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.
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.
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.
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.
Abstract
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.
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.
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.
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.
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.
Abstract
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.
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