Oligonucleotide Enhancing Compound Increases Tricyclo-DNA Mediated Exon-Skipping Efficacy in the Mdx Mouse Model.

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.

Enhanced delivery of peptide-morpholino oligonucleotides with a small molecule to correct splicing defects in the lung.

Pulmonary diseases offer many targets for oligonucleotide therapeutics. However, effective delivery of oligonucleotides to the lung is challenging. For example, splicing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) affect a significant cohort of Cystic Fibrosis (CF) patients. These individuals could potentially benefit from treatment with splice switching oligonucleotides (SSOs) that can modulate splicing of CFTR and restore its activity. However, previous studies in cell culture used oligonucleotide transfection methods that cannot be safely translated in vivo. In this report, we demonstrate effective correction of a splicing mutation in the lung of a mouse model using SSOs. Moreover, we also demonstrate effective correction of a CFTR splicing mutation in a pre-clinical CF patient-derived cell model. We utilized a highly effective delivery strategy for oligonucleotides by combining peptide-morpholino (PPMO) SSOs with small molecules termed OECs. PPMOs distribute broadly into the lung and other tissues while OECs potentiate the effects of oligonucleotides by releasing them from endosomal entrapment. The combined PPMO plus OEC approach proved to be effective both in CF patient cells and in vivo in the mouse lung and thus may offer a path to the development of novel therapeutics for splicing mutations in CF and other lung diseases.

Chemical Manipulation of the Endosome Trafficking Machinery: Implications for Oligonucleotide Delivery.

Antisense oligonucleotides (ASOs), siRNA and splice switching oligonucleotides (SSOs) all have immense potential as therapeutic agents, potential that is now being validated as oligonucleotides enter the clinic. However, progress in oligonucleotide-based therapeutics has been limited by the difficulty in delivering these complex molecules to their sites of action in the cytosol or nucleus of cells within specific tissues. There are two aspects to the delivery problem. The first is that most types of oligonucleotides have poor uptake into non-hepatic tissues. The second is that much of the oligonucleotide that is taken up by cells is entrapped in endosomes where it is pharmacologically inert. It has become increasingly recognized that endosomal trapping is a key constraint on oligonucleotide therapeutics. Thus, many approaches have been devised to address this problem, primarily ones based on various nanoparticle technologies. However, recently an alternative approach has emerged that employs small molecules to manipulate intracellular trafficking processes so as to enhance oligonucleotide actions. This review presents the current status of this chemical biology approach to oligonucleotide delivery and seeks to point out possible paths for future development.

Addressing cancer signal transduction pathways with antisense and siRNA oligonucleotides.

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.

Impact of the Endosomal Escape Activity of Cell-Penetrating Peptides on the Endocytic Pathway.

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.

Cytosolic Delivery of Macromolecules in Live Human Cells Using the Combined Endosomal Escape Activities of a Small Molecule and Cell Penetrating Peptides.

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.

Retro-1-Oligonucleotide Conjugates. Synthesis and Biological Evaluation.

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⁻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.

Structure-activity relationships and cellular mechanism of action of small molecules that enhance the delivery of oligonucleotides.

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.

A Novel Family of Small Molecules that Enhance the Intracellular Delivery and Pharmacological Effectiveness of Antisense and Splice Switching Oligonucleotides.

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 delivery of therapeutic oligonucleotides.

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.

DNA Three-Way Junctions Stabilized by Hydrophobic Interactions for Creation of Functional Nanostructures.

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 (<20 nm) with shorter double-stranded regions. UV thermal melting studies show that lipophilic spacers strategically placed within the junction space significantly increased thermal stability. For a 3WJ with eight base pair arms, thermal stability was increased from 30.5 °C for the unmodified junction to a maximum stability of 55.0 °C. The stability of the junction can be modulated within this temperature range by using the appropriate combinations of spacers.

Conditional control of alternative splicing through light-triggered splice-switching oligonucleotides.

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 oligonucleotides (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.

Multicellular tumor spheroids as a model for assessing delivery of oligonucleotides in three dimensions.

Oligonucleotides have shown promise in selectively manipulating gene expression in vitro, but that success has not translated to the clinic for cancer therapy. A potential reason for this is that cells behave differently in monolayer than in the three-dimensional tumor, resulting in limited penetration and distribution of oligonucleotides in the tumor. This may be especially true when oligonucleotides are associated with nanocarriers such as lipoplexes and polyplexes, commonly used delivery vehicles for oligonucleotides. The multicellular tumor spheroid (MCTS), a three-dimensional model that closely resembles small avascular tumors and micrometastases, has been utilized as an intermediate between monolayer culture and in vivo studies for the screening of small-molecule drugs. However, spheroids have been little used for the study of various oligonucleotide delivery formulations. Here, we have evaluated the uptake and efficacy of splice-switching antisense oligonucleotides using various delivery modalities in two- and three-dimensional culture models. We find that the size of the delivery agent dramatically influences penetration into the spheroid and thus the biological effect of the oligonucleotides. We hypothesize that the MCTS model will prove to be a useful tool in the future development of oligonucleotide delivery formulations.Molecular Therapy-Nucleic Acids (2014) 3, e153; doi:10.1038/mtna.2014.5; published online 11 March 2014.

The small molecule Retro-1 enhances the pharmacological actions of antisense and splice switching oligonucleotides.

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 retrograde trafficking pathway. We demonstrate that Retro-1 can also substantially enhance the effectiveness of antisense and splice switching oligonucleotides 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.

Aptamer-mediated delivery of splice-switching oligonucleotides to the nuclei of cancer cells.

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.

Cellular uptake and intracellular trafficking of antisense and siRNA oligonucleotides.

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.

A molecular umbrella approach to the intracellular delivery of small interfering RNA.

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.

Unconventional internalization mechanisms underlying functional delivery of antisense oligonucleotides via cationic lipoplexes and polyplexes.

There is mounting interest in developing antisense and siRNA oligonucleotides into therapeutic entities; however, this potential has been limited by poor access of oligonucleotides to their pharmacological targets within cells. Transfection reagents, such as cationic lipids and polymers, are commonly utilized to improve functional delivery of nucleic acids including oligonucleotides. Cellular entry of large plasmid DNA molecules with the assistance of these polycationic carriers is mediated by some form of endocytosis; however, the mechanism for delivery of small oligonucleotide molecules has not been well established. In this study, splice-shifting oligonucleotides have been formulated into cationic lipoplexes and polyplexes, and their internalization mechanisms have been examined by using pharmacological and genetic inhibitors of endocytosis. The results showed that intercellular distribution of the oligonucleotides to the nucleus governs their pharmacological response. A mechanistic study revealed that oligonucleotides delivered by lipoplexes enter the cells partially by membrane fusion and this mechanism accounts for the functional induction of the target gene. In contrast, polyplexes are internalized by unconventional endocytosis pathways that do not require dynamin or caveolin. These studies may help rationally design novel delivery systems with superior transfection efficiency but lower toxicity.

Intracellular delivery of an antisense oligonucleotide via endocytosis of a G protein-coupled receptor.

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.

Targeted intracellular delivery of antisense oligonucleotides via conjugation with small-molecule ligands.

Selective delivery of antisense or siRNA oligonucleotides to cells and tissues via receptor-mediated endocytosis is becoming an important approach for oligonucleotide-based pharmacology. In most cases receptor targeting has been attained using antibodies or peptide-type ligands. Thus, there are few examples of delivering oligonucleotides using the plethora of small-molecule receptor-specific ligands that currently exist. In this report we describe a facile approach to the generation of mono- and multivalent conjugates of oligonucleotides with small-molecule ligands. Using the sigma-receptor ligand anisamide as an example, we describe conversion of the ligand to a phosphoramidite and direct incorporation of this moiety into the oligonucleotide by solid-phase DNA synthesis. We generated mono- and trivalent conjugates of anisamide with a splice switching antisense oligonucleotide (SSO) and tested their ability to modify splicing of a reporter gene (luciferase) in tumor cells in culture. The trivalent anisamide-SSO conjugate displayed enhanced cellular uptake and was markedly more effective than an unconjugated SSO or the monovalent conjugate in modifying splicing of the reporter. Significant biological effects were attained in the sub-100 nM concentration range.