Intracellular Trafficking and Endosomal Release of Oligonucleotides: What We Know and What We Don't.

Understanding the cellular uptake and intracellular trafficking of oligonucleotides provides an important basic underpinning for the developing field of oligonucleotide-based therapeutics. Whether delivered as "free" oligonucleotides, as ligand-oligonucleotide conjugates, or in association with various nanocarriers, all forms of oligonucleotide enter cells by endocytosis and are initially ensconced within membrane-limited vesicles. Accordingly, the locus and extent of release to the cytosol and nucleus are key determinants of the pharmacological actions of oligonucleotides. A number of recent studies have explored the intracellular trafficking of various forms of oligonucleotides and their release from endomembrane compartments. These studies reveal a surprising convergence on an early-intermediate compartment in the trafficking pathway as the key locus of release for oligonucleotides administered in "free" form as well as those delivered with lipid complexes. Thus, oligonucleotide release from multivesicular bodies or from late endosomes seems to be the crucial endogenous process for attaining pharmacological effects. This intrinsic process of oligonucleotide release may be amplified by delivery agents such as lipid complexes or small molecule enhancers.

RGD Conjugated Dendritic Polylysine for Cellular Delivery of Antisense Oligonucleotide.

Dendritic polylysines (DPL) are highly branched nano-sized spherical polymer with positively charged primary amino groups on surface. This structural feature is useful for a delivery of antisense oligonucleotide or siRNA. In this study, we modified the surface of DPL with cyclic RGD (and iRGD) peptide by conjugation reaction generating RGD (and iRGD) peptide conjugated dendritic poly-lysines, RGD-DPL or iRGD-DPL. The prepared conjugates were evaluated for integrin receptor-mediated cellular delivery of antisense oligonucleotide. The conjugation of RGD or iRGD peptide on DPL was monitored by measuring the retention time in capillary zone electrophoresis and the absorbance at UV-Vis spectroscopy. Cellular delivery by DPL-RGD (or -iRGD)/antisense oligonucleotide complex was examined by antisense splicing correction assay on integrin alpha v/beta 3 positive A375B3-Luc cells, which were stably transfected with plasmid pLuc/705. DPL-RGD (or -iRGD)/antisense oligonucleotide complexes exhibited integrin receptor mediated uptake on A375B3 cells without inducing cellular toxicity. In addition, the delivery of antisense oligonucleotide was integrin receptor-dependent with moderate efficiency.

Cellular uptake and intracellular trafficking of oligonucleotides.

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.

High-throughput screening identifies small molecules that enhance the pharmacological effects of oligonucleotides.

The therapeutic use of antisense and siRNA oligonucleotides has been constrained by the limited ability of these membrane-impermeable molecules to reach their intracellular sites of action. We sought to address this problem using small organic molecules to enhance the effects of oligonucleotides by modulating their intracellular trafficking and release from endosomes. A high-throughput screen of multiple small molecule libraries yielded several hits that markedly potentiated the actions of splice switching oligonucleotides in cell culture. These compounds also enhanced the effects of antisense and siRNA oligonucleotides. The hit compounds preferentially caused release of fluorescent oligonucleotides from late endosomes rather than other intracellular compartments. Studies in a transgenic mouse model indicated that these compounds could enhance the in vivo effects of a splice-switching oligonucleotide without causing significant toxicity. These observations suggest that selected small molecule enhancers may eventually be of value in oligonucleotide-based therapeutics.

Cellular uptake and intracellular trafficking of oligonucleotides: implications for oligonucleotide pharmacology.

One of the major constraints on the therapeutic use of oligonucleotides is inefficient delivery to their sites of action in the cytosol or nucleus. Recently it has become evident that the pathways of cellular uptake and intracellular trafficking of oligonucleotides can strongly influence their pharmacological actions. Here we provide background information on the basic processes of endocytosis and trafficking and then review recent literature on targeted delivery and subcellular trafficking of oligonucleotides in that context. A variety of approaches including molecular scale ligand-oligonucleotide conjugates, ligand-targeted nanocarriers, and the use of small molecules to enhance oligonucleotide effects are discussed.

Covalent conjugation of oligonucleotides with cell-targeting ligands.

