peptide-oligonucleotide conjugates, as well as to the Ant peptide alone. Besides the expected reduction in MDR1 message level, 37 other genes (∼2% of those tested) showed changes of comparable magnitude. The validity of the array results was confirmed for selected genes using Northern blots to assess messenger RNA levels. These results suggest that studies using antisense oligonucleotide technology to modulate gene expression need to be interpreted with caution.

Integrin-mediated adhesion to the extracellular matrix regulates the cellular response to mitogens. Anchorage-dependent growth factor activation of the extracellular signal-regulated kinase (ERK) is an intensely studied example of this regulation. Given the central role of ERK in mediating cell migration, division, and survival, it is also an extremely important example. Recent work has demonstrated that cell adhesion can regulate ERK signaling at several checkpoints and has begun to define the mechanism and consequences associated with anchorage-dependent effects on the ERK cascade.

The rapidly changing world of modern biomedical research is raising important new issues for traditional PhD training programs and is creating concern among young PhD scientists about their futures. Specifically, the United States is producing substantially more biomedical PhDs than can be accommodated in professional positions that truly require the PhD as a credential. The "surplus" PhD population is being relegated to poorly paid, unstable, and increasingly unsatisfying jobs. In addition, many current graduate and postdoctoral training programs may not be adequately preparing young scientists for the more complex, more quantitative biological science of the future. Finally, many current graduate training programs are not attracting a sufficient portion of the most talented young people in the nation. To ameliorate these problems in the training and early career paths of basic biomedical scientists, the authors make specific recommendations, such as urging (1) that graduate trainees should be supported exclusively by competitive individual fellowships, training grants, or institutional funds and not by RO1s or similar research awards; (2) that graduate and postdoctoral stipends be increased so that they provide a reasonable living wage; and (3) that research-intensive academic institutions create a career path for biomedical PhDs other than that designed for the traditional tenure-track, grant-funded principal investigator and faculty member. They conclude that it is in the interest of faculty and institutions to make these and other drastic changes because the current system is both inherently unfair and self-destructive.

Members of the integrin family of cell adhesion receptors influence several important aspects of cancer cell behavior, including motility and invasiveness, cell growth, and cell survival. Engagement of integrins with extracellular matrix (ECM) proteins can activate members of the Rho-family of small GTPases; conversely, Rho- and Ras-family proteins can influence the ability of integrins to bind their ligands. These events impinge on the control of cell motility, and ultimately on invasive and metastatic behavior. Integrin engagement with ECM also has important effects on cell survival, particularly for cells of epithelial origin. In some cases, specific integrins have selective effects on the efficiency of signal transduction in cell survival pathways.

This review critically examines current understanding of the kinetics and biodistribution of antisense oligonucleotides, both at the cellular level and at the level of the intact organism. The pharmacodynamic relationships between biodistribution and the ultimate biological effects of antisense agents are considered. The problems and advantages inherent in the use of delivery systems are discussed in the light of further enhancing in vivo pharmacological actions of oligonucleotides.

Vascular endothelial cells are important in a variety of physiological and pathophysiological processes. The growth and functions of vascular endothelial cells are regulated both by soluble mitogenic and differentiation factors and by interactions with the extracellular matrix; however, relatively little is known about the role of the matrix. In the present study, we investigate whether integrin-mediated anchorage to a substratum coated with the extracellular matrix protein fibronectin regulates growth factor signaling events in human endothelial cells. We show that cell adhesion to fibronectin and growth factor stimulation trigger distinct initial tyrosine phosphorylation events in endothelial cells. Thus, integrin-dependent adhesion of endothelial cells leads to tyrosine phosphorylation of both focal adhesion kinase and paxillin, but not of several growth factor receptors. Conversely, EGF stimulation causes receptor autophosphorylation, with no effect on focal adhesion kinase or paxillin tyrosine phosphorylation. Adhesion to fibronectin, in the absence of growth factors, leads to activation of MAPK. In addition, adhesion to fibronectin also potentiates growth factor signaling to MAPK. Thus, polypeptide growth factor activation of MAPK in anchored cells is far more effective than in cells maintained in suspension. Other agonists known to activate MAPK were also examined for their ability to activate MAPK in an anchorage-dependent manner. The neuropeptide bombesin, the bioactive lipid lysophosphatidic acid (LPA), and the cytokine tumor necrosis factor α, which signal through diverse mechanisms, were all able to activate MAPK to a much greater degree in fibronectin-adherent cells than in suspended cells. In addition, tumor necrosis factor α activation of c-Jun kinase (JNK) was also much more robust in anchored cells. Together, these data suggest a cooperation between integrins and soluble mitogens in efficient propagation of signals to downstream kinases. This cooperation may contribute to anchorage dependence of mitogenic cell cycle progression.