A continuing problem in the area of oligonucleotide-based therapeutics is the poor access of these molecules to their sites of action in the nucleus or cytosol. A number of approaches to this problem have emerged. One of the most interesting is the use of ligand-oligonucleotide conjugates to promote receptor mediated cell uptake and delivery. Here we provide an overview of recent developments regarding targeted conjugates, including use of peptides, carbohydrates and small molecules as ligands. Additionally we discuss our own experience with this approach and point out both advantages and limitations.

Receptors, endocytosis, and trafficking: the biological basis of targeted delivery of antisense and siRNA oligonucleotides.

The problem of targeted delivery of antisense and siRNA oligonucleotides can be resolved into two distinct aspects. The first concerns devising ligand-oligonucleotide or ligand-carrier moieties that bind with high selectivity to receptors on the cell type of interest and that are efficiently internalized by endocytosis. The second concerns releasing oligonucleotides from pharmacologically inert endomembrane compartments so that they can access RNA in the cytosol or nucleus. In this review, we will address both of these aspects. Thus, we present information on three important receptor families, the integrins, the receptor tyrosine kinases, and the G protein-coupled receptors in terms of their suitability for targeted delivery of oligonucleotides. This includes discussion of receptor abundance, internalization and trafficking pathways, and the availability of suitable high affinity ligands. We also consider the process of oligonucleotide uptake and intracellular trafficking and discuss approaches to modulating these processes in a pharmacologically productive manner. Hopefully, the basic information presented in this review will be of value to investigators involved in designing delivery approaches for oligonucleotides.

The chemistry and biology of oligonucleotide conjugates.

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.

Targeted albumin-based nanoparticles for delivery of amphipathic drugs.

We report the preparation and physical and biological characterization of human serum albumin-based micelles of approximately 30 nm 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 α(v)ß(3) integrin. Multiple poly(ethylene glycol)s (PEGs) with molecular weight of 3400 Da 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 α(v)ß(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.

Role of DLC-1, a tumor suppressor protein with RhoGAP activity, in regulation of the cytoskeleton and cell motility.

DLC-1 was originally identified as a potential tumor suppressor. One of the key biochemical functions of DLC-1 is to serve as a GTPase activating protein (GAP) for members of the Rho family of GTPases, particularly Rho A-C and Cdc 42. Since these GTPases are critically involved in regulation of the cytoskeleton and cell migration, it seems clear that DLC-1 will also influence these processes. In this review we examine basic aspects of the actin cyoskeleton and how it relates to cell motility. We then delineate the characteristics of DLC-1 and other members of its family, and describe how they may have multiple effects on the regulation of cell polarity, actin organization, and cell migration.

Intracellular delivery of oligonucleotide conjugates and dendrimer complexes.

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.

Epigenetic modulation of gene expression in mammalian cells.

The precisely controlled regulation of gene expression plays an important role in normal cellular function, whereas abnormalities of gene expression can contribute to cancer and other diseases. Thus, the ability to selectively regulate gene expression is of tremendous importance, both as a basic research tool and as a potential therapeutic approach. Over the last two decades, several different strategies have been used for controlling expression of specific genes at the transcriptional and post-transcriptional levels. The best-known approach involves antisense oligonucleotides that downregulate gene expression through various mechanisms, most importantly by triggering the degradation of mRNA. The use of designed transcription factors has also developed into an interesting technology since it allows either the enhancement or the reduction of expression of a targeted gene. Recently RNA interference has emerged as a powerful tool for selective inhibition of gene expression. This review examines the basic aspects, advantages, and liabilities of these three approaches to gene modulation.

Delivery of MDR1 small interfering RNA by self-complementary recombinant adeno-associated virus vector.

Small interfering RNAs (siRNAs) are potentially powerful tools for therapeutic gene regulation. DNA cassettes encoding RNA polymerase III promoter-driven hairpin siRNAs allow long-term expression of siRNA in targeted cells. A variety of viral vectors have been used to deliver such cassettes to cells. Here we report on the development and use of a self-complementary recombinant adeno-associated virus (scAAV) vector for siRNA delivery into mammalian cells. We demonstrate that this modified vector efficiently delivers siRNA into multidrug-resistant human breast and oral cancer cells and suppresses MDR1 gene expression. This results in rapid, profound, and durable reduction in the expression of the P-glycoprotein multidrug transporter and a substantial reversion of the drug-resistant phenotype. This research suggests that scAAV-based vectors can be very effective agents for efficient delivery of therapeutic siRNA.