Integrins are adhesion receptors thought to be important in the process of cancer cell invasion and metastasis. Unlike other integrins, which attach a cell to extracellular matrix molecules, the alpha6beta4 integrin participates in the formation of hemidesmosomes, attaching epithelial cells to the basement membrane. Investigations of the alpha6beta4 integrin in human prostatic carcinoma have yielded conflicting results and have been primarily qualitative rather than quantitative. Expression of the beta4 integrin subunit was determined using rat monoclonal antibody 439-9B and image analysis in regions of benign prostatic epithelium (BPE), high-grade prostatic intraepithelial neoplasia (PIN), and prostatic carcinoma (CaP) in 38 patients treated by radical prostatectomy for clinically localized CaP. The beta4 integrin subunit was significantly downregulated in CaP compared with BPE; PIN stained intermediate in intensity between BPE and CaP. Thirty-four of 35 patients showed downregulation of the beta4 integrin subunit, and all 15 patients with PIN had downregulation of beta4 in PIN as compared with BPE. Degree of downregulation of the beta4 integrin subunit did not add prognostic significance to the information present at initial biopsy (age, clinical stage, clinical grade, and serum prostate-specific antigen level). There was no correlation between intensity of staining of CaP, absolute change in staining, or percent loss of beta4 integrin subunit staining with age, pathological stage, or Gleason's score. Downregulation of the beta4 integrin in CaP and PIN compared with BPE may be correlated with neoplastic transformation of the prostate and loss of hemidesmosomes or basal epithelial cells.

Purpose. This study was conducted to investigate the impact of backbone modifications on the hepatobiliary disposition of oligonucleotides.

Recently it has become clear that integrins and other adhesive receptors play an important role in the control of cell growth and differentiation. In various cell types, anchorage to the extracellular matrix via integrins strongly influences the ability of the cell to respond to soluble mitogens or to differentiation factors. Thus adhesive receptors must generate signals that influence cell behavior. Some of the pathways of adhesion receptor signaling are now beginning to be worked out, but there is still much to learn. In particular, the mechanistic basis for the cooperation between anchorage signals and signals from soluble growth and differentiation factors remains ill-defined. This review will examine some of the current information linking adhesion receptors to control of mitogenesis and differentiation.

International audience; The attainment of strong pharmacological effects with oligonucleotides is hampered by inefficient access of these molecules to their sites of action in the cytosol or nucleus. Attempts to address this problem with lipid or polymeric delivery systems have been only partially successful. Here, we describe a novel alternative approach involving the use of a non-toxic small molecule to enhance the pharmacological effects of oligonucleotides. The compound Retro-1 was discovered in a screen for small molecules that reduce the actions of bacterial toxins and has been shown to block the retro-grade trafficking pathway. We demonstrate that Retro-1 can also substantially enhance the effectiveness of antisense and splice switching oligo-nucleotides in cell culture. This effect occurs at the level of intracellular trafficking or processing and is correlated with increased oligonucleotide accumulation in the nucleus but does not involve the perturbation of lysosomal compartments. We also show that Retro-1 can alter the effectiveness of splice switching oligonucleotides in the in vivo setting. These observations indicate that it is possible to enhance the pharmacological actions of oligonucleotides using non-toxic and non-lysosomotropic small molecule adjuncts.

Purpose. The therapeutic use of antisense oligonucleotides will likely involve their administration over protracted periods of time. The oral route of drug dosing offers many advantages over other possible routes when chronic drug administration is necessary. However, little is known about the potential for oligonucleotide uptake from the gastrointestinal tract. This issue is addressed in the current work.

Significant progress is being made concerning the development of oligonucleotides as therapeutic agents. Studies with antisense, siRNA, and other forms of oligonucleotides have shown promise in cellular and animal models and in some clinical studies. Nonetheless, our understanding of how oligonucleotides function in cells and tissues is really quite limited. One major issue concerns the modes of uptake and intracellular trafficking of oligonucleotides, whether as "free" molecules or linked to various delivery moieties such as nanoparticles or targeting ligands. In this review, we examine the recent literature on oligonucleotide internalization and subcellular trafficking in the context of current insights into the basic machinery for endocytosis and intracellular vesicular traffic.

Short DNA or RNA oligonucleotides have tremendous potential as therapeutic agents. Because of their ability to engage in Watson-Crick base pairing, they can interact with mRNA or pre-mRNA targets with high selectivity. As a result, they could precisely manipulate gene expression. This possibility has engendered extensive efforts to develop oligonucleotides as drugs, and many candidates are already in clinical trials. However, a major impediment to the maturation of this field of oligonucleotide-based therapeutics remains: these relatively large and often highly charged molecules don't easily cross cellular membranes, making it difficult for them to reach their sites of action in the cytosol or nucleus. In this Account, we summarize some basic features of the biology of antisense and siRNA oligonucleotides. We then discuss chemical conjugation as an approach to improving the intracellular delivery and therapeutic potential of these agents. Instead of focusing on the details of conjugation chemistry, we emphasize the pharmacological ramifications of oligonucleotide conjugates. In one important approach to improving delivery and efficacy, researchers have conjugated oligonucleotides with ligands designed to bind to particular receptors and thus provide specific interactions with cells. In another strategy, researchers have coupled antisense or siRNA with agents such as cell penetrating peptides that are designed to provoke escape of the conjugate from intracellular vesicular compartments. Although both of these strategies have had some success, further research is needed before oligonucleotide conjugates can find an important place in human therapeutics.

To reduce the adverse effects of cancer therapies and increase their efficacy, new delivery agents that specifically target cancer cells are needed. We and others have shown that aptamers can selectively deliver therapeutic oligonucleotides to the endosome and cytoplasm of cancer cells that express a particular cell surface receptor. Identifying a single aptamer that can internalize into many different cancer cell-types would increase the utility of aptamer-mediated delivery of therapeutic agents. We investigated the ability of the nucleolin aptamer (AS1411) to internalize into multiple cancer cell types and observed that it internalizes into a wide variety of cancer cells and migrates to the nucleus. To determine if the aptamer could be utilized to deliver therapeutic oligonucleotides to modulate events in the nucleus, we evaluated the ability of the aptamer to deliver splice-switching oligonucleotides. We observed that aptamer-splice-switching oligonucleotide chimeras can alter splicing in the nuclei of treated cells and are effective at lower doses than the splice switching oligonucleotides alone. Our results suggest that aptamers can be utilized to deliver oligonucleotides to the nucleus of a wide variety of cancer cells to modulate nuclear events such as RNA splicing.

A series of diwalled and tetrawalled molecular umbrellas have been synthesized using cholic acid, spermidine, and lysine as starting materials. Coupling of these molecular umbrellas to an octaarginine peptide afforded agents that were capable of promoting the transport of small interfering RNA to HeLa cells, as judged by the knockdown of enhanced green fluorescent protein expression. The efficiency of this knockdown was found to increase with an increasing number of facially amphiphilic walls present, and also when a cleavable disulfide linker was replaced with a noncleavable, maleimido moiety; i.e., a group that is not susceptible to thiolate-disulfide interchange. The knockdown efficiency that was observed for one tetrawalled molecular umbrella-octaargine conjugate was comparable to that observed with a commercially available transfection agent, Lipofectamine 2000, but the conjugate showed less cytotoxicity.

This review will discuss the basic concepts concerning the use of cell-targeting peptides (CTPs) and cell-penetrating peptides (CPPs) in the context of nanocarrier technology. It deals with the discovery and subsequent evolution of CTPs and CPPs, issues concerning their interactions with cells and their biodistribution in vivo, and their potential advantages and disadvantages as delivery agents. The article also briefly discusses several specific examples of the use of CTPs or CPPs to assist in the delivery of nanoparticles, liposomes, and other nanocarriers.

Smad4 is a key tumor suppressor that is frequently deleted or inactive in pancreatic and colon tumors. In this report, we describe an approach for attaining selective killing of Smad4-deficient tumor cells. Using a vector system involving a designed repressor with zinc finger binding domains and the herpes simplex virus thymidine kinase (HSV-TK) "suicide gene," we demonstrate Smad4-responsive regulation of HSV-TK expression and consequent altered susceptibility to the prodrug ganciclovir (GCV). In pancreatic tumor cell lines stably transfected with the vector system, a robust differential of HSV-TK expression and GCV toxicity was attained depending on the presence or absence of cotransfected Smad4. In matched colon tumor cell lines lacking Smad4 or expressing physiological levels of Smad4, an adenoviral version of the vector system attained a significant degree of preferential killing of Smad4-negative tumor cells in response to GCV. These findings demonstrate the possibility of achieving selective killing of pancreatic and colon cells depending on their Smad4 status.

ispartof: pages:300-301 ispartof: CHEMISTRY OF NUCLEIC ACID COMPONENTS vol:10 pages:300-301 ispartof: symposium on chemistry of nucleic acid components location:Cesky Krumlov date:8 Jun - 13 Jun 2008 status: published

Expression of the tumor suppressor deleted in liver cancer-1 (DLC-1) is lost in non-small cell lung (NSCLC) and other human carcinomas, and ectopic DLC-1 expression dramatically reduces proliferation and tumorigenicity. DLC-1 is a multi-domain protein that includes a Rho GTPase activating protein (RhoGAP) domain which has been hypothesized to be the basis of its tumor suppressive actions. To address the importance of the RhoGAP function of DLC-1 in tumor suppression, we performed biochemical and biological studies evaluating DLC-1 in NSCLC. Full-length DLC-1 exhibited strong GAP activity for RhoA as well as RhoB and RhoC, but only very limited activity for Cdc42 in vitro. In contrast, the isolated RhoGAP domain showed 5- to 20-fold enhanced activity for RhoA, RhoB, RhoC, and Cdc42. DLC-1 protein expression was absent in six of nine NSCLC cell lines. Restoration of DLC-1 expression in DLC-1-deficient NSCLC cell lines reduced RhoA activity, and experiments with a RhoA biosensor demonstrated that DLC-1 dramatically reduces RhoA activity at the leading edge of cellular protrusions. Furthermore, DLC-1 expression in NSCLC cell lines impaired both anchorage-dependent and -independent growth, as well as invasion in vitro. Surprisingly, we found that the anti-tumor activity of DLC-1 was due to both RhoGAP-dependent and -independent activities. Unlike the rat homologue p122RhoGAP, DLC-1 was not capable of activating the phospholipid hydrolysis activity of phospholipase C-delta1. Combined, these studies provide information on the mechanism of DLC-1 function and regulation, and further support the role of DLC-1 tumor suppression in NSCLC.

Liposome delivery technology was used to permit the utilization of valinomycin as a novel, membrane-active antitumor drug. The incorporation of valinomycin in liposomes profoundly reduces the toxicity of the drug to mice. Liposomal valinomycin, however, retains significant antitumor activity. Thus the use of liposomes permits an improvement in the therapeutic index of valinomycin when used as an individual antitumor drug. Perhaps of even greater interest, it was shown that valinomycin can have profound effects on the phenomenon of membrane-based drug resistance. Thus very low doses of valinomycin, in either free or liposomal form, reverse doxorubicin resistance. The mechanism involved is unclear at this time, but seems to be different from effects mediated by calcium channel blockers which work by hindering the action of the P-glycoprotein efflux pump. Since liposomal valinomycin is relatively nontoxic one may be able to modulate doxorubicin resistance of tumors in vivo, thus permitting more effective therapy with minimal toxicity to the host.

A simple and efficient method for the preparation of pyrimidine 2 ′-O-hydroxyethoxymethylribonucleosides and 2 ′-O-hydroxypropoxymethylribonucleosides has been developed. These modified nucleosides were incorporated into oligoribonucleotides, which were shown to form stable RNA/RNA duplexes. The effect of 2 ′ -O-modification in the antisense and sense strands of small interference RNA was evaluated in multi-drug resistant NIH 3T3 cells.

CeNA is an oligonucleotide where the (deoxy)ribose sugars have been replaced by cyclohexenyl moieties. We have determined the NMR structure of a CeNA:RNA duplex and have modeled this duplex in the crystal structure of a PIWI protein. An N puckering of the ribose nucleosides, a 2H3 conformation of the cyclohexenyl nucleosides, and an A-like helix conformation of the backbone, which deviates from the standard A-type helix by a larger twist and a smaller slide, are observed. The model of the CeNA:RNA duplex bound to the PIWI protein does not show major differences in the interaction of the guide CeNA with the protein when compared with dsRNA, suggesting that CeNA modified oligonucleotides might be useful as siRNAs. Incorporation of one or two CeNA units in the sense or antisense strands of dsRNA led to similar or enhanced activity compared to unmodified siRNAs. This was tested by targeting inhibition of expression of the MDR1 gene with accompanying changes in P-glycoprotein expression, drug transport, and drug resistance.

Altritol-modified nucleic acids (ANAs) support RNA-like A-form structures when included in oligonucleotide duplexes. Thus altritol residues seem suitable as candidates for the chemical modification of siRNAs. Here we report that ANA-modified siRNAs targeting the MDR1 gene can exhibit improved efficacy as compared to unmodified controls. This was particularly true of ANA modifications at or near the 3' end of the sense or antisense strands, while modification at the 5' end of the antisense strand resulted in complete loss of activity. Multiple ANA modifications within the sense strand were also well tolerated. Duplexes with ANA modifications at appropriate positions in both strands were generally more effective than duplexes with one modified and one unmodified strand. Initial evidence suggests that the loss of activity associated with ANA modification of the 5'-antisense strand may be due to reduced phosphorylation at this site by cellular kinases. Treatment of drug resistant cells with MDR1-targeted siRNAs resulted in reduction of P-glycoprotein (Pgp) expression, parallel reduction in MDR1 message levels, increased accumulation of the Pgp substrate rhodamine 123, and reduced resistance to anti-tumor drugs. Interestingly, the duration of action of some of the ANA-modified siRNAs was substantially greater than that of unmodified controls. These observations suggest that altritol modifications may be helpful in developing siRNAs with enhanced pharmacological effectiveness. ispartof: Nucleic acids research vol:35 issue:4 pages:1064-1074 ispartof: location:England status: published

Enhancing the delivery of antisense and siRNA molecules to cells and tissues is a key issue for oligonucleotide therapeutics. Cell-penetrating peptides (CPPs) have the ability to convey linked "cargo" molecules into the cytosol; thus we have explored the use of CPPs as delivery agents for oligonucleotides. We have extensively evaluated CPP-oligonucleotide conjugates, and have recently begun to explore the use of CPP-dendrimer-oligonucleotide complexes. We have found that CPP-antisense oligonucleotide conjugates can be taken up by cells and can effectively modify gene expression in cell culture and in tissues. Although not as potent in cell culture as cationic lipid delivery agents, CPP-oligonucleotide conjugates offer the advantage of being molecules rather than particles, and may have substantial advantages over particle-based delivery in the in vivo setting.

Cyclohexenyl nucleic acids and altritol nucleic acids were evaluated as RNA modifications in an siRNA experiment targeting the P-glycoprotein expression. Both modified oligonucleotides are well accepted as RNA mimics leading to an increase in potency and duration of action of the gene-silencing effect.

The delivery of therapeutic, detection and imaging agents for the diagnosis and treatment of cancer patients has improved dramatically over the years with the development of nano-carriers such as liposomes, micelles, dendrimers, biomolecules, polymer particles, and colloidal precipitates. While many of these carriers have been used with great success in vitro and in vivo , each suffers from serious drawbacks with regard to stability, flexibility, or functionality. To date, there has been no general particle fabrication method available that afforded rigorous control over particle size, shape, composition, cargo and chemical structure. By utilizing the method we has designed referred to as P article R eplication I n N on-wetting T emplates, or PRINT , we can fabricate monodisperse particles with simultaneous control over structure ( i.e. shape, size, composition) and function ( i.e. cargo, surface structure). Unlike other particle fabrication techniques, PRINT is delicate and general enough to be compatible with a variety of important next-generation cancer therapeutic, detection and imaging agents, including various cargos (e.g. DNA, proteins, chemotherapy drugs, biosensor dyes, radio-markers, contrast agents), targeting ligands (e.g. antibodies, cell targeting peptides) and functional matrix materials (e.g. bioabsorbable polymers or stimuli responsive matrices). PRINT makes this possible by utilizing low-surface energy, chemically resistant fluoropolymers as molding materials and patterned substrates to produce functional, harvestable, monodisperse polymeric particles.

PAMAM G5 dendrimer (P) was conjugated to Tat peptide (T), a cell penetrating peptide, in search of an efficient cellular delivery vehicle for antisense and siRNA oligonucleotides.PAMAM G5 dendrimer was reacted with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, sulfosuccinimidyl ester, sodium salt (BODIPY) for visualization to yield the conjugate BP. Bifunctional sulfosuccinimidyl 6-[alpha-methyl-alpha-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-SMPT) was then used to conjugate primary amino groups of BP to cysteine derivatized Tat peptide to give the designed conjugate, BPT. This conjugate was complexed with antisense and siRNA oligonucleotides designed to inhibit MDR1 gene expression. NIH 3T3 MDR cells were used for the evaluation of biological activity of the conjugate.Both antisense and siRNA readily formed complexes with the synthesized BPT, introduced into NIH 3T3 MDR cells, and primarily accumulated in intracellular vesicles. MDR1 gene expression was partially inhibited by the antisense-BPT complex and weakly inhibited by the siRNA-BPT complex when both were tested at nontoxic levels of dendrimer. Conjugation with Tat peptide did not improve the delivery efficiency of the dendrimer.Dendrimer-oligonucleotide complexes were moderately effective for delivery of antisense and only poorly effective for delivery of siRNA. Conjugation of the dendrimer with the Tat cell penetrating peptide failed to further enhance the effectiveness of the dendrimer.

Conjugation of oligonucleotides to certain types of peptides provides an interesting approach for enhancing delivery of antisense and siRNA to cells and tissues. This article will provide a perspective on issues in oligonucleotide delivery, and will examine recent literature on the preparation and use of several types of peptide-oligonucleotide conjugates.

Hexitol nucleic acids (HNAs) are nuclease resistant and provide strong hybridization to RNA. However, there is relatively little information on the biological properties of HNA antisense oligonucleotides. In this study, we compared the antisense effects of a chimeric HNA 'gapmer' oligonucleotide comprising a phosphorothioate central sequence flanked by 5' and 3' HNA sequences to conventional phosphorothioate oligonucleotides and to a 2'-O-methoxyethyl (2'-O-ME) phosphorothioate 'gapmer'. The antisense oligomers each targeted a sequence bracketing the start codon of the message of MDR1, a gene involved in multi-drug resistance in cancer cells. Antisense and control oligonucleotides were delivered to MDR1-expressing cells using transfection with the cationic lipid Lipofectamine 2000. The anti-MDR1 HNA gapmer was substantially more potent than a phosphorothioate oligonucleotide of the same sequence in reducing expression of P-glycoprotein, the MDR1 gene product. HNA and 2'-O-ME gapmers displayed similar potency, but a pure HNA antisense oligonucleotide (lacking the phosphorothioate 'gap') was ineffective, indicating that RNase H activity was likely required. Treatment with anti-MDR1 HNA gapmer resulted in increased cellular accumulation of the drug surrogate Rhodamine 123 that correlated well with the reduced cell surface expression of P-glycoprotein. Thus, HNA gapmers may provide a valuable additional tool for antisense-based investigations and therapeutic approaches.