Selective transcription of p53 target genes by zinc finger-p53 DNA binding domain chimeras.

Active p53 stimulates the transcription of a number of key genes, including the pro-apoptotic gene bax, as well as p21, a cell cycle regulator. In this study we constructed novel chimeric zinc finger-p53 DNA binding domain (DBD) transcription factors designed to bind to the promoters of specific p53 regulated genes. In order to selectively increase the expression of Bax, we coupled a pre-selected three-zinc finger (Zif) peptide targeted to a sequence in the bax promoter to a minimal p53 DBD. This chimeric protein could increase reporter gene transcription from a minimal bax promoter (up to 10-fold) but not from a minimal p21 promoter in p53-deficient Saos-2 cells. However, fusion proteins carrying longer p53 DBDs displayed entirely different selectivity and potency. Thus, Zif-p53 DBD chimeras containing N- and C-terminal extensions of the minimal DBD could increase transcription driven by a minimal p21 promoter up to 800-fold. These chimeras preferred the minimal p21 promoter up to 500-fold over the minimal bax promoter. Additionally, endogenous p21 message and protein levels were increased in cells expressing the p21 selective Zif-p53 DBD chimera and expression of the chimeric proteins resulted in partial cell cycle arrest. Cell fractionation experiments indicated that the Zifs enhanced nuclear localization of the Zif-p53 DBD chimera. These studies suggest that it is possible to create chimeric transcription factors able to strongly and selectively activate genes downstream of p53.

Strategies for inhibition of MDR1 gene expression.

Several distinct strategies have been used to modulate the expression of cancer-associated genes, including antisense oligonucleotides, small interfering RNAs (siRNAs), and artificial transcriptional factors. One major cause for chemotherapeutic treatment failure in cancer is the overexpression of P-glycoprotein, the product of the multidrug resistance gene MDR1. In this study, we tested the ability of siRNAs to inhibit MDR1 gene expression. We evaluated the efficiency of chemically synthesized dsRNAs as well as vector-based hairpin siRNAs and investigated the behavior of clones of multidrug-resistant NCI/ADR-RES breast carcinoma cells stably transfected with hairpin siRNA vectors. The effects of siRNA on the MDR phenotype were compared with those elicited by antisense oligonucleotides or by designed transcription factors targeting the MDR1 promoter. These studies suggest that there are several comparably effective strategies for inhibiting MDR1 expression.

Integrin regulation of cell signalling and motility.

Integrins clearly play a key role in regulating both mitogenic signalling and cell migration. Thus integrins modulate the efficiency of the Erk (extracellular-signal-regulated kinase)/MAP kinase (mitogen-activated protein kinase) pathway, acting at several distinct levels. We have shown that both cAMP-dependent protein kinase and PAKs (p21-activated kinases) play a role in integrin regulation of the Erk pathway, acting primarily at the level of Raf-1. Integrins and PAKs also play a role in the control of cell migration. Thus we have discovered a novel protein that links the alpha5beta1 integrin to migration controlled by Rho-family GTPases. This protein, termed Nischarin, is a large cytosolic macromolecule that is not related to well-known protein families. The N-terminus of Nischarin interacts with a short segment of the cytoplasmic domain of the alpha5 integrin subunit. Overexpression of Nischarin alters actin organization and inhibits Rac-driven cell migration and tumour cell invasion. Use of effector domain mutants of Rac suggest that Nischarin acts downstream of Rac, probably at the level of PAK-family kinases. These studies emphasize the intricate connection between integrins and Rho-family GTPases and their effectors in controlling both mitogenesis and migration.

Regulation of signal transduction by integrins.

The integrin family of cell membrane receptors plays an important role in signal transduction cascades. Ligation of integrins by extracellular matrix proteins can lead to direct activation of Rho-family GTPases and MAP kinase pathways. However, perhaps the most significant signaling function of integrins is to modulate signal transduction events initiated by receptor tyrosine kinases and G protein-coupled receptors. This probably plays a role in coordinating information about cell shape and position with information about the availability of soluble growth factors